FLUOROUS ASSISTED ONE-POT OLIGOSACCHARIDE SYNTHESIS AND
CHEMICAL SYNTHESIS OF HOMOGENEOUS HEPARAN SULFATE
PROTEOGLYCAN
By
Bo Yang

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Chemistry
2012

ABSTRACT
FLUOROUS ASSISTED ONE-POT OLIGOSACCHARIDE SYNTHESIS AND
CHEMICAL SYNTHESIS OF HOMOGENEOUS HEPARAN SULFATE
PROTEOGLYCAN
By
Bo Yang
Pre-activation based glycosylation is a powerful method for synthesizing
bioloigically important oligosaccharides. Chapter 1 summarized the progress in this area
in recent years. This approach has unique characteristics compared to traditional
oligosaccharide synthetic methods. This chapter discussed its chemoselectivity and
stereoselectivity in details. Pre-activation based iterative one-pot synthesis greatly
accelerated the oligosaccharide assembly process. To further improve the overall
synthetic efficiency, we developed a new method which was described in Chapter 2.
This new method combines one-pot synthesis and fluorous separation. After one-pot
glycosylation is complete, a fluorous tag is introduced into the reaction mixture to
selectively “catch” the desired oligosaccharide to form fluorous tagged product, which is
isolated from other impurities by fluorous solid phase extraction (F-SPE). Followed by
“release” and another “F-SPE”, the purification of desired product is achieved. A
trisaccharide and a tetrasaccharide are synthesized using this method. Hpearan sulfate
proteoglycans

(HSPGs),

including

syndecans

and

glypicans,

are

ubiquitous

components of mammalian cell surface and extracellular matrix and they play important
roles in various biological processes such as cancer development, angiogenesis. viral
infection and wound repair. They are composed of heparan sulfate (HS),
tetrasaccharide linkage and core protein and increasing evidence shows both HS and
core protein can direct the function of HSPGs. To establish structure-activity relationship,

it is important to obtain homogeneous HSPGs. However, naturally existing HSPGs are
higly heterogeneous and chemical synthesis has never been reported. To address this
issue, we chemically synthesized the ectodomain of human syndecan-1. Chapter 3
described an efficient synthetic route which has been established to synthesize a HS
bearing glycopeptide. The success of the synthesis will lay the foundation for accessing
other proteoglycan structures.

DEDICATION
I would like to dedicate this thesis to my father Wanxiang Yang and my mother
Taohong Wu their endless support, love and faith in me in the past years and to my
loved wife, Xiaomin Shi, who always supported me and stood by me through difficult
times during my graduate studies. They all taught me how to believe in myself, work
hard for my dreams and achieve success.

iv

ACKNOWLEDGEMENTS
I received lots of help from different people, including professors and labmates.
Without them, I could not accomplish anything during the past few years.
First of all, I would like to thank my advisor, Prof. Xuefei Huang who prepared my
life for my future career. When I came to the States in 2006, I barely knew what
“Science” is. He gave me a lot of training and taught me how to do research. His
patience, dedication and intelligence impressed me. Under his guidance, now I became
a more mature and independent person.
I would also like to thank Dr. Daniel Holmes and Mr. Kermit Johnson for training
me how to do NMR experiments. Also, Dr. Daniel Jones and Ms. Lijun Chen helped me
a lot in how to use mass spectrometer.
Special thanks to my previous labmates, Dr. Bin Sun and Dr. Zhen Wang (Jason).
When I was a first year graduate student, they taught me a lot of sugar chemistry and
basic organic synthetic skills.
Thanks for Prof. Xudong Fan for teaching me how to operate Transmission
Electron Microscope (TEM) when I was working on nanoparticle project.
Also, I would like to acknowledge all the people I met during my graduate studies,
and all former and present labmates who gave me help in one way or another. In no
particular order those people are: Dr. Qi Chen, Dr. Mohamad El-Dakdouki, Dr. Medha
Kamat, Dr. Adeline Miermont, Balasubramanian Srinivasan (Bala), Dr. Gopinath
Tiruchinapally, Dr. Jingguang Xia, Dr. Zhaojun Yin, Dr. Keisuke Yoshida, Dr. Youlin
Zeng, Gilbert Wasonga, Luyuan Zhou, Nardos Teumelsan, Herbert Kavunja, Qian Qin,

v

Peng Wang, Xiaowei Lu, Yuqing Jing, Philip Bentley, Steven DuLaney, and Hovig
Kouyoumdjian. I have learnt a lot from discussions with them either on group meetings
or at casual time and I really enjoyed my past few years working with these guys.
Thanks for Dr. Steven Sucheck for his assistance and guidance in the courses I
took with him.
Thanks for my committee members: Dr. William Wulff, Dr. Gregory Baker and Dr.
Gary Blanchard.

vi

TABLE OF CONTENTS
LIST OF TABLES………………………………………………………………………..x
LIST OF FIGURES …………………………………………………………………......xi
LIST OF ABBREVIATIONS ……………………………………………………………xiv
LIST OF SCHEMES …………………………………………………………………....xxi
CHAPTER 1 Pre-activation Based Glycosylation Strategy ……………………1
1.1. Introduction ……………………………………………………………….1
1.2. Selectivity in Glycosidic Bond Formation………………………………3
1.2.1. Chemoselectivity ……………………………………………….. 3
1.2.2. Stereoselectivity …………………………………………………5
1.2.3. Regioselectivity ………………………………………………….8
1.3. Chemoselectivity in Pre-activation Based Glycosylation ……………9
1.3.1. Chemoselective Glycosylation of Glycal ………………….…9
1.3.2. Chemoselective Dehydrative Glycosylation …………………11
1.3.3. Bromoglycoside-Mediated Iterative Glycosylation of
Selenoglycoside ………………………………………………17
1.3.4. Chemoselecitve Glycosylation of
Thioglycoside ………………………………………………… 20
1.4. Stereoselectivity in Pre-activation Based
Glycosylation ………………………………………………………… 31
1.4.1. Protecting Groups on Donor ……………………………… …32
1.4.1.1. Anomeric Leaving Group Effect ………………… 32
1.4.1.2. Vicinal Neighboring Group Participation …………36
1.4.1.3. Remote Neighboring Group Participation …… …45
1.4.1.4. 4, 6-O-Benzylidene Acetal Effect …………… ……48
1.4.1.5. Cyclic Carbonate Effect ……………………………55
1.4.1.6. Oxazolidinone Effect …………………………………59
1.4.1.7. Inductively Disarming Effect…………………………62
1.4.1.8. Steric Effect……………………………………………66
1.4.1.9. 3, 4-Bisacetal Effect …………………………………68
1.4.1.10. 2, 6-Anhdrosugar as Donor …………………………71
1.4.2. Acceptor Effect ……………………………………………………73
1.4.3. Promoter Effect ……………………………………………………74
1.4.4. Solvent and Additive Effect ………………………………………75
1.5. Conclusions ………………………………………………………………80
References …………………………………………………………………83
CHAPTER 2 Fluorous-Assisted One-pot Oligosaccharide Synthesis …….99
2.1. Introduction ………………………………………………………………99

vii

2.2.

2.3.
2.4.

2.5.

Results and Discussion ……………………………………………103
2.2.1. Designing a New “Catch” and “Release” Strategy…………..103
2.2.2. Screening Different Linkers for “Catch” and Release” Strategy
…………………………………………………………………………106
2.2.3. Synthesis of a Disaccharide as a Model Study …….………..109
2.2.4. Synthesis of LewisX Trisaccharide
………………………..111
2.2.5. Synthesis of a Linear Tetrasaccharide ……………………..112
2.2.6. Stability of Glycosidic Linkage toward Acidic Conditions ….113
Conclusions ………………….……………….……………….………114
Experimental Section …………………………………………………114
2.4.1. General Experimental Procedure ……………………………..114
2.4.2. Characterization of Anomeric Chemistry ……………………...115
2.4.3. General Procedure for “Catch” and “Release” …………….. 115
2.4.4. Procedures and NMR Data for New Compounds ……….….116
TLC, NMR and MS Data ………………………………………………130
Appendix A ……………………………………………………………..133
References ……………………………………………………………..170

CHAPTER 3 Chemical Synthesis of Homogeneous Heparan Sulfate
Proteoglycan
…………………………………………………………………………177
3.1. Introduction …………………………………………………………... 177
3.2. Synthetic Design-First Generation ……………………………….......190
3.3. Synthesis of Monosaccharide Building Blocks ……………………...192
3.3.1. Synthesis of Glucosamine Building Block
…………………192
3.3.2. Synthesis of Glucose Building Block …………………………194
3.3.3. Synthesis of Galactose Building Block ………………………195
3.3.4. Synthesis of Xylose Building Block …………………………198
3.3.5. Idose Building Block
…………………………………………201
3.4. Evalution of “1+1” glycosylation
…………………………...……….201
3.4.1. “Gal + Gal” Coupling …………………………………...………201
3.4.2. “Xyl + Serine” Coupling
……………………………...………203
3.4.3. “Gal + Xyl” Coupling
………………………………...………204
3.4.4. “Ido + GluN + Glu” Coupling
………………………...………208
3.5. Synthetic Design-Second Generation …………………………………209
3.6. Synthesis of Building Blocks ………………………...…………………211
3.6.1. Synthesis of Trisaccharide 124
……………………...……211
3.6.2. Screening Different Conditions for the Synthesis of
Disaccharide 122 ………………………………………………………214
3.7. Synthetic Design-Third Generation
…………………………..…….216
3.8. Synthesis of Fully Protected Octasaccharide ………………..…….217
3.8.1. Synthesis of Pentasaccharide
………………………………217
3.8.2. Investigation of “2 + 5” Coupling
……………………………220
3.8.3. Investigation of “2 + 6” Coupling
……………………………222
3.9. Synthetic Design-Fourth Generation
……………………………....226

viii

3.10. Synthesis of Glycopeptide 175 ………..…………………….………228
3.10.1. Synthesis of Trisaccharide 177
…………………………228
3.10.2. Synthesis of Disaccharide 178 ………………………………230
3.10.3. Synthesis of Trisaccharide 179 ………………………………231
3.10.4. Synthesis of Octasaccharide 176 ………………………………232
3.10.5. Synthesis of Glycopeptide 17……………………………………233
3.11. Deprotection of Glycopeptide 175 ……………………………………..236
3.11.1. Deprotection of Octasaccharide 195 ………………………..237
3.11.2. Deprotection of Glycopeptide 175………………………………247
3.12. N-Sulfation ……………………………………………………………….249
3.13. Synthesis of Glycopeptide 233 ………………..…………………………250
3.14. Conclusions………………………………………………………………...267
3.15. Experimental Section ……………………………………………………. 268
3.15.1. General Experimental Procedures
………………………… 268
3.15.2. Characterization of Anomeric Stereochemistry ……………...269
3.15.3. General Procedure for Pre-activation Based Single-Step
Glycosylation ……………………………………………………………. 270
3.15.4. General Procedure for Protection of 6-OH with Lev ………… 270
3.15.5. General Procedure for Deprotection of Lev
……..…………270
3.15.6. General Procedure for O-Sulfation
………………………… 271
3.15.7. General Procedure for Global Debenzylation………………… 271
3.15.8. General Procedure for Solid Phase Peptide Synthesis……… 272
3.15.9. General Procedure for HPLC Analysis………………………… 275
3.15.10. Detailed Procedures and NMR, MS Data …………………… 276
Appendix B ……………………………………………………………….397
References ………………………………………………………………..793

ix

LIST OF TABLES
Table 1.1. Effect of solvent ratio on stereoselectivity .………………………………….77
Table 1.2. Modulating effect of DMF on stereoselectivity………………………………79
Table 3.1. Screening donor-acceptor pairs for Gal-Gal disaccharide synthesis…….202

x

LIST OF FIGURES
Figure 1.1. Four different types of glycosidic linkages………………………….……….6
Figure 1.2. 1, 2-cis glycoside……………………………………………………….………7
Figure 1.3. Tetrasaccharide 97…………………………………………………….…..…14
Figure 1.4. Retrosynthetic analysis of pentasaccharide 109…………………….……..16
Figure 1.5. Combinatorial synthesis of oligoglucoside library……………………….….20
1

13

Figure 1.6. H NMR and C NMR of the intermediate after pre-activation of donor
178…………………………...………………………………………………………………..23
Figure 1.7. HMBC of the intermediate a) before and b) after pre-activation of donor
180…………………………………………………………………………………………….24
Figure 1.8. Fully protected decasaccharide 174…………………………………………27
Figure 1.9. Micrococcus luteus teichuronic acid………………………………………….34
Figure 1.10. Mannosazide methyl uronate donors with different anomeric leaving
groups………………………………………………………………………………………….34
o

Figure 1.11. 1H NMR of donors after pre-activation under -80 C ……………………35
Figure 1.12. Influence of DTBMP on the outcome of glycosylation……………………38
1

1

Figure 1.13. (a) H NMR of donor 251; (b) H TOCSY 1 D on irradiation of H4 of 252; (c)
o

o

HMBC of 252. Reagents and conditions: (a) TMSOTf, DCM, -78 C to 0 C; (b) MeOH, o

o

78 C to 0 C………………………………………………………………………………….44
Figure 1.14. Bacteria capsular polysaccharide 382 ……………………….……………65
Figure 1.15. Donor and acceptor pairs resulting low selectivity………………………..68
Figure 1.16. Staggered conformations about O-2 bond ………………………………68
1

Figure 1.17. Partial H NMR of donor 415 after pre-activation (a) 1 min after Tf2O
o

o

addition (-78 C); (b) 60 min after Tf2O addition (-40 C)……………….………………73

xi

Figure 2.1. (a) a cartoon of F-SPE; (b) fluorous silica gel …………………………….101
Figure 2.2. Fluorous-assisted synthesis of β-(1-6) linked D-glucopyranoside
homotetramer………………………………………………………………………………..102
Figure 2.3. While glycosylation of trisaccharide donor 10 failed with (a) the fluorous
acceptor 11a, its glycosylation with (b) acceptor 11b was successfully performed under
identical reaction conditions…………………………….………………………………….104
Figure 2.4. Fluorous-assisted one-pot oligosaccharide synthesis……………………105
Figure 2.5. Compounds used for Staudinger reaction…………………………………106
Figure 2.6. (a) HPLC chromatogram of the crude reaction mixture of compound 30 after
the glycosylation reaction. The peak marked with an * was ditolyl disulfide, which was a
side product from donor activation and has a strong UV absorbance band. This
chromatogram corresponds to lane 1 of the TLC shown in Scheme 5; (b) HPLC
chromatogram of the organic fraction after the second F-SPE, which corresponds to
lane 3 of the TLC shown in Scheme 5. (HPLC mobile phase: hexane/ethyl acetate = 2:1,
flow rate 1 mL/min, UV monitoring at 256 nm, HPLC column: SupelCOSIL LC-Si, 25 cm
X 4.6 mm, 5-μm particle size……………………………………………………………….111
Figure 2.7. TLC data of compounds 35 and 37………………………………………….130
Figure 2.8. HPLC data of compounds 35 and 37 ……………………………………….131
1

Figure 2.9. H-NMR (CDCl3, 500 MHz) of 17............................................................134
13

Figure 2.10. C-NMR (CDCl3, 125 MHz) of 17.........................................................135
Figure 2.11. gCOSY (CDCl3, 500 MHz) of 17............................................................136
1

Figure 2.12. H-NMR (CDCl3, 500 MHz) of 18...........................................................137
1

Figure 2.13. H-NMR (CDCl3, 500 MHz) of 18...........................................................138
Figure 2.14.

13

C-NMR (CDCl3, 125 MHz) of 18.........................................................139

1

Figure 2.15. H-NMR (CD3OD, 500 MHz) of 22…………………………………………140
13

Figure 2.16. C-NMR (CD3OD, 125 MHz) of 22…………………………………………141
Figure 2.16.

19

F-NMR (CD3OD, 282 MHz) of 22.........................................................142

xii

1

Figure 2.17. H-NMR (CDCl3, 500 MHz) of 24………………………………………….143
Figure 2.18.

13

C-NMR (CDCl3, 125 MHz) of 24 ….…………………………………….144

Figure 2.19. gCOSY (CDCl3, 500 MHz) of 24……………………………………………145
1

Figure 2.20. H-NMR (CD3OD, 500 MHz) of 27 ….……………………………………146
Figure 2.21.
Figure 2.22.

13

C-NMR (CD3OD, 125 MHz) of 27 ……………………………………….147

19

F-NMR (CD3OD, 282 MHz) of 27 ………………………………………..148

1

Figure 2.23. H-NMR (CDCl3, 500 MHz) of 30 ………………………………………….149
Figure 2.24.

13

C-NMR (CDCl3, 125 MHz) of 30 …………………………………………150

Figure 2.25. gCOSY (CDCl3, 500 MHz) of 30 …………………………………………...151
Figure 2.26. HMQC (CDCl3, 500 MHz) of 30 ..............................................................152
1

Figure 2.27. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 30……………….153
Figure 2.28. gHMBC (CDCl3, 500 MHz) of 30……………………………………………154
1

Figure 2.29. H-NMR (CDCl3, 500 MHz) of 33 …………………………………………155
Figure 2.30.

13

C-NMR (CDCl3, 125 MHz) of 33 …………………………………………156

Figure 2.31. gCOSY (CDCl3, 500 MHz) of 33 ………………………………………...…157
1

Figure 2.32. H-NMR (CDCl3, 500 MHz) of 35 …………………………………………158
Figure 2.33.

13

C-NMR (CDCl3, 125 MHz) of 35 …………………………………………159

Figure 2.34. gCOSY (CDCl3, 500 MHz) of 35 …………………………………………...160
Figure 2.35. gHMQC (CDCl3, 500 MHz) of 35 …………………………………………..161
1

Figure 2.36. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 35……………….162

xiii

Figure 2.37. gHMBC (CDCl3, 500 MHz) of 35……………………………………………163
1

Figure 2.38. H-NMR (CDCl3, 500 MHz) of 37…………………………………………..164
Figure 2.39.

13

C-NMR (CDCl3, 125 MHz) of 37………………………………………….165

Figure 2.40. gCOSY (CDCl3, 500 MHz) of 37……………………………………………166
Figure 2.41. gHMQC (CDCl3, 500 MHz) of 37……………………………………………167
1

Figure 2.42. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 37………………168
Figure 2.43. gHMBC (CDCl3, 500 MHz) of 37……………………………………………169
Figure 3.1. Structure of HSPG and its biosynthetic pathway …………………………..178
Figure 3.2. Biological distributions of syndecans………………………………………...181
Figure 3.3. Structure of the four vertebrate syndecans ………………………………..181
Figure 3.4. Shedding of syndecans by metzincin proteinases………………………….182
Figure 3.5. Sequence of human syndecan-1 ………………………………..…………183
Figure 3.6. Arixtra, a fully synthetic pentasaccharide drug for treatment of deep
vein thrombosis …………………………………………………………………..…………185
Figure 3.7. Structure of our synthetic target HSPG 1…………………………...……….187
Figure 3.8. Structures of 12 synthetic HS hexasaccharides……………………..……..188
Figure 3.9. FGF-2 (1ug) was incubated with 10 (empty bar) or 40 ug (filled bar)
ublabeled hexasaccharides 6-17 or HS (1.6 ug) in PBS buffer (200 uL) at room
35
temperature for 30 min. Then, [ SHS (4500 cpm) was added to the mixture followed by
o

incubation at 37 C for 90 min. The data represent the average of four or more
experiments with the error bars showing standard deviations. The control was the
sample without any hexasaccharides or unlabled HS. The percentage of residual
35
35
[ S]HS binding was calculated by dividing the residual S counts on the membrane
with sample incubation by control counts……………………………………………..…..189
Figure 3.10. Retrosynthetic analysis of tetrasaccharide 18 …………….…………..…190
Figure 3.11. Structure of our current synthetic target 24………………………………..190

xiv

Figure 3.12. Retrosynthetic analysis of glycopeptide 24………………………………192
Figure 3.13. Idose building blocks 79 and 80………………………………………….201
Figure 3.14. Retrosynthetic analysis of glycopeptide 24………………………………211
Figure 3.15. Retrosynthetic analysis of glycopeptide 24………………………………217
Figure 3.16. Retrosynthetic analysis of glycopeptide 24………………………………227
Figure 3.17. Steps required for deprotection of octasaccharide 195………………..236
Figure 3.18. Side product 201 generated from Staudinger reaction…………………238
Figure 3.19. Possible structures of hydrolysis products……………………………….242
Figure 3.20. Retrosynthetic analysis of glycopeptide 233……………………….…….250
Figure 3.21. Retrosynthetic analysis of glycopeptide 234……………………………..254
1

Figure 3.22. H-NMR (CDCl3, 500 MHz) of 35………………………………………….398
1

Figure 3.23. H-NMR (CDCl3, 500 MHz) of 36………………………………………….399
1

Figure 3.24. H-NMR (CDCl3, 500 MHz) of 37………………………………………….400
1

Figure 3.25. H-NMR (CD3Cl, 500 MHz) of 38…………………………………………..401
Figure 3.26. H-NMR (CDCl3, 500 MHz) of 39…………………………………………….402
1

Figure 3.27. H-NMR (CD3Cl, 500 MHz) of 40………………………………………..…403
1

Figure 3.28. H-NMR (CDCl3, 500 MHz) of 41…………………………………………..404
1

Figure 3.29. H-NMR (CDCl3, 500 MHz) of 43…………………………………………..405
1

Figure 3.30. H-NMR (CDCl3, 500 MHz) of 44……………………..……………………406
1

Figure 3.31. H-NMR (CDCl3, 500 MHz) of 45…………………………………………..407
1

Figure 3.32. H-NMR (CDCl3, 500 MHz) of 46…………………………………………..408

xv

1

Figure 3.33. H-NMR (CDCl3, 500 MHz) of 47…………………………………………409
1

Figure 3.34. H-NMR (CDCl3, 500 MHz) of 48………………………………………….410
1

Figure 3.35. H-NMR (CDCl3, 500 MHz) of 50…………………………………………..411
1

Figure 3.36. H-NMR (CDCl3, 500 MHz) of 51…………………………………………..412
1

Figure 3.37. H-NMR (CDCl3, 500 MHz) of 52…………………………………………..413
Figure 3.38.

13

C-NMR (CDCl3, 125 MHz) of 52…………………………………………414

Figure 3.39. gCOSY (CDCl3, 500 MHz) of 52……………………………………………415
Figure 3.40. gHMQC (CDCl3, 500 MHz) of 52……………………………………………416
Figure 3.41. gHMBC (CDCl3, 500 MHz) of 52……………………………………………417
1

Figure 3.42. H-NMR (CDCl3, 500 MHz) of 53…………………………………………..418
Figure 3.43.

13

C-NMR (CDCl3, 125 MHz) of 53………………………………………….419

1

Figure 3.44. H-NMR (CDCl3, 500 MHz) of 54…………………………………………..420
Figure 3.45.

13

C-NMR (CDCl3, 125 MHz) of 54…………………………………………421

Figure 3.46. gCOSY (CDCl3, 500 MHz) of 54……………………………………………422
1

Figure 3.47. H-NMR (CDCl3, 500 MHz) of 55…………………………………………..423
Figure 3.48.

13

C-NMR (CDCl3, 125 MHz) of 55…………………………………………424

Figure 3.49. gCOSY (CDCl3, 500 MHz) of 55……………………………………………425
1

Figure 3.50. H-NMR (CDCl3, 500 MHz) of 56…………………………………………..426
Figure 3.51.

13

C-NMR (CDCl3, 125 MHz) of 56…………………………………………427

Figure 3.52. gCOSY (CDCl3, 500 MHz) of 56……………………………………………428

xvi

1

Figure 3.53. H-NMR (CDCl3, 500 MHz) of 57………………………………………….429
Figure 3.54.

13

C-NMR (CDCl3, 125 MHz) of 57………………………………………...430

Figure 3.55. gCOSY (CDCl3, 500 MHz) of 57……………………………………………431
1

Figure 3.56. H-NMR (CDCl3, 500 MHz) of 58…………………………………………..432
1

Figure 3.57. H-NMR (CDCl3, 500 MHz) of 59…………………………………………..433
1

Figure 3.58. H-NMR (CDCl3, 500 MHz) of 60…………………………………………..434
1

Figure 3.59. H-NMR (CDCl3, 500 MHz) of 61…………………………………………..435
Figure 3.60.

13

C-NMR (CDCl3, 125 MHz) of 61…………………………………………436

Figure 3.61. gCOSY (CDCl3, 500 MHz) of 61……………………………………………437
1

Figure 3.62. H-NMR (CDCl3, 500 MHz) of 64…………………………………………..438
Figure 3.63.

13

C-NMR (CDCl3, 125 MHz) of 64………………………………………….439

Figure 3.64. gCOSY (CDCl3, 500 MHz) of 64……………………………………………440
1

Figure 3.65. H-NMR (CD3OD, 500 MHz) of 65…………………………………………441
Figure 3.66.

13

C-NMR (CD3OD, 125 MHz) of 65………………………………………..442

1

Figure 3.67. H-NMR (CDCl3, 500 MHz) of 66…………..………………………………443
Figure 3.68.

13

C-NMR (CDCl3, 125 MHz) of 66………………………………………….444

Figure 3.69. gCOSY (CDCl3, 500 MHz) of 66……………………………………………445
Figure 3.70. gHMQC (CDCl3, 500 MHz) of 66……………………………………………446
Figure 3.71. gHMBC (CDCl3, 500 MHz) of 66……………………………………………447
1

Figure 3.72. H-NMR (CDCl3, 500 MHz) of 67…………………………………………..448

xvii

1

Figure 3.73. H-NMR (CDCl3, 500 MHz) of 68…………………………………………449
Figure 3.74.

13

C-NMR (CDCl3, 125 MHz) of 68………………………………………..450

Figure 3.75. gCOSY (CDCl3, 500 MHz) of 68……………………………………………451
Figure 3.76. gHMQC (CDCl3, 500 MHz) of 68……………………………………………452
1

Figure 3.77. H-NMR (CDCl3, 500 MHz) of 69…………………………………………..453
Figure 3.78.

13

C-NMR (CDCl3, 125 MHz) of 69………………………………………….454

Figure 3.79. gCOSY (CDCl3, 500 MHz) of 69……………………………………………455
1

Figure 3.80. H-NMR (CDCl3, 500 MHz) of 70…………………………………………..456
Figure 3.81.

13

C-NMR (CDCl3, 125 MHz) of 70………………………………………….457

Figure 3.82. gCOSY (CDCl3, 500 MHz) of 70……………………………………………458
1

Figure 3.83. H-NMR (CDCl3, 500 MHz) of 71…………………………………………..459
Figure 3.84.

13

C-NMR (CDCl3, 125 MHz) of 71………………………………………….460

Figure 3.85. gCOSY (CDCl3, 500 MHz) of 71……………………………………………461
1

Figure 3.86. H-NMR (CDCl3, 500 MHz) of 72…………………………………………..462
Figure 3.87.

13

C-NMR (CDCl3, 125 MHz) of 72………………………………………….463

Figure 3.88. gCOSY (CDCl3, 500 MHz) of 72……………………………………………464
1

Figure 3.89. H-NMR (CDCl3, 500 MHz) of 73………………..…………………………465
Figure 3.90.

13

C-NMR (CDCl3, 125 MHz) of 73……………….…………………………466

Figure 3.91. gCOSY (CDCl3, 500 MHz) of 73……………………………………………467
1

Figure 3.92. H-NMR (CDCl3, 500 MHz) of 74…………………………………………..468

xviii

Figure 3.93.

13

C-NMR (CDCl3, 125 MHz) of 74…………………………………………469

Figure 3.94. gCOSY (CDCl3, 500 MHz) of 74……………………………………………470
1

Figure 3.95. H-NMR (CDCl3, 500 MHz) of 75…………………………………………..471
1

Figure 3.96. H-NMR (CDCl3, 500 MHz) of 76…………………………………………..472
1

Figure 3.97. H-NMR (CDCl3, 500 MHz) of 77…………………………………………..473
1

Figure 3.98. H-NMR (CDCl3, 500 MHz) of 78…………………………………………..474
Figure 3.99.

13

C-NMR (CDCl3, 125 MHz) of 78………………………………………….475

Figure 3.100. gCOSY (CDCl3, 500 MHz) of 78…………………………………………..476
Figure 3.101. gHMQC (CDCl3, 500 MHz) of 78………………………………………….477
Figure 3.102. gHMBC (CDCl3, 500 MHz) of 78…………………………………………..478
1

Figure 3.103. H-NMR (CDCl3, 500 MHz) of 81…………………………………………479
Figure 3.104.

13

C-NMR (CDCl3, 125 MHz) of 81………………………………………..480

Figure 3.105. gCOSY (CDCl3, 500 MHz) of 81…………………………………………..481
1

Figure 3.106. H-NMR (CDCl3, 600 MHz) of 82…………………………………………482
Figure 3.107.

13

C-NMR (CDCl3, 150 MHz) of 82………………………………………..483

Figure 3.108. gCOSY (CDCl3, 600 MHz) of 82…………………………………………..484
1

Figure 3.109. H-NMR (CDCl3, 500 MHz) of 83…………………………………………485
1

Figure 3.110. H-NMR (CDCl3, 500 MHz) of 84…………………………………………486
1

Figure 3.111. H-NMR (CDCl3, 500 MHz) of 87…………………………………………487
Figure 3.112.

13

C-NMR (CDCl3, 125 MHz) of 87………………………………………..488

xix

Figure 3.113. gCOSY (CDCl3, 500 MHz) of 87………………………………………….489
Figure 3.114. gHMQC (CDCl3, 500 MHz) of 87…………………………………………490
1

Figure 3.115. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 87…………….491
1

Figure 3.116. H-NMR (CDCl3, 500 MHz) of 89…………………………………………492
Figure 3.117.

13

C-NMR (CDCl3, 125 MHz) of 89………………………………………..493

Figure 3.118. gCOSY (CDCl3, 500 MHz) of 89…………………………………………..494
Figure 3.119. gHMQC (CDCl3, 500 MHz) of 89………………………………………….495
1

Figure 3.120. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 89……………..496
1

Figure 3.121. H-NMR (CDCl3, 500 MHz) of 91…………………………………………497
1

Figure 3.122. H-NMR (CDCl3, 500 MHz) of 92………………………………………...498
Figure 3.123.

13

C-NMR (CDCl3, 150 MHz) of 92………………………………………..499

Figure 3.124. gCOSY (CDCl3, 500 MHz) of 92…………………………………………..500
Figure 3.125. gHMQC (CDCl3, 600 MHz) of 92………………………………………….501
1

Figure 3.126. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 92……………..502
Figure 3.127. gHMBC (CDCl3, 600 MHz) of 92………………………………………….503
1

Figure 3.128. H-NMR (CDCl3, 500 MHz) of 93…………………………………………504
Figure 3.129.

13

C-NMR (CDCl3, 125 MHz) of 93………………………………………..505

Figure 3.130. gCOSY (CDCl3, 500 MHz) of 93…………………………………………..506
1

Figure 3.131. H-NMR (CDCl3, 500 MHz) of 94…………………………………………507
1

Figure 3.132. H-NMR (CDCl3, 600 MHz) of 96…………………………………………508

xx

Figure 3.133.

13

C-NMR (CDCl3, 150 MHz) of 96………………………………………..509

Figure 3.134. gCOSY (CDCl3, 600 MHz) of 96…………………………………………..510
Figure 3.135. gHMQC (CDCl3, 600 MHz) of 96………………………………………….511
1

Figure 3.136. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 96……………..512
1

Figure 3.137. H-NMR (CDCl3, 500 MHz) of 97…………………………………………513
Figure 3.138.

13

C-NMR (CDCl3, 125 MHz) of 97………………………………………..514

Figure 3.139. gCOSY (CDCl3, 500 MHz) of 97…………………………………………..515
1

Figure 3.140. H-NMR (CDCl3, 500 MHz) of 98……………………………..…………..516
Figure 3.141.

13

C-NMR (CDCl3, 125 MHz) of 98………………………………………..517

1

Figure 3.142. H-NMR (CDCl3, 500 MHz) of 100………………………………………..518
1

Figure 3.143. H-NMR (CDCl3, 600 MHz) of 101………………………………………..519
Figure 3.144.

13

C-NMR (CDCl3, 150 MHz) of 101………………………………………520

Figure 3.145. gCOSY (CDCl3, 600 MHz) of 101…………………………………………521
Figure 3.146. gHMQC (CDCl3, 600 MHz) of 101………………..………………………522
1

Figure 3.147. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 101..................523
Figure 3.148. gHMBC (CDCl3, 600 MHz) of 101…………………………………………524
1

Figure 3.149. H-NMR (CDCl3, 500 MHz) of 102………………………………………..525
1

Figure 3.150. H-NMR (CDCl3, 500 MHz) of 103………………………………………..526
Figure 3.151.

13

C-NMR (CDCl3, 125 MHz) of 103……………………………………....527

Figure 3.152. gCOSY (CDCl3, 500 MHz) of 103…………………………………………528

xxi

Figure 3.153. gHMQC (CDCl3, 500 MHz) of 103………………………………………...529
1

Figure 3.154. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 103……………530
Figure 3.155. gHMBC (CDCl3, 500 MHz) of 103…………………………………………531
1

Figure 3.156. H-NMR (CDCl3, 500 MHz) of 105………………………………………..532
Figure 3.157.

13

C-NMR (CDCl3, 150 MHz) of 105......................................................533

Figure 3.158. gCOSY (CDCl3, 600 MHz) of 105…………………………………………534
Figure 3.159. gHMQC (CDCl3, 600 MHz) of 105………………………………………..535
1

Figure 3.160. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 105……………536
1

Figure 3.161. H-NMR (CDCl3, 600 MHz) of 106………………………………………..537
Figure 3.162.

13

C-NMR (CDCl3, 150 MHz) of 106………………………………………538

Figure 3.163. gHMQC (CDCl3, 600 MHz) of 106………………………………………...539
1

Figure 3.164. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 106……………540
1

Figure 3.165. H-NMR (CDCl3, 600 MHz) of 107………………………………………..541
Figure 3.166.

13

C-NMR (CDCl3, 150 MHz) of 107………………………………………542

Figure 3.167. gCOSY (CDCl3, 600 MHz) of 107…………………………………………543
Figure 3.168. gHMQC (CDCl3, 600 MHz) of 107………………………………………...544
1

Figure 3.169. H-NMR (CDCl3, 600 MHz) of 108………………………………………..545
Figure 3.170.

13

C-NMR (CDCl3, 150 MHz) of 108………………………………………546

Figure 3.171. gCOSY (CDCl3, 600 MHz) of 108…………………………………………547
Figure 3.172. gHMQC (CDCl3, 600 MHz) of 108………………………………………..548

xxii

1

Figure 3.173. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 108……………549
1

Figure 3.174. H-NMR (CDCl3, 600 MHz) of 109……………………………………….550
Figure 3.175.

13

C-NMR (CDCl3, 150 MHz) of 109………………………………………551

Figure 3.176. gCOSY (CDCl3, 600 MHz) of 109…………………………………………552
1

Figure 3.177. H-NMR (CDCl3, 500 MHz) of 111………………………………………..553
Figure 3.178.

13

C-NMR (CDCl3, 125 MHz) of 111………………………………………554

Figure 3.179. gCOSY (CDCl3, 500 MHz) of 111…………………………………………555
1

Figure 3.180. H-NMR (CDCl3, 500 MHz) of 114………………………………………..556
Figure 3.181.

13

C-NMR (CDCl3, 125 MHz) of 114………………………………………557

Figure 3.182. gCOSY (CDCl3, 600 MHz) of 114….……………………………………..558
1

Figure 3.183. H-NMR (CDCl3, 600 MHz) of 115………………………………………..559
Figure 3.184.

13

C-NMR (CDCl3, 125 MHz) of 115………………………………………560

Figure 3.185. gCOSY (CDCl3, 600 MHz) of 115…….…………………………………..561
1

Figure 3.186. H-NMR (CDCl3, 600 MHz) of 116………………………………………..562
Figure 3.187.

13

C-NMR (CDCl3, 125 MHz) of 116………………………………………563

Figure 3.188. gCOSY (CDCl3, 600 MHz) of 116…………………………………………564
Figure 3.189. gHMQC (CDCl3, 500 MHz) of 116………………………………………..565
1

Figure 3.190. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 116……………566
1

Figure 3.191. H-NMR (CDCl3, 500 MHz) of 117……………………………………….567
1

Figure 3.192. H-NMR (CDCl3, 600 MHz) of 124……………………………………….568

xxiii

Figure 3.193.

13

C-NMR (CDCl3, 150 MHz) of 124………………………………………569

Figure 3.194. gCOSY (CDCl3, 600 MHz) of 124…………………………………………570
Figure 3.195. gHMQC (CDCl3, 600 MHz) of 124………………………………………...571
1

Figure 3.196. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 124……………572
Figure 3.197. gHMBC (CDCl3, 600 MHz) of 124………………………………………..573
1

Figure 3.198. H-NMR (CDCl3, 500 MHz) of S1………………………………………...574
Figure 3.199.

13

C-NMR (CDCl3, 125 MHz) of S1……………………………………….575

Figure 3.200. gCOSY (CDCl3, 500 MHz) of S1………………………………………….576
1

Figure 3.201. H-NMR (CDCl3, 500 MHz) of 126……………………………………….577
Figure 3.202.

13

C-NMR (CDCl3, 125 MHz) of 126………………………………………578

Figure 3.203. gCOSY (CDCl3, 500 MHz) of 126…………………………………………579
1

Figure 3.204. H-NMR (CDCl3, 600 MHz) of 127………………………………………..580
Figure 3.205.

13

C-NMR (CDCl3, 150 MHz) of 127………………………………………581

Figure 3.206. gCOSY (CDCl3, 600 MHz) of 127…………………………………………582
Figure 3.207. gHMQC (CDCl3, 600 MHz) of 127………………………………………...583
1

Figure 3.208. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 127……………584
Figure 3.209. gHMBC (CDCl3, 600 MHz) of 127…………………………………………585
1

Figure 3.210. H-NMR (CDCl3, 600 MHz) of 128………………………………………..586
Figure 3.211.

13

C-NMR (CDCl3, 150 MHz) of 128………………………………………587

Figure 3.212. gCOSY (CDCl3, 600 MHz) of 128…………………………………………588

xxiv

Figure 3.213. gHMQC (CDCl3, 600 MHz) of 128………………………………………...589
1

Figure 3.214. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 128……………590
Figure 3.215. gHMBC (CDCl3, 600 MHz) of 128………………………………………..591
1

Figure 3.216. H-NMR (CDCl3, 600 MHz) of 130……………………………………….592
Figure 3.217.

13

C-NMR (CDCl3, 150 MHz) of 130………………………………………593

Figure 3.218. gCOSY (CDCl3, 600 MHz) of 130…………………………………………594
Figure 3.219. gHMQC (CDCl3, 600 MHz) of 130………………………………………..595
1

Figure 3.220. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 130……………596
Figure 3.221. gHMBC (CDCl3, 600 MHz) of 130…………………………………………597
1

Figure 3.222. H-NMR (CDCl3, 500 MHz) of 131………………………………………..598
1

Figure 3.223. H-NMR (CDCl3, 500 MHz) of 132………………………………………..599
1

Figure 3.224. H-NMR (CDCl3, 600 MHz) of 133………………………………………..600
Figure 3.225.

13

C-NMR (CDCl3, 150 MHz) of 133………………………………………601

Figure 3.226. gCOSY (CDCl3, 600 MHz) of 133…………………………………………602
Figure 3.227. gHMQC (CDCl3, 600 MHz) of 133………………………………………...603
1

Figure 3.228. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 133…………...604
Figure 3.229. gHMBC (CDCl3, 600 MHz) of 133………………………………………...605
1

Figure 3.230. H-NMR (CDCl3, 600 MHz) of 134………………………………………..606
Figure 3.231.

13

C-NMR (CDCl3, 150 MHz) of 134………………………………………607

Figure 3.232. gCOSY (CDCl3, 600 MHz) of 134…………………………………………608

xxv

Figure 3.233. gHMQC (CDCl3, 600 MHz) of 134………………………………………..609
1

Figure 3.234. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 134……………610
Figure 3.235. gHMBC (CDCl3, 600 MHz) of 134…………………………………………611
1

Figure 3.236. H-NMR (CDCl3, 500 MHz) of 135………………………………………..612
Figure 3.237.

13

C-NMR (CDCl3, 125 MHz) of 135………………………………………613

Figure 3.238. gCOSY (CDCl3, 500 MHz) of 135…………………………………………614
Figure 3.239. gHMQC (CDCl3, 500 MHz) of 135………………………………………...615
1

Figure 3.240. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 135……………616
Figure 3.241. gHMBC (CDCl3, 500 MHz) of 135………………………………………..617
1

Figure 3.242. H-NMR (CDCl3, 500 MHz) of 136………………………………………..618
1

Figure 3.243. H-NMR (CDCl3, 500 MHz) of 137………………………………………..619
1

Figure 3.244. H-NMR (CDCl3, 500 MHz) of 140………………………………………..620
Figure 3.245.

13

C-NMR (CDCl3, 125 MHz) of 140………………………………………621

Figure 3.246. gCOSY (CDCl3, 500 MHz) of 140…………………………………………622
1

Figure 3.247. H-NMR (CDCl3, 600 MHz) of 142………………………………………..623
Figure 3.248.

13

C-NMR (CDCl3, 150 MHz) of 142………………………………………624

Figure 3.249. gCOSY (CDCl3, 600 MHz) of 142…………………………………………625
1

Figure 3.250. H-NMR (CDCl3, 500 MHz) of 143………………………………………..626
Figure 3.251.

13

C-NMR (CDCl3, 125 MHz) of 143………………………………………627

Figure 3.252. gCOSY (CDCl3, 500 MHz) of 143…………………………………………628

xxvi

1

Figure 3.253. H-NMR (CDCl3, 500 MHz) of 144………………………………………..629
Figure 3.254.

13

C-NMR (CDCl3, 125 MHz) of 144………………………………………630

Figure 3.255. gCOSY (CDCl3, 500 MHz) of 144………………………………………...631
Figure 3.256. gHMQC (CDCl3, 500 MHz) of 144………………………………………...632
1

Figure 3.257. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 144……………633
Figure 3.258. gHMBC (CDCl3, 500 MHz) of 144…………………………………………634
1

Figure 3.259. H-NMR (CDCl3, 500 MHz) of 145………………………………………..635
Figure 3.260.

13

C-NMR (CDCl3, 125 MHz) of 145………………………………………636

Figure 3.261. gCOSY (CDCl3, 500 MHz) of 145…………………………………………637
Figure 3.262. gHMQC (CDCl3, 500 MHz) of 145………………………………………...638
1

Figure 3.263. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 145……………639
Figure 3.264. gHMBC (CDCl3, 500 MHz) of 145………………………………………...640
1

Figure 3.265. H-NMR (CDCl3, 500 MHz) of 146………………………………………..641
Figure 3.266.

13

C-NMR (CDCl3, 125 MHz) of 146………………………………………642

Figure 3.267. gCOSY (CDCl3, 500 MHz) of 146…………………………………………643
1

Figure 3.268. H-NMR (CDCl3, 500 MHz) of 149………………………………………..644
Figure 3.269.

13

C-NMR (CDCl3, 125 MHz) of 149………………………………………645

Figure 3.270. gCOSY (CDCl3, 500 MHz) of 149…………………………………………646
1

Figure 3.271. H-NMR (CDCl3, 500 MHz) of 150………………………………………..647
1

Figure 3.272. H-NMR ((CD3)2CO, 500 MHz) of 125………………………………...…648

xxvii

Figure 3.273.

13

C-NMR ((CD3)2CO, 150 MHz) of 125………………………………….649

Figure 3.274. gCOSY ((CD3)2CO, 600 MHz) of 125…………………………………….650
Figure 3.275. gHMQC ((CD3)2CO, 600 MHz) of 125……………………………………651
1

Figure 3.276. gHMQC (without H decoupling) ((CD3)2CO, 600 MHz) of 125………652
Figure 3.277. gHMBC ((CD3)2CO, 600 MHz) of 125……………………………………653
1

Figure 3.278. H-NMR (CDCl3, 500 MHz) of 154……………………………………….654
Figure 3.279.

13

C-NMR (CDCl3, 125 MHz) of 154……………………………………...655

Figure 3.280. gCOSY (CDCl3, 500 MHz) of 154………………………………………...656
Figure 3.281. gHMQC (CDCl3, 500 MHz) of 154………………………………………..657
1

Figure 3.282. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 154…………...658
Figure 3.283. gHMBC (CDCl3, 500 MHz) of 154………………………………………...659
1

Figure 3.284. H-NMR (CDCl3, 500 MHz) of 155………………………………………..660
Figure 3.285.

13

C-NMR (CDCl3, 125 MHz) of 155………………………………………661

Figure 3.286. gCOSY (CDCl3, 500 MHz) of 155…………………………………………662
1

Figure 3.287. H-NMR (CDCl3, 500 MHz) of 156………………………………………..663
Figure 3.288.

13

C-NMR (CDCl3, 125 MHz) of 156………………………………………664

Figure 3.289. gCOSY (CDCl3, 500 MHz) of 156…………………………………………665
1

Figure 3.290. H-NMR (CDCl3, 500 MHz) of 159………………………………………..666
Figure 3.291.

13

C-NMR (CDCl3, 150 MHz) of 159………………………………………667

Figure 3.292. gCOSY (CDCl3, 600 MHz) of 159…………………………………………668

xxviii

1

Figure 3.293. H-NMR (CDCl3, 600 MHz) of 160………………………………………..669
Figure 3.294.

13

C-NMR (CDCl3, 150 MHz) of 160………………………………………670

Figure 3.295. gCOSY (CDCl3, 600 MHz) of 160…………………………………………671
1

Figure 3.296. H-NMR (CDCl3, 500 MHz) of 162………………………………………..672
Figure 3.297.

13

C-NMR (CDCl3, 125 MHz) of 162………………………………………673

Figure 3.298. gCOSY (CDCl3, 500 MHz) of 162…………………………………………674
1

Figure 3.299. H-NMR (CDCl3, 600 MHz) of 163………………………………………..675
Figure 3.300.

13

C-NMR (CDCl3, 150 MHz) of 163………………………………………676

Figure 3.301. gCOSY (CDCl3, 600 MHz) of 163…………………………………………677
1

Figure 3.302. H-NMR (CDCl3, 500 MHz) of 165………………………………………..678
Figure 3.303.

13

C-NMR (CDCl3, 125 MHz) of 165………………………………………679

Figure 3.304. gCOSY (CDCl3, 500 MHz) of 165…………………………………………680
1

Figure 3.305. H-NMR (CDCl3, 500 MHz) of 167………………………………………..681
1

Figure 3.306. H-NMR (CDCl3, 600 MHz) of 168………………………………………..682
1

Figure 3.307. H-NMR (CDCl3, 500 MHz) of 172………………………………………..683
1

Figure 3.308. H-NMR (CDCl3, 500 MHz) of 174………………………………………..684
1

Figure 3.309. H-NMR (CDCl3, 500 MHz) of 276………………………………………..685
Figure 3.310. gCOSY (CDCl3, 500 MHz) of 276…………………………………………686
1

Figure 3.311. H-NMR (CDCl3, 500 MHz) of 183………………………………………..687
Figure 3.312.

13

C-NMR (CDCl3, 125 MHz) of 183………………………………………688

xxix

Figure 3.313. gCOSY (CDCl3, 500 MHz) of 183…………………………………………689
1

Figure 3.314. H-NMR (CDCl3, 500 MHz) of 184………………………………………..690
Figure 3.315.

13

C-NMR (CDCl3, 125 MHz) of 184………………………………………691

Figure 3.316. gCOSY (CDCl3, 500 MHz) of 184…………………………………………692
1

Figure 3.317. H-NMR (CDCl3, 500 MHz) of 185……………………………………..…693
Figure 3.318.

13

C-NMR (CDCl3, 125 MHz) of 185………………………………………694

Figure 3.319. gCOSY (CDCl3, 500 MHz) of 185………………………………………...695
1

Figure 3.320. H-NMR (CDCl3, 500 MHz) of 186........................................................696
Figure 3.321.

13

C-NMR (CDCl3, 125 MHz) of 186………………………………………697

Figure 3.322. gCOSY (CDCl3, 500 MHz) of 186…………………………………………698
1

Figure 3.323. H-NMR (CDCl3, 500 MHz) of 187………………………………………..699
Figure 3.324.

13

C-NMR (CDCl3, 125 MHz) of 187………………………………………700

Figure 3.325. gCOSY (CDCl3, 500 MHz) of 187…………………………………………701
1

Figure 3.326. H-NMR (CDCl3, 500 MHz) of 188………………………………………..702
Figure 3.327.

13

C-NMR (CDCl3, 125 MHz) of 188………………………………………703

Figure 3.328. gCOSY (CDCl3, 500 MHz) of 188…………………………………………704
1

Figure 3.329. H-NMR (CDCl3, 500 MHz) of 269………………………………………..705
Figure 3.330.

13

C-NMR (CDCl3, 125 MHz) of 269………………………………………706

1

Figure 3.331. H-NMR (CDCl3, 500 MHz) of 120………………………………………..707
Figure 3.332.

13

C-NMR (CDCl3, 125 MHz) of 120………………………………………708

xxx

1

Figure 3.333. H-NMR (CDCl3, 500 MHz) of 177………………………………………..709
Figure 3.334.

13

C-NMR (CDCl3, 125 MHz) of 177………………………………………710

Figure 3.335. gCOSY (CDCl3, 500 MHz) of 177…………………………………………711
Figure 3.336. gHMQC (CDCl3, 500 MHz) of 177…………………………………..……712
1

Figure 3.337. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 177……………713
Figure 3.338. gHMBC (CDCl3, 500 MHz) of 177…………………………………………714
1

Figure 3.339. H-NMR (CDCl3, 600 MHz) of 191………………………………………..715
Figure 3.340.

13

C-NMR (CDCl3, 150 MHz) of 191………………………………………716

Figure 3.341. gCOSY (CDCl3, 600 MHz) of 191………………………………………...717
1

Figure 3.342. H-NMR (CDCl3, 500 MHz) of 178………………………………………..718
Figure 3.343.

13

C-NMR (CDCl3, 125 MHz) of 178………………………………………719

Figure 3.344. gCOSY (CDCl3, 500 MHz) of 178…………………………………………720
1

Figure 3.345. H-NMR (CDCl3, 600 MHz) of 179……………………………………….721
Figure 3.346.

13

C-NMR (CDCl3, 150 MHz) of 179………………………………………722

Figure 3.347. gCOSY (CDCl3, 600 MHz) of 179…………………………………………723
Figure 3.348. gHMQC (CDCl3, 600 MHz) of 179………………………………………...724
1

Figure 3.349. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 179…………...725
Figure 3.350. gHMBC (CDCl3, 600 MHz) of 179…………………………………………726
1

Figure 3.351. H-NMR (CDCl3, 600 MHz) of 180………………………………………..727
Figure 3.352.

13

C-NMR (CDCl3, 150 MHz) of 180………………………………………728

xxxi

Figure 3.353. gCOSY (CDCl3, 600 MHz) of 180…………………………………………729
Figure 3.354. gHMQC (CDCl3, 600 MHz) of 180………………………………………...730
1

Figure 3.355. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 180……………731
Figure 3.356. gHMBC (CDCl3, 600 MHz) of 180…………………………………………732
1

Figure 3.357. H-NMR (CDCl3, 500 MHz) of 181………………………………………..733
Figure 3.358.

13

C-NMR (CDCl3, 150 MHz) of 181………………………………………734

Figure 3.359. gCOSY (CDCl3, 500 MHz) of 181…………………………………………735
1

Figure 3.360. H-NMR (CDCl3, 600 MHz) of 182……………………………………….736
Figure 3.361.

13

C-NMR (CDCl3, 150 MHz) of 182……………………………………...737

Figure 3.362. gCOSY (CDCl3, 600 MHz) of 182………………………………………...738
Figure 3.363. gHMQC (CDCl3, 600 MHz) of 182………………………………………...739
1

Figure 3.364. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 182…………...740
Figure 3.365. gHMBC (CDCl3, 600 MHz) of 182…………………………………………741
1

Figure 3.366. H-NMR (CDCl3, 600 MHz) of 176………………………………………..742
Figure 3.367.

13

C-NMR (CDCl3, 150 MHz) of 176………………………………………743

Figure 3.368. gCOSY (CDCl3, 600 MHz) of 176…………………………………………744
Figure 3.369. gHMQC (CDCl3, 500 MHz) of 176………………………………………...745
1

Figure 3.370. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 176……………746
Figure 3.371. gHMBC (CDCl3, 500 MHz) of 176…………………………………………747
1

Figure 3.372. H-NMR (CDCl3, 500 MHz) of 192………………………………………..748

xxxii

Figure 3.373.

13

C-NMR (CDCl3, 125 MHz) of 192………………………………………749

Figure 3.374. gCOSY (CDCl3, 500 MHz) of 192…………………………………………750
1

Figure 3.375. H-NMR (CDCl3, 500 MHz) of 194………………………………………..751
Figure 3.376.

13

C-NMR (CDCl3, 125 MHz) of 194………………………………………752

Figure 3.377. gCOSY (CDCl3, 500 MHz) of 194…………………………………………753
1

Figure 3.378. H-NMR (CDCl3, 500 MHz) of 195………………………………………..754
Figure 3.379.

13

C-NMR (CDCl3, 125 MHz) of 195………………………………………755

Figure 3.380. gCOSY (CDCl3, 500 MHz) of 195…………………………………………756
1

Figure 3.381. H-NMR (CDCl3, 500 MHz) of 196………………………………………..757
1

Figure 3.382. H-NMR (CDCl3, 500 MHz) of 175………………………………………..758
Figure 3.383.

13

C-NMR (CDCl3, 125 MHz) of 175………………………………………759

1

Figure 3.384. H-NMR (CDCl3, 500 MHz) of 220………………………………………..760
Figure 3.385.

13

C-NMR (CDCl3, 125 MHz) of 220………………………………………761

Figure 3.386. gCOSY (CDCl3, 500 MHz) of 220…………………………………………762
1

Figure 3.387. H-NMR (CDCl3, 500 MHz) of 198………………………………………..763
1

Figure 3.388. H-NMR (CDCl3, 500 MHz) of 205………………………………………..764
1

Figure 3.389. H-NMR (CDCl3, 500 MHz) of 221………………………………………..765
Figure 3.390.

13

C-NMR (CDCl3, 125 MHz) of 221………………………………………766

1

Figure 3.391. H-NMR (CDCl3, 500 MHz) of 222………………………………………..767
1

Figure 3.392. H-NMR (CDCl3, 500 MHz) of 223………………………………………..768

xxxiii

Figure 3.393.

13

C-NMR (CDCl3, 125 MHz) of 223………………………………………769

1

Figure 3.394. H-NMR (D2O, 600 MHz) of 226…………………………………………..770
1

Figure 3.395. H-NMR (CDCl3, 600 MHz) of 227………………………………………..771
Figure 3.396.

13

C-NMR (CDCl3, 150 MHz) of 227………………………………………772

1

Figure 3.397. H-NMR (CDCl3, 500 MHz) of 228………………………………………..773
Figure 3.398.

13

C-NMR (CDCl3, 125 MHz) of 228………………………………………774

1

Figure 3.399. H-NMR (D2O, 600 MHz) of 24……………………………………………775
1

Figure 3.400. H-NMR (CDCl3, 600 MHz) of 239………………………………………..776
Figure 3.401.

13

C-NMR (CDCl3, 125 MHz) of 239………………………………………777

Figure 3.402. gCOSY (CDCl3, 600 MHz) of 239…………………………………………778
1

Figure 3.403. H-NMR (CDCl3, 500 MHz) of 243………………………………………..779
Figure 3.404.

13

C-NMR (CDCl3, 125 MHz) of 243………………………………………780

1

Figure 3.405. H-NMR (CDCl3, 500 MHz) of 251………………………………………..781
1

Figure 3.406. H-NMR (CDCl3, 500 MHz) of 252………………………………………..782
Figure 3.407.

13

C-NMR (CDCl3, 125 MHz) of 252………………………………………783

Figure 3.408. gCOSY (CDCl3, 500 MHz) of 252…………………………………………784
1

Figure 3.409. H-NMR (CDCl3, 500 MHz) of 253………………………………………..785
Figure 3.410.

13

C-NMR (CDCl3, 125 MHz) of 253………………………………………786

Figure 3.411. gCOSY (CDCl3, 500 MHz) of 253…………………………………………787
1

Figure 3.412. H-NMR (CDCl3, 500 MHz) of 254………………………………………..788

xxxiv

1

Figure 3.413. H-NMR (CDCl3, 500 MHz) of 254………………………………………..789
1

Figure 3.414. H-NMR (D2O, 600 MHz) of 233…………………………………………..790
Figure 3.415. HPLC curve for crude peptide 244 before purification...........................791
Figure 3.416. HPLC curve for crude glycopeptide 238 before purification..................792

xxxv

LIST OF ABBREVIATIONS
Acetic acid (HOAc)
Acetic anhydride (Ac2O)
Acetonitrile (MeCN)
Acetyl (Ac)
Ammonium acetate (NH4OAc)
Ammonium formate (HCOONH4)
(7-aza-1H-benzotriazole-1-yl)-1, 1, 3, 3-tetramethyluronium hexafluorophosphate
(HATU)
Benzenesulfinyl morpholine (BSM)
1-Benzenesulfinyl piperidine (BSP)
Benzenesulfenyl triflate (PhSOTf)
Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP)
Benzoyl (Bz)
Benzoyl chloride (BzCl)
Benzyl (Bn)
Benzyl bromide (BnBr)
[Bis(acetoxy)iodo]benzene (BAIB)
Boron trifluoride etherate (BF3-Et2O)
Camphorsulfonic acid (CSA)
Cerium (Ce)
Cesium fluoride (CsF)

xxxvi

Chloroform (CHCl3)
Chloroacetyl (ClAc)
Chondroitin sulfate (CS)
Concentrated (conc.)
Copper sulfate (CuSO4)
Correlation spectroscopy (COSY)
°

Degree celsius ( C)
Delta (δ)
Dichloromethane (DCM)
Deuterated chloroform (CDCl3)
Deuterated methanol (CD3OD)
Deuterated water (D2O)
Dibutyltin oxide (Bu2SnO)
2, 3-Dichloro-5, 6-dicyano-1, 4-benzoquinone (DDQ)
Diisopropylethylamine (DIPEA)
4-Dimethylaminopyridine (DMAP)
Dimethyldioxirane (DMDO)
Dimethylformamide (DMF)
2, 6-Dimethylphenyl (DMP)
2, 2-Dimethyltrimethylene (DMTM)
Dimethyl sulfoxide (DMSO)
Diphenylsulfoxide (Ph2SO)

xxxvii

2, 6-di-tert-butyl-4-methylpyridine (DTBMP)
Doublet (d)
Electrospray ionization (ESI)
Equivalent (equiv.)
Ethyl acetate (EtOAc)
1-Ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride (EDC)
Exotosin (EXTL)
Fluorenylmethyloxycarbonyl chloride (FmocCl)
Fluorous solid-phase extraction (F-SPE)
Galactose (Gal)
Galactosyltransferases (GLCATs)
Glucosamine (GluN)
Glucronic acid (GlcA)
Glucuronyltransferase (GLCAT)
Glycosaminoglycan (GAG)
Gram (g)
High performance liquid chromatography (HPLC)
Heparin (HP)
Heparan sulfate (HS)
Heparan sulfate proteoglycans (HSPGs)
Heteronuclear multiple bond coherence (HMBC)
Heteronuclear multiple quantum coherence (HMQC)
Hyaluronic acid (HA)

xxxviii

Hydrazine hydrate (NH2NH2-H2O)
Hydrochloric acid (HCl)
Hydrogen fluoride (HF)
Hydrogen peroxide (H2O2)
Idose (Ido)
Iduronic acid (Idu)
Levulinoyl (Lev)
Lithium hydroxide (LiOH)
Magnesium methoxide (Mg(OMe)2)
Mass spectrometry (MS)
Mega Hertz (MHz)
Methanol (MeOH)
Microliter (μL)
Milligram (mg)
Millimolar (mmol)
Multiplet (m)
N-Acetylglucosamine (GlcNAc)
N-iodosuccinimide (NIS)
N, N’-Dicyclohexylcarbodiimide (DCC)
Nuclear magnetic resonance (NMR)
O-benzotriazole-N, N, N’, N’-tetramethyl-uronium hexafluoro-phosphate (HBTU)
O, O-Dimethylthiophosphonosulfenyl bromide (DMTPSB)
Palladium chloride (PdCl2)

xxxix

Palladium hydroxide (Pd(OH)2)
Palladium on activated carbon (Pd/C)
p-Methoxybenzyl (PMB)
Phthalimido (Phth)
Potassium bicarbonate (KHCO3)
Potassium carbonate (K2CO3)
Proteoglycans (PGs)
p-Toluenesulfenyl chloride (p-TolSCl)
p-Toluenesulfonic acid (p-TsOH)
Pyridine (Py)
Relative Reactivity Values (RRVs)
Room temperature (r.t.)
Silver triflate (AgOTf)
Singlet (s)
Size-exclusion column (LH-20)
Size-exclusion column (G-15)
S-(4-methoxyphenyl) benzene-thiosulfinate (MBPT)
Sodium bicarbonate (NaHCO3)
Sodium carbonate (Na2CO3)
Sodium hydride (NaH)
Sodium hydroxide (NaOH)
Sodium methoxide (NaOMe)

xl

Sodium sulfate (Na2SO4)
Sodium thiosulfate (Na2S2O3)
Sodium bis(trimethylsilyl)amide (NaHMDS)
Solid phase supported triphenylphosphine (PS-PPh3)
Sulfur trioxide (SO3)
t-Butanol (t-BuOH)
Tert-butyl diphenyl silyl (TBDPS)
Tetra-butyl ammonium fluoride (TBAF)
2, 2, 6, 6-Tetramethylpiperidin-1-oxyl (TEMPO)
Tetrahydrofuran (THF)
Tetra-n-butyl ammonium bromide (TBAB)
Thin layer chromatography (TLC)
Thioacetic acid (HSAc)
Tin chloride (SnCl4)
Trichloroethoxycarbonyl (Troc)
Triflic acid (TfOH)
Trifluoromethanesulfonic anhydride (Tf2O)
Triisopropylsilyl (TIPS)
Trimethylphosphine (Me3P)
Trimethylsilyl triflate (TMSOTf)
Tri-tert-butyl pyrimidine (TTBP)
Triphenylphosphine (Ph3P)

xli

Triplet (t)
Trichloroacetyl (TCA)
Triethylamine (Et3N)
Triethyl phosphite (TEP)
Triethylsilane (Et3SiH)
Trifluoroacetic acid (TFA)
Tri-tert-butyl silyl (TBS)
Tri-tert-butylsilyltrifluoromethanesulfonate (TBSOTf)
Water (H2O)
Xylose (Xyl)
Xylotransferase (XYLT)
Zinc (Zn)

xlii

LIST OF SCHEMES
Scheme 1.1. Pre-activation based glycosylation strategy.………………………………..3
Scheme 1.2. Glycosylation of unreactive substrate

.…………………………………….3

Scheme 1.3. Chemoselective glycosylation .……..……….………..…………….……….4
Scheme 1.4. Orthoganol synthesis of trisaccharide 14

…………………………………5

Scheme 1.5. One-pot synthesis of tetrasaccharide 30 …………………………………...5
Scheme 1.6. Active-latent strategy for the synthesis of disaccharide 34…..…………….5
Scheme 1.7. Neighbouring group participation by C-2 ester….……………………………6
Scheme 1.8. Representative approaches for synthesizing 1, 2-cis linkages .................8
Scheme 1.9. One-pot synthesis of heptasaccharide 55……………...…………….………9
Scheme 1.10. Glycosylation of glycals.……………………………………………………..10
Scheme 1.11. General strategy for solution-phase oligosaccharide synthesis ………..10
Scheme 1.12. Solid-phase synthesis of tetrasaccharide 82 ………………………….….11
Scheme 1.13. Chemoselective dehydrative glycosylation …….………………………...13
Scheme 1.14. Possible mechanism for dehydrative glycosylation ……………..……….13
Scheme 1.15. Possible reactive intermediates in dehydrative glycosylation ......……...13
Scheme 1.16. Chemoselective iterative dehydrative glycosylation .…………………...14
Scheme 1.17. Chemoselective iterative dehydrative glycosylation ..………………..….16
Scheme 1.18. Chemical synthesis of HA trimer 108 ……………………………………16
Scheme 1.19. Effects of anomeric leaving group on glycosylation outcomes ………...17
Scheme 1.20. Effects of activator system on glycosylation outcomes ………………....17
Scheme 1.21. Synthesis of 1, 2-orthoester from glycosyl bromide ……....….….….…18
Scheme 1.22. Bromoglycoside-mediated glycosylation .…….….….….….…………..…19

xliii

Scheme 1.23. Reactivity based one-pot synthesis of oligosaccharides …………….…22
Scheme 1.24. Iterative one-pot synthesis of oligosaccharides …………………….……23
Scheme 1.25. Proposed mechanism for pre-activation glycosylation strategy ………23
Scheme 1.26. Pre-activation of donor 166 ….................................………….….……..24
Scheme 1.27. One-pot synthesis of HA pentasaccharide 173 …………….…..….…..26
Scheme 1.28. Retrosynthetic analysis of HA decasaccharide 175 ………………...…27
Scheme 1.29. One-pot synthesis of HP-like hexasaccharide 185 ……..….….….….…27
Scheme 1.30. Synthesis of hexasaccharide 190 with preinstalled sulfates……………28
Scheme 1.31. BSP/Tf2O promoted synthesis of 193………………………..……………30
Scheme 1.32. Weak promoting ability of BSP/Tf2O………………..……………………..30
Scheme 1.33. BSP/Tf2O promoted one-pot synthesis of tetrasaccharide 202……..….31
Scheme 1.34. Possible pathway leading to the formation of orthoester…..……………37
Scheme 1.35. Possible effects of anomeric leaving group on orthoester formation…..38
Scheme 1.36. Control experiments………………………..………………………………..38
Scheme 1.37. Formation of oxazoline upon pre-activation

…………….……………..39

Scheme 1.38. Synthesis of fully protected decasaccharide 156………………..……….40
Scheme 1.39. Participation of DMTM in glycosylation and subsequent cleavage ...…41
Scheme 1.40. Plausible reaction mechanism…………………….……………………….41
Scheme 1.41. Chiral auxiliary assisted stereoselective glycosylation via preactivation………………………………………………………………………………………..43
Scheme 1.42. Unexpected low anomeric selectivity from donor 254……..…………….45
Scheme 1.43. Equilibrium between oxacarbenium ion and α-sulfonium ion……..…….45
Scheme 1.44. Participation of 2-O-Boc group ….………………………………………..47

xliv

Scheme 1.45. Participation of axial 3-O-Boc group.……………………………………..47
Scheme 1.46. Isotopic labeling experiment……….………………………………………47
Scheme 1.47. Disaccharide 275 was isolated in (1) 65% (α:β = 10:1) when steps A and
B were combined. (2) 93% (α:β = 1:10.5) when steps A and B were carried out in a
sequential manner………………………….…………………………………………………49
Scheme 1.48. Proposed mechanism leading to high β-selectivity……….…………......49
Scheme 1.49. Detection of α-triflate………………….…………………………………….50
Scheme 1.50. Highly stereoselective synthesis of β-mannoside………..………………50
Scheme 1.51. Pre-activation of 289 led to anomeric mixture…………………..………..51
Scheme 1.52. Equilibrium between reactive intermediates upon pre-activation of
289..................................................................................................................................51
Scheme 1.53. Strikingly different stereoselectivity for glucose series………..…………52
Scheme 1.54. Torsional angle values for reactive intermediates of mannose and
glucose………………………………………………………………………………………….52
Scheme 1.55. Loss of stereoeselectivity in 2-deoxy and 3-deoxy donor
series ………………………………………………………………………………………...53
Scheme 1.56. Loss of stereoeselectivity in 2-deoxy-2-fluoro and 3-deoxy-3-fluoro donor
series ………………………………………………………………………………………...54
Scheme 1.57. Poor stereoeselectivity for glucose serie………………………………….55
Scheme 1.58. Similar stereoeselectivity in galactose series.……………………………55
Scheme 1.59. Similar stereoeselectivity in mannose series…..…………………………55
Scheme 1.60. Stereo-directing effect of 2, 3-carbonate protecting group….………….56
Scheme 1.61. Stereo-directing effect of 3, 4-carbonate protecting group……..……….57
Scheme 1.62. Reversal of stereoselectivity in the case of 3, 4-isopropylidene acetal
group…..………………………………………………………………………………………..57
Scheme 1.63. Stereo-directing effect of 2, 3-carbonate protecting group……..……….58
Scheme 1.64. Loss of stereoselectivity for 3, 4-carbonate protected glucose donor…58

xlv

Scheme 1.65. Stereoselective glycosylation of 2-deoxygalactose/glucose…………….59
Scheme 1.66. β-selective glycosylation of oxazolidinone protected donor 350………..60
Scheme 1.67. Effect of donor protecting groups on stereoselectivity….………………..61
Scheme 1.68. Synthesis of trisaccharide 367…………………………...………………...61
Scheme 1.69. Stereo-directing effect of 2-O-sulfonate group………………..………….63
Scheme 1.70. Stereo-directing effect of a second electron-withdrawing group…..……63
Scheme 1.71. Poor stereoselectivity for mannoside donor 373…………………..……..64
Scheme 1.72. Stereo-directing effects of different electron-withdrawing groups..…….64
Scheme 1.73. Highly α-selective synthesis of phosphosugars………….………………66
Scheme 1.74. Stereoselective glycosylation of 2-deoxygalactose/glucose.….……….66
Scheme 1.75. Steric effect on β-mannosylation…………….…………………………….67
Scheme 1.76. α-Selective glycosylation with 3, 4-bisacetal protected donor 403……..69
Scheme 1.77. α-Selective glycosylation with 3, 4-bisacetal protected donor 405…..…69
Scheme 1.78. β-Selective glycosylation of donor 407 ……………………..……………70
Scheme 1.79. Loss of stereoselectivity in the case of glycosylation of donor 409….…70
Scheme 1.80. Stereoselective synthesis of arabinofuranoside …………..……………72
Scheme 1.81. Proposed mechanism for arabinofuranosylation……..…………………..73
Scheme 1.82. Stereoselective synthesis of arabinofuranoside ………………...………73
Scheme 1.83. Effect of acceptor on stereoselective glycosylation.…………………….74
Scheme 1.84. Effect of acceptor on stereoselective glycosylation………..…………….74
Scheme 1.85. Effect of promoter on stereoselective glycosylation……………….…….75
Scheme 1.86. Effect of solvent on stereoselective glycosylation………………..………76
Scheme 1.87. Effect of AgOTf on stereoselective glycosylation ……………………….76

xlvi

Scheme 1.88. Proposed mechanism for effects of solvents and AgOTf on
stereoselectivity………………………………………………………………………………76
Scheme 1.89. Evidence for solvent participation ………………………………………..78
Scheme 1.90. Possible mechanism for DMF-modulated glycosylation ……………….79
Scheme 1.91. Detection of α-glycosyl imidate 448……………………..…………….…80
Scheme 1.92. Generation of sulfenate from sulfoxide

..………………………………81

Scheme 2.1. Synthetic application of fluorous tagged glycosyl donor ……….….……102
Scheme 2.2. Application of fluorous thiol in oligosaccharide synthesis ………..……102
Scheme 2.3. Synthesis of aldehyde 18, fluorous hydrazine 22 and ketone 15……….108
Scheme 2.4. Synthesis of fluorous hydrazine 27 and its application in “catch and
release” of 24….……………………………………………………………………………..109
Scheme 2.5. Synthesis of disaccharide 31. TLC: Lane 1, reaction mixture after
glycosylation; Lane 2, reaction mixture after “catch”, first F-SPE, and “release”; Lane 3,
organic fraction after the second F-SPE………………………………………………….110
Scheme 2.6. Synthesis of LewisX trisaccharide 35………………………………..…..112
Scheme 2.7. Synthesis of tetrasaccharide 37...………………………………..………113
Scheme 3.1. HSPGs contain diverse HS chain structures due to modifications by
multiple enzymes……………………………………………………………………………184
Scheme 3.2. Synthesis of glucosamine building block 41…………………………….188
Scheme 3.3. Synthesis of glucose building block 48 …………………………………195
Scheme 3.4. Synthesis of galactose building block 57 ………………………………197
Scheme 3.5. Synthesis of galactose building block 61 ………………………………198
Scheme 3.6. Synthesis of xylose building block 67…………………………………….199
Scheme 3.7. Synthesis of xylose building block 72……………………………………200
Scheme 3.8. Screening donor-acceptor pairs for Xyl-Serine synthesis……………..204
Scheme 3.9. Synthesis of disaccharide 97……………………………………..……...205
xlvii

Scheme 3.10. Synthesis of disaccharide 101…………………………………………..206
Scheme 3.11. Synthesis of trisaccharide 103…………………………………………..206
Scheme 3.12. Synthesis of trisaccharide 104…………………………………………..206
Scheme 3.13. Synthesis of trisaccharide 108…………………………………………..207
Scheme 3.14. Outcome of the coupling between donor 109 and acceptor 40 ……209
Scheme 3.15. Synthesis of trisaccharide 117………………………………………….209
Scheme 3.16. Synthesis of trisaccharide 128………………………………………….212
Scheme 3.17. Synthesis of trisaccharide 128………………………………………….213
Scheme 3.18. Synthesis of disaccharide 144………………………………………….214
Scheme 3.19. Synthesis of disaccharide 149………………………………………….216
Scheme 3.20. Synthesis of pentasaccharide 157……………………………………..218
Scheme 3.21. Oxidation of pentasaccharide 156……………………………………...219
Scheme 3.22. Synthesis of pentasaccharide 160……………………………………..220
Scheme 3.23. Synthesis of heptasaccharide 161……………………………………..221
Scheme 3.24. Synthesis of heptasaccharide 164……………………………………..221
Scheme 3.25. Synthesis of pentasaccharide 166……………………………………..222
Scheme 3.26. Synthesis of hexasaccharide 168………………………………………223
Scheme 3.27. Possible pathway for the formation of 169…………………………….224
Scheme 3.28. Synthesis of octasaccharide 173…………………………….…………225
Scheme 3.29. Synthesis of hexasaccharide 174………………………………………225
Scheme 3.30. Synthesis of hexasaccharide 177………………………………………239
Scheme 3.31. Synthesis of disaccharide 178…………………………………………..230
Scheme 3.32. Synthesis of trisaccharide 179………………………………………….231

xlviii

Scheme 3.33. Synthesis of octasaccharide 176……………………………………….232
Scheme 3.34. Synthesis of dipeptide 120………………………………………………233
Scheme 3.35. Possible pathway for the formation of side product 187……………..233
Scheme 3.36. Synthesis of octasaccharide 194……………………………………….234
Scheme 3.37 Synthesis of glycopeptide 175…………………………………………..235
Scheme 3.38. Synthesis of octasaccharide 200………………………………………..238
Scheme 3.39. Synthesis of octasaccharide 204………………………………………..239
Scheme 3.40. Synthesis of octasaccharide 207………………………………………..241
Scheme 3.41. Hydrolysis of compound 94 ……………………………………………243
Scheme 3.42. Hydrolysis of compound 220…………………………………………….243
Scheme 3.43. Synthesis of octasaccharide 224………………………………………..244
Scheme 3.44. Synthesis of octasaccharide 226………………………………………..246
Scheme 3.45. Synthesis of glycopeptide 229…………………………………………..247
Scheme 3.46. Synthesis of glycopeptide 24……………………………………………248
Scheme 3.47. N-Sulfation of octasaccharide 203……………………………………..249
Scheme 3.48. Solid phase synthesis of glycopeptide 239……………………………252
Scheme 3.49. Solid phase synthesis of glycopeptide 241……………………………253
Scheme 3.50. Synthesis of glycopeptide 243………………………………………….255
Scheme 3.51. Synthesis of peptide 245…………………………………………………256
Scheme 3.52. Synthesis of glycopeptide 251…………………………………………..257
Scheme 3.53. Synthesis of glycopeptide 255…………………………………….……258
Scheme 3.54. Synthesis of glycopeptide 258………………………………………….259
Scheme 3.55. N-Sulfation of glycopeptide 258………………….……………………..260

xlix

Scheme 3.56. Synthesis of glycopeptide 262………………………………………….261
Scheme 3.57. Synthesis of glycopeptide 233…………………………………………..262
Scheme 3.58. Synthesis of octasaccharide 265………………………………………..265
Scheme 3.59. Synthesis of octasaccharide 266………………………………………..266
Scheme 3.60. Synthesis of glycopeptide 233…………………………………………..267

l

Chapter 1
Pre-activation Based Glycosylation Strategy
1.1. Introduction
Carbohydrates are widely present in biological systems and many of them
have important functions, such as anticoagulation, inflammation and bacterial
infection.

1-2

In order to explore their biological functions, large quantities of

oligosaccharides with high purity are required. However, this is hampered by limited
availability of oligosaccharides from nature. Thus, chemical synthesis becomes a
powerful tool providing high quality samples for biological studies. Traditional
oligosaccharide synthesis is a time-consuming process due to the need for protecting
group manipulations and aglycon adjustments on reaction intermediates. A typical
procedure starts from the coupling of a donor to an acceptor to yield a disaccharide,
which can be converted to another acceptor after protecting group manipulation.
Then this disaccharide acceptor can be coupled to another donor to give a
trisaccharide. This process can be repeated until the desired oligosaccharide
structure is assembled.

3

To expedite oligosaccharide assembly, many novel

strategies have been developed, such as automated solid phase synthesis,
orthoganol glycosylation,
active-latent activation,

5-6

11-13

reactivity-based armed-disarmed glycosylation,

and iterative glycosylation.

14-17

4

7-10

Among these methods,

iterative glycosylation is particularly noteworthy. This strategy utilizes one type of
glycosyl donor and acceptor such as thioglycoside along with a single set of
glycosylation

conditions,

therefore

eliminating

extensive

protecting

group

manipulations and aglycon adjustments. The overall efficiency has greatly improved

1

via this strategy, which is achieved by pre-activation based glycosylation.
Pre-activation means the donor is activated in the absence of acceptor to form a
reactive intermediate, followed by addition of acceptor to the activated donor to form
the desired glycosylation product. as demonstrated in Scheme 1.1,

18-19

donor 1 was

first activated to give a reactive intermediate 2, which was converted to glycoside 4
upon addition of acceptor 3. For a pre-activation based glycosylation reaction to be
successful, intermediate 2 has to be stable prior to the addition of acceptor 3 and yet
reactive enough to electrophilically attack acceptor 3. Once converted to its activated
form 2, donor 1 can directly react with acceptor 2 without the need for other activating
reagents. The first pre-activation based glycosylation reaction was reported by Kahne
and coworkers in 1989.

20

They found by simply changing the sequence of the

reagents added into the reaction flask, they were able to glycosylate very unreactive
substrate as shown in Scheme 1.2. The C-7 hydroxyl in sterol 7 is extremely
sterically hindered due to unfavorable 1, 3-diaxial interaction with the C-4 methylene.
Traditional glycosylation procedures need extended reaction time and usually give
o

low yield. In this paper, they pre-mixed 5 with Tf2O under -78 C, followed by addition
of sterol 7 and 2, 6-di-tert-butyl-4-methylpyridine (DTBMP) which works as an acid
scavenger. After aqueous workup and column purification they were able to get
compound 6 in 86% yield. Ever since then, pre-activation strategy has been widely
applied in oligosaccharide synthesis. Compared to traditional oligosaccharide
assembly approach, pre-activation has unique reaction selectivities. This review will
focus on chemoselectvity and stereoselectivity brought by pre-activation approach.

2

O
PGO
1

X

Activate X

O
HO PGO
3

O
PGO
2

X
O
PGO

Q

O
O PGO

X

4

Scheme 1.1. Pre-activation based glycosylation strategy.
PivO
PivO
PivO

O
S
OPiv Ph

O

a
7

PivO
PivO
PivO

H

5

O

OR
6 OPiv

CO2CH3
ROH = H

H
OH 7

EtO 2CO

Scheme 1.2. Glycosylation of unreactive substrate. Reagents and conditions: (a)
o

Tf2O, - 78 C, DCM, then 7, DTBMP.
1.2. Selectivity in Glycosidic Bond Formation
Glycosylation is one of the most important reactions in carbohydrate
21

chemistry.

Selectivity,

including

chemoselectivity,

stereoselectvity

and

regioselectivity, is the most important issue in glycosylation reaction. Although a lot of
advances have been achieved in this area in the past few decades, selective
glycosylation remains unresolved and needs further exploration.
1.2.1. Chemoselectivity
In a glycosylation reaction, if both donor and acceptor have the potential of
getting activated by promoter, specific reaction conditions have to be used to
selectively activate donor, followed by nucleophilic attack of acceptor to form the
product (Scheme 1.3). For this chemoselective glycosyaltion, only compound 1 can
get activated by the promoter, which further coupled with compound 8 to form
compound 9. To achieve this, several methods are available.

3

O
PGO
1

O
X + HO
PGO
8

Y

promoter

O
PGO

O
O
PGO
9

Y

Scheme 1.3. Chemoselective glycosylation.
In 1994, Ogawa and coworkers developed orthogonal glycosylation
approach, which utilizes two different leaving groups in the two coupling partners and
each one can be selectively activated over the other one by a promoter.

6

Donor 10

was activated over acceptor 11 to form disaccharide 12, which was further activated
over acceptor 13 to give trisaccharide 14. In this example, thioglycoside 10 and
fluoride 12 were activated over each other by different promoters (Scheme 1.4).
Wong and coworkers made tremendous contribution to reactivity based glycosylation
approach. In 1999, they quantified the reactivity of a large library of p-methylphenyl
thioglycosides.

7

Based on this database, they were able to synthesize different

oligosaccharides via one-pot approach. With Relative Reactivity Values (RRVs) of all
the monosaccharides in hand, they synthesized tetrasaccharide 30 in 40% yield
(Scheme 1.5). To achieve chemoselectivity, active-latent activation is another
alternative approach. Boons and coworkers started from allyl glycoside 31, which
was converted to donor 32 and acceptor 33. Glycosylation of 32 to 33 afforded 34 in
12

89%, which was ready for another round of glycosylation (Scheme 1.6).

4

AcO
AcO

OAc
O

HO
SPh+ BnO
NPhth
10
b

HO
BnO

OBn
O

SPh
NPhth
13

OBn
O

OBn
OAc
O
O O
F
F
BnO
90%
NPhth
NPhth
NPhth
12
11
OBn
OBn
OAc
O
O O
O O
AcO
SPh
BnO
BnO
AcO
NPhth
NPhth
NPhth
81%
14
a

AcO
AcO

Scheme 1.4. Orthogonal synthesis of trisaccharide 14. Reagents and conditions: (a)
o

o

NIS, AgOTf, DCM, -50 C-r.t.; (b) Cp2HfCl2, AgClO4, DCM, -50 C-r.t.

HO
BzO

OBz
O

BzO

OBn
O

OH
O

STol BzO
STol HO
AcO
NHTroc
AcO
OBz
OMe
27 (162.9)
28 (13.1)
29
BnO
STol
c
a
b
OBn
15 (1.7 x 104) BnO OBn
O
BnO
OBz
BnO
O
O
O
BzO
OBn
TrocHN BzO
O
O O
30 BzO
AcO
40%
AcO
OBz
OMe
BnO

OBn
O

Scheme 1.5. One-pot synthesis of tetrasaccharide 30. Reagents and conditions: (a)
NIS, TfOH, DCM; (b) NIS; (c) NIS.

BnO
O
AcO
BnO
BnO O
31

a

b

BnO
O
AcO
BnO
BnO O
32
BnO
HO
BnO

BnO
BnO
O O
AcO
c
O
89% BnO BnO BnO
BnO O
34

O

BnO O
33

Scheme 1.6. Active-latent strategy for the synthesis of disaccharide 34. Reagents
and conditions: (a) (PhP)3RhCl; (b) NaOMe; (c) TMSOTf, CH3CN.
1.2.2. Stereoselectivity
In general, there are four different types of glycosidic linkages: α-1,2-cis,
22

α-1,2-trans, β-1,2-cis, β-1,2-trans (Figure 1.1).
5

Neighbouring group participation or

anchimeric assistance has been widely used for installing 1, 2-trans glycosidic
linkages such as 36 and 38.
for this type of linkage.

23

2-O-carboxylate esters is of fundamental importance

22,24-28

The participating of 2-O-carboxylate esters is

supported by the isolation of crystalline dioxalenium ions in some reactions,

29

by the

spectroscopic data of the same species as intermediates in some other examples,

30

31

by computational work , by rate acceleration in the case of weakly activated
donors,

29,32

and also supported by the highly 1,2-trans-selectivity in these reactions
33

(Scheme 1.7).
such

Besides 2-O-carboxylate esters, other types of participating groups

as

2-O-(2-pyridyl)methyl

2-deoxy-2-dibenzylamino systems,
39

benzyl ethers

O

PGO

2-O-(2-thio)ethers,
37-38

2-O-phosphate esters,

33-35

and other bulky

have been used for installing 1, 2-trans linkages.
R 1O
O

R1O
O
O
OR2
PGO
OR2PGO
R1O
OR2
β-1, 2-trans
α-1, 2-trans β-1, 2-cis

PGO

R1OOR
2
α-1, 2-cis

36

ethers,

Figure 1.1. Four different types of glycosidic linkages.
O

RO

O
R

A+
X

O
39

O

RO
AX

O
R

O
40
oxo-carbenium ion

RO

O

H

O

R'

O

RO

O

OO
41 R
acetoxonium ion

OR'

R

O
42
1,2-tr ans-glycoside

Scheme 1.7. Neighbouring group participation by C-2 ester.
Compared to 1, 2-trans linkage, 1, 2-cis linkage is much more challenging
and so far no simple, robust methodology has been established. Figure 1.2 shows
several widely used monosaccharide building blocks with α-linkage at anomeric

6

position.

40

Except mannoside 46, there are some methods available for construction

of other 1, 2-cis linked glycosides based on traditional non-preactivation approach. To
introduce 1, 2-cis glycosidic linkage, nonassisting protecting group at C-2 position
such as 2-O-benzyl, 2-azido is required. However, the use of these glycosyl donors
usually leads to the formation of anomeric mixtures and separation of these mixtures
33

is a painful process.

To further increase α selectivity, different approaches have
40-42

been developed as summarized in Scheme 1.8.

However, none of these

methods are general and sometimes they can only give moderate stereoselectivity.
For some specific methods like intramolecular aglycon delivery, extra synthetic steps
have to be carried out before and after glycosylation which decreases the overall
efficiency. Synthesis of β mannoside, considered as one of the most difficult
glycosidic linkage to be formed and very few methods are available,
solved by Crich and coworkers,

43

42

was later

which will be discussed in the later section.

Figure 1.2. 1, 2-cis glycoside.

7

Et4NBr
O
Br
in situ
PO
PO
Br anomerization
R'

Structure, Temperature BnO Li
O
Pressure, Promoter
Additives, Concentration

O

O

O
O

H
OR
PO
Long-Range
Participation
O

BnO OBn
Complexation of
Lithium Cation
Ph
Ph
O Ph
O

O

PO

POOR

O

OR
H
Steric Hindrance
PO

O

H

O
R'
O
R'' O
Intramolecular
Aglycon Delivery

O

H
O

X
LH

PO
OR
Leaving Group

O

O

PO

OR
H
Solvent Effect

Scheme 1.8. Representative approaches for synthesizing 1, 2-cis linkages.
1.2.3. Regioselectivity
When a glycosyl acceptor has more than one hydroxyl groups, all of these
free hydroxyls can potentially attack the glycosyl donor to form a new glycoside. The
regioselectivity of the reaction depends on the reactivity of different hydroxyls, which
can be affected by different factors such as protecting groups, sterics, conformations
18-19

of sugar, etc.

44

(Scheme 1.9).

One example is one-pot synthesis of heptasaccharide 55

Regioselective glycosylation of acceptor 49 and 55 was achieved

by higher reactivity of primary hydroxyl groups at the 6 position. To achieve high
regioselectivity, preactivation based glycosylation approach also utilizes the different
reactivities of free hydroxyl groups in the acceptor, which is the same as traditional
non-preactivation based approach. Therefore, this review will only focus on
chemoselectivity and stereoselectivity.

8

HO
BnO
HO
O
O
MBzO
O
BnO
BnO
F
SEt MBzO
SPh
SEt HO
BnO
MBzO
BzO
PivO
52
50
51
c
d
b
MBzO
O O
MBzO
MBzO MBzO
O O
BnO
BnO
BnO
O
O BzO
BnO
O
O O
SPh BnO
BnO
MBzO
BnO PivO
MBzO MBzO
54 PivO
O O
f
BnO
BnO
O BzO
O
BnO
O
BnO PivO 55 AcO
AcO AcO
OMe
24%

HO
O
BnO
MBzO
HO
O
MBzO
49 BzO
MBzO
a
MBzO
48
Br
HO
O
AcO
AcO
AcO
53
OMe
e

Scheme 1.9. One-pot synthesis of heptasaccharide 55. Reagents and conditions: (a)
AgOTf, MS 4Å, DCM, -20

o

C; (b) MeOTf, DCM, r.t.; (c) DCM, r.t.; (d) AgOTf,

o

o

o

HfCp2Cl2, DCM, 0 C; (e) DMTST, DCM, 0 C; (f) DCM, 0 C.
1.3. Chemoselectivity in Pre-activation Based Glycosylation
Pre-activation approach achieves chemoselectivity via the usage of a single
set of glycosylation conditions and aglycon. Different glycosyl donor and promoter
combinations have been developed to achieve this chemoselectivity.
1.3.1. Chemoselective Glycosylation of Glycal
Glycals can be activated by a variety of protocols to give reactive glycosylabe
species (Scheme 1.10). (Methylthio)-sulfonium salt 58,
46

perchlorate 63

47

and NIS 67

45

iodonium di-sym-collidine

have been used to activate glycals. All these

reactions were done by pre-mixing with acceptors and stereisomers were generated
in the case of using 58 and 63 as promoters. Danishefsky and coworkers developed
a preactivation based strategy for solution-phase oligosaccharide synthesis with
48

glycals (Scheme 1.11).

Glycal 69 was first activated to give intermediate 70, which

glycosylated with acceptor 71 to produce glycoside 72. This disaccharide could be
further activated and the whole process can be reiterated to yield the desired
9

oligosaccharide sequence. They applied this strategy to solid-phase oligosaccharide
synthesis (Scheme 1.12). Epoxidizing agent 3, 3-dimethyldioxirane (DMDO) was
used to activate glycal 76 to give epoxide 77, which was attacked by acceptor 75 to
yield 78. Reiteration gave tetrasaccharide 81 followed by cleaving from solid support
to give final target 82.

BnO
BnO

OBn
O

OSnBu3
a

+

56

AcO
AcO

57

OAc
OAc
O
O + AcO
OH
AcO
62 OAc
61

OAc Ph
O +

AcO
AcO

O
O
HO
66

61

Scheme

BnO
BnO
BnO

O

O
59 SMe
42%
AcO
AcO
AcO

AcO
AcO

-

of

I

OAc
O

O
O

OAc
64
76%

AcO
AcO
AcO

c
54%
OMe

O

Glycosylation

1.10.
+

b

BnO
BnO
+ BnO

I

SMe
O
60 O
14%

AcO
O
AcO
O
AcO
OAc I
+
O
OAc
AcO
AcO 65
17%

O

Ph
O
O
O
68

glycals.

O
OMe

Reagents

and

conditions:

(a)

o

(MeSSMe2) BF4 58, DCM, -20 C; (b) iodonium di-sym-collidine perchlorate 63,
DCM; (c) NIS 67, CH3CN.

RO
RO
RO
RO
RO
RO

O

RO
RO
RO
70

E

69

O

HO
R'O
R'O

E

O
71

RO
RO
RO
72

O

O
E R'O
R'O

O

O

O
E RO
RO

O

73

O
E R'O
n R'O

O

Scheme 1.11. General strategy for solution-phase oligosaccharide synthesis with
+

glycals. Here E can be DMDO.

10

OSiPh2 S
OSiPh2 S
OH
Ph
b O O
O
a O O
c
O
O
Si Cl +
S
O
O
O
O
Ph
O
74
76
77 O
75
OR
OR
O
O
O
O
O
OR
OH
O
O
O
O
O
O
O
OH
e
d
O
O
OH
O
BnO
O
O
O
BnO
O
O
OH
O
80
O
O
O
O
O
O
O
OH
O
OH
O
O
O
O
O
O
78
O
O
O
O
OH
79
O
BnO
BnO
81: R = SiPh2 S
f
82: R = H

Scheme 1.12. Solid-phase synthesis of tetrasaccharide 82. Reagents and conditions:
(a) Hunig’s base, DCM; (b) DMDO, DCM; (c) 75, ZnCl2, THF; (d) DMDO, DCM, then
75, ZnCl2, THF; (e) DMDO, DCM, then 80, ZnCl2, THF; (f) TBAF, HOAc, THF.
1.3.2. Chemoselective Dehydrative Glycosylation
Traditional glycosidic coupling requires functionalization of the anomeric
position to form an isolable donor followed by reaction with a promoter to induce
49-53

irreversible transfer to an acceptor.

The direct substitution of anomeric hydroxyl

with the desired acceptor could be more efficient as this route obviates the need of
anomeric functionalization.

54-56

Gin and coworkers reported a one-step glycosylation

procedure starting from the free hemiacetal of the glycosyl donor which involves the
in situ activation of 1-hydroxy donors with trifluoromethanesulfonic anhydride (Tf2O)
and diphenylsulfoxide (Ph2SO).

57

In a representative procedure, Tf2O (1.4 equiv.)

was added into the mixture of donor 83 (1 equiv.) and Ph2SO (2.8 equiv.) in toluene
o

o

and dichloromethane (DCM) at -78 C. The reaction mixture was stirred at -40 C for

11

1 h followed by slow addition of acid scavenger 2-chloropyridine (5 equiv.) and
o

acceptor isopropyl alcohol (3 equiv.). The solution was stirred at 0 C for 15 mins and
o

1 h at 23 C to afford compound 84 in 86% yield (α:β = 27:73) (Scheme 1.13). This
glycosylation strategy can be applied to a variety of glycosyl acceptors besides
isopropyl alcohol, including oxygen, sulfur, carbon and nitrogen nucleophiles. Even
the

most

unreactive

N-(trimethylsilyl)trimethylacetamide

could

be

efficiently

glycosylated to afford the corresponding glycosyl amide. Based on the experimental
results, they proposed two possible reaction mechanisms (Scheme 1.14).

58

In

pathway 1, hemiacetal 85 attacked the sulfonium center of the in situ formed diphenyl
sulfide bis(triflate) to give the glycosyl oxosulfonium intermediate 86, which later
glycosylated the acceptor to yield the product 87. Alternatively, in pathway 2,
hemiactal 85 attacked the sulfonyl center of diphenyl sulfide bis(triflate) to give the
glycosyl triflate intermediate 88, followed by glycosylation to give 87. To distinguish
between these two possible reaction pathways, an
out.

18

O-labled hemiacetal 85 was prepared and

18

O-labeling study was carried

18

O-labeled diphenyl sulfoxide was

detected which meant pathway 1 was the operative reaction mechanism. Although 86
was considered as the first reactive species based on

18

O-labeling experiments,

other intermediates might also exist in the system, such as glycosyl triflate 88 formed
via dissociation of diphenyl sulfoxide from 86 and glycosyl pyridinium intermediate 89
in the presence of acid scanvenger 2-chloropyridine (Scheme 1.15). This
glycosylation strategy to iterative one-pot glycosylation for oligosaccharide assembly
was shown in Scheme 1.16.

17

Donor 94 is pre-activated, followed by addition of

acceptor 95 to afford disaccharide 96 and this process can be repeated to give the
12

desired oligosaccharide sequence. The fact that C-1 hydroxyl in acceptor 95 did not
react was due to its low nucleophilicity since 95 was in equilibrium with it is open form.
To demonstrate the feasibility of this approach, they synthesized a 1, 4-α-linked
tetrasaccharide 97 (Figure 1.3).
BnO
BnO
BnO

BnO
a
O
BnO
86% BnO BnO OCH(CH3)2
BnO OH
83
84
O

Scheme 1.13. Chemoselective dehydrative glycosylation. Reagents and conditions:
o

(a) Ph2SO, Tf2O, 2-chloropyridine, (CH3)2CHOH, -40 C.
TfO O O
Ph
S CF3
S O
Ph

Pathway 1
RO
RO
RO

O
OR 18OH
85

Pathway 2
RO
RO
RO

O
OR 18OH
85

RO
RO
RO

RO
Nu-H
Ph
O
RO
RO
18O S Ph
OR
OR Nu
86
TfO Ph S18O + TfOH 87
2
O

TfOH
Ph
O S TfO
Ph RO
F3C S
O O
RO
RO

RO
Nu-H
O
O O
RO
RO
18O S CF
OR
OR
3
88
87
18
Tf OH
O

Ph2SO + TfOH

Nu

Scheme 1.14. Possible mechanism for dehydrative glycosylation.
RO
RO
RO

RO
RO
RO

a

O
OR OH
89

TfO
Ph
S
OR O Ph
90

RO
RO
RO

O

RO
RO
RO

O
OR Nu
93

RO
RO
RO

O
OR OH
91

O N
OR Cl TfO
92

Scheme 1.15. Possible reactive intermediates in dehydrative glycosylation.
o

Reagents and conditions: (a) Ph2SO, Tf2O, 2-chloropyridine, (CH3)2CHOH, -40 C.
O
OR OHHO
n
94

a

O
O

OR n OH
95

OR n

O
96

O

repeat

OR n OH

13

oligosaccharide

Scheme 1.16. (Cont’d). Chemoselective iterative dehydrative glycosylation.
o

Reagents and conditions: (a) Ph2SO, Tf2O, 2-chloropyridine, (CH3)2CHOH, -40 C.
AcO
O

O

O
O

O
O

O

O
O

O

O
O

O

OH

O

O

Figure 1.3. Tetrasaccharide 97.
Inspired by Gin’s work, van der Marvel and coworkers developed a novel
sequential one-pot glycosylation strategy which combines the use of 1-hydroxyl and
59

thiodonors, as illustrated in Scheme 1.17.

Compared to Gin’s approach, they used

thioglycoside as the acceptor for the first coupling. And this thioglycoside 100 was
pre-activated by Ph2SO/Tf2O, followed by addition of acceptor to furnish
trisaccharide 102. This process was repeated to give the longer oligosaccharide
sequence. This approach was applied to the synthesis of hyaluronic acid (HA)
oligomers.

60

Starting from three monosaccharide building blocks 103 and 104, 105,

they synthesized HA trimer 106 (Scheme 1.18). It should be pointed out that the
glycosylation yield was low and the overall yield for two coupling reactions was only
about 26%. One-pot synthesis of this trisaccharide gave only 12% yield. They
ascribed this to the combination of acid-labile benzylidene protective group and base
catalyzed formation of orthoester. In a similar fashion, they constructed HA tetramer
and pentamer in moderate yields. Despite the success of this approach for the
assembly of HA oligomers, alternative strategies have to be developed with higher
glycosylation yields. They further applied their strategy to the synthesis of heparin
(HP) and heparan sulfate (HS), which are the most complex members of the
glycosaminoglycan family.

61

A model molecule pentasaccharide 109 was chosen as
14

the synthetic target and the first modular approach which employed only
monosaccharide building blocks was developed toward HP and HS synthesis (Figure
1.4). Two types of synthons were used: 1-hydroxyl azido glucoside and 1-thio uronic
acid. One main challenge of HP and HS synthesis lies in the coupling of azido
glucoside to uronic acid in α selective fashion. A variety of azido glucoside donor and
uronic acid acceptor pairs were screened under pre-activation conditions and most
reactions gave good αselectivity. However, they found anomeric leaving group and
activating system also had profound effects on glycosylation outcomes (Scheme
1.19). Donor 115 was first pre-activated by Ph2SO/Tf2O. When iduronic acid 116 was
used as acceptor, disaccharide 117 was obtained only in 31% yield (α:β = 2.5:1)
along with side product 119 (24%) and aglycon transfer product 118 (19%). The
rather low yield resulted from the low nucleophilicity of 4-OH as compared to
thioether function, which would lead to the formation of 118. In contrast, when they
changed the thioether function to thiophenyl function, no aglycon transfer product
was isolated and disaccharide 120 was obtained in 43% yield. The improvement was
presumably due to the steric effect of phenyl group, which blocked the nucleophilic
attack by the thio function. However, they did not further optimize the reaction to
improve the yield. Another factor which affected the outcomes of their glycosylation
reactions was the activator system as shown in Scheme 1.20. When donor 121 was
activated by Ph2SO/Tf2O followed by the addition of acceptor 122, no desired
trisaccharide was formed although prior to the addition of acceptor, TLC showed
clean activation of donor 121. As a comparison, when donor 121 was activated by
BSP/Tf2O, trisaccharide 123 was isolated in 76% yield after addition of acceptor 122
(Scheme 1.20).

15

HO
O

a

O
OR n
99

SR'

HO
O

OR n OH
98

OR n

O

O
100

OR n

SR' a

O
OR n
101

O
OR n

O

O
OR n
102

O

O
OR n

Scheme 1.17. Chemoselective iterative dehydrative glycosylation. Reagents and
o

conditions: (a) Ph2SO, Tf2O, -40 C.
MeOOC
MeOOC
a, 56%
O
O
O Ph O
LevO
LevO
O
BzO
OH Ph
O
BzO
O
OBz
OBz
O
SPh
103
HO
106
104 NHTCA
b, 47%
MeOOC
O
O MeOOC
O Ph O
LevO
O
O
MeOOC
BzO
BzO
O
OBz
TCAHN
HO
O
N3
BzO
107
105 OBz
HOOC
HO
HO

c
54%

HO
HO
O
OH
108

O

HOOC
O
NHAc HO

O

O
OH

O

SPh
NHTCA

O

O
OBz

O

N3

N3

Scheme 1.18. Chemical synthesis of HA trimer 108. Reagents and conditions: (a)
o

o

o

Ph2SO, TTBP, DCM, -60 C, then Tf2O, 104, -60 C-0 C; (b) Ph2SO, TTBP, DCM,
o

o

o

-60 C, then Tf2O, 105, -60 C-0 C; (c) p-TsOH, MeOH, then H2O, THF, KOH, then
Ac2O, MeOH.

BnO
BnO

BnO
BnO

OAc
O
110

N3

OH

OAc
O

OAc
O

O
O
OBn BnO
COOMeAcO MeOOC O
N3
N3
O
O
O
O
BnO
OBz
OBz BzO
N3
109 O
P = Orthoganol protecting group

HO
BnO

COOMe
O

SPh
OBz
111

AcO
HO
BzO

SPh
OAc
OBn
MeOOC
O
O
O
HO
O
OP
BnO
N3
OH OBz
N3
114
112
113

Figure 1.4. Retrosynthetic analysis of pentasaccharide 109.

16

OBn
O

SEt

OBn SEt
OAc
AcO MeOOC O
O MeOOC OBn OBz
LevO
O
OH OBz LevO
O
+
BzO + BzO
116
BzO
N3
OH OH
OAc
N 3O
SEt
b
a
O
117 ( 31%)
118 (19%)
119 (24%)
LevO
OH
BzO
SPh
OBn
N3
b
MeOOC O
115
AcO
SPh LevO
O
OBn
BzO
BzO
MeOOC
O
N3 O
OH OBz
120 (43%)
113
MeOOC

Scheme 1.19. Effects of anomeric leaving group on glycosylation outcomes.
o

Reagents and conditions: (a) Ph2SO, Tf2O, TTBP, DCM, -40 C; (b) 116 or 113, -40
o

C-r.t.
O
OBn
O

BnO
BnO

a

OAc
O
COOMe
N3
O
O
SPh
BnO
OBz
121

b

OH N3
122
c

No product

BnO
BnO

122
c

OAc
O
N3 COOMe
O
O
O
BnO
OBz
123
76%

O
OBn
O
N3

Scheme 1.20. Effects of activator system on glycosylation outcomes. Reagents and
o

o

conditions: (a) Ph2SO, Tf2O, TTBP, DCM, -40 C; (b) BSP, Tf2O, TTBP, DCM, -40 C;
o

(c) 122, -40 C-r.t.
1.3.3. Bromoglycoside-Mediated Iterative Glycosylation of Selenoglycoside
In 1965, Lemieux and coworkers reported the formation of 1, 2-orthoester
62-63

by treating glucosyl bromide with ethanol (Scheme 1.21).

By treatment with

tetra-n-butyl ammonium bromide (TBAB), there was an equilibrium between
α-bromide 124 and β-bromide 126, the latter was in another equilibrium with 1,
2-acetoxonium ion 127. Upon addition of ethanol, 127 was converted orthoester 125.
17

The equilibrium was shifted toward the formation of 125.
AcO
AcO

OAc
O

AcO
AcO

sym-collidine, TBAB
dry ethanol, 50 oC

AcO
124 Br

125
AcO
AcO

OAc
O
AcO
126

Br

OAc
O
+ Br
O
127 O

OAc
O
O
O
OEt

AcO
AcO

Scheme 1.21. Synthesis of 1, 2-orthoester from glycosyl bromide.
Based on this pioneering work, Jun-ichi Yoshida and coworkers developed
an iterative glycosylation approach for assembling complex oligosaccharides.

64

Selenoglycoside 128 was quantitatively converted toβ-bromide 129 by 0.5 equiv. Br2
and 129 was reactive enough so that without an additional activator it was converted
to orthoester 130 upon addition of selenoglycoside acceptor and a base. It was found
that the coupling did not proceed without base in many cases.
formation of β-bromide was affected by several factors.

65

65

The stereoselective

First, donors bearing

C2-acyl group gave β-isomer while those with C2-benzyl group gave α-isomer.
Second, the stereochemistry of selenoglycoside affected the outcome. When
α-isomer was used, they isolated anomeric mixture of glycosyl bromides. Third, the
chalcogen atom was also important. Thioglycosides were converted to glycosyl
bromides in a very slow fashion while the telluroglycosides worked as well as the
selenoglycosides. But due to their slight sensitivity to oxygen, telluroglycosides were
not commonly used for glycosylation reactions. In situ isomerization of 130 by
catalytic amount of TMSOTf gave disaccharide 131. In this process, the anomeric
seleno group of the acceptor was retained, which opened the door to iterative
glycosylation using this system (Scheme 1.22). Upon addition of 0.5 equiv. of Br2,
half of the donor 128 would be activated to form 129, accompanied by the generation
18

of PhSeBr which can further activate the other half of 128 to ensure the quantitative
conversion of 128 to 129. In this process, diselenide was generated as a side product.
Addition of the acceptor afforded orthoester 130 which was rearranged in situ to
disaccharide 131. By repeating this same reaction sequence, more complex
oligosaccharides could be obtained. The main advantage of this approach is that it
can be applied for combinatorial synthesis to achieve structural diversity. In the
7

previously reported chemoselective glycosylation approach, after n repetitions, only
(n+2) compounds could be generated. In this approach, for the glycosylation pair, the
more reactive one would act as donor with the less reactive one acting as acceptor.
In contrast, a huge oligosaccharide library can be generated after n repetitions using
this iterative glycosylation approach. In this approach, either one in the glycosylation
pair can act as donor or acceptor. As an example, they built up a library of the
phytoalexin elicitor-active oligosaccharides

66

starting from a monosaccharide 132

and a disaccharide 133 building block (Figure 1.5). Besides compounds shown here,
they also synthesized dfferent hexasaccharides and heptasaccharides. Although
highly diverse, this approach requires one additional step after each glycosylation,
which is in situ isomerization of orthoester to the desired product.

PO
PO

OP
O
PO
128

P'O
P'O

OP
O
Br 2 (0.5 equiv.) PO
SePh
Br
PO
PO
129
PhSeSePh

cat. TMSOTf PO
PO

OP
O

O
PO P'O
131
P'O

O

OH
O
P'O

SePh
PO
PO
130
R'

P = Bz or Ac
R' = Ph or Me
P' = Protecting
Group

SePh

P'O

Scheme 1.22. Bromoglycoside-mediated glycosylation.

19

OP
O
OO
O
P'O
P'O

O
P'O

SePh

RO
RO
AcO
O
O
RO
BnO
O HO
AcO
SePh
SePh+ AcO
O
AcO
O
BnO
O
AcO
AcO132
AcO 133
AcO
AcO
AcO
O
O HO
AcO
SePh
O
AcO
RO
AcO
AcO
AcO
O
134
HO
O
AcO
O
O
AcO
AcO
AcO
RO
AcO
O
BnO
AcO
O HO
O
135
SePh
AcO
O
AcO
O
AcO
AcO AcO
RO
AcO
BnO
O
O HO
AcO
O
AcO
O
O
SePh AcO
RO
AcO
AcO
AcO
AcO
O
BnO
136
O
O HO
AcO
O
AcO
O
O
AcO
AcO AcO
AcO
AcO AcO
O
RO
O
O HO
AcO
O HO
AcO
O
O
SePh
BnO
O
O
AcO
AcO
AcO
O
AcO
AcO
AcO
AcO
O
BnO
AcO
139
137 AcO
SePh
RO
O
BnO
AcO
O
O
BnO
AcO
O
AcO
AcO
AcO
AcO
O
O HO
AcO
O
SePh
BnO
O
AcO
O
AcO
AcO 140 AcO
AcO
AcO
O
O HO
AcO
SePh
O
AcO
AcO
AcO 138

Figure 1.5. Combinatorial synthesis of oligoglucoside library.
1.3.4. Chemoselective Glycosylation of Thioglycoside
7

The reactivity-based armed-disarmed one-pot glycosylation is a powerful
strategy to streamline the oligosaccharide synthesis process. It refers to one in which
glycosyl donors with decreasing reactivities can sequentially react in a single flask,
the armed (more reactive) donor 141 will react first, followed by less armed one until
the disarmed one (reducing end 145), which avoids the purification step after each
coupling cycle and thus greatly improves the overall efficiency (Scheme 1.23).
However, the drawback of this approach is that extensive protecting group
manipulations have to be carried out to fine-tune the anomeric reactivities. To further
improve the overall synthetic efficiency, Huang and coworkers developed a new
14

iterative one-pot glycosylation strategy (Scheme 1.24).

20

In this approach, donor 147

was first pre-activated by stoichiometric amount of promoter to yield intermediate 148
o

under low temperature (-70 C), followed by addition of 0.9 equiv. of acceptor 149 to
give disaccharide 150. The slightly excess activated donor was decomposed by
increasing the temperature to room temperature. Then the reaction flask was cooled
o

back to -70 C and it is ready for the second round of glycosylation with acceptor 152.
This process can be repeated until the desired sequence is acquired. Several
prerequisites have to be satisfied for such an operation: first, the promoter must be
able to activate a wide range of donors in stoichiometric amount; second, the reactive
intermediate should be stable prior to the addition of acceptor and reactive enough to
couple with the acceptor; third, the side products generated should not interfere with
glycosylation reactions. Huang and coworkers proposed a possible mechanism for
their pre-activation based glycosylation protocol (Scheme 1.25). Upon addition of
p-TolSCl to the mixture of donor 155 and AgOTf, p-TolSOTf is formed, which
electrophilically attacks anomeric sulfur atom of 155 to form disulfonium ion 156 (Step
1 in Scheme 1.25). Along with the formation of disulfide side product, 156 can further
evolve into several reactive species, such as oxacarbenium ion 158, α-triflate 159,
disulfonium ion 160, dioxalenium ion 161. Nucleophilic attack of acceptor will
generate desired glycoside 157. To figure out which intermediate was the dominant
species in their glycosylation protocol, low-temperature NMR experiment was carried
out.

67 1

H NMR revealed that one major new carbohydrate species was formed upon
o

addition of p-TolSCl to a mixture of donor 162 and AgOTf in CDCl3 at -60 C. Its
anomeric proton signal was a doublet at δ 6.57 ppm with J1,2 = 3.2 Hz.

13

C NMR also

shows a new carbohydrate species with its anomeric carbon signal residing at δ
104.4 ppm. All these data indicate the presence of α-triflate 163 as the major
21

intermediate

68-70

(Figure

When

1.6).

more

electron-rich

donor

164

was

pre-activated, another new carbohydrate species was formed indicated by NMR. To
better probe the intermediate by

13

C NMR,

13

C labeled 164 was used (Figure 1.7).

Before pre-activation, there was no correlation between
however, a new correlation with

13

13

C and anomeric proton;

C and anomeric proton was observed which

suggests the formation of dioxalenium ion 165 via the participation of 2-benzoyl (Bz)
group.

Interestingly, when 166 was pre-activated, two major intermediates were

formed (α-triflate 167 and dioxalenium ion 168) indicated by NMR (Scheme 1.26).
The different outcome upon pre-activation can be explained in terms of different
electron-withdrawing properties of protective groups on these three donors. For 162,
Bz greatly disfavors the formation of positively charged dioxalenium ion while
electron-donating benzyl (Bn) group can stabilize dioxalenium ion. Donor 166
presents the intermediate case between the two above.

Scheme 1.23. Reactivity based one-pot synthesis of oligosaccharides.

22

PG 2O
O
HO
STol
149

promoter

O
PG1O
STol
147
148
x'
PG: protective roup
reactive intermediate
PG3O
O
PG 2O
HO
STol
promoter
O
O O
152
PG 1O
PG1O
151
X'
reactive intermediate
O

PG1O

repeat

O

PG1O

PG1O

O

PG2O
O
O O
150

PG 3O
PG2O
O
O O
O

STol

STol

153

PG n+1O
PGnO
O
O
O
O
OR
n
154

Scheme 1.24. Iterative one-pot synthesis of oligosaccharides.

S S

p-TolSCl + AgOTf
PGO

O

S
155

p-TolSOTf
Step 1

PGO

O

Step 3

O

PGO

OTf
158

159

S

O

PGO

157

Possible reactive intermediates:
PGO

S
Step 2

OTf
156

Acceptor

Reactive intermediates(s)

O

OR

Glycoside product

O

PGO

S S

OTf

160

OTf

PGO

O
OO

161

OTf

R

Scheme 1.25. Proposed mechanism for pre-activation based glycosylation strategy.

1

Figure 1.6. H NMR and

13

C NMR of the intermediate after pre-activation of donor

178.
23

BzO OBz
BnO OBn
p-TolSCl, AgOTf
O
O
BnO
STol CDCl , -60 oC BzO
3
OO
O
13
13
O C
Ho
C
Ho'
Ho'

Ho
165

164

Figure 1.7. HMBC of the intermediate a) before and b) after pre-activation of donor
180. Adapted with permission from ref 76. Copyright 2008 American Chemical
Society.

Scheme 1.26. Pre-activation of donor 166.
This novel strategy has been applied for the assembly of various complex
oligosaccharides,
chitotetrose,

74

as

witnessed

oligomanose,

dodecasaccharide,

77

75

by
Lewis

HP and HS.

78

Globo
X

H,

and

71

HA oligosaccharides,

Lewis

X

dimer,

76

72-73

N-glycan

One good example to demonstrate this
72-73

approach is the chemical synthesis of HA oligosaccharide (Scheme 1.27).

In

this one-pot operation, monosaccharides 169, 170, 172 and disaccharide 171 were

24

used as building blocks. The whole process took about 6 h and the overall yield after
final column purification was 55%. As a comparison, in van der Marel’s synthesis of
HA trisaccharide 107, when one-pot operation was carried out, the isolated yield was
only 12%. However, in the case of 173 synthesis, no orthoester was isolated when
bezoyl group was used as C-2 protecting group. This protocol was further applied to
the fully protected HA decasaccharide 174 (Figure 1.8). However, deprotection of
174 turned out to be problematic. Oxidative removal of p-methoxybenzyl (PMB) and
cleaving phthalimido (Phth) are not compatible with protective group on 174. To solve
this problem, a second-generation synthetic route was designed (Scheme 1.28). In
this route benzyl esters were installed at the disaccharide stage to avoid the problem
of PMB removal on the decasaccharide and trichloroacetyl (TCA) was used to mask
the amino group instead of Phth. The main challenge for this route comes from
formation of trichloromethyl oxazoline side product due to the participation of TCA.
Addition of trimethylsilyl triflate was found to be efficient to suppress the formation of
this side product and the yield for “4+6” glcosylation which leads to 176 increased
from 10% to 77%. Another good example is the combinatorial, one-pot synthesis of
HP and HS hexasaccharides.

78

Since α-selective linkage is challenging to synthesize

stereospecifically, disaccharides with pre-installed α-1, 4-linkage were used as
building blocks (Scheme 1.29). By alternating sugar backbone and sulfation patterns,
a library of up to 12 HP/HS-like hexasaccharides were synthesized and used for
HP/HS-basic fibroblast growth factor binding studies. The yields for one-pot
operations for all the hexasaccharides range from 50% to 70%. In this modular
approach, α-stereoselectivity only had to be taken care of during preparation of
disaccharides building blocks since all the required disaccharides can be derivatized
61

from the common precursor. As a comparison, in van der Marel’s approach,
25

they

need consider α-selectivity whenever the azido glucoside was used as the glycosyl
donor, Moreover, they used glucronic acid/iduronic acid as acceptors. Due to the low
nucleophilicity of these acceptors, coupling yields were low along with other side
products. In Huang’s approach, hexasaccharide backbone was constructed followed
by oxidative transformation, which circumvented the low reactivity of glucuronic
acid/iduronic acid acceptors. Typically, to access divergent HP/HS oligosaccharide
structures with different sulfation patterns, different protecting groups were utilized for
masking hydroxyl and amino groups, which can be selectively removed for sulfation.
However, it is often very difficult to completely sulfate all the required positions when
multiple sulfates are present in the structure. Alternative to post-glycoassembly
sulfation strategy, Huang and coworkers tested the possibility of using pre-sulfated
disaccharide as building blocks for assembling larger oligosaccharides, as shown in
Scheme 1.30.

79

Donor 186 with preinstalled sulfate was coupled to 187 smoothly

to give tetrasaccharide 188 followed by removing silyl ether to give acceptor 189,
which was coupled with 186 with simultaneous cleavage of silyl ether to yield
hexasaccharide 190 in 70%. The success in assembling 190 with two preinstalled
sulfates highlights the powerfulness of this pre-activation based glycosylation
strategy since these preinstalled TCE sulfate esters did not significantly disarm
donors and acceptors tested for HP/HS assembly.
p-TolSCl acceptor 170 p-TolSCl acceptor 171 p-TolSCl
acceptor 172
(1 equiv.) (0.9 equiv)
(0.9 equiv.) (0.81 equiv) (0.81 equiv.) (0.72 equiv.)
donor 169
(1 equiv.)
173
+
90 min (55%)
AgOTf
5 min 90 min 30 min 5 min 90 min 30 min 5 min
(3 equiv)
- 78 oC - 78 oC
rt
- 78 oC - 78 oC rt - 78 oC - 78 oC
rt
OPMB
OPMB
PMBO Ph O
Ph
O
O
O
O
HO
O
TBSO
O
O O
STol HO
OCH3
STol BnO
STol
O
HO
BnO
BnO
170 NPhth
172 OBn
169 OBz
OBz 171 NPhth
PMBO
OPMB
PMBO Ph O
O
O
O O
O Ph O
TBSO
O
O O
O
O
OCH3
O
BnO
BnO
BnO
PhthN
OBz
OBn
OBz PhthN
173
26

Scheme 1.27. (Cont’d). One-pot synthesis of HA pentasaccharide 173.
PMBO
TBSO
BnO

O
BzO

Ph O
OO

O
PhthN

PMBO
O
BnO
174

PMBO
Ph O
O O
O O
O
BnO
OBz PhthN

Ph
O
O O
O
BzO n=2

O

OMe
NPhth

Figure 1.8. Fully protected decasaccharide 174.
OH
O
AcHN

OH
HO2C
O
O HO
O
O
HO
AcHN
175 OH

BnO 2C
Ph
O
O
O
TBSO
O
O
BnO
OBz
TCAHN

BnO2C Ph O
O
O O
O
O
BnO
176 OBz TCAHN

BnO 2C
O
HO
HO
OH

HO
O

HO2C
O
HO

O HO
O
OH n=2

BnO2C Ph O
O O
O
O
BnO
BzO n=2

BnO2C
Ph O
BnO2C
Ph O
O
O
O
TBSO
O
O
O
STol + HO
O
O
BnO
BnO
TCAHN
OBz
TCAHN
OBz
177
178
BnO 2C
Ph O
O
O
O
HO
OMe
O
BnO
TCAHN
OBz
179
PMBO
TBSO
BnO

O

STol
OBz
169

Ph

O
O
HO

OH
O

OMe
NHAc

O

OMe
NHTCA

+
STol

Ph
O
O
O
OMe
STol
HO
NHTCA
NHTCA
181
180
O

Scheme 1.28. Retrosynthetic analysis of HA decasaccharide 175.
p-TolSCl 183 (0.9 equiv.) 184 (0.8 equiv.) p-TolSCl
(1 equiv.) TTBP (1 equiv.) TTBP (1 equiv.) (0.81 equiv.)
donor 182
(1 equiv.) +
185 (50%)
90 min
5 min
90 min 30 min
5 min
AgOTf
oC
oC
oC
o
rt
- 78
- 78
- 78
(4 equiv.) - 78 C
rt
OLev
OLev
OLev
OBn
OBn
OBn
O
O
O
OBn
OBn
OBn OM
STol HO
STol HO
O
TBSO
O
O
N3 O
N3 O
N3 O
BnO
BnO
BnO
184 BzO
182 BzO
183 BzO
OBn
OLev
OBn
OLev
OBn
O
OLev
OBn OMe
O
O
OBn O
O
OBnO
N3 O
BnO
O
TBSO
O BnO
N3 O
N3 O
BnO
BzO
185 BzO
BzO

Scheme 1.29. One-pot synthesis of HP-like hexasaccharide 185.

27

TBSO
BnO

OSO3TCE
O
N3
O
BnO
186

a, 82%
OAc
O

OLev
HO
O
STol BnO
OBz

OSO3TCE
O

N3
O
187 BnO

OAc
O

OLev
O
O
OBz

Cbz
N
Bn
Cbz
N Bn

OLev
OLev
O O
O O
N3
N3
BnO
O
O
c, 70%
OBz
BnO
BnO
OBz
188 R = TBS b, 84%
189 R = H
OSO3TCE
OSO3TCE
OAc
O
O
O
OLev
OLev
OLev
RO
O O
O O
O O
N3
BnO
N3
BnO
N3
BnO
O
O
O
BnO
OBz 190 BnO
OBz
OBz
BnO
RO
BnO

Cbz
N Bn

Scheme 1.30. Synthesis of hexasaccharide 190 with preinstalled sulfates.
Although this pre-activation based one-pot approach greatly accelerated
oligosaccharide assembly, final column purification is needed to get rid of all the side
products generated during one-pot operation, which can be time- and solventconsuming. To further improve overall efficiency, Huang and coworkers reported a
new

fluorous-assisted

one-pot

synthesis

method,

with

which

no

column

chromatography is required and the whole process can be completed in just a few
80

hours (Chapter 2).

Besides AgOTf/p-TolSCl, other promoter systems have been developed for
pre-activating

thioglycosides,

(BSP)/trifluoromethanesulfonic

such

as

anhydride
82

benzene-thiosulfinate

(MBPT)/Tf2O,

1-benzenesulfinyl
81

(Tf2O),

S-(4-methoxyphenyl)

Ph2SO/Tf2O,

O-Dimethylthiophosphonosulfenyl bromide (DMTPSB)/AgOTf
morpholine

85

(BSM)/Tf2O.

For

instance,

84

57,83

O,

and benzenesulfinyl

BSP/Tf2O has been applied for

combinatorial synthesis of linear oligoglucosamine library,
28

piperidine

16

Ph2SO/Tf2O was used

for construction of all possible trisaccharide repeating units of the Zwitterionic
polysaccharide Sp1

86

60

and HA oligomers.

To synthesize oligoglucosamine library, Yoshida and coworkers developed an
iterative approach utilizing BSP/Tf2O and after the coupling reaction was complete
under low temperature, they had to quench the reaction before warming up to room
temperature.

16

The same procedure was carried out by Sakairi and coworkers to

synthesize a fully protected Lewis Y trisaccharide.

87

Thus, this approach is not

suitable for one-pot operation since the temperature has to be increased to
decompose slightly excess activated donor as demonstrated by Huang and
coworkers.

14

van der Marel and coworkers

15

found that when donor 191 was

pre-activated by BSP/Tf2O followed by addition of acceptor 192, the reaction was
clean based of TLC judgment. However, when the reaction was quenched under
room temperature, the isolated yield was only 44% (α:β = 2:1) (Scheme 1.31). They
attributed the low yield to the formation of (N-piperidino)phenyl(S-thioethyl)sulfide
triflate. It is capable of activating thioglycosides like 193, albeit under higher
temperature. This finding explains the need for low temperature quenching operation
in oligoglucosamine library synthesis.

16

To avoid the side reaction caused by this

triflate, a scavenger triethyl phosphite (TEP) was used to timely quench it. Repeating
the reaction shown in Scheme 1.31 with addition of TEP gave a satisfactory
glycosylation yield (78%). Another issue associated with this promoter was that it
could not promote some “well-documented” glycosylations (Scheme 1.32). Armed
donor 190 failed to couple with armed acceptor 194 and disarmed donor 196 failed to
couple with disarmed acceptor 192. By contrast, very similar couplings promoted by
29

AgOTf/p-TolSCl gave desired disaccharides in satisfactory yields.

88

The weak

promoting ability of BSP/Tf2O might be due to the presence of electron-donating
1-piperidino group, which makes sulfonium not a good electrophile.

BnO
BnO

OBn
O

BSP, Tf 2O,
TTBP, -60 oC

SEt
OTf
OBn
191
EtS S N
Ph

BzO
BzO

OH
O

BnO
BnO

SEt
192 OBz

OBn
O

O
OBn
BzO
BzO
193
44% (2:1)

O

SEt
OBz

Scheme 1.31. BSP/Tf2O promoted synthesis of 193.

Scheme 1.32. Weak promoting ability of BSP/Tf2O. Reagents and conditions: (a)
o

BSP, Tf2O, DCM, TTBP, -60 C; (b) 192 or 194; (c) TEP.
Other promoting systems including MBPT/Tf2O and Ph2SO/ Tf2O also have
drawbacks and limitations when pre-activation based one-pot glycosylation was
applied.

85

Ye and coworkers developed a new promoter system (benzenesulfinyl

morphline/Tf2O), which can potentially work as an alternative to AgOTf/p-TolSCl,
since p-TolSCl is not quite stable and requires in situ preparation. This success of this
promoter was evidenced by one-pot synthesis of tetrasaccharide 202 (Scheme
85

1.33).

30

Scheme 1.33. BSP/Tf2O promoted one-pot synthesis of tetrasaccharide 202.
1.4. Stereoselectivity in Pre-activation Based Glycosylation
Pre-activation approach achieves stereoselectivity via the electronic and
steric properties of the donor and different reaction conditions. The following factors
tend to have great effects on the stereoselectivity of the α/β ratio: (1) protecting
groups on glycosyl donor, such as anomeric leaving group, non-anomeric protecting
group; (2) Solvent and reagent used for glycosylation. Among these two, protecting
group pattern on the donor dominates the stereochemistry. In pre-activation based
glycosylation strategy, activation of glycosyl donor and coupling of activated donor to
acceptor are carried out in two distinct steps, which makes it possible to study the
reactive intermediates upon pre-activation spectroscopically and rationalize the
stereochemical outcome of a glycosylation reaction. The intermediates are allowed to
exist in the solution for long time at low temperature and could significantly impact the
stereochemical outcome of the glycosylation reaction. Low-temperature NMR
provides a powerful tool to carbohydrate chemists for characterizing the
intermediates formed upon pre-activation. This section will cover different effects
exerted by protecting groups and briefly discuss the effect of solvent and reagent.
1.4.1. Protecting groups on donor
31

Protecting groups at different positions tend to have different effects on
stereochemistry and the can significantly change the electronic and steric properties
of the donor. Protecting group pattern will affect the reactive intermediates generated
89-90

upon pre-activation, which can be the glycosyl triflate or oxacarbneium ion.
1.4.1.1. Anomeric leaving group effect

Micrococcus luteus teichuronic acid 203 is a polysaccharide which is
composed of alternating N-acetyl-D-mannosaminuronic acid (ManNAcA) and glucose
91-93

unit in the form of 1,2-cis linkage (Figure 1.9).

Among these two linkages, the

one between ManNAcA and glucose is more challenging to synthesize. To build up
this linkage, van der Marel and coworkers tested seven different azido mannoside
94

methyl uronate donors with different anomeric leaving groups (Figure 1.10).

For
1

donors 205, 207 and 208, they exist as a mixture of two different conformations ( C4
and

4

C1) based on

1

H NMR. Sulfoxide donors 209 and 210 exist in
1

diastereomerically pure but undefined form. Both 210a and 210b have exclusive C4
57

conformation. Donors 204, 205 and 208 were pre-activated by Ph2SO/Tf2O,

donors 206 and 207 were pre-activated by trifluoromethanesulfonic acid (TfOH) while
donors 209 and 210 were pre-activated by Tf2O. Upon activation, low-temperature
NMR was used to detect the possible reactive intermediates (Figure 1.11). Upon
pre-activation of donor 204, a mixture of two major intermediates was formed. With
the help of 2D NMR, they assigned all the protons on the sugar ring. One
intermediate was α-triflate 211 and the other one is β-triflate 212. The presence of
212 was indicated by H1, H2 coupling (8.8 Hz) as well as the coupling constants of

32

the other ring protons. They attributed the prevalence of 212 to the significant amount
of positive charge on the anomeric carbon atom, which in turn is caused by the
presence of the anomeric triflate, C-5 ester and C-2 azide. Consequently, the
intermediate preferred to exist as 212 to accommodate the positive charge on the
ring.

95

This was the first time that equatorial anomeric triflates have ever been

detected spectroscopically. Upon addition of CD3OD as the acceptor, the product
was formed as a mixture of two isomers (α:β = 1:5). The selectivity originates from
SN2-like attack of CD3OD on the anomeric carbon. Pre-activation of donors 205, 206
and 207 led to the formation of the same intermediates as compared to 204 based on
low-temperature NMR. Followed by addition of acceptor, β-glycoside was formed
predominately (205: α:β= 1:6; 206:α:β = 1:>10; 207:α:β = 1:>10). When donor 208
was subjected to pre-activation, another reactive species different from triflate was
3

detected. The anomeric proton (δ = 6.16 ppm) appeared as a doublet ( JH1-H2 = 8.3
58

Hz) and the reactive species was assigned to be oxosulfonium triflate 213.

Upon

addition of acceptor, β-isomer was formed exclusively. Pre-activation of donor 209
gave rise to a mixture of three major intermediates, two of which were anomeric
triflates. Based on the chemical shifts of the third intermediate (H-1:δ = 6.16 ppm, C-1:
δ = 91.4 ppm), they presumed that it corresponded to β-sulfonium bistriflate species
214.

69

Upon addition of acceptor, anomeric mixture formed with the same selectivity

as donor 204. Pre-activation of donor 210a led to the overall downfield shift of the
3

sugar ring protons, with H-1 as a doublet (δ = 5.38 ppm, JH1-H2 = 10.8 Hz) and C-1
indicating anomeric thio functionality (δ = 86.2 ppm). This reactive species was
considered as equatorial α-anomeric sulfonium bistriflate 215. Interestingly, addition
33

of the acceptor did not result in any desired product. In contrast, pre-activation of the
other diastereomer 210b resulted in the formation of anomeric triflates and another
new species, which was different from 215 but very similar. They assumed this
species to be the other diastereomeric sulfonium bistriflate 216. Addition of acceptor
gave the same selectivity as donor 204.

Figure 1.9. Micrococcus luteus teichuronic acid.
MeOOC
MeOOC N3
OBn
MeOOC N3
O
AcO
SPh
O
O
AcO
N3
BnO
O
SPh
BnO
NPh
OAc 205
206
O
NPh
4
1
F3C
C1 : C4 = 1 : 10
F3C
MeOOC
MeOOC
O
OBn
OBn
MeOOC N3 O
207
SPh
OH AcO
O
O
O
4
1
N3
N3
SPh
BnO
C1 : C4 = 1.3 : 1
OAc 210a, 210b
OAc 208
209
4
1
4
1
C1 : C4 = 0 : 1
C1 : C4 = 1 : 1.7
MeOOC N3
O
AcO
BnO
204

Figure 1.10. Azido mannoside methyl uronate donors with different anomeric leaving
groups.

MeOOC N3
MeOOC N3 MeOOCOBn
Ph2SO/Tf 2O AcO
O
O
AcO
OTf
O
SPh
BnO
BnO
N3
or TfOH
204, 205
211
OAc 212
OTf
206, 207

Figure 1.11. 1H NMR of donors after pre-activation under -80

o

C. Adapted with

permission from ref 94. Copyright 2010 American Chemical Society.

34

Figure 1.11. (Cont’d). For interpretation of the references to color in this and all other
figures, the reader is referred to the electronic version of this dissertation.

35

Figure 1.11. (Cont’d).
van der Marvel’s work revealed that donors having different anomeric leaving
group could be converted to different active species upon pre-activation. Addition of
acceptors would generate glycosides with different anomeric selectivities.
1.4.1.2. Vicinal Neighboring Group Participation
Conventional

glycosylation

approach

achieved

stereoselectivity

via

neighboring group participation as mentioned in Section 1.2.2. This approach has
been widely applied in pre-activation based glycosylation approach to ensure the
formation of 1, 2-trans linkage. For example, Bz and NPhth have been used to mask
hydroxyl and amino groups at C-2 position respectively to form 1, 2-trans linkage in
the case of HA oligosaccharide synthesis as depicted in Scheme 1.24.

72

Huang and

coworkers has conducted studies to reveal the mechanisms for this participation in
67

thioglycosides as mentioned in Section 1.3.4.

However, one major drawback of this approach is that orthoesters can form
as frequent side product, and in some cases the only product. This happens by
trapping the intermediate bridging cation as opposed to attacking the anomeric
carbon (Scheme 1.34). Pre-activation of 217 gives oxacarbenium ion 218, which can
be in equilibrium with bridging cation 220. Addition of acceptor can directly convert
218 to 219 or it can react with 220 to form either desired product 219 or orthoester
221. So here the key question will be: which condition will favor the formation of
desired product and which condition will give rise to orthoester? A lot of efforts have
been carried out to suppress the formation of orthoester when donors and acceptors

36

are pre-mixed, for example, bulky ester such as pivalate ester has been used,
however, they cannot eliminate orthoester formation and in some cases, orthoesters
even predominate.

96-98

To answer this fundamental question, Crich and coworkers

did systematic studies using xyloside as glycosyl donor.

30

Pre-activation of sulfoxide

donor 222 with Tf2O in the presence of DTBMP followed by addition of cyclohexanol
gave orthoester 223 only while glycosylation of bromide 224 with cyclohexanol by
AgOTf exclusively yield β-xyloside 225 (Scheme 1.35). This dichotomy might be due
to the different anomeric leaving groups on 222 and 224. To test this hypothesis,
sulfoxide donor 222 was pre-activated by Tf2O in the absence of DTBMP (Scheme
1.36). To their surprise, only β-xyloside 226 was obtained. This clearly demonstrates
that anomeric leaving group does not have effect on the outcome of the reaction.
Instead, base additive (DTBMP) is the determining factor since the acid sensitive
orthoester can be buffered in the presence of DTBMP. Without DTBMP, it can be
readily rearranged toβ-xyloside. To further get insight into the reaction mechanism,
they carried out low-temperature NMR studies. Pre-activation of

13

C-labeled xyloside

227 revealed a new instense signal residing at 180.3 ppm, which was assigned as
the bridging cation 228.

99

A smaller peak at 120.9 ppm may be orthoester carbon in

triflate 229 or hemi-ortho ester 230 (Figure 1.12a). Upon addition of methanol, the
peak at 180.3 ppm was displaced by a strong signal at 121.1 ppm, which
corresponded to the orthoester carbon of 231 (Figure 1.12b). By contrast, when 227
was pre-activated in the absence of DTBMP, no orthoester was detected by NMR
upon addition of methanol.

37

Scheme 1.34. Possible pathway leading to the formation of orthoester.

Scheme 1.35. Possible effects of anomeric leaving group on orthoester formation.
o

Reagents and conditions: (a) Tf2O, DTBMP, DCM, -78 C, then C5H11OH; (b) AgOTf,
o

DCM, C5H11OH, -78 C.

o

Scheme 1.36. Control experiments. Reagents and conditions: (a) Tf2O, DCM, -78 C,
then MeOH.

38

Figure 1.12. Influence of DTBMP on the outcome of glycosylation. Adapted with
permission from ref 30. Copyright 1999 American Chemical Society.
In the case of 2-amino sugar, oxazoline can form during neighboring group
participation when TCA was used to mask amino group, which is well illustrated by
the synthesis of HA decasaccharide.

73

Once pre-activated, donor 232 was converted

to oxacarbenium ion 233, which was in equilibrium with the oxazolinium ion 234.
Addition of acceptor 235 gave the desired product 236, which will shift the equilibrium
toward pathway a. However, deprotonation of 234 will form 237, shifting the
equilibrium toward pathway b. To suppress oxazoline formation, the key step is to
minimize deprotonation from 234 to 237. Based on this rationale, Huang and
coworkers made adjustments to improve the yield for “4 + 6” coupling (Scheme 1.38).
With TTBP, only oxazoline 240 was isolated in quantitative yield while without TTBP,
156 was isolated in 10% together with 85% 240. When TfOH was added, 156 was

39

isolated in 40%. The best glycosylation yield was achieved (77%) when TMSOTf was
used as additive. The result clearly demonstrated that addition of acid could inhibit
the deprotonation of 234 and shift the equilibrium toward the formation of desired
product.
1
PG O

O
HN

PG1O

2
OPG
O
HO
STol
1
PG O
235
pathway a

O

233 CCl3
STol Pre-activation
NHTCA
1
O
PG O
232
HN O
O

234

O

O
NHTCA
236

1
PG O

-H
pathway b

OPG
O

2
STol

O
N
237

CCl3

O
CCl3

Scheme 1.37. Formation of oxazoline upon pre-activation.

Scheme 1.38. Synthesis of fully protected decasaccharide 156. (a) AgOTf, p-TolSCl,
o

DCM, -78 C, then 239.
To

completely

suppress

orthoester

formation

during

glycosylation,

participating groups other than 2-acyl groups were developed. Yamago and
coworkers reported dialkylphosphates as stereodirecting groups for oligosaccharide
synthesis.

37

After screening different phosphate protecting groups, they chose the 2,

40

2-dimethyltrimethylene (DMTM) phosphate group to assist the formation of 1, 2-trans
linkage (Scheme 1.39). Donor 241 was pre-activated by BSP/Tf2O, followed by
addition of cyclohexanol to yield 242 with excellent β-selectivity. Removal of this
phosphate protecting group was achieved by treating with NaOH in EtOH/H2O under
o

60 C for 1 h. They probed the potential reactive intermediates by low-temperature
NMR and they were surprised to find the α-triflate 244 as the only detectable
16,69

intermediate, as indicated by chemical shifts of anomeric carbon (105 ppm)

and

proton (6.4 ppm, J1,2 = 2.4 Hz). This conclusion was also supported by the
phosphorus signal (-8.9 ppm) in

31

P NMR, indicating that the phosphorus atom in the

intermediate did not have a positive charge, which was responsible for minimizing the
formation of orthophosphate product.

100

They proposed a plausible reaction

mechanism based on low-temperature NMR studies (Scheme 1.40). Upon
pre-activation, α-triflate 244 was formed which underwent “SN2-like” attack by
cyclohexanol to afford glycoside 246. Although 244 was the only detectable
intermediate, 245 may also exist which was short-lived and in equilibrium with 244.
Attack of cyclohexanol from less hindered side in 245 would lead to the formation of
246. This protecting group strategy was later applied to the synthesis of a series of
oligosaccharides. However, this strategy has not seen applications in literature so far.

Scheme 1.39. Participation of DMTM in glycosylation and subsequent cleavage.

41

o

Reagents and conditions: (a) BSP, Tf2O, DCM, -60 C, then cyclohexanol; (b) NaOH,
o

EtOH/H2O, 60 C, 1 h.
BnO
BnO
BnO
BnO
BnO
BnO

O
O O
P
O O
241

SPh
BSP, Tf 2O, DTBMP
DCM, -60 oC

BnO
BnO
BnO

O
O O
P OTf
O O
244

O
O O
O P
O

HOC6H11
BnO
BnO
BnO
HOC6H11

O

O O
P
O O
246

OC6H11

245

Scheme 1.40. Plausible reaction mechanism.
In 2005, Boons and coworkers reported a novel strategy for creating 1, 2-cis
linkage.

34

They took advantage of ethyl 2-hydroxy-2-phenyl acetate as chiral

auxiliary to mask C-2 hydroxyl group, which enabled them to achieve high
α-selectivity by mixing donor and acceptor together. However, this group was difficult
was introduce and remove. To achieve exclusive α-selectivity, they developed a
second generation chiral auxiliary the (1 S)-phenyl-2-(phenylsulfanyl)ethyl moiety.

33

Upon pre-activation by TMSOTf, donor 247 was converted to a reactive intermediate,
which was transformed to disaccharide 250 in 88% yield with addition of acceptor
249 (Scheme 1.41). To figure out the possible reactive intermediates 248 and further
shed light on the mechanism of this transformation, they carried out low-temperature
NMR studies, based on which the mechanism was proposed (Figure 1.13). Upon
pre-activation, the anomeric proton of donor 251 showed upfield shift from 6.58 ppm
to 5.30 ppm with a larger vicinal coupling constant (J1,2 = 9.5 Hz) (Figure 1.13a,
1.13b) and HMBC showed a correlation between C-1 and H8eq (Figure 1.13c). All

42

these data suggested the formation of trans-decalin system 252. In this conformation,
there was no unfavorable gauche interaction. The chiral center of the auxiliary also
played an important role in determining selectivity. Changing the configuration from R
to S resulted in lower stereoselectivity. Besides chiral auxiliary, protecting group
pattern was also found to effect the outcome of glycosylation.

101

When electron-rich

donor 254 was coupled to acceptor 255, disaccharide 256 was isolated in 70% with
no stereoselectivity (Scheme 1.42). Although β-sulfonium ion 259 was the only
detectable intermediate, small amount of oxacarbenium ion 258 could exist upon
pre-activation, with an equilibrium between these two species favoring the sulfonium
ion 259. For donor with electron-withdrawing groups, oxacarbenium ion 258 was
further destabilized so that the reaction will go through sulfonium ion 259
intermediate to generate α-glycoside. By contrast, for donors with electron-donating
groups, oxacarbenium ion 258 could be stabilized and couple to subsequent acceptor
to afford anomeric mixtures (Scheme 1.43). To demonstrate the applicability of this
approach, they synthesized branched α-glucan which bears 1, 2-cis linkage between
each sugar unit.

Scheme

102

1.41.

Chiral

auxiliary

assisted

stereoselective

glycosylation
o

o

via

pre-activation. Reagents and conditions: (a) TMSOTf, DCM, -78 C to 0 C; (b) 249,
o

o

-78 C to 0 C.

43

1

1

Figure 1.13. (a) H NMR of donor 251; (b) H TOCSY 1 D on irradiation of H4 of 252;
o

o

(c) HMBC of 252. Reagents and conditions: (a) TMSOTf, DCM, -78 C to 0 C; (b)
MeOH, -78

o

C to 0

o

C. Adapted with permission from ref 33. Copyright 2005

American Chemical Society.

44

Scheme 1.42. Unexpected low anomeric selectivity from donor 254. Reagents and
o

o

o

o

conditions: (a) TMSOTf, DCM, -78 C to 0 C; (b) 255, DTBMP, -78 C to 0 C.

Scheme 1.43. Equilibrium between oxacarbenium ion and α-sulfonium ion. Reagents
o

o

o

o

and conditions: (a) TMSOTf, DCM, -78 C to 0 C; (b) 249, -78 C to 0 C.

1.4.1.3.

Remote Neighboring Group Participation
Different research groups have suggested remote neighboring group

participation as one way to control the stereoselectivity in glycosylation under
103-113

traditional coupling conditions (pre-mixing donor and acceptor).

However,

selectivities were not good in many cases and there was no conclusive evidence to
support this type of participation. Pre-activation approach provides a good
opportunity to investigate this type of participation since upon pre-activation, it is
possible to trap the intermediate prior to addition of acceptor. Crich and coworkers
have carried out systematic studies in this area. They used a tert-butoxycarbonyl

45

(Boc) group as an acyl type participating group since the loss of a tert-butyl-cation
could lead to the formation of a cyclic carbonate ester. They installed a Boc group at
different positions of the glycosyl donor and pre-activated the donor to check the
intermediate. Pre-actvation of donor 262 led to the formation of cyclic carbonate 263,
which clearly indicated the participating ability of 2-O-Boc (Scheme 1.44).
Pre-activation of donor 264 with axial 3-O-Boc gave cyclic carbonate 265 (Scheme
1.45). When acceptor such as cyclohexanol was added, desired glycoside with high
β-selectivity was isolated, which demonstrated the participation of axial 3-O-Boc. In
contrast, when donor with equatorial 3-O-Boc was pre-activated, no cyclic carbonate
formed. After aqueous workup, they got a complex mixture which retained 3-O-Boc,
ruling out the possibility of participation by this group. For donors with equatorial
4-O-Boc and 6-O-Boc, they obtained similar results as compared to equatorial
3-O-Boc. For donor bearing axial 4-O-Boc, they also failed to isolate any cyclic
structure. However, Boons and coworkers suggested the possible participation from
110

4-benzoyl ester group based on the high α-selectivity.

To confirm whether the high

selectivity came from participation of 4-benzoyl group, Crich and coworkers
developed a new isotopic labeling probe as outlined in Scheme 1.46. Donor 266 was
pre-activated by BSP/Tf2O to form reactive intermediates. If 4-benzoyl ester can
18

participate, intermediate 268 will dominate instead of 267. Quenching with H O will
generate either 269 or 270, with the latter rearranged to 272. Upon acetylation and
thioglycoside formation, 271 or 273 will form with only 273 isotopically labeled. After
these transformations, they were only able to isolate 271, indicating of absence of

46

4-benzoyl ester participation. Although these experimental data can give us some
sense about remote neighboring group participation, we cannot draw such
conclusions that some positions can participate while others cannot. It is simply
because that we cannot detect by experiments does not mean they do not exist.

Scheme 1.44. Participation of 2-O-Boc group. Reagents and conditions: (a) NIS,
o

AgOTf, DCM, -10 C.

Scheme 1.45. Participation of axial 3-O-Boc group. Reagents and conditions: (a)
o

BSP, Tf2O, DCM, -60 C.

Scheme 1.46. Isotopic labeling experiment. Reagents and conditions: (a) BSP, Tf2O,

47

o

DCM, -60 C; (b) Ac2O, pyridine; (c) BF3-Et2O, PhSH.
1.4.1.4. 4, 6-O-Benzylidene Acetal Effect
Being

one

of

the

most

difficult

glycosidic

bonds

to

form,

1,

2-cis-β-D-mannopyranosyl linkage has attracted a lot of attention from the
carbohydrate community. The introduction of glycosylation method utilizing sulfoxide
20

donor by Kahne and coworkers

triggered Crich to investigate the applicability of

this approach. Crich and coworkers discovered that 4, 6-benzylidene protected
mannosyl sulfoxide had unique behavior upon pre-activation and gave excellent
β-selectivity.

89

When donor 273 was coupled to acceptor 274 in the presence of Tf2O,

disaccharide 275 was isolated in 65% yield (Protocol A) (Scheme 1.47). However,
Crich and coworkers found that by performing the pre-activation procedure, they
were able to get high β-selectivity (α:β= 1:10.5) (Protocol B).

114

For donors bearing 4,

6-benzylidene protecting group, they could achieve high stereoselectivity with a wide
range of acceptors including primary and secondary alcohols.

115

Later, this method

was also extended to the pre-activation of thioglycoside donors,
activated by benzenesulfenyl triflate (PhSOTf).

117-118

116

which were

Based on the high β-selectivity,
115

they proposed a possible mechanism for this transformation (Scheme 1.48).

Upon pre-activation with Tf2O, donor 273 was converted to 274, which could evolve
into reactive species such as oxacarbenium ion 277 and α-triflate 279. These two
species are in dynamic equilibrium and the presence of 4, 6-benzylidene protecting
group would shift the equilibrium toward 279. They attributed this to electronic and
steric effects. Electronically, this cyclic protecting group can lock the C5-C6 bond in

48

trans-gauche conformation. As a result, C6-O6 bond is antiperiplanar to C5-O5 bond,
maximizing its electron-withdrawing effect on 277.

89,119

Sterically, as 276 collapses

to oxacarbenium ion 277, the torsional strain on this bicyclic system would
increase.

89,120

To support this mechanism, they carried out low-temperature NMR
69

studies to probe α-triflate.

o

Pre-activation of donor 281 under -78 C with Tf2O led to

the formation of a new carbohydrate species (Scheme 1.49), which was assigned as
α-triflate 282 based on its anomeric proton and carbon signals (broad singlet at 6.2
ppm and 104.6 ppm respectively).

121

Besides, the new signal at -0.037 ppm in

19

F

NMR was also attributed to the presence of 282.

Scheme 1.47. Disaccharide 275 was isolated in (1) 65% (α:β = 10:1) when steps A
and B were combined. (2) 93% (α:β = 1:10.5) when steps A and B were carried out in
a sequential manner.

Scheme 1.48. Proposed mechanism leading to high β-selectivity.

49

Scheme 1.49. (Cont’d). Detection of α-triflate. Reagents and conditions: (a) Tf2O,
o

DTBMP, CD2Cl2, -78 C.
Inspired by Crich’s work, Kim and coworkers developed a new type of
glycosyl

donor,

namely,

2-(hydroxycarbonyl)benzyl
13

selectiveβ-mannopyranosylation (Scheme 1.50).

glycoside,

for

highly

Donor 283 was pre-activated by

Tf2O followed by addition of acceptor 284 to yield disaccharide 285 in 91% yield. In
this transformation, donor 283 was first transformed to anhydride 286, which further
evolved to oxacarbenium ion 287 and α-triflate 288 along with the generation of
phthalide. Based on Crich’s rationale, 288 would exist as the dominate species. Then
SN2-like substitution by acceptor 284 would afford disaccharide 285.

Scheme 1.50. Highly stereoselective synthesis of β-mannoside.
To further confirm the β-directing effect from 4, 6-benzylidene group, Crich
and coworkers also carried out low-temperature NMR studies by pre-activating more
conformationally labile donor 289 (Scheme 1.51). Although α–triflate was detected as
the main reactive species, addition of methanol as acceptor gave anomeric mixture
290 with no stereoselectivity. The loss of stereoselectivity was explained in terms of
reactive intermediates for donor 289. Although not detected by NMR, more reactive
intermediate 292 can exist in small amount due to the more flexible conformation

50

compared to 281 and coupling to methanol will convert α–triflate 291 to 292 (Scheme
69

For oxacarbenium ion 292, nucleophilic attack took place in a SN1-like

1.52).

manner, resulting in loss of stereoselectivity.

Scheme 1.51. Pre-activation of 289 led to anomeric mixture. Reagents and
o

o

o

conditions: (a) Tf2O, DTBMP, DCM, -78 C, then MeOH, -78 C to 0 C.

Scheme 1.52. Equilibrium between reactive intermediates upon pre-activation of
289.
With mannose series giving excellent stereoselectivity, Crich and coworkers
extended this method to glucose series.

122

Pre-activation of thioglycoside 293

followed by addition of different acceptor led to the isolation of different glycosides in
execellent β-selectivity (Scheme 1.53), standing in contrast to mannose series.
Low-temperature NMR studies were carried out to identify the reactive intermediate
upon pre-activation. The newly formed species showed a distinct anomeric proton
resonance at 6.3 ppm (J1,2 = 3.5 Hz) with anomeric carbon signal at 100.6 ppm,
indicating the formation of α-triflate as intermediate. They rationalized that contrasting
stereoselectivity was caused by the influence of O2-C2-C3-O3 interaction (Scheme
123

1.54).

For mannose series, when theα-triflate 288 collapses to oxacarbenium ions,
4

124

either the H3 chair 287 or the B2,5 conformer 299,
B

the O2-C2-C3-O3 torsional

angle decreases while O2-C2-C1-O5 torsional angle increases, maximizing the
51

C2-O2 electron-withdrawing effect. As a result, the oxacarbenium ion will be
destabilized, leading to high β-selectivity. For the glucose series, when the α-triflate
4

4

300 collapses to oxacarbenium ion, either the H3 chair 301 or the E conformer
302,

124

the O2-C2-C3-O3 torsional angle increases while O2-C2-C1-O5 torsional

angle decreases, thus stabilizing the oxacarbenium ion, contributing to the loss of
stereoselectivity.

Scheme 1.53. Strikingly different stereoselectivity for glucose series. Reagents and
o

o

o

conditions: (a) PhSOTf, DTBMP, DCM, -78 C, then ROH, -78 C to 0 C.

Scheme 1.54. Torsional angle values for reactive intermediates of mannose and
glucose.
To further prove the importance of O2-C2-C3-O3 interaction for highly
stereoselective glycosylation, Crich and coworkers synthesized 2-deoxy donor 303
52

and 3-deoxy donor 304, which do not have O2-C2-C3-O3 interaction. Based on the
discussion above, this type of donor should not have any stereoselectivity upon
glycosylation. Indeed, coupling of donor 303 to acceptor 306 gave disaccharide 307
in 75% yield (α:β = 1:1.5), coupling of donor 304 to acceptor 306 gave disaccharide
308 in 85% yield (α:β= 1.9:1), while coupling of donor 305 to acceptor 306 gave
125

disaccharide in 95% yield (α:β = 1.2:1) (Scheme 1.55).

In the case of donor 303,

low-temperature NMR only shows minor signal forα-triflate (anomeric proton signal:
6.0-6.3 ppm), indicating that α-triflate is too unstable for

glycosylation in the

2-deoxy donor series. In the case of donors 304 and 305, clean formation of α-triflate
was observed by low-temperature NMR. However, the loss of stereoselectivity can
also result from absence of the electron-withdrawing C2-O2 bond (donor 303) or
C3-O3 bond (donors 304 and 305). To exclude this possibility, Crich and coworkers
synthesized 2-deoxy-2-fluoro and 3-deoxy-3-fluoro donor series. Here fluoro
substituent functioned as an electron-withdrawing group. Repeating the same
glycosylation reactions as in Scheme 1.55, high yields were obtained with no
126

stereoselectivity (Scheme 1.56).

In all these three cases, α-triflates were detected

as the major reactive intermediated by low-temperature NMR. This experiment
clearly demonstrated the importance of O2-C2-C3-O3 interaction for the observed
selectivity.

53

Scheme 1.55. (Cont’d). Loss of stereoeselectivity in 2-deoxy and 3-deoxy donor
o

o

series. Reagents and conditions: (a) Tf2O, BSP, DCM, -78 C, then 306, -78 C to 0
o

C.

Scheme 1.56. Loss of stereoeselectivity in 2-deoxy-2-fluoro and 3-deoxy-3-fluoro
o

donor series. Reagents and conditions: (a) Tf2O, BSP, DCM, -78 C, then 306, -78
o

o

C to 0 C.
Inspired by the profound influence of 4, 6-O-benzylidene acetal on

stereoselectivity, Crich and coworkers tested the effect of two analogous protecting
groups (4, 6-O-phenylboronate ester and 4, 6-O-polystyrylboronate ester) on the
127-128

stereoselectivity in galacto-, gluco- and mannopyranosyl thioglycoside series.

In the case of glucose series, to get a better comparison with previous results,

122

1-adamantanol was used as acceptor. Pre-activation of donor 316 followed by
addition of 1-adamantanol led to the isolation of α/β mixture (2.8:1) (Scheme 1.57).
They attributed the loss of stereoselectivity to the shift of equilibrium of
α-triflate/oxacarbenium ion toward oxacarbenium ion. In the case of galactose series,
both donors 318 and 319 gave similar stereoselectivity (Scheme 1.58). In the case of
mannose series, they got similar results as compared to the galactose series
(Scheme 1.59). Both 4, 6-O-phenylboronate ester and 4, 6-O-polystyrylboronate
54

ester protected donors 323 and 324 gave β-isomer exclusively. The contrasting
behavior among these three donor series awaits further explanation.

Scheme 1.57. Poor stereoeselectivity for glucose series. Reagents and conditions: (a)
o

o

o

Tf2O, BSP, DCM, -60 C, then 1-adamantanol, -60 C to 0 C.

Scheme 1.58. Similar stereoeselectivity in galactose series. Reagents and conditions:
o

o

o

(a) Tf2O, BSP, DCM, -60 C, then 306, -60 C to 0 C.

Scheme 1.59. Similar stereoeselectivity in mannose series. Reagents and conditions:
o

o

o

(a) Tf2O, BSP, DCM, -60 C, then 1-adamantanol, -60 C to 0 C.
1.4.1.5. Cyclic Carbonate Effect
When screening the substrate scope for the 4, 6-benzylidene acetal effect on
stereoselective synthesis of β-mannosides, Crich and coworkers tested the
55

stereochemical outcome using 2, 3-O-carbonate protected donor 328.

129

Donor 328

was pre-activated followed by addition of acceptor 329 to give exclusively the
α-isomer in 60% yield (Scheme 1.60). To explain this unexpected stereoselectivity,
they suggested that pre-activation of donor 328 will first generate α-triflate, which
0

adopts H5 conformation as indicated by NMR of similar substrates.

130

Due to the

presence of 2, 3-O-carbonate group, the energy gap was small between the α-triflate
collapses and the sofa shaped reactive oxacarbenium ion. This shifts the equilibrium
toward the oxacarbenium ion, resulting highly α-selective glycosylation.

131

In this

specific case, the α-directing effect of 2, 3-carbonate group overrode the β-directing
effect of 4, 6-benzylidene acetal. They further expanded the substrate scope from
mannose series to the rhamnose series.

Scheme 1.60. Stereo-directing effect of 2, 3-carbonate protecting group. Reagents
o

o

o

and conditions: (a) AgOTf, PhSCl, TTBP, DCM, -60 C, then 329, -60 C to 0 C.
However, in the case of 3, 4-O-carbonate protected donor, they observed
completely different stereoselectivity.

131

Coupling of donor 331 to glucose acceptor

332 gave disaccharide 333 in 77% favoring the β-isomer while coupling to
1-adamantanol led to 334 in β-isomer exclusively (Scheme 1.61). The 3, 4-carbonate
protecting group may exert its directing effects in two ways: conformational effect and
electron-withdrawing effect. To figure out the dominant effect in the observed
stereoselectivity, for donor 331, they replaced the 3, 4-O-carbonate group with 3,
56

4-O-isopropylidene acetal group which should only have the conformational effect
(donor 335). The same pre-activation protocol led to the isolation of disaccharide 336
only in α-form (Scheme 1.62). Based on this result, they concluded that the
stereoselectivity comes from electron-withdrawing effect, which can stabilize α-triflate
intermediate followed by SN2 reaction to give β-isomer.

Scheme 1.61. Stereo-directing effect of 3, 4-carbonate protecting group. Reagents
o

o

and conditions: (a) Tf2O, BSP, DCM, -60 C, then 332 or 1-adamantanol, -60 C to 0
o

C.

Scheme 1.62. Reversal of stereoselectivity in the case of 3, 4-isopropylidene acetal
o

o

group. Reagents and conditions: (a) Tf2O, BSP, DCM, -60 C, then 332, -60 C to 0
o

C.
Crich and coworkers further evaluated the stereoselectivity for glucose donor

bearing cyclic carbonate group.

132

For 2, 3-O-carbonate protected glucose donor,

they got moderate to good β-selectivity, in contrast to the high α-selectivity observed
in the mannose series (Scheme 1.63). For 3, 4-O-carbonate protected glucose donor,
the stereoselectivity was lost under the pre-activation protocol, as shown by the
57

example given in Scheme 1.64. Coupling of donor 342 to acceptor 340 gave an α/β
mixture (1:1). Low-temperature NMR studies showed that glucosides also went
through α-triflate intermediate upon activation. Further work has to be done to
illustrate the reason for this unique selectivity of the glucose series.

Scheme 1.63. Stereo-directing effect of the 2, 3-carbonate protecting group.
o

o

o

Reagents and conditions: (a) Tf2O, BSP, DCM, -60 C, then ROH, -60 C to 0 C.

Scheme 1.64. Loss of stereoselectivity for 3, 4-carbonate protected glucose donor.
o

o

o

Reagents and conditions: (a) Tf2O, BSP, DCM, -60 C, then 340, -60 C to 0 C.
In 2008, Ye and coworkers reported a new method based on the
pre-activation approach for highly α-selective glycosylation of 2-deoxysugars and 2,
6-dideoxysugars.

133

Under the pre-activation protocol, donor 345 can be coupled to

different acceptors in high yields and stereoselectivity (Scheme 1.65). Similar results
were achieved for 2, 6-dideoxysugars. Control experiments were carried out to show
the unique selectivity brought by pre-activation approach. For instance, when donor
344 was coupled to acceptor 348 by the non-preactivation approach, disaccharide
58

was isolated in 5% (α:β = 1:1). To expain the stereoselectivity, the activated species
can exist as α–triflate or β–triflate, with the latter being more reactive. The acceptor
will undergo SN2-like reaction with relatively more reactive β–triflate to give
α–glycoside. This process will also shift the equilibrium toward β–triflate from
α–triflate. Under non-preactivation protocol, there is no time for this equilibrium shift
and the acceptor will also react with α–triflate, leading to the formation of anomeric
mixture.
O

O

O

ROH = BnO
BnO

OBz
O

SPh

344
OH
O

BnO OMe
346

HO
BnO

a

O

ROH
OBn
O Ph

BnO OMe
347

OBz
O

O

O

345 OR
80%-90%
O
O
HO

O

Ph

BnO OMe
348

O
O
BnO

O
HO OMe
349

Scheme 1.65. Stereoselective glycosylation of 2-deoxygalactose/glucose. Reagents
o

o

o

and conditions: (a) Tf2O, BSP, TTBP, DCM, -72 C, then ROH, -72 C to 0 C.
1.4.1.6. Oxazolidinone Effect
Similar to the cyclic carbonate group, oxazolidinone group has also found to
affect the stereochemistry under pre-activation conditions. In the course of
developing a novel method for synthesizing 1, 2-cis-2-deoxy-2-amino sugars, Ye and
coworkers established a protocol for controllable glycosylations of oxazolidinone
protected glucosamines.

134

Pre-activation of donor 350 in the presence of TTBP

followed by addition of acceptor produced β-glycoside in high yields. This protocol
was applied to a wide range of acceptors as shown in Scheme 1.66. Since 2-N-acyl
135

group cannot have neighboring group participation effect,

they reasoned that

glycosylation took place through SN2-like mechanism via the α-triflate intermediate.
To their surprise, they found that when this operation was carried out in the absence
59

of TTBP, the stereochemistry was completely reversed to give α-glycoside. They
proposed that this phenomenon was a consequence of acid catalyzed in situ
anomerization of β-glycoside.

136-137

Another possible explanation would be SN2-like

reaction via β-glycoside.

Scheme 1.66. β-selective glycosylation of oxazolidinone protected donor 350.
o

o

Reagents and conditions: (a) Tf2O, BSP, TTBP, DCM, -73 C, then ROH, -73 C to 0
o

C.
Since donor 350 can give high stereoselectivity under pre-activation

condition, analogous donors bearing different protecting groups were examined to
explore the influence of protective groups on stereochemistry.

138

As shown in

Scheme 67, for donor 359, covalent triflates (α and β) would exist as the major
reactive intermediates due of the electron-withdrawing acetyl groups, which
underwent SN2-like reaction to give anomeric mixtures. For donor 360, the benzyl
group would facilitate the formation of oxacarbenium ion as the reactive intermediate.
The 2-N-benzyl group further blocked the α-face, leaving only β-face accessible for
acceptor attack, resulting in exclusive β-selectivity. Pre-activation of donor 361 or 362
would also generate oxacarbenium ion as reactive intermediate due to similar
60

electronic properties as compared to donor 360 with both faces accessible for
acceptor attack, yielding anomeric mixtures. With donor 360 giving the best
stereoselectivity, they further tested different acceptors besides 348 and most of
them gave good to excellent stereoselectivities.

Scheme 1.67. Effect of donor protecting groups on stereoselectivity. Reagents and
o

o

o

conditions: (a) Tf2O, BSM, DCM, -73 C, then 348, -73 C to 0 C.
Repeating α-(1-4)-linked N-acetyl-galactosamine units are present in a wide
range of oligosaccharides and glycoconjugates.

139-140

Ye and coworkers took

advantage of the directing effect of the oxazolidinone group to facilitate the synthesis
141

of a trisaccharide repeating unit (Scheme 1.68).
gave exclusive α-isomer.

61

In this example, each coupling

HO OBn
O
OTBDMS
STol
OBn
TBDMSO OTBDMS O
O
STol
TBDMSO
O
NAc
O
O
b
c
N
d
STol
O 364
O
Ac
NAc
O
NAc
70% over three steps
a
O
O
O 363
O
365
53%
OBn
O
OBn
STol
HO
O
N O
Ac
NAc
O
O 366 O
O

363

e
68%

OTBDMS
O
OBn
OBn
TBDMSO
O
STol
O
N O
O
Ac
N
O
NAc
Ac
O
O
367
O
O
O

Scheme 1.68. (Cont’d). Synthesis of trisaccharide 367. Reagents and conditions: (a)
o

o

o

Tf2O, Ph2SO, TTBP, DCM, -72 C, then 364, -72 C to 0 C; (b) TBAF, THF, r.t.; (c)
o

PhCH(OMe)2, CH3CN, r.t.; (d) Et3SiH, TfOH, DCM, -72 C; (e) Tf2O, Ph2SO, TTBP,
o

o

o

DCM, -72 C, then 366, -72 C to 0 C.
Another example of stereo-directing effect of oxazolidinone is α-sialylation
reaction. In 2011, Sun and coworkers reported pre-activation based highly α-selective
sialylation with oxazolidinone protected thiosialoside donor.

142

They screened a

variety of acceptor including primary and secondary alcohols and most of these
reactions led to α-sialosides with good to excellent yield.
1.4.1.7. Inductively Disarming Effect
4, 6-O-benzylidene acetal and cyclic carbonates affect the stereoselectivty
mainly via torsional disarming effect. Disarming effect can also happen inductively.
Schuerch and coworkers found that 2-O-sulfonyl group could stabilize α-mannosyl
143-144

and α-rhamnosyl sulfonate esters and direct β-selective glycosylation.

Inspired by this pioneering work, Crich and coworkers set out to test the possibility of
direct formation of β-L-rhamnopyranosides from 2-O-sulfonate ester protected
62

thioglycoside donor.

145

For example, coupling of donor 368 to 3β-cholestanol gave

glycoside 369 with moderate anomeric selectivity. (Scheme 1.69) Other sulfonate
esters with different substituents on the aromatic ring were also screened, giving
similar or lower β-selectivity. To further improve β-selectivity, they installed a second
electron-withdrawing ester group on the donor to enhance the disarming effect, as
shown in Scheme 70. By simply replacing the benzyl group at 4-O position with the
benzoyl group, they were able to increase the β-selectivity up to 90%. However,
when carbohydrate was applied as acceptor, the selectivity decreased as in the case
of coupling donor 370 to acceptor 332. And when acceptors with lower reactivity were
used, the selectivity was worse.

Scheme 1.69. Stereo-directing effect of 2-O-sulfonate group. Reagents and
o

o

o

conditions: (a) Tf2O, BSP, TTBP, DCM, -60 C, then 3β-cholestanol, -60 C to 0 C.

O
SPh
BzO
BnO O O
CF3 a
370 S
O

O
O
BzO
BnO O O
CF3
3 -Cholestanol
S
371
b
71% O
OH
: = 1:9
O
BnO
BnO
O
O
BzO
332 BnOOMe
O
BnO O BnO
BnO
O
372
S
CF3 BnOOMe
68% O
: = 1:5

Scheme 1.70. Stereo-directing effect of a second electron-withdrawing group.
o

Reagents and conditions: (a) Tf2O, BSP, TTBP, DCM, -60 C (b) 3β-cholestanol or
o

o

332, -60 C to 0 C.
63

With the moderate selectivity achieved by 2-O-sulfonate ester in the
rhamnose series, they further extended this strategy to the mannose series (Scheme
146

1.71).

Although high β-selectivity was achieved when acceptor 340 was coupled

to donor 373, α-isomer dominated in the case of acceptor 347, even in the presence
of the second electron-withdrawing acetyl group in donor 373. Apparently,
introduction of a second electron-withdrawing group does not help improving
stereoselectivity. They also investigated other electron-withdrawing groups at 2-O
position in mannose series as potential stereo-directing group, such as vinylogous
esters, phosphates and cyanates (Scheme 1.72). Donor 376 with vinylogous ester
and 377 with phosphates gave rather poor stereoselectivity. Although donor 378 with
cyanate gave high stereoselectivity, coupling to carbohydrate acceptors was
accompanied

by

completely

opposite

stereoselectivity.

So

all

these

electron-withdrawing groups led to worse selectivities compared to the sulfonate
ester.

Scheme 1.71. Poor stereoselectivity for mannoside donor 373. Reagents and
o

o

o

conditions: (a) Tf2O, BSP, TTBP, DCM, -60 C (b) 340 or 332, -60 C to 0 C.

64

Scheme 1.72. Stereo-directing effects of different electron-withdrawing groups.
o

Reagents and conditions: (a) Tf2O, BSP, TTBP, DCM, -60 C (b) 3β-cholestanol, -60
o

o

C to 0 C.
147

The capsular polysaccharide from Campylobacter jejuni RM 1221

is

composed of a linear chain of trisaccharide repeating units (Figure 1.14). In this
molecule, two α- and one β-6-deoxy-D-manno-hepto-pyranose residues are
punctuated by a phosphodiester linkage. Synthesis of β-linkage could be easily
achieved by applying Crich’s approach as described in Section 1.4.1.4. To building up
the α-linkage, Crich and coworkers further introduced electron-withdrawing acetyl
group in their mannoside donor, which was found to glycosylate different
phosphosugar acceptors with high α-selectivity (Scheme 1.73). All the acceptors they
tried here led to high α-selectivity, which might be caused by the disarming effect of
acetyl group overriding the β-directing effect of 4, 6-benzylidene acetal group. Due to
the disarming effect, α- and β-triflates would exist as the major intermediates, with the
latter being more reactive leading to α-glycoside. This process would shift the
equilibrium toward β-triflate from α-triflate.

65

Figure 1.14. Bacteria capsular polysaccharide 382.

Scheme 1.73. Highly α-selective synthesis of phosphosugars. Reagents and
o

o

o

conditions: (a) Tf2O, BSP, TTBP, DCM, -60 C, then ROH, -60 C to 0 C.
In 2010, Ye and coworkers reported an alternative approach for highly
89

α-selective glycosylation of 2-deoxysugars and 2, 6-dideoxysugars.

Instead of

using torsionally strained protecting groups as discussed in Section 1.4.1.5,

133

they

installed inductively disarming protecting groups to affect the stereochemistry. As
shown in Scheme 1.74, acetylated glucoside and galactoside were used as donors,
which were coupled to different acceptors as shown in Scheme 1.65 to afford the
corresponding product. Most of these coupling reactions led to high α-selectivity. The
selectivity can be explained in the same manner as in the case of donor 383.

66

Scheme 1.74. Stereoselective glycosylation of 2-deoxygalactose/glucose. Reagents
o

o

o

and conditions: (a) Tf2O, BSM, DCM, -72 C, then ROH, -72 C to 0 C. ROH = 346,
347, 348, 349.
1.4.1.8. Steric Effect
The 4, 6-benzylidene acetal directed β-mannosylation developed by Crich
and coworkers as discussed earlier is applicable to a wide range of acceptors.
However, in some cases, difficulties were encountered and low selectivity was
obtained. For example, when Crich and coworkers tried coupling donor 394 to
acceptor 395 using their standard pre-activation protocol, the desired disaccharide
was isolated in 77% with a rather disappointing anomeric ratio (α:β = 1.8:1), while
coupling of donor 396 to acceptor 395 gave 72% yield with slightly better selectivity
148

(α:β = 1:3) (Figure 1.15).

They attributed this to steric buttressing due to the

presence of the bulky substituent on O-2 and O-3 postions. To better explain this
effect, they analyzed the possible staggered conformations of the triflate intermediate
upon pre-activation (Figure 1.16).

149

397a, 397b, 397c are the three possible

staggered conformations at the O-2 bond. Due to the steric hindrance, the population
of conformations 397a, 397b was presumably low, leading to the increase in the
population of 397c. For conformation 397c, the presence of benzyl group would
shield the β-face from nucleophilic attack, which was responsible for loss of
β-selectivity. This problem may be circumvented by the usage of less bulkier
protecting groups at O-2 and O-3 positions. During the course of synthesis of a
mannohexaose, which is a representative structure of the mannan from Rhodotorula
glutinis,

150-151

Crich and coworkers found that coupling of donor 398 to acceptor 399

67

led to trisaccharide 400 with no stereoselectivity even in the presence of 4,
152

6-benzylidene acetal (Scheme 1.75).

Again, this can be attributed to steric

hindrance described earlier. In contrast, when donor 401 was used as the donor,
selectivity was greatly enhanced (α:β =1:5) due to the presence of the less bulkier
2-O-propargyl ether group.

Figure 1.15. Donor and acceptor pairs resulting low selectivity.

Figure 1.16. Staggered conformations about O-2 bond.

PMP

PMP

OBn
O

O
O
BnO

O
Ph
O
OBn O
O
398

O
O
BnO

BnO
HO
BnO

Ph
O O
O
OBn O
O O
SPh
401

SPh

OBn
O

OMe
PMP

O
O
BnO

PMP

399

O
O
BnO

a
399
a

OBn
OBn
Ph
O
OBn
O
OBn O
O
O
O O
OMe
BnO
400
86%, :
OBn
Ph
O OBn
OBn
O
O
OBn
O
O
O
O
OMe
BnO
402
80%, :

Scheme 1.75. Steric effect on β-mannosylation. Reagents and conditions: (a) Tf2O,
o

o

o

BSP, TTBP, DCM, -60 C, then 399, -60 C to 0 C.
1.4.1.9. 3, 4-Bisacetal Effect
3, 4-Bisacetal is another type of trans-fused bicyclic protecting group which
can selectively protect 3, 4-hydroxyls. Crich and coworkers reported that donors with
this type of protecting group can also couple to various acceptors in a stereoselective
manner.

129

For instance, coupling of donor 403 to acceptor 329 led to disaccharide
68

404 only in a very α-selective manner (Scheme 1.76). Their original consideration
was that the axial methoxy group on the α-face may destabilize α-face by
electrostatic interaction. To give direct support for this thought, they prepared donor
405 without methoxy groups and tried coupling with acceptor 329, which also
proceeded in a highly α-selective manner (Scheme 1.77). This result clearly ruled out
the

possibility

that

the

axial

methoxy

group

controlled

stereoselectivity.

Low-temperature NMR studies showed the presence of α-triflate as the major
intermediate (δH:6.03, δC:105.0). In the equilibrium of α-triflate and oxacarbenium ion,
the presence of 3, 4-bisacetal group did not destabilize the oxacarbenium ion,

120

which underwent coupling with acceptor to give α-glycoside, further shifting the
equilibrium away from α-triflate.

Scheme 1.76. α-Selective glycosylation with 3, 4-bisacetal protected donor 403.
o

o

Reagents and conditions: (a) PhSOTf, DTBMP, DCM, -78 C, then 329, -78 C to 0
o

C.

Scheme 1.77. α-Selective glycosylation with 3, 4-bisacetal protected donor 405.
o

o

Reagents and conditions: (a) PhSOTf, DTBMP, DCM, -78 C, then 329, -78 C to 0
69

o

C.
The dichotomy between glucose and mannose continued for the 3,

4-bisacetal protecting group. Pre-activation of donor 407 followed by coupling to
1-isopropanol or 1-adamantanol led to β-glycoside exclusively in high yields, while
coupling to glucose acceptor 347 gave anomeric mixtures favoring the β-isomer
70

(Scheme 1.78).

Once pre-activated, donor 407 was first converted to the α-triflate,

which was in equilibrium with the oxacarbenium ion. The conformation of the latter
4

was like a H3 half-chair.

153-154

In this conformation, C2-O2 bond rotated down

below the sugar ring plane, resulting in an increased steric interaction with the axial
methoxy group on the bisacetal group, which shifted the equilibrium toward the
α-triflate. To prove this hypothesis, a control experiment was carried out. Donor 409
without methoxy group on its bisacetal group was coupled to acceptor 346 under the
same condition and disaccharide was isolated in 50% yield (α:β = 1:1) (Scheme
1.79). In this reaction, steric repulsion is absent due to the removal of methoxy group.
Therefore, the intermediate can exist as oxacarbenium ion, leading to the loss of
stereoselectivity.
OMe
OBn
O
O
SPh
O
OMe BnO
407

ROH =

HO

a
ROH

HO
BnO

HO

72%
only

OMe
OBn
O
O
OR
O
BnO
OMe
408
OBn
O

BnOOMe
347
80%
: = 4:1

60%
only

Scheme 1.78. β-Selective glycosylation of donor 407. Reagents and conditions: (a)
o

o

o

Tf2O, BSP, TTBP, DCM, -60 C, then ROH, -60 C to 0 C.

70

O
O

OBn
O
BnO
409

SPh

a

O
O

50%, : = 1:1

OBn
O

OBn
O
O
BnO BnO
BnOOMe
410

Scheme 1.79. Loss of stereoselectivity in the case of glycosylation of donor 409.
o

o

Reagents and conditions: (a) Tf2O, BSP, TTBP, DCM, -60 C, then 347, -60 C to 0
o

C.

1.4.1.10. 2, 6-Anhydrosugar as Donor
In

2003,

Lowary

and

coworkers

reported

the

synthesis

of

an

arabinofuranosyl hexasaccharide which is a motif in two mycobacterial cell wall
polysaccharides.

155

In their synthesis, they found it was difficult to install the

β-arabinofuranosyl residue stereoselectively. To solve this problem, they developed a
new method using epoxy thioglycoside or glycosyl sulfoxide 411 as the donor
156

(Scheme 1.80).

Pre-activation of donor 411 followed by addition of n-octanol led

to β-arabinofuranoside 412 exclusively. The stereochemistry of 412 was independent
of the anomeric configuration of donor 411. Both α and β isomers led to 412 in 82%
yield. Similarly, pre-activation of donor 413 (α or β) resulted in α-arabinofuranoside
414 in 83%. Excited by this result, they proposed a possible mechanistic pathway for
this transformation (Scheme 1.81). Reaction of 415 with Tf2O leads to the formation
of intermediate 416, which would evolve to oxacarbenium ion 417. This species
would be in equilibrium with α-triflate 418 and β-triflate 419. Once the acceptor was
added, it could attack 417 to give α and β mixture in a SN1-like manner or attack 418
or 419 in a SN2-like manner to yield β-arabinofuranoside 420 or α-arabinofuranoside
421 respectively. To figure out the most reasonable pathway, they carried out
computational and low-temperature NMR studies. They used ab initio and density
71

functional theory calculations to determine the relative energies of 9 conformers for
α-triflate 418 and β-triflate 419. Calculation showed for each specific conformer, the
energy level of 418 was lower than that of 419. ΔE values ranged between 1.3 and
5.2 kcal/mol, which validated the preference of 418 as the major intermediate.
Pre-activation of donor 415 gave the presence of multiple intermediates revealed by
low-temerature NMR, which equilibrated into one major species over time (Figure
1.17). Based on anomeric proton chemical shift (around 6.4 ppm) and anomeric
carbon chemical shift (around 110 ppm), they assigned this major species as α-triflate
(Figure 1.17b).

69

Upon addition of acceptor, α-triflate would undergo SN2-like

substitution of give β-isomer. As many as 9 intermediates existed at the initial stage
of pre-activation, which could be 416, 419, sulfurane (formed by reaction of 416 with
-

TfO ), or various rotamers. The major intermediate generated by pre-activating donor
413 was identified as β-triflate determined by computational and low-temperature
NMR studies. This intermediate then underwent SN2-like substitution to generate
α-arabinofuranoside. As revealed by NMR (Figure 1.17), the multiple reactive
species would not equilibrate into α-triflate until 60 minutes after Tf2O addition (-40
o

C). They modified their reaction protocol to further improve their glycosylation

selectivity. After pre-activation of donor 415, if they only waited 10 minutes before
adding acceptor 347, they isolated disaccharide 422 in 71% (β:α = 5:1). If after 10
o

minutes, they warmed up the reaction to -40 C and waited another 20 minutes, they
were able to isolate pure β-glycoside in 77% yield (Scheme 1.82). If the
pre-activation protocol were not adapted for this reaction, with the nucleophilic
acceptor present the intermediates would not have time to evolve to the α-triflate.

72

These results highlighted the importance of mechanistic studies and the advantage of
the pre-activation protocol.

Scheme 1.80. (Cont’d). Stereoselective synthesis of arabinofuranoside. Reagents
o

o

o

and conditions: (a) Tf2O, DTBMP, DCM, -78 C, then n-Octanol, -60 C to 0 C.
OBz
O
Tf 2O
O
S(O)Tol
415
OBz
O OR ROH
O
420

OBz
O
O
418 OTf

OBz
O
O TfO
416 S

OBz
O OTf
O

OBz
O
O TfO
417

419

OBz
O
O
OR
421

Scheme 1.81. Proposed mechanism for arabinofuranosylation.

1

Figure 1.17. Partial H NMR of donor 415 after pre-activation (a) 1 min after Tf2O
o

o

addition (-78 C); (b) 60 min after Tf2O addition (-40 C).

Scheme 1.82. Stereoselective synthesis of arabinofuranoside. Reagents and
o

o

o

conditions: (a) Tf2O, DTBMP, DCM, -60 C, then n-Octanol, -60 C to 0 C.
1.4.2. Acceptor Effect
73

The stereoselectivity of a glycosylation reaction is also affected by reactivity
of the acceptor. Different acceptors can give different anomeric selectivities when
coupled to the same donor. For example, Crich and coworkers found when different
acceptors were coupled to donor 422, they obtained glycosides with different
157

anomeric selectivities (Scheme 1.83).

It seems that the more reactive acceptor

gives better selectivity than less reactive acceptor. However, Ye and coworkers
reported

that

for

oxazolidinone

protected

donor

system,
138

stereoselectivity for less reactive acceptor (Scheme 1.84).

it

gave

higher

So acceptor affects

stereoselectivity no only by their reactivity. Instead, it is a combination of different
factors, including the property of the donor as discussed in previous sections,
promoter, solvent and additives. Another possible explanation is stereochemical
matching/mismatching (donor dependence is stereochemical matching).

158

Scheme 1.83. Effect of acceptor on stereoselective glycosylation. Reagents and
o

o

o

conditions: (a) Tf2O, BSP, DCM, -60 C, then ROH, -60 C to 0 C.

74

BnO
O

OBn
O

BnO
O

OBn
O

STol a
OR
N Bn
ROH
N Bn
O 360
O 425
HO
O
BnO
O Ph O
O
Ph OO
O
OMe CH3(CH2)7OH
BnO
BnO
ROH =
HO
BnO OMe
HO
BnO OMe
348
348
346
95%
85%
85%
85%
: = 1:3.5
only
: = 1:2
: = 1:6

Scheme 1.84. Effect of acceptor on stereoselective glycosylation. Reagents and
o

o

o

conditions: (a) Tf2O, BSM, DCM, -73 C, then ROH, -73 C to 0 C.
1.4.3. Promoter Effect
As mentioned in earlier sections, different promoters are available for
pre-activation based glycosylation approach. For example, Ye and coworkers
reported different anomeric ratios when pre-activating donor 360 with different
138

promoter systems (Scheme 1.85).

Scheme 1.85. Effect of promoter on stereoselective glycosylation.
1.4.4. Solvent and Additive Effect
Solvents are well-known to affect stereochemistry of glycosylation
reactions.

159-162

In 2011, Huang and coworkers reported the effect of solvent in their

pre-activation strategy.

88

When the reaction was carried out in DCM, disaccharide

429 was isolated in 69% favoring β. By contrast, when diethyl ether was used as

75

solvent, 429 was isolated in 90% favoring α (Scheme 1.86). This trend applies to a
wide range of donor/acceptor pairs. Here they used small amount of toluene instead
of CH3CN to dissolve AgOTf due to the known nitrile effect.

159,162-163

By varying the

amount of AgOTf used for pre-activation, they observed different stereoselectivity
with more AgOTf favoring β (Scheme 1.87). To explain their results, they proposed
the possible mechanism for these two effects (Scheme 1.88). As discussed in
Scheme 1.28, pre-activation of donor would generate α-triflate as the major
intermediate.

67

When diethyl ether was used as solvent, it would act as a nucleophile

to afford intermediate 430, and subsequent displacement of the ether molecule by
acceptor in SN2-like fashion would lead to α-glycoside as the major product (Pathway
a). When DCM is used as solvent, the reaction would directly go through SN2-like
pathway to afford β-glycoside as the major product (Pathway c). In the presence of
excess AgOTf, it is likely that AgOTf will coordinate with the oxygen atom of the
triflate, leading to its activation to favor the formation of oxacarbenium ion 433. So the
reaction would undertake SN1 like pathway (Pathway b). Besides DCM and diethyl
ether, they also screened other solvents such as tetrahydrofuran (THF), toluene,
toluene/1, 4-dioxane, and neat acetonitrile. However, none of these solvents gave
productive coupling.

Scheme 1.86. Effect of solvent on stereoselective glycosylation.

76

Scheme 1.87. Effect of AgOTf on stereoselective glycosylation.

Scheme 1.88. Proposed mechanism for effects of solvents and AgOTf on
stereoselectivity.
In 2006, Crich and coworkers reported the nitrile effect on stereoselective
β-rhamnopyranosylation.

163

As shown in Table 1.1, when varying the ratio of CH3CN

(EtCN) and DCM, they got different anomeric ratios, with entry 4 giving the best
selectivity. Further decreasing or increasing the amount of CH3CN (EtCN) led to low
coupling yield or stereoselectivity. Applying this solvent ratio to other coupling
reactions slightly increased the β-selectivity. Interestingly, when donor 422 was
coupled to acceptor 346 in DCM/CH3CN (7:3), compound 438 was isolated in 30%
after column purification (Scheme 1.89). This reaction presumably went through 439,
which was caused by nucleophilc attack of CH3CN at the anomeric center, followed
by nucleophilic attack of acceptor 346 at the nitrilium ion. Compound 438 provided
direct evidence for the participation of CH3CN during glycosylation.

77

Table 1.1. Effect of solvent ratio on stereoselectivity.

Scheme 1.89. Evidence for solvent participation.
To build up 1, 2-cis linkage, Mong and coworkers developed a novel strategy
using dimethylformamide (DMF) as a modulating molecule to deliver α-selectivity in
2011.

164

Table 1.2 shows the effect of DMF on stereochemistry in formation of

disaccharide 441. Under the pre-activation condition, as the amount of DMF
increased from 1.5 equiv. to 6.0 equiv., the α:β selectivity increased from 1:1 to 19:1.
Also, traditional glycosylation approach (mixing donor, acceptor and promoter
together) gave lower anomeric selectivity (Entries 2 and 3). This trend turned out to
be general for a variety of donor/acceptor pairs. The modulating effect of DMF
worked for different thioglycosides except for 2-azido-2-deoxythioglucoside, for which

78

further optimization was required. Encouraged by this result, they further tested the
possibility of using dimethylacetamide (DMA) as the modulating reagent using the
same pre-activation condition shown in Table 1.2. Disappointingly, the selectivity was
not attractive (α:β = 4:1). To explain this excellent stereoselectivity, they proposed a
possible mechanism for this transformation (Scheme 1.90). Pre-activation of the
thioglycoside led to reactive intermediates such as oxacarbenium ion 442, addition of
acceptor in the absence of DMF gave anomeric mixtures of 445. In the presence of
DMF, it could act as a nucleophile to attack the anomeric carbon to form α-triflate 443
or β-triflate 444, with the latter being more reactive toward the acceptor to form
α-glycoside 446. To provide evidence for this mechanism, low-temperature NMR
studies were carried out to detect the major intermediate upon pre-activation
(Scheme 1.91). Pre-activation of donor 447 at -10

o

C in CDCl3 led to a new
a

carbohydrate species, which they assigned as the α-glycosyl imidate 448 (H : 6.39
b

c

d

ppm, J1,2 = 3 Hz; H : 5.60 ppm; H : 8.90 ppm; H : 3.40, 3.22 ppm). Addition of the
acceptor resulted in the disappearance of these signals. Although they were not able
to detect β-glycosyl imidate by NMR, it is reasonable that the acceptor preferentially
reacts with more reactive β-glycosyl imidate existing in small amounts to give
α-glycoside. This would shift α/β-glycosyl imidate toward β-glycosyl imidate.

Table 1.2. Modulating effect of DMF on stereoselectivity. Entries 1 and 2:
Non-preactivation; Entries 3, 4 and 5: Pre-activation.
79

Scheme 1.90. Possible mechanism for DMF-modulated glycosylation.

Scheme 1.91. Detection of α-glycosyl imidate 448.
As discussed in Section 1.4.1.6, TTBP is a good β-directing reagent for
donor 350.

134

Besides TTBP, Ye and coworkers also tested other additives as

potential stereo-directing reagents.

165

They coupled donor 350 to acceptor 348

under their pre-activation condition discussed in Section 1.4.1.6 to get disaccharides
with different anomeric ratios when different additives were used. In general,
thiophene and catalytic amount of tetra-butyl ammonium iodide (TBAI) led to
excellent α-selectivity, while stoichiometric amount of TBAI led to β-selectivity.
Low-temperature NMR showed the presence of α-triflate as the major intermediate.
To rationalize their results, they proposed that β-selectivity was a result of direct
SN2-like substitution and α-selectivity was due to in situ anomerization after
substitution.
1.5. Conclusions
During the past two decades, pre-activation based glycosylation approach

80

has found wide applications in the construction of complex oligosaccharides and
glycoconjugates due to its unique chemoselectivity and stereoselectivity compared to
traditional glycosylation approach. However, there are still limitations to be overcome.
In terms of chemoselectivity, for sulfoxide donor, glycosyl sulfenate could be
the major species formed upon pre-activation, which could be unreactive toward the
166

acceptor, lowering the glycosylation yield (Scheme 1.92).

The formation of

sulfenate involves coupling of unreacted sulfoxide 450 with activated species 451 to
form sulfonium ion 452, which ejected sulfenate 453 to regenerate 451. The amount
of 453 depended on the relative rates of triflation and sulfoxide glycosylation. To
suppress sulfenate formation, Kahne and coworkers changed the order of adding
reagents. Instead of using pre-activation approach, they added sulfoxide donor slowly
to a solution containing acceptor, Tf2O and base. For thioglycosides, aglycon transfer
is a widely present problem in many glycosylation systems. After donor is activated,
another thioglycoside is added as acceptor. Thioether can function as a nucleophile
to compete with hydroxyl group. When this happens, the thioglycoside donor will be
regenerated. Whether transfer happens or not mainly depends on two factors: 1)
relative reactivity of sulfur atom and oxygen atom; 2) electronic properties of donor
and acceptor. To solve this problem, Gildersleeve and coworkers developed a new
aglycon, namely, 2, 6-dimethylphenyl (DMP).

167

The bulkiness of this group blocked

the sulfur atom from attacking activated donor. However, this strategy cannot be
applied for iterative one-pot synthesis since the thioether function cannot be activated
by promoter due to sterics.

81

S Ph
O
Sulfoxide 450
O

Glycosylation

TfO
O
Activated
species 451
TfO
Ph
S
O
O
O
Sulfonium
ion 452

O SPh
O
Sulfenate 453
Ejection

Scheme 1.92. Generation of glycosyl sulfenate from glycosyl sulfoxide donor.
In terms of stereoselectivity, stereochemistry is determined by all the factors
discussed in the earlier sections. The outcome of the reaction will need to be
determined in each individual reaction.
With continual development, the pre-activation strategy will enjoy more and
more applications in carbohydrate synthesis. It complements well with the traditional
approach of mixing donor and acceptor together for glycosylation and is a powerful
tool to prepare complex oligosaccharides for tackling important problems in the area
of glycobiology.

82

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83

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98

CHAPTER 2
Fluorous-assisted One-Pot Oligosaccharide Synthesis
2.1. Introduction
Carbohydrates are widely expressed in biological systems with their unique
functions such as inflammation, bacterial infection and blood coagulation.

1-2

To

illustrate structure-activity relationship, biologists need oligosaccharides with sufficient
quantity and high purity. To achieve this goal, synthetic chemists have been actively
involved in carbohydrate synthesis area over the several decades. Traditional
oligosaccharide synthesis is time-consuming, due to the requirement for protecting
group manipulation and aglycon adjustments at the oligosaccharide intermediate stage.
To improve the synthetic efficiency, many novel methods have been developed,
including orthogonal glycosylation,
armed-disarmed
glycosylation,

17-20

glycosylation,

9-10

active-latent activation,

14-16

11-13

reactivity-independent

and automated solid-phase synthesis.

21

3-8

reactivity-based
chemoselective

Several of these strategies

have been adopted into one-pot protocols, by which a series of glycosylation reactions
can be carried out in a single flask without the need to purify the intermediates.

22-23

Many complex oligosaccharides have been assemble through this one-pot protocol,
such as the phytoalexin elicitor heptasaccharide,
Globo-H Hexasaccharide,

27-28

24

dimeric LewisX antigens,

the Lewis Y carbohydrate hapten,

fucosylated bi-antennary N-glycan dodecasaccharide.

99

30

29

25-26

the core-

For all these one-pot

methodologies, upon completion of the synthesis, silica gel chromatography is required
to purify the final product from side products such as disulfide and decomposed donors
generated in the reaction, which can be time and solvent consuming. It would be
desirable that purification of the final product from the one-pot reactions can also be
expedited to improve the overall synthetic efficiencies.
Recently, fluorous chemistry has become popular to facilitate organic synthesis
and catalysis.

31-34

Highly fluorinated (fluorous) compounds have strong affinities with

fluorinated silica gel and can be easily separated from nonfluorous compounds through
32,35

F- SPE (Figure 2.1a).

A mixture of organic and fluorous compounds is loaded onto

fluorous silica gel, a type of silica gel whose surface is modified with a fluorous chain
(Figure 2.1b). In order to take advantage of this unique property in carbohydrate
synthesis, a fluorous tag can be introduced onto a building block, typically a glycosyl
acceptor prior to glycosylation.

35-42

For example, Seeberger and coworkers coupled

fluorous acceptor 3 (n = 0) to the glycosyl donor 1 to yield a fluorous disaccharide 2
(n=1), which was purified by F-SPE. After deprotection, the fluorous disaccharide
acceptor 3 (n=1) was ready for another cycle of coupling. This process was repeated
until the fluorous tagged tetrasaccharide 2 (n=4) was obtained (Figure 2.2). Or, fluorous
43-

tags can also be introduced into glycosyl donors as protective groups (Scheme 2.1).
45

Silyl ether tagged donor 4 was coupled to acceptor, followed by F-SPE to yield

product 5 in high yield. Excess acceptor was readily recovered from organic fraction. Or
fluorous tags can be introduced to the aglycon leaving group. In 2004, our group
designed and synthesized a new fluorous thiol 7, which was introduced to glycosyl
100

46

donor 9 as an aglycon leaving group (Scheme 2.1).

This donor was coupled to

various acceptors in high yields with generation of fluorous disulfide, from which
fluorous thiol can be easily regenerated. Recently, another method has been developed
by Seeberger and coworkers where a fluorinated silyl protective group has been used to
selectively tag the desired product after oligosaccharide assembly on solid phase.

47

After the same coupling procedure shown in Figure 2.2 was complete, they used a
fluorous tag selectively “catch” the desired oligosaccharide, followed by cleaving off the
resin and removing the fluorous tag, they were able to isolate the product without
column purification.

Figure 2.1. (a) a cartoon of F-SPE; (b) fluorous silica gel. For interpretation of the
references to color in this and all other figures, the reader is referred to the electronic
version of this dissertation.
101

Figure

2.2.

Fluorous-assisted

synthesis

of

β-(1-6)

linked

D-glucopyranoside

homotetramer.

Scheme 2.1. Synthetic application of fluorous tagged glycosyl donor.
OAc HS
O
AcO
+
AcO
AcO
Br
6
c
83 %

BzO
BzO

O
N
CH3
7
OBz
O

S

BzO
9

C8H17

a, b
100 %

HO
HO

OH
O

S

O

HO

N
CH3

8

O
N
CH3

d
BzO
68%-93 % BzO
C8H17

OBz
O
BzO
10

C8H17

OR

Scheme 2.2. Application of fluorous thiol in oligosaccharide synthesis. Reagents and
conditions: (a) 1 M aq Na2CO3, EtOAc, r.t.; (b) CH3ONa, CH3OH, rt; (c) BzCl, DMAP,
o

pyridine, r.t.; (d) NIS, AgOTf, ROH, DCM, MS-AW 300, -78 C.
102

Our group is interested in developing new methodologies for expediting
oligosaccharide assembly. Our initial foray into fluorous chemistry involved the
development of fluorous thiol as the aglycon leaving group for thioglycoside building
blocks as shown in Scheme 2.2. In this project, we developed a novel method to aid
one-pot oligosaccharide assembly.
2.2. Results and discussion
2.2.1. Designing a new “catch” and “release” strategy
Based on the success of the fluorous thiol chemistry as shown in Scheme 2.2,
we further applied this strategy to larger oligosaccharide synthesis. However, serious
difficulties were encountered with this approach,

48

as the fluorous disaccharide

acceptor 11a reacted poorly with trisaccharide donor 10 (Figure 2.3a); Main side
products resulted from donor decomposition or hydrolysis. In contrast, the reaction of
the corresponding non-fluorous lactoside 11b with 10 produced pentasaccharide 12b in
70% yield (Figure 2.3a). Although other fluorous acceptors have been reported for
successful glycosylations,

35-38,42

the failure of our reaction was presumably due to the

low solubility of the fluorous acceptor. Another potential complication of this preglycosylation fluorous tag introduction strategy is that in synthesis of large
oligosaccharides, the original fluorous chain at the reducing end may not be sufficient to
retain the growing oligosaccharides on fluorous silica gel during F-SPE with the
increasing sizes of the organic components. These considerations led us to explore an
alternative strategy for fluorous-assisted carbohydrate synthesis.

103

Figure 2.3. While glycosylation of trisaccharide donor 10 failed with (a) the fluorous
acceptor 11a, its glycosylation with (b) acceptor 11b was successfully performed under
identical reaction conditions.
As outlined in Figure 2.4, this new approach combines a fluorous “catch and
release” protocol

46-54

with the pre-activation based one-pot synthesis, which begins by

activation of a thioglycosyl donor by the promoter p-TolSCl/AgOTf generating a reactive
intermediate

55

20

in the absence of an acceptor.
104

Upon addition of a thioglycosyl

acceptor to the pre-activated donor, a disaccharide will be formed, which can be
activated for the next round of glycosylation. This process can be repeated in the same
flask until the desired oligosaccharide length is achieved. The last acceptor at the
reducing end of the oligosaccharide will bear a functionalized linker, which can react
with a fluorous tag. After one-pot glycosylation reactions are completed, the fluorous tag
is added into the reaction mixture to selectively “catch” the desired oligosaccharide,
which is easily separated from non-fluorous impurities by F-SPE. Subsequent release of
the tag and F-SPE will lead to the pure desired oligosaccharide product without any
silica gel column purification.

Figure 2.4. Fluorous-assisted one-pot oligosaccharide synthesis.
We envisioned that this fluorous “catch and release” protocol could potentially
address the aforementioned limitations of the pre-glycosylation tag introduction

105

approach. As no fluorous groups are present during glycosylations, high solubilities of
the building blocks can be maintained especially at low temperatures required by some
glycosylation reactions. Furthermore, the post- glycosylation introduction of the fluorous
tag bestows the flexibility of choosing the appropriate fluorous groups to “catch” the
oligosaccharides.
2.2.2. Screening different linkers for “catch” and “release” strategy
In order for the fluorous “catch and release” protocol to be successful, the
functionalized linker should be inert under glycosylation conditions and must not
interfere with glycosylations. Furthermore, the “catch” and “release” reactions must be
highly chemoselective in the presence of the heavily functionalized oligosaccharides
and the reactions should be rapid ideally completed within minutes.
The first reaction we examined is the Staudinger reaction between mannosyl
56

azide 13

and the commercially available fluorous phosphine 14. However, the

resulting aza-ylide 15 was hydrolyzed during the F-SPE after the “catch” reaction
(Figure 2.5).

Figure 2.5. Compounds used for Staudinger reaction.
Hydrazone formation was explored next with aldehyde 18 and fluorous hydrazine
22. Aldehyde 18 was synthesized from compound 16,
106

14

which was first glycosylated

with 4-penten-1-ol to give compound 17 followed by ozonolysis to introduce the
aldehyde moiety (Scheme 2.3a). Triphenylphosphine was used initially to reduce the
trioxolane intermediate from ozonolysis. Although the reaction was successful, it turned
out to be difficult to cleanly separate the desired product from triphenylphosphine oxide
generated in the reaction. This problem was solved by employing the solid phase
supported triphenylphosphine (PS-PPh3), which was easily removed after the reaction
by simple filtration. Removal of TBDPS group in 17 by HF in pyridine led to aldehyde 18.
57

Hydrazine 22 was obtained by EDC-HCl mediated coupling of compound 19

and

compound 20 and subsequent deprotection with TFA (Scheme 2.1b). The “catch and
release” reactions between 18 and 22 were then performed. Unfortunately, aldehyde 18
was found to be unstable due to air oxidization. To overcome this obstacle, ketone 24
was prepared.

Glycosylation of compound 16 with 4-hydroxy-2-butanone gave

compound 23, which was treated with HF in pyridine to yield ketone 24 (Scheme 2.3c).
However, with increased steric hindrance around the carbonyl group in ketone 24, its
“catch” reaction with fluorous hydrazine 22 turned out to be very slow, requiring more
than 24 hours to complete, rendering it an unattractive candidate.

107

Scheme 2.3. Synthesis of aldehyde 18, fluorous hydrazine 22 and ketone 15. Reagents
o

and conditions: (a) AgOTf, p-TolSCl, MS 4Å, 4-penten-1-ol, -78 C- r.t.; (b) O3,DCM,
PS-PPh3; (c) HF/Pyridine; (d) EDC, DMF; (e)TFA, DCM; (f) AgOTf, p-TolSCl, MS 4Å, 4o

hydroxy-2-butanone, -78 C- r.t.; (g) HF/Pyridine.
The slow reaction rate of 24 with hydrazine 22 prompted us to the design of
fluorous hydrazide 27. The preparation of hydrazide 27 started from fluorous carboxylic
acid 25, which was first coupled to tert-butyl carbazate to provide compound 26 in 74%
yield. Removal of the Boc group from 26 by TFA gave fluorous tag 27 in 100% yield
(Scheme 2.4). To our delight, hydrazide 27 was much more reactive, reacting with
ketone 24 in less than 10 minutes leading to 28. 28 was stable under neutral condition,
the hydrazone bond of which can be cleaved with aqueous acid releasing ketone 24 in
quantitative yield.

108

Scheme 2.4. Synthesis of fluorous hydrazine 27 and its application in “catch and
release” of 24. Reagents and conditions: (a) BocNHNH2, EDC, DMF; (b) TFA/DCM; (c)
24, DCM, MeOH, then F-SPE; (d) acetone, TFA, then F-SPE.
2.2.3. Synthesis of a disaccharide as a model study
With compound 27 in hand, its utility in oligosaccharide synthesis was examined
14

first in a single step glycosylation reaction. Glycosylation of acceptor 24 by donor 29

promoted by p-TolSCl/AgOTf led to disaccharide 30. Upon completion of the reaction,
solvent was removed and the reaction mixture (Scheme 2.5, TLC Lane 1, and Figure
2.6a) was redispersed in DCM and methanol (1:1), followed by addition of fluorous tag
27. The “catch” reaction was completed in less than 10 minutes, followed by F-SPE
removing all non-fluorous compounds. The fluorous fraction was then dissolved in
acetone with 0.5% trifluoroacetic acid to cleave the hydrazone bond in 10 minutes (Lane
2). After a subsequent F-SPE, pure disaccharide 30 was obtained in 80% yield after the
overall purification process (Lane 3, and Figure 2.6b). The purity and structure of the
product were confirmed by NMR spectroscopy, MS, and HPLC analysis. The success
of this reaction proved that the ketone moiety is stable and it does not interfere with the
glycosylation reaction. The whole “catch and release” purification process took less than
1 h to complete with less than 30 mL organic solvent. In contrast, a typical silica gel

109

chromatography separation requires a few hours and consumes several hundred
milliliters of solvent. The speed in obtaining 30 highlights the advantage of our approach.
The linker in 30 was removed through a reverse Michael reaction under basic conditions,
together with the benzoyl groups, to produce 31 in 80% yield.

Scheme 2.5 Synthesis of disaccharide 31. TLC: Lane 1, reaction mixture after
glycosylation; Lane 2, reaction mixture after “catch”, first F-SPE, and “release”; Lane 3,
organic fraction after the second F-SPE. Reagents and conditions: (a) AgOTf, p-TolSCl,
o

MS 4Å, -78 C-r.t.; (b) DCM/MeOH, then F-SPE; (c) 0.5% TFA in acetone, then F-SPE;
(d) NaOMe/MeOH.

110

Figure 2.6. (a) HPLC chromatogram of the crude reaction mixture of compound 30 after
the glycosylation reaction. The peak marked with an

* was ditolyl disulfide, which was a

side product from donor activation and has a strong UV absorbance band. This
chromatogram corresponds to lane 1 of the TLC shown in Scheme 2.5; (b) HPLC
chromatogram of the organic fraction after the second F-SPE, which corresponds to
lane 3 of the TLC shown in Scheme 2.5. (HPLC mobile phase: hexane/ethyl acetate =
2:1, flow rate 1 mL/min, UV monitoring at 256 nm, HPLC column: SupelCOSIL LC-Si,
25 cm X 4.6 mm, 5-μm particle size).
2.2.4. Synthesis of LewisX trisaccharide
Based on the success of disaccharide synthesis, we moved on to test the
feasibility of the fluorous assisted assembly of a branched oligosaccharide, LewisX
58-59

trisaccharide. LewisX is biologically important,

testing candidate for new synthetic methodologies.
111

and has been shown to be a good

25-26,60-61

For our synthesis, donor

32 was first pre-activated by p-TolSCl/AgOTf, which regiospecifically glycosylated
glucosamine diol 33 at its 4-hydroxyl group.

Upon complete consumption of the

acceptor 33, the resulting disaccharide was immediately subject to fucosylation by
14

fucosyl donor 34

in the same reaction flask, which led to the formation of LewisX

trisaccharide 35 in three hours (Scheme 2.6).

One potential obstacle of fluorous

assisted one-pot synthesis was the incomplete consumption of acceptors leading to
multiple products bearing the functionalized linker. To solve this problem, we found by
raising the reaction temperature to room temperature after each glycosylation, the
acceptor was completely consumed and no deletion sequences were observed. The
fluorous hydrazide 27 then successfully caught and released pure trisaccharide 35 from
the reaction mixture within one hour. The overall process of acquiring trisaccharide 35
through a one-pot synthesis and “catch and release” purification took 4 h with an overall
yield of 62% starting from donor 32.

Scheme 2.6. Synthesis of LewisX trisaccharide 35. Reagents and conditions: (a) AgOTf,
o

o

p-TolSCl, MS 4Å, -78 C; (b) -78 oC-r.t.; (c) AgOTf, p-TolSCl, MS 4Å, -78 C-r.t.; (d)
DCM/MeOH, then F-SPE; (e) 0.5% TFA in acetone, then F-SPE.
2.2.5. Synthesis of a linear tetrasaccharide
112

To further examine the scope of our method, a linear tetrasaccharide was
synthesized via a four component one-pot synthesis. Sequential pre-activation based
one-pot glycosylations of 29,

14

36,

62

36 and 24 gave tetrasaccharide 37 (Scheme 2.7).

The temperature cycling protocol was adopted to minimize deletion sequences.
Fluorous purification with hydrazide 16 generated pure tetrasaccharide 28 in 61% yield
from galactoside donor 21 within just a few hours.
BzO
BzO

BzO

OBz
O

BzO
29

36
b

OBz
24
e

d

BzO

a

STol

36

BzO

OBz
O

OH
O

O
OBz

STol

OBz

g

27
f

BzO

O

O

OBz

BzO

37

BzO

c

61% over seven steps

O

O
OBz

BzO

BzO
BzO

O

O

O

OBz

Scheme 2.7. Synthesis of tetrasaccharide 37. Reagents and conditions: (a) AgOTf, po

o

o

o

TolSCl, MS 4Å, -78 C; (b) -78 C-r.t.; (c) AgOTf, p-TolSCl, MS 4Å, -78 C; (d) -78 Co

r.t.; (e) AgOTf, p-TolSCl, MS 4Å,-78 C-r.t.; (f) DCM/MeOH, then F-SPE; (g) 0.5% TFA
in acetone, then F-SPE.
2.2.6. Stability of glycosidc linkage toward acidic conditions
A potential concern with the acidic conditions employed for detagging is acidmediated glycosidic linkage cleavage. Although, in general, glycosidic linkages can be
sensitive to acids, the cleavage rate is dependent on the amount of acid used. The
LewisX trisaccharide 35 with the acid-labile fucosyl linkage was stable under the mild
113

acidic conditions employed for tag removal (0.5% TFA in acetone, 10 min). Control
experiments have shown that 35 was un-affected in 5% TFA over 30 min. Thus, the
fluorous “catch and release” protocol can be of general utility.
2.3. Conclusions
We have developed a new fluorous “catch” and “release” protocol, which facilitate
the purification of the desired oligosaccharide after one-pot synthesis without any silica
gel chromatography.

Among several reactions examined, the easily accessible

hydrazide 27 was found to be most suitable, which rapidly reacted with ketone
functionalized oligosaccharides. Compared to silica gel column purification, fluorous
separation is fast, convenient and saves organic solvent. Both linear and branched
oligosaccharides can be constructed using this strategy as exemplified by the one-pot
assemblies of LewisX trisaccharide 35 and the galactose containing tetrasaccharide 37.
Due to the rapid reaction rates and much reduced volume of organic solvents employed
for purification, this post-glycosylation tagging strategy provides an attractive alternative
to the current practice of pre-glycosylation introduction of fluorous tags.
2.4. Experimental Section
2.4.1. General experimental procedures
All reactions were carried out under nitrogen with anhydrous solvents in flamedried glassware, unless otherwise noted. Glycosylation reactions were performed in the
presence of molecular sieves, which were flame–dried right before the reaction under
high vaccum. Glycosylation solvents were dried using a solvent purification system and
used directly without further drying. Chemicals used were reagent grade as supplied
except where noted. Compound 5 and the fluorous silica gel were purchased from
114

Fluorous Technologies Incorporated.

Analytical thin-layer chromatography was

performed using silica gel 60 F254 glass plates. Compounds were visualized by UV
light (254 nm) and by staining with a yellow solution containing Ce(NH4)2(NO3)6 (0.5 g)
and (NH4)6Mo7O24 4H2O (24.0 g) in 6% H2SO4 (500 mL).
chromatography was performed on silica gel 60 (230-400 Mesh).

Flash column
Fluorous column

chromatography was performed on 40 µm fluorous silica gel purchased from Fluorous
1

Technologies. NMR spectra were referenced using residual CHCl3 (δ H-NMR 7.26
ppm,
1

13

C-NMR 77.0 ppm). Peak and coupling constants assignments are based on
1

1

1

13

1

13

H-NMR, H- H gCOSY and (or) H- C gHMQC and H- C gHMBC experiments. All
o

optical rotations were measured at 25 C using the sodium D line.
2.4.2. Characterization of anomeric stereochemistry
The stereochemistries of the newly formed glycosidic linkages in disaccharide 31,
3

LewisX trisaccharide 35 and tetrasaccharide 37 were determined by JH1,H1 through
1

1

1

H-NMR and/or JC1,H1 through gHMQC 2-D NMR (without H decoupling). Smaller
3

coupling constants of JH1,H2 (around 3 Hz) indicate α linkages and larger coupling
3

constants JH1,H1 (7.2 Hz or larger) indicate β linkages.
suggests α linkages and 160 Hz suggests β linkages.

1

JC1,H1 around 170 Hz

63

2.4.3. General procedure for “catch” and “release”
The reaction mixture was dispersed in DCM/MeOH (1:1). Fluorous hydrazide
(1.5 equiv.) was added into the solution (pH around 8). After stirring under room
115

temperature for 10 minutes, the solution was neutralized by NaHCO3 to pH 6-7.
Following removal of the solvents, the residue was loaded into fluorous silica gel column
with minimum amount of MeOH. The column was first flushed with MeOH/H2O (4:1)
with all the non-fluorous compounds coming out immediately. This was followed by
flushing with MeOH to elute fluorous compounds. The fluorous fraction was collected
and concentrated to dryness. The residue was further dissolved in acetone (0.6 mL),
followed by addition of 0.5 equiv. of TFA. After stirring under room temperature for 10
minutes, solvent was removed and the residue was loaded into a fluorous column. After
flushing with MeOH/H2O (4:1), the desired product came out immediately, which was
concentrated and dried under high vacuum to provide the pure product.
2.4.4. Procedures and NMR data for new compounds
4-Oxobutyl 2,3,4-tri-O-benzoyl-β-D-galactopyranoside (18). p-Tolyl 2,3,4-tri14

O-benzoyl-6-O-tert-butyl-diphenylsilyl-1-thio-β-D-galactopyranoside 16
mmol) was dissolved in DCM (5 mL) and stirred at -78
molecular sieves MS 4Å (1 g) for 30 minutes.

(540 mg, 0.65

o

C with freshly activated

Silver triflate (498 mg, 1.94 mmol)

dissolved in acetonitrile (0.3 mL) was added to the reaction mixture. Five minutes later,
orange-colored p-TolSCl (102 μL, 0.65 mmol) was added directly into the reaction
mixture. This needs to be performed quickly in order to prevent the p-TolSCl from
freezing inside the syringe tip or on the flask wall. The yellow color of the solution
quickly dissipated within a few seconds, indicating the complete consumption of pTolSCl. 4-penten-1-ol (80 μL, 0.7746 mmol, dried by dissolving in DCM and stirring with
molecular sieves MS 4Å overnight before use) was then added along the wall of the
116

flask slowly. The reaction mixture was warmed to room temperature under stirring in 2
hours. The reaction mixture was then diluted with DCM and filtered through Celite. The
Celite was washed with DCM until no organic compounds were present in the filtrate.
The filtrate was extracted with saturated solution of NaHCO3. The organic layer was
then dried over Na2SO4 and concentrated to dryness. The residue was purified by
silica gel column chromatography (Hexanes/EtOAc, 4:1) to give compound 4-oxobutyl 2,
3, 4-tri-O-benzoyl-6-tert-butyl-diphenylsilyl-β-D-galactopyranoside 17 (464 mg, 90%).
20

[α]

o
1
D +83 (c = 1.0, CH2Cl2); H-NMR (500 MHz, CDCl3): δ 0.98 (s, 9 H, C(CH3)3),

1.55-1.65 (m, 2 H, OCH2CH2CHCHCH2), 1.91-1.97 (m, 2 H, OCH2CH2CH2CHCH2),
3.47-3.52 (m, 1 H, OCH2CH2CH2CHCH2), 3.81 (d, 2 H, J5,6 = 7 Hz, H-6), 3.87-3.91 (m,
1 H, OCH2CH2CH2CHCH2), 4.03 (dt, 1 H, J4,5 = 1 Hz, J5,6 = 7 Hz, H-5), 4.71 (d, 1 H,
J1,2 = 7.5 Hz, H-1), 4.76-4.81 (m, 2 H, OCH2CH2CH2CHCHH), 5.59 (dd, 1H, J2,3 =
10.5 Hz J3,4 = 3.5 Hz, H-3), 5.60-5.64 (m, 1 H, OCH2CH2CH2CHCH2), 5.68 (dd, 1H,
J1,2 = 7.5 Hz, J2,3 = 10.5 Hz, H-2), 6.01 (dd, 1 H, J3,4 = 3.5 Hz, J4,5 = 1 Hz, H-4), 7.088.02 (m, 25 H, COPh, Ph);

13

C-NMR (125 MHz, CDCl3): δ18.9 (C(CH3)3), 26.6

(C(CH3)3), 28.5, 29.7, 61.4, 67.9, 69.3, 70.1, 71.9, 73.8, 101.5, 114.7, 127.5, 127.6,
127.7, 128.2, 128.3, 128.4, 129.0, 129.3, 129.5, 129.5, 129.6, 129.6, 129.7, 129.8,
129.9, 130.1, 132.6, 132.9, 133.0, 133.2, 135.4, 135.5, 136.4, 137.7, 165.2 (3 C, COPh),
+

165.4, 165.6,. HRMS [M+Na] m/z: calcd for C48H50O9SiNa 821.3122, found 821.3162.

117

This compound was dissolved in pyridine (4 mL) in a plastic flask followed by addition of
65-70% HF·pyridine solution (4 mL) at 0 oC. The solution was stirred overnight until
complete disappearance of the starting material as judged from TLC analysis. The
reaction mixture was diluted with EtOAc and washed with 10% aqueous CuSO4 solution.
The aqueous phase was extracted with EtOAc twice and the combined organic layers
were washed with saturated aqueous NaHCO3 solution. After drying over Na2SO4 and
solvent removal, the residue was purified by silica gel column chromatography
(Hexanes/EtOAc, 5:2 then 2:1). The product was dissolved in DCM (3 mL) in a threeo

neck flask, stirred at -78 C and O3 was bubbled into the solution. The solution turned
blue in a few seconds and polymer-supported triphenylphosphine (3.0 mmol/g, 600 mg)
was added into the flask. The reaction mixture was further stirred overnight under room
temperature. Polymer was removed by filtration and the solution was concentrated and
purified by silica gel chromatography(Hexanes/EtOAc, 1:1) to give compound 9 (228 mg,
80%).

However, this compound was not very stable and was partially oxidized to
1

carboxylic acid over time as evidenced by H-NMR and

13

1

C-NMR. H-NMR (500 MHz,

CDCl3): δ 1.80-1.89 (m, 2 H, OCH2CHHCH2CHO), 3.59-3.66 (m, 2 H, H-6’,
OCHHCH2CH2CHO), 2.35-2.47 (m, 2 H, OCH2CH2CHHCHO), 3.80-3.83 (m, 1H, H-6),
3.95-4.01 (m, 2 H, H-5, OCHHCH2CH2CHO), 4.77 (dd, 1 H, J1,2 = 8 Hz, H-1), 5.58 (dd,
1H, J2,3 = 10 Hz, J3,4 = 3 Hz, H-3), 5.79-5.83 (m, 2 H, H-2, H-4), 7.22-7.81 (m, 15 H,
COPh), 9.53 (t, 1H, J = 1 Hz, CHO, this peak became smaller over time);

118

13

C-NMR (125

MHz, CDCl3): δ 21.7, 24.5, 29.7, 58.9, 68.1, 68.2, 68.3, 70.1, 71.9, 72.9, 99.7, 128.6,
128.7, 128.8, 128.8, 128.8, 128.9, 129.0, 129.1, 129.1, 133.6, 133.6, 133.7, 164.6 (3 C,
COPh), 164.9, 165.0, 173.9 (COOH, formed from oxidation of aldehyde), 202.5 (CHO).
57

Fluorous alkyl hydrazine (22) Tri-Boc-protected α-hydrazinoacetic acid 19

(378 mg, 0.97 mmol) and 3-(perfluorooctyl)propylamine 20 (463 mg, 0.97 mmol) were
dissolved in DMF (6 mL), EDC-HCl (278 mg, 1.46 mmol) and DMAP (177 mg, 1.46
mmol) were added to the solution. The reaction mixture was stirred under room
temperature overnight. DMF was removed and the residue was purified by silica gel
column chromatography (Hexanes/EtOAc, 3:1) to yield tri-Boc protected ester, which
was dissolved in DCM (3 mL), followed by treatment with TFA (3 mL) overnight at room
temperature. The solvent was removed and dried under high vacuum to provide
compound 22 (450 mg, 70 % over two steps) in the form of a salt.

1

H-NMR (500 MHz,

CD3OD): δ 1.85-1.99 (m, 2 H, NHCH2CH2CH2C8F17), 2.16-2.27 (m, 2 H, CONHCH2),
3.33 (t, 2 H, CH2C8F17), 3.51, 3.64 (s, 2 H, NH2NHCH2CO);

13

C-NMR (125 MHz,

CD3OD): δ 20.3, 28.3, 38.1, 50.5, 169.3 (CONH), 170.5 (CO of TFA).

19

F-NMR (282

MHz, CD3OD): -127.31 (m, 2F), -124.40 (m, 2F), -123.76 (m, 2F), -122.91 (m, 6F), +

115.31 (m, 2 F), -82.43 (m, 3F), -77.18 (s, 3F). HRMS [M+H] m/z: calcd for
C13H13F17N3O 550.0800, found 550.0787.
3-Oxobutyl 2, 3, 4-tri-O-benzoyl-β-D-galactopyranoside (24). p-Tolyl 2, 3, 464

tri-O-benzoyl-6-O-tert-butyl-diphenylsilyl-1-thio-β-D-galactopyranoside 16

119

(299 mg,

0.36 mmol) and 4-hydroxy-2-butanone (37 μL, 0.43 mmol, dried by dissolving in DCM
and stirring with molecular sieves MS 4Å overnight before use) were dissolved in DCM
o

(5 mL) and stirred at -78 C with freshly activated molecular sieves MS 4 Å (600 mg) for
30 minutes. Silver triflate (276 mg, 1.07 mmol) dissolved in acetonitrile (0.3 mL) was
added to the reaction mixture. Five minutes later, orange-colored p-TolSCl (56.6 μL,
0.36 mmol) was added directly into the reaction mixture. This needs to be performed
quickly in order to prevent the p-TolSCl from freezing inside the syringe tip or on the
flask wall.

The yellow color of the solution quickly dissipated within one minute,

indicating the complete consumption of p-TolSCl. The reaction mixture was warmed to
room temperature under stirring in 2 h. Then the reaction mixture was diluted with DCM
and filtered through Celite. The Celite was washed with DCM until no organic
compounds were present in the filtrate.

The filtrate was extracted with saturated

solution of NaHCO3. The organic layer was then dried over Na2SO4 and concentrated
to dryness. The residue was purified by silica gel column chromatography
(Hexanes/EtOAc, 4:1). The product was dissolved in pyridine (4 mL) in a plastic flask
o

followed by addition of 65-70% HF·pyridine solution (2.5 mL) at 0 C. The solution was
stirred overnight until complete disappearance of the starting material as judged from
TLC analysis. The reaction mixture was diluted with EtOAc and washed with 10%
aqueous CuSO4 solution. The aqueous phase was extracted with EtOAc twice and the
combined organic layers were washed with saturated aqueous NaHCO3 solution. After
drying over Na2SO4 and concentrated, the residue was purified by silica gel column

120

chromatography (Hexanes/EtOAc, 1:1 then 1:2) to give compound 15 (148 mg, 74%
20

o

over two steps). [α] D +162 (c = 0.2, CH2Cl2); 1H-NMR (500 MHz, CDCl3): δ 1.99 (s, 1
H, COCHH2), 2.61-2.66 (m,1 H, OCH2CHHCO), 2.69-2.76 (m,1H, OCH2CHHCO), 2.82
(m, 1H, OH), 3.65-3.69 (m, 1 H, H-6), 3.82-3.84 (m, 1 H, H-6), 3.92-3.97 (m, 1 H,
OCHHCH2CO), 4.05 (dt, 1 H, J4,5 = 1 Hz, J5,6 = 9 Hz, H-5), 4.15-4.18 (m, 1 H,
OCHHCH2CO), 4.86 (d, 1 H, J1,2 = 7.5 Hz, H-1), 5.59 (dd, 1 H, J2,3 = 10.5 Hz, J3,4 = 3
Hz, H-3), 5.75 (dd, 1 H, J1,2 = 7.5 Hz, J2,3 = 10.5 Hz, H-2), 5.81-5.83 (m, 1 H, H-4),
7.18-8.07 (m, 15 H, COPh);

13

C-NMR (125 MHz, CDCl3): δ 14.0, 20.9, 30.3, 43.3, 60.5,

65.3, 68.8, 69.8, 71.7, 74.0, 101.8, 128.2, 128.2, 128.5, 128.7, 128.7, 129.2, 129.6,
129.9, 133.1, 133.2, 133.6, 165.3 (3 C, COPh), 165.4, 165.5, 206.6 (COCH3). HRMS
+

[M+H] m/z: calcd for C13H13F17N3O 585.1737, found 585.1726.
Fluorous acyl hydrazide (27). 2H, 2H, 3H, 3H-Perfluoroundecanoic acid 25
(241 mg, 0.49 mmol) and tert-butyl carbazate (77.6 mg, 0.59 mmol) were dissolved in
DMF (5 mL), followed by addition of EDC (141 mg, 0.74 mmol). The resulting solution
was stirred overnight under room temperature. DMF was removed and the residue was
purified by silica gel column chromatography (Hexanes/EtOAc, 3:1) to give Boc
protected 26, which was further treated with TFA (3 mL) in DCM (3 mL). After stirring
under room temperature overnight, solvent was removed and the residue was dried
under high vacuum to give compound 27 (225 mg, 74% over two steps) in the form of
salt.

1

H-NMR (500 MHz, CD3OD): δ 2.60-2.63 (m, 4 H);

121

13

C-NMR (125 MHz, CD3OD):

δ 25.3, 27.1, 162.6 (CONHNH2), 171.4 (CO of TFA).

19

F-NMR (282 MHz, CD3OD): δ -

127.44--127.42 (m, 1 F), -124.64--124.62 (m, 1 F), -123.87--123.86 (m, 1 F), -123.01-123.00 (m, 3 F), -115.84--115.83 (m, 1 F), -82.59--82.58 (m, 3 F), -77.01--77.00 (m, 2 F).
+

HRMS [M+H] m/z: calcd for C11H8F17N2O 507.0365, found 507.0369.
3-Oxobutyl 2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-Obenzoyl-β-D-galactopyranoside
galactopyranoside

14

(30).

p-Tolyl

2,3,4,6-tetra-O-benzoyl-1-thio-β-D-

(29 mg, 0.041 mmol) and compound 24 (19 mg, 0.034 mmol) were
o

dissolved in DCM (3 mL) and stirred at -78 C with freshly activated molecular sieves
MS 4Å (150 mg) for 30 minutes.

Silver triflate (32 mg, 0.12 mmol) dissolved in

acetonitrile (30 μL) was added to the reaction mixture. Five minutes later, orangecolored p-TolSCl (6.53 μL, 0.041 mmol) was added directly into the reaction mixture.
This needs to be performed quickly in order to prevent the p-TolSCl from freezing inside
the syringe tip or on the flask wall. The yellow color of the solution quickly dissipated
within a few seconds, indicating the complete consumption of p-TolSCl. The reaction
mixture was warmed to room temperature under stirring in 2 h. Then the reaction
mixture was diluted with DCM and filtered through Celite. The Celite was washed with
DCM until no organic compounds were present in the filtrate. The filtrate was extracted
with a saturated solution of NaHCO3. The organic layer was then dried over Na2SO4
and concentrated to dryness and redispersed in 0.8 mL DCM/MeOH (1:1). Compound
27 (51 mg, 0.0826 mmol) was added into the solution. Following the general procedure
20

for “catch” and “release”, compound 30 (31 mg, 80%) was obtained. [α] D +100o (c =

122

0.1, CH2Cl2); 1H-NMR (500 MHz, CDCl3): δ 1.87 (s, 3 H, COCH3), 2.27-2.31 (m, 1 H,
OCH2CH2CO), 2.50-2.55 (m, 1 H, OCH2CH2CO), 3.61-3.66 (m, 1 H, OCH2CH2CO),
3.78-3.88 (m, 2 H, H-6, OCH2CH2CO), 4.12-4.16 (m, 2 H, H-5, H-6), 4.24-4.29 (m, 2 H,
H-5’, H-6’), 4.44 (m, 1 H, H-6’), 4.68 (d, 1 H, J1,2 = 8 Hz, H-1), 4.93 (d, 1 H, J1’,2’ = 8 Hz,
H-1’), 5.47 (dd, 1 H, J2,3 = 10 Hz, J3,4 = 3.5 Hz, H-3), 5.57-5.65 (m, 2 H, H-2, H-3’),
5.76 (dd, 1 H, J1’,2’ = 8 Hz, J2’,3’ = 10.5 Hz, H-2’), 5.81 – 5.87 (m, 1 H, H-4), 5.91 – 5.96
(m, 1 H, H-4’), 7.19-8.07 (m, 35 H, COPh);

13

C-NMR (125 MHz, CDCl3): δ 29.6, 30.4,

43.0, 61.7, 64.9, 65.9, 67.9, 68.0, 68.6, 69.7, 69.8, 71.2, 71.5, 71.5, 73.2, 101.1 (1 C,
JC1’,H1’ = 161.5 Hz), 101.6 (1 C, JC1,H1 = 160.9 Hz), 128.1, 128.2, 128.3, 128.4, 128.4,
128.5, 128.6, 128.7, 128.9, 129.0, 129.1, 129.3, 129.3, 129.3, 129.7, 129.7, 129.7,
129.9, 130.0, 133.1, 133.1, 133.2, 133.2, 133.4, 133.5, 165.0 (7 C, COPh), 165.2, 165.3,
165.3, 165.5, 165.5, 206.6 (COCH3). HRMS [M+Na]

+

m/z: calcd for C65H56O19Na

1163.3314, found 1163.3339.
6-O-β-D-Galactopyranosyl-D-galactose (31). To compound 30 (38 mg, 0.033
mmol) dissolved in MeOH (4 mL) was added dropwise 0.1 M NaOMe/MeOH until the
pH was 11. The reaction mixture was stirred at room temperature for 4 h. After the
reaction was complete, the solution was neutralized by acidic resin (Amberlite). After
filtration and size-exclusion chromatography by using Sephadex G-15, compound 31
was isolated in 80% yield. The identity of compound 31 was confirmed by comparison
with literature NMR spectroscopic data and MS analysis.

123

64

3-Oxobutyl 6-O-benzyl-2-deoxy-2-N-phthalimido-β-D-glucopyranoside (33).
p-Tolyl 6-O-benzyl-2-deoxy-2-N-phthalimido-1-thio-β-D-glucopyranoside

65

(292 mg,

0.58 mmol) and 4-hydroxy-2-butanone (498 μL, 5.78 mmol, dried by dissolving in DCM
and stirring with molecular sieves MS 4Å overnight before use) were dissolved in DCM
o

(5 mL) and stirred at -78 C with freshly activated molecular sieves MS 4Å (800 mg) for
30 minutes. Silver triflate (445 mg, 1.73 mmol) dissolved in acetonitrile (0.3 mL) was
added to the reaction mixture. Five minutes later, orange-colored p-TolSCl (91.3 μL,
0.58 mmol) was added directly into the reaction mixture. This needs to be performed
quickly in order to prevent the p-TolSCl from freezing inside the syringe tip or on the
flask wall. The yellow color of the solution quickly dissipated within a few seconds,
indicating the complete consumption of p-TolSCl. The reaction mixture was warmed to
room temperature under stirring in 2 h. Then the reaction mixture was diluted with DCM
and filtered through Celite. The Celite was washed with DCM until no organic
compounds were present in the filtrate. The filtrate was extracted with a saturated
solution of NaHCO3. The organic layer was then dried over Na2SO4 and concentrated
to dryness. The residue was purified by silica gel column chromatography
20

o

(Hexanes/EtOAc, 1:2) to give compound 33 (243 mg, 90%). [α] D = -45 (c = 0.1,
1

CH2Cl2); H-NMR (500 MHz, CDCl3): δ 1.87 (s, 3 H, COCH3), 2.43-2.48 (m, 1H,
OCH2CH2CO), 2.54-2.60 (m, 1H, OCH2CHHCO), 2.82 (d, 1 H, JH-3,OH-3 = 4.5 Hz, OH3), 3.24 (d, 1 H, JH-4,OH-4 = 2.5 Hz, OH-4), 3.55-3.62 (m, 2 H, H-4, H-6), 3.68-3.80 (m,
3 H, H-5, H-6, OCH2CH2CO), 3.94-3.99 (m, 1 H, OCH2CH2CO), 4.06 (d, 1 H, J1,2 = 8.5
124

Hz, J2,3 = 11 Hz, H-2), 4.29-4.33 (m, 1 H, H-3), 4.54-4.63 (m, 2 H, CH2Ph), 5.16 (d, 1 H,
J1,2 = 8.5 Hz, H-1), 7.24-7.82 (m, 9 H, CH2Ph, H of NPhth);

13

C-NMR (125 MHz,

CDCl3): δ 30.2, 43.1, 56.1, 64.8, 70.3, 71.5, 73.8, 98.5, 123.3, 127.7, 127.9, 128.5,
+

131.7, 134.0, 137.6, 168.3 (1 C, COPh), 206.5 (COCH3). HRMS [M+Na] m/z: calcd for
C25H27NO8Na 492.1634, found 492.1612.
3-Oxobutyl

2-O-benzoyl-3,4,6-tri-O-benzyl-β-D-galactopyranosyl-(1→4)-

[2,3,4-tri-O-benzyl-α-L-fucopyranosyl-(1→3)]-6-O-benzyl-2-deoxy-2-N-phthalimidoβ-D-glucopyranoside
27

galactopyranoside 32

(35).

p-Tolyl

2-O-benzoyl-3,4,6-tri-O-benzyl-1-thio-β-D-

(40.3 mg, 0.061 mmol) was dissolved in DCM (5 mL) and

o

stirred at -78 C with freshly activated molecular sieves MS 4Å (300 mg) for 30 minutes.
Silver triflate (47 mg, 0.19 mmol) dissolved in acetonitrile (50 μL) was added to the
reaction mixture. Five minutes later, orange-colored p-TolSCl (9.65 μL, 0.061 mmol)
was added directly into the reaction mixture. This needs to be performed quickly in
order to prevent the p-TolSCl from freezing inside the syringe tip or on the flask wall.
The yellow color of the solution quickly dissipated within a few seconds, indicating the
complete consumption of p-TolSCl. The glucosamine acceptor 33 (23 mg, 0.049 mmol)
dissolved in DCM (0.2 mL) was then added dropwise to the reaction mixture. This was
stirred for another one hour at which point the temperature reached room temperature
and glucosamine acceptor 33 was completely consumed. The fucose donor 34 (53.5
mg, 0.099 mmol) dissolved in DCM (1 mL) was added to the mixture. The solution was
o

cooled back to -78 C and silver triflate (50.3 mg, 0.196 mmol) dissolved in acetonitrile
125

(50 μL) was added to the reaction mixture. Five minutes later, orange-colored p-TolSCl
(15.6 μL, 0.099 mmol) was added directly into the reaction mixture.

The reaction

mixture was warmed to room temperature under stirring in 1 h. Then the reaction
mixture was diluted with DCM and filtered through Celite. The Celite was washed with
DCM until no organic compounds were present in the filtrate. The filtrate was extracted
with a saturated solution of NaHCO3. The organic layer was then dried over Na2SO4
and concentrated to dryness and redispersed in 0.8 mL DCM/MeOH (1:1). Compound
27 (61 mg, 0.098 mmol) was added into the solution. Following the general procedure
20

for fluorous catch and release, pure compound 35 (44 mg, 62%) was obtained. [α] D =
o

1

-49 (c = 0.3, CH2Cl2); H-NMR (500 MHz, CDCl3): δ 1.21 (d, 3 H, J = 6.5 Hz, CH3 of
fucose), 1.78 (s, 3 H, COCH3), 2.45-2.51 (m, 1 H, OCH2CH2CO), 3.17-3.64 (m, 7 H,
OCH2CH2CO), 3.72-3.86 (m, 6 H, OCH2CH2CO), 4.04-4.17 (m, 3 H), 4.26-4.67 (m, 15
H, H-1’, H-1’’), 4.80-4.90 (m, 3 H, H-1), 5.11 (dd, 1 H, J1’,2’ = 10 Hz, J2’,3’ = 8 Hz, H-2’),
6.87-7.93 (m, 44 H, CH2Ph, COPh, H of NPhth);

13

C-NMR (125 MHz, CDCl3): δ 16.3,

29.6, 30.2, 43.0, 56.3, 64.6, 66.5, 67.7, 71.1, 71.6, 71.9, 72.0, 72.4, 72.7, 72.9, 73.4,
73.5, 73.8, 74.2, 74.8, 74.9, 75.4, 78.5, 79.4, 80.2, 96.9 (1 C, JC1’’,H1’’ = 170.2 Hz), 98.5
(1 C, JC1,H1 = 163.8 Hz), 99.8 (1 C, JC1’,H1’ = 162.2 Hz), 123.4, 126.9, 126.9, 127.0,
127.1, 127.4, 127.6, 127.7, 127.7, 127.8, 127.8, 127.8, 127.8, 128.0, 128.1, 128.3,
128.4, 128.4, 128.5, 128.7, 129.7, 129.8, 133.0, 133.9, 137.7, 137.8, 138.1, 138.2,

126

+

138.6, 139.1, 139.3, 164.6 (1 C, COPh), 206.3 (COCH3),. HRMS [M+Na] m/z: calcd
for C86H87NO18Na 1444.5821, found 1444.5787.
3-Oxobutyl

(2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl)-(1→6)-[(2,3,4,6-

tetra-O-benzoyl-β-D-galactopyranosyl)-(1→6)]-[(2,3,4,6-tetra-O-benzoyl-β-Dgalactopyranosyl) -(1→6)]-2,3,4,6-tetra-O-benzoyl- β-D-galactopyranoside (37). p14

Tolyl 2,3,4,6-tetra-O-benzoyl-1-thio-β-D- galactopyranoside 29

(50 mg, 0.071 mmol)

o

was dissolved in DCM (5 mL) and stirred at -78 C with freshly activated molecular
sieves MS 4Å (400 mg) for 30 minutes. Silver triflate (55 mg, 0.21 mmol) dissolved in
acetonitrile (50 μL) was added to the reaction mixture. Five minutes later, orangecolored p-TolSCl (11.25 μL, 0.071 mmol) was added directly into the reaction mixture.
This needs to be performed quickly in order to prevent the p-TolSCl from freezing inside
the syringe tip or on the flask wall. The yellow color of the solution quickly dissipated
within a few seconds, indicating the complete consumption of p-TolSCl. Acceptor 36
(34 mg, 0.057 mmol) dissolved in DCM (1 mL) was then added dropwise to the reaction
mixture. This was warmed up to room temperature under stirring in one hour. The
reaction mixture was kept under room temperature for half an hour to completely
o

decompose the excess activated donor. Then the solution was cooled back to -78 C.
Silver triflate (29 mg, 0.11 mmol) dissolved in acetonitrile (50 μL) was added to the
reaction mixture. Five minutes later, orange-colored p-TolSCl (9 μL, 0.057 mmol) was
added directly into the reaction mixture. After 5 minutes, acceptor 36 (26.8 mg, 0.045
mmol) dissolved in DCM (1 mL) was added dropwise to the reaction mixture. The
reaction mixture was warmed up to room temperature under stirring in one hour, and
127

kept under room temperature for half an hour to completely decompose the excess
activated donor. Acceptor 24 (14.7 mg, 0.026 mmol) dissolved in DCM (1 mL) was then
o

added to the reaction mixture. The solution was cooled back to -78 C. Silver triflate
(23 mg, 0.090 mmol) dissolved in acetonitrile (50 μL) was added to the reaction mixture.
Five minutes later, p-TolSCl (7 μL, 0.045 mmol) was added directly into the reaction
mixture. This was warmed up to room temperature under stirring in one hour. Then the
reaction mixture was diluted with DCM and filtered through Celite. The Celite was
washed with DCM until no organic compounds were present in the filtrate. The filtrate
was extracted with saturated solution of NaHCO3. The organic layer was then dried
over Na2SO4 and concentrated to dryness and redispersed in 0.8 mL DCM/MeOH (1:1).
Compound 27 (34 mg, 0.052 mmol) was added into the solution. Following the general
procedure for fluorous catch and release, pure compound 37 (34 mg, 61%) was
20

o

1

obtained. [α] D = +68 (c 0.1, CH2Cl2); H-NMR (500 MHz, CDCl3): δ 1.84 (s, 3 H,
COCH3), 2.25-2.30 (m, 1 H, OCH2CH2CO), 2.47-2.52 (m, 1 H, OCH2CH2CO), 3.313.34 (m, 1 H, H-6), 3.52-3.56 (m, 1 H, H-6’), 3.60-3.65 (m, 1 H, OCH2CH2CO), 3.724.22 (m, 11 H, H-5, H-5’, H-5’’, H-5’’’, H-6, H-6’, H-6’’, H-6’’, H-6’’’, H-6’’’,
OCHHCH2CO), 4.49-4.53 (m, 2 H, H’, H’’), 4.66 (d, 1 H, J = 8 Hz, H-1), 4.79 (d, 1 H, J
= 8 Hz, H-1’’’), 5.38-5.66 (m, 8 H, H-2, H-2’, H-2’’, H-2’’’, H-3, H-3’, H-3’’, H-3’’’), 5.795.89 (m, 4 H, H-4, H-4’, H-4’’, H-4’’’), 7.17-8.06 (m, 65 H, COPh);

13

C-NMR (125 MHz,

CDCl3): δ 24.6, 29.6, 30.3, 36.6, 43.0, 61.3, 64.9, 66.2, 66.3, 67.5, 67.7, 67.8, 67.8,
128

68.5, 69.7, 69.8, 69.8, 69.9, 71.1, 71.5, 71.6, 72.2, 72.5, 72.9, 100.7 (2 C, JC1’,H1’ =
160.1 Hz, JC1’’,H1’’ = 160.1 Hz), 100.8, 101.0 (1 C, JC1,H1 = 162.5 Hz), 101.6 (1 C,
JC1’’’,H1’’’ = 162.5 Hz), 128.1, 128.1, 128.2, 128.3, 128.3, 128.4, 128.4, 128.4, 128.5,
128.7, 128.9, 128.9, 129.0, 129.2, 129.3, 129.3, 129.4, 129.4, 129.6, 129.7, 129.7,
129.7, 129.7, 129.8, 129.9, 130.0, 130.0, 130.1, 132.9, 133.0, 133.0, 133.1, 133.1,
133.2, 133.4, 164.9 (13 C, COPh), 165.0, 165.1, 165.2, 165.2, 165.3, 165.3, 165.4,
165.5, 165.7, 206.3 (COCH3).

+

HRMS [M+Na]

2111.5943, found 2111.5969.

129

m/z: calcd for C119H100O35Na

2.5. TLC and HPLC Data
TLC for compounds 35 and 37
Compound 35

Compound 37

Figure 2.7. TLC data for compounds 35 and 37. Lane 1: reaction mixture after
glycosylation. Lane 2: reaction mixture after “catch”, first F-SPE and “release”.Lane 3:
organic fraction after the second F-SPE.

130

HPLC chromatogram for compounds 35 and 37

Figure 2.8. (a) HPLC chromatogram of the crude reaction mixture of compound 35 after
the one-pot glycosylation reaction. The peak marked with an

*

was ditolyl disulfide,

which was a side product from donor activation and has a strong UV absorbance band.
(b) HPLC chromatogram of the organic fraction after the second F-SPE.

131

Figure 2.8. (Cont’d) (a) HPLC chromatogram of the crude reaction mixture of
compound 35 after the one-pot glycosylation reaction. The peak marked with an

* was

ditolyl disulfide, which was a side product from donor activation and has a strong UV
absorbance band. (b) HPLC chromatogram of the organic fraction after the second FSPE.

132

Appendix A

133

NMR Data

1

Figure 2.9. H-NMR (CDCl3, 500 MHz) of 17

134

BzO OTBDPS
O
BzO
O
OBz
17

13

Figure 2.10. C-NMR (CDCl3, 125 MHz) of 17

135

BzO OTBDPS
O
BzO
O
OBz
17

Figure 2.11. gCOSY (CDCl3, 500 MHz) of 17

136

BzO
BzO

OH
O

O
OBz
18

CHO

1

Figure 2.12. H-NMR (CDCl3, 500 MHz) of 18

137

BzO
BzO

BzO

OH
O

O
OBz
18

CHO

and

BzO

OH
O

O
OBz

COOH

1

Figure 2.13. H-NMR (CDCl3, 500 MHz) of 18

138

BzO
BzO

BzO

OH
O

O
OBz
18

Figure 2.14.

CHO

and

BzO

OH
O

O
OBz

COOH

13

C-NMR (CDCl3, 125 MHz) of 18

139

O
C8F17

N
H 22

H
N

NH2

1

Figure 2.15. H-NMR (CD3OD, 500 MHz) of 22

140

O
C8F17

N
H 22

H
N

NH2

13

Figure 2.16. C-NMR (CD3OD, 125 MHz) of 22

141

O
C8F17

Figure 2.16.

N
H 22

H
N

NH2

19

F-NMR (CD3OD, 282 MHz) of 22

142

1

Figure 2.17. H-NMR (CDCl3, 500 MHz) of 24

143

Figure 2.18.

13

C-NMR (CDCl3, 125 MHz) of 24

144

Figure 2.19. gCOSY (CDCl3, 500 MHz) of 24

145

1

Figure 2.20. H-NMR (CD3OD, 500 MHz) of 27

146

Figure 2.21.

13

C-NMR (CD3OD, 125 MHz) of 27

147

Figure 2.22.

19

F-NMR (CD3OD, 282 MHz) of 27

148

1

Figure 2.23. H-NMR (CDCl3, 500 MHz) of 30

149

Figure 2.24.

13

C-NMR (CDCl3, 125 MHz) of 30

150

Figure 2.25. gCOSY (CDCl3, 500 MHz) of 30

151

Figure 2.26. HMQC (CDCl3, 500 MHz) of 30

152

1

Figure 2.27. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 30

153

Figure 2.28. gHMBC (CDCl3, 500 MHz) of 30

154

1

Figure 2.29. H-NMR (CDCl3, 500 MHz) of 33

155

Figure 2.30.

13

C-NMR (CDCl3, 125 MHz) of 33

156

Figure 2.31. gCOSY (CDCl3, 500 MHz) of 33

157

1

Figure 2.32. H-NMR (CDCl3, 500 MHz) of 35

158

Figure 2.33.

13

C-NMR (CDCl3, 125 MHz) of 35

159

Figure 2.34. gCOSY (CDCl3, 500 MHz) of 35

160

Figure 2.35. gHMQC (CDCl3, 500 MHz) of 35

161

1

Figure 2.36. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 35

162

Figure 2.37. gHMBC (CDCl3, 500 MHz) of 35

163

1

Figure 2.38. H-NMR (CDCl3, 500 MHz) of 37

164

Figure 2.39.

13

C-NMR (CDCl3, 125 MHz) of 37

165

Figure 2.40. gCOSY (CDCl3, 500 MHz) of 37

166

Figure 2.41. gHMQC (CDCl3, 500 MHz) of 37

167

1

Figure 2.42. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 37

168

Figure 2.43. gHMBC (CDCl3, 500 MHz) of 37

169

References

170

References
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176

CHAPTER 3
Chemical Synthesis of Homogeneous Heparan Sulfate Proteoglycan
3.1. Introduction
PGs are a family of molecules containing one or more GAG chains covalently
attached to the core protein. PGs are known to play vital functional roles in different
biological processes, such as blood coagulation, cell proliferation, differentiation,
adhesion, and wound repair. A major type of PG is the HSPGs, where the GAG chains
are HS.

1-3

4

A typical structure of HSPG is shown in Figure 3.1. HS chain is linked to

the core protein via a tetrasaccharide linkage region. All HSPGs share this linkage,
which is composed of GlcA, Ga, Gal and Xyl. Xyl moiety is linked to serine residue in
the core protein. HS is one type of polyanionic polysaccharide which is composed of
disaccharide repeating unit D-GluN and uronic acid (either L-Idu or D-GlcA) joined by α
(1-4) and β (1-4) linkages. HS can have various sulfation patterns. For example, 2-O
position in uronic acid unit can be sulfated and 3-O, 6-O positions in GluN unit can be
sulfated. The 2-amino group in glucosmine unit can be either sulfated or acetylated.

177

Figure 3.1. Structure of HSPG and its biosynthetic pathway
Various enzymes are involved in the biosynthetic process of assembling HSPG.
In mammals, as many as 26 enzymes are involved in the formation of HS chains. HSPG
core proteins are first xylosylated at specific serine residues by XYLT1 and XYLT2. Two
Gal residues are added sequentially by GLCATs 1 and 2 in the Golgi, followed by
addition of GlcA by GLCAT1. The first N-acetylglucosamine (GlcNAc) unit is added by
EXTL3. EXT1/EXT2 co-polymerize GlcNAc and GlcA units into linear HS chains of 40 to
100 residues. This HS chain undergoes extensive enzymatic modifications, which will
be discussed later.
HSPGs are ubiquitous components of mammalian cell surface and the
3

extracellular matrix. This type of molecule can serve as receptors for many adhesion
178

molecules and growth factors, such as fibroblast growth factor, integrin, Wnt proteins
and herpes simplex virus glycoprotein D. Therefore, HSPGs play important roles in a
variety of biological processes including cancer development, angiogenesis, viral
infection, and wound repair,

1-2,5-7

which render them as potential targets for the

development of novel therapeutic interventions.

8

Most of the above-mentioned biological activities of HSPGs are due to the
presence of HS chain. However, there are increasing evidences showing that both the
HS structures and the core protein can be important for directing the biological functions
of HSPGs.

5,9

Disappointingly, naturally existing HSPGs are a highly heterogeneous

mixture containing diverse HS structures. This renders it extremely challenging to
isolate HSPGs bearing homogeneus HS chains, which seriously hinders the thorough
understanding of the structure-function relationship of HSPGs. Synthesis of HSPGs can
reduce the structural heterogeneity and provide sufficient quantity of pure compounds
for biological evaluation, which is crucial for advancing glycobiology. However, no
synthetic methods are available to date to prepare the highly complex HSPGs. In this
project, we want to develop effective methods towards the assembly of homogeneous
HSPGs bearing defined HS sequences.
10

The major cell membrane HSPGs are syndecans and glypicans (Figure 3.2).

Syndecan-1 is a member of the syndecan family, which plays important roles in
controlling cell proliferation, differentiation, adhesion, and migration.

1-2,5-6

7,11

Syndecan-1 has one of the representative HSPG structures, consisted of a short
cytoplasmic signaling domain, a transmembrane domain, and a large extracellular
179

domain (ectodomain) where many receptors bind. The molecular weight of the core
12

protein is about 33 kDa.
13

and 4 (Figure 3.3).

Besides sydecan-1, syndecan family contains syndecans 2, 3

The ectodomain of syndecan-1 can be shed from the cell surface

through protease cleavage.

13

Several metzincin enzymes such as MMP9 can cleave

the syndecan core proteins at the site close to cell membrane. Shedding is reported to
be enhanced if the HS chains are first cleaved by heparanase. Found in acute dermal
14

wound fluids, the shed syndecan-1 regulates growth factor activity
balance.

15

16

tumors

and proteolytic

Furthermore, it can facilitate the growth, angiogenesis, and metastasis of

and serum level of syndecan-1 has been used as an independent prognostic

marker for cancer.

17

The remnant core protein at the cell surface may be further

processed by intermembrane cleavage by the presenilin/γ-secretase complex. There
12

may be signalling through MAP kinases (Figure 3.4).

In this project, we will focus on

developing an effective method to construct syndecan-1 ectodomain.

180

Figure 3.2. Biological distributions of syndecans. Adapted with permission from ref 10.
Copyright 2009 Springer.

Figure 3.3. Structure of the four vertebrate syndecans. Adapted with permission from
ref 12. Copyright 2010 John Wiley and Sons.

181

Figure 3.4. Shedding of syndecans by metzincin proteinases. Adapted with permission
from ref 12. Copyright 2010 John Wiley and Sons.
The ectodomain of syndecan-1 is a glycoprotein. In its N-terminal, several GAG
chains such as HS and CS can be attached to serine-glycine residues flanked by acdic
and aromatic amino acids in the core protein.

18

The amino acid sequence of human

syndecan-1 was determined by Jalkanen and coworkers in 1990.

19

This core protein is

composed of 310 amino acids with five potential serine residues for HS or CS chain
attachment (Figure 3.5).

182

Figure 3.5. Sequence of human syndecan-1. Circled serine residues demonstrate
possible GAG side chain attachment sites. Underlined part indicates the hydrophobic
transmembrane domain. The red part will be our synthetic sequence.
Syndecan-1 contains very diverse glycoform HS structures due to modification by
HS biosynthetic enzymes. Nature synthesizes the HS glycan of HSPGs by first installing
a homopolymer of heparosan GlcNAc-α-1,4-GlcUA onto the tetrasaccharide linker as
mentioned earlier. This is then modified by the sequential actions of many enzymes,
including N-deacetylase/N-sulfotransferase, C5-epimerase, 2-O-sulfotransferase, 6-Osulfotransferase, 3-O-sulfotransferase using 3’-phosphoadenosine-5’-phosphosulfate
20-22

as the sulfate donor (Scheme 3.1).

Most of these reactions are incomplete thus

creating tremendous structural heterogeneity of glycoforms on syndecan-1. This
structural diversity greatly expands the ligand-binding properties of syndecan-1.

183

Scheme 3.1. HSPGs contain diverse HS chain structures due to modifications by
multiple enzymes.
The HS glycan chain on syndecan-1 can interact with many proteins and the
ligand specificity can be encoded by the precise structure of HS. The most thoroughly
studied example is the serine protease inhibitor antithrombin III (AT III) involved in the
blood coagulation cascade.

23-24

Structural analysis demonstrated that the HS

sequence responsible for AT III binding is a rare pentasaccharide containing a 3-O
sulfate in the middle GlcN unit. Removal of the crucial 3-O sulfate reduced its AT III
affinity by about 10000 folds.

25

This understanding led to the development of Arixtra, a

fully synthetic pentasaccharide drug approved by FDA for treatment of deep vein
thrombosis (Figure 3.6). Another example is the binding of fibronectin with HS. This
184

interaction is important for mediating cell adhesion, which requires N-sulfation, but not
26

2-O sulfation of the HS chain.
OSO3
O
HO
HO
O3SHN O2C
O
HO

OSO3
OSO3
O
O
O
O2C OH O
OH HO
HO
O
O3SHN
O3SHN
OMe
O
OSO3

O

Figure 3.6. Arixtra, a fully synthetic pentasaccharide drug for treatment of deep vein
thrombosis.
In contrast to the myriad functions of HS, the core protein of syndecan-1 was
previously thought to be merely providing a scaffold for HS display without specific
functions by itself. However, there is increasing evidence suggesting that the
ectodomain core protein itself can have biological functions independent of HS.

5,9,27

For example, the syndecan-1 core protein can regulate invasive properties of cancer
cells. Mutagenesis of a 5-residue sequence in the ectodomain resulted in a loss of
28

invasive migration properties of the cells.

Binding of syndecan-1 ectodomain with

antibodies can lead to integrin dependent cell adhesion and migration.

29-30

A fragment

of the ectodomain of syndecan-1 has been shown to bind with αvβ3 and α5β3 integrins,
which inhibited tumor angiogenesis in vivo.

31

On the other hand, HS oligosaccharides

longer than hexasaccharides can also bind with integrin.

32

Besides integrin, midkine

and pleiotrophin, which are growth factors involved in oncogenesis and inflammation,
have been shown to interact with both HS and the core protein of syndecan-1.
185

33

Therefore, HS chain and the core protein in HSPGs can cooperate in binding their
biological targets in vivo. The synergistic effect in core protein and glycan chain binding
adds an important additional level to the unique functional diversity of syndecan-1 as
receptors.
In order to thoroughly understand and decipher the structure functions
relationship of HSPGs, HSPGs bearing homogeneous HS chains are required. However,
this type of studies has been severely hampered by the lack of access to homogeneous
HSPGs. Due to the diverse glycoforms present on the core protein, it is possible to
isolate pure HSPG such as syndecan-1 from nature. Consequently, the availability of
effective synthetic methods allowing access to homogeneous HSPGs becomes highly
desirable. Based on the amino acid sequence shown in Figure 3.5, our synthetic target
will be glycopeptide 1, which corresponds to the red part shown in Figure 3.5 bearing
two HS chains (Figure 3.7). Two HS chains are attached to serine residues 37 and 47,
respectively. The preparation of this molecule represents a significant challenge, as this
endeavor involves difficult glycosylation reactions, stereochemical control, regiospecific
sulfation, peptide synthesis and union of complex oligosaccharides with peptides. With
the presence of so many different types of functional groups, it is highly demanding to
design a suitable strategy to incorporate all the structural requirements.

186

Figure 3.7. Structure of our synthetic target HSPG 1.
A lot of work has been done in synthesis of HS oligosaccharides.

25,34-44

For

example, our group developed a pre-activation based, one-pot combinatorial strategy
for assembling HS hexasaccharides with systematically varied and precisely controlled
34

backbone structures.

Pre-activation of donor 2 followed by addition of acceptor 3 gave

a tetrasaccharide intermediate, which was subjected to another coupling with acceptor 4
leading to the formation of hexasaccharide 5 in 50% in a one-pot operation.
Deprotection of 5 gave fully deprotected hexasaccharide 6. The same one-pot strategy
was applied to access 12 other hexasccharides (Figure 3.8) which were used for
probing the effect of backbone structure on binding with the fibroblast growth factor-2
(Figure 3.9). The binding results show that N-sulfation and the trisaccharide motif
GlcNS-IdoA2S-GlcNS are important for FGF-2 binding and the presence of IdoA2S is
crucial.

187

Scheme 3.2. One-pot synthesis of hexasaccharide 5.

Figure 3.8. Structures of 12 synthetic HS hexasaccharides.

188

7

8

9

10

11

12

13

14

15

16

17

6

HS Control

Figure 3.9. FGF-2 (1ug) was incubated with 10 (empty bar) or 40 ug (filled bar)
ublabeled hexasaccharides 6-17 or HS (1.6 ug) in PBS buffer (200 uL) at room
35

temperature for 30 min. Then, [ S]HS (4500 cpm) was added to the mixture followed
o

by incubation at 37 C for 90 min. The data represent the average of four or more
experiments with the error bars showing standard deviations. The control was the
sample without any hexasaccharides or unlabled HS. The percentage of residual
35

[ S]HS binding was calculated by dividing the residual

35

S counts on the membrane

with sample incubation by control counts.
The assembly of the tetrasaccharide linkage part has also been reported
before.

44-50

One most recent example came from the work of Hung and coworkers, in

which they developed a one-pot glycosylation strategy to assemble the fully protected
44

tetrasaccharide (Figure 3.10).

Monosaccharide building blocks 20, 21, 22 were

synthesized via one-pot regioselective strategy developed in their laboratory.

51

glycosylation and subsequent deprotection led to compound 18 in high yields.
189

One-pot

Figure 3.10. Retrosynthetic analysis of tetrasaccharide 18.
Despite the success in HS chain and tetrasaccharide linker synthesis, synthesis
of homogeneous HS containing syndecan-1 glycopeptides and glycoproteins have not
been accomplished. To achieve the synthesis of HS glycopeptide 1, our current
synthetic target will be glycopeptide 24 (Figure 3.11), which is part of the structure of
glycopeptide 1. Successful synthesis of this molecule will pave the way for accessing
glycopeptide 1.

Figure 3.11. Structure of our current synthetic target 24.
3.2. Synthetic design – First Generation
Glycopeptide 24 can be accessed from fully protected form 25. In order to have
high overall synthetic efficiency, our first generation approach was to couple

190

heptasaccharide 27 with tripeptide 26. The heptasaccharide 27 contains HS residues
and the non-reducing end trisaccharide from the linker region. The tripeptide 26 will be
prepared with its serine residue functionalized with xylose. This HS oligosaccharide +
xylose glycopeptide coupling approach allows great flexibility in varying the structures of
both the core peptides and the oligosaccharides suitable for convergent preparation of
different HSPG structures. Heptasaccharide 27 will be synthesized by coupling
disaccharide donor 28 to pentasaccharide acceptor 29. And pentasaccharide 29 will be
assembled by the union of trisaccharide 30 with disaccharide 31. To differentiate
different hydroxyl groups, we will use ClAc group to mask primary hydroxyl groups in
sugar units A, C and E. Selective removal of ClAc followed by oxidation will expose
carboxylic acid at these positions. Lev group will be used to mask the hydroxyl groups
which will be sulfated during the deprotecting stage. Other hydroxyl groups will be
permanently protected by Bn group, PMB group or Bz group. These permanent
protecting groups will be removed by catalytic hydrogenation and ester hydrolysis in the
last stage of deprotection. Disaccharide and trisaccharide building blocks will be
synthesized

from

commercially

available

monosaccharides.

Five

different

monosaccharides are required for assembling this molecule, which are Ido, GluN, Glu,
Gal and Xyl.

191

Figure 3.12. Retrosynthetic analysis of glycopeptide 24.
3.3. Synthesis of monosaccharide building blocks
3.3.1. Synthesis of GluN building block

192

Synthesis of GluN building block started from commercially available compound
34,52-54

32 based on literature procedures and standard manipulations (Scheme 3.2).

Compound 32 was first neutralized by aqueous NaHCO3 and the resulting compound
with free amine was protected by Troc group to give compound 33, which was
acetylated to give compound 34 in 78% over two steps. Subsequent introduction of pthiotolyl group using SnCl4 as the activating agent afforded the GluN thioglycoside 35 in
1

81% yield as an anomeric mixture indicated by H NMR. Other activating reagents such
as BF3-Et2O were screened and led to similar results. Simultaneous removal of Troc
and Ac groups in compound 35 with NaOH in THF/H2O led to compound 36 in 80%
yield. Previously, we used a two-step deprotection procedure for conversion of 35 to 36:
first, the Troc group was removed by Zn dust in HOAc; second, Ac group was removed
by NaOMe. However, a quick silica gel column was needed and deacetylation step has
to be carried out immediately to prevent the acyl transfer side reaction.

34

Diazao

transfer to compound 36 afforded the azido glucose 37 in 70% yield, which was
followed by benzylidene formation to furnish compound 38. At this point, we were able
to separate α/β mixture by silica gel column purification. Pure β product 38 was isolated
in 68% along with 7% α anomer. Benzylation by NaH and BnBr led to compound 39 in
92% yield. To regioselectively open the benzylidene ring, we first tried the combination
of NaCNBH3 and TFA. However, this reaction is rather slow and even after two days,
there was still starting material 39 left based on TLC analysis. To speed up this reaction,
we tried an alternative reaction condition reported by Wong and coworkers.
193

54

Compound 39 was treated with Et3SiH and TFA in the presence dehydrating agent (MS
AW 300) to afford compound 40 in 98% in 1 hour. Installation of TBS was accomplished
by treatment with TBSOTf under low temperature.

Scheme 3.2. Synthesis of glucosamine building block 41. Reagents and conditions: (a)
TrocCl, NaHCO3/H2O, r.t., 2h; (b) Ac2O, pyridine, r.t., overnight; (c) p-TolSH, SnCl4,
MS AW 300, DCM, r.t., overnight; (d) NaOH, THF/H2O; (e) K2CO3/ZnCl2, TfN3,
DCM/MeOH, H2O, r.t.; (f) PhCH(OCH3)2, CSA, CH3CN, r.t.; (g) BnBr, DMF, NaH, r.t., 2
o

o

h; (h) Et3SiH, TFA, DCM, 0 C, MS AW 300; (i) TBSOTf, 2, 6-lutidine, DCM, -40 C to
r.t., 5 h.
3.3.2. Synthesis of glucose building block
Glucose building block 48 was synthesized according to literature procedures
34,55

(Scheme 3.3).

β-D-Glucose pentaacetate 42 was treated with p-toluenethiol in the

presence of BF3-Et2O to give glucose thioglycoside 43 in 92% yield. Deacetylation of
194

compound 43 by NaOMe in DCM/MeOH followed by regioselective benzylidene
formation afforded compound 45 in 72% yield. Regioselective benzylation at 3-O
position by Bu2SnO, followed by benzoylation led to compound 47 in 62% yield over
two steps. Regioselective opening of benzylidene by TFA and NaCNBH3 gave
compound 48 in 85% yield.

Scheme 3.3. Synthesis of glucose building block 48. Reagents and conditions: (a)
BF3/Et2O, p-TolSH, r.t.; (b) NaOMe, DCM/MeOH, r.t.; (c) p-methoxybenzylidene
o

dimethyl acetal, CSA, DMF, 50-70 C; (d) Bu2SnO, toluene, reflux, 3 h, then BnBr, CsF,
o

DMF, 140 C; (e) BzCl, DMAP, DCM, r.t.; (f) NaCNBH3, TFA, DMF, r.t., 48 h.
3.3.3. Synthesis of galactose building block
Synthesis of galactose building block 57 started from the commercially available
glucose pentaacetate 49 (Scheme 3.4). It was first treated with p-toluenethiol in the
presence of BF3-Et2O to give galactose thioglycoside 50 in 85% yield. Deacetylation of
compound 51 by NaOMe in DCM/MeOH led to compound 51 in 94% yield.
Regioselective allylation at 3-O position by treating with Bu2SnO in toluene/THF

195

followed by reaction with allyl bromide in the presence of TBAB converted compound 51
to 52 in 70% yield. This transformation at 1 g scale worked quite well. However, larger
scale (10 g) turned out to be messy as indicated by TLC. To circumvent this obstacle,
we did several smaller scale reactions and combined the reaction mixtures for
purification. We were able to isolate compound 52 in satisfactory yield. Benzylidene
formation using p-methoxybenzylidene dimethyl acetal in the presence of CSA afforded
compound 53 in 82% yield, followed by benzoylation to furnish compound 54 in 90%
yield.

56

Removal of benzylidene by p-toluenesulfonic acid in DCM/MeOH led to diol 55

in 80%. To install the benzyl group, we treated compound 55 with NaH and BnBr in
DMF. However, 2-OBz was partially cleaved after aqueous workup. Since we used
slightly excess NaH (1.3 equiv.) for the reaction, during aqueous workup, excess NaH
was converted to NaOH, which cleaved benzoyl ester. We also tried sodium
o

bis(trimethylsilyl)amide and BnBr under low temperature (-78 C), disappointingly, the
reaction was too slow. Finally, we solved the benzoylation problem by running the
reaction in the presence of dehydrating agents (MS 4Å) followed by quenching with
10% HCl solution. Under this condition, excess NaH was quenched and not converted
to NaOH. To expose 3-OH, we treated compound 56 with PdCl2. However, STol and
allyl were cleaved together with methyl glycoside and glycal detected as the major
products. We tested an alternative condition using [Ir(COD)(PMePh2)2PF6], which
cleanly converted compound 56 to compound 57.

196

49

Scheme 3.4. Synthesis of galactose building block 57. Reagents and conditions: (a)
BF3/Et2O, p-TolSH, r.t.; (b) NaOMe, DCM/MeOH, r.t.; (c) Bu2SnO, toluene/THF, reflux,
3 h, then AllBr, Bu4NBr, THF, reflux; (d) benzaldehyde dimethyl acetal, CSA, CH3CN; (e)
o

BzCl, DMAP, pyridine, 50 C; (f) p-TsOH, DCM/MeOH, r.t.; (g) NaH, BnBr, DMF; (h)
o

[Ir(COD)(PMePh2)2PF6], THF, then H2O, I2, H2, 0 C-r.t..
To synthesize 4, 6-benzylidene protected galactose building block, we started
from compound 51, which was protected by 4, 6-benzylidene acetal regioselectively to
give compound 58. Regioselective protection of 3-OH was achieved by reacting with
levulinic acid using DCC as the activating agent. To achieve better regioselectivity, the
amount of levulinic acid and DCC used has to be controlled (1.2 equiv.). We found
when using more than 2 equiv. of DCC, di-Lev protected product was isolated with small
amount of 2-OLev protected compound. Benzoylation of compound 59 followed by
removing Lev with hydrazine led to compound 61 in good yield.
group in 54 was removed to yield 61 (Scheme 3.5).

197

57

Alternatively, allyl

Scheme 3.5. Synthesis of galactose building block 61. Reagents and conditions: (a) pmethoxybenzylidene dimethyl acetal, CSA, CH3CN; (b) DCC, LevOH, DMAP, DCM; (c)
BzCl,

DMAP,

DCM,

50

o

C;

(d)

NH2NH2,

HOAc,

DCM/MeOH;

(e)

o

[Ir(COD)(PMePh2)2PF6], H2, THF, then H2O, I2, 0 C-r.t..
3.3.4. Synthesis of xylose building block 67
Synthesis of xylose building block 67 started from the commercially available
xylose 62. Global acetylation gave compound 63 in 93%, which was treated with ptoluenethiol in the presence of BF3-Et2O to give xylose thioglycoside 64 in 62% yield.
After recrystallization, compound 64 was obtained in its pure β form. Deacetylation of
compound 64 afforded compound 65 in 90% yield, which was selectively protected by
ClAc at 4-O position by treating with Bu2SnO in dioxane followed by ClAcCl.

58

To install

benzoyl group at the remaining hydroxyl groups, compound 66 was treated with BzCl
and DMAP in DCM at room temperature. However, the desired product 67 was isolated
in only 5% yield with 80% mono-benzoylated product (Scheme 3.6). To improve the
yield, we heated the reaction to reflux to convert the mono-benzoylated compound to

198

the desired product 67. But ClAc protecting group was found to be unstable under this
condition.

Scheme 3.6. Synthesis of xylose building block 67. Reagents and conditions: (a) Ac2O,
pyridine; (b) BF3/Et2O, p-TolSH, r.t.; (c) NaOMe, DCM/MeOH, r.t.; (d) Bu2SnO, dioxane,
then ClAcCl, DCM; (e) BzCl, DMAP, DCM, r.t..
To solve this problem, we took another route starting from compound 65.
Regioselective installation of isopropylidene acetal was achieved by 2-methoxypropene
o

and camphorsulfonic acid at 60 C. For this transformation, we found that using more
reagents can improve they yield. However, longer reaction time lowered the yields.
Treatment of compound 68 with NaH and para-methoxybenzyl chloride afforded
compound 69 in 85%, followed by benzoylation and PMB removal to furnish building
block 72 (Scheme 3.7a). Besides the PMB protecting group, we also tested Bn and Lev
as potential protecting groups to mask 4-OH. Compound 68 was first converted to
compound 73 by treating with NaH and BnBr, followed by isopropylidene removal and
benzoylation to afford compound 75. In order to remove Bn protecting group, we used
large excess amount of DDQ (50 equiv.). However, the reaction was very slow. By
heating the reaction to reflux overnight, we were able to improve the yield to 60%
(Scheme 3.7b). Due to the harsh reaction conditions employed in this transformation,
this route was abandoned. The third route we tried was to use Lev to mask 4-OH
199

(Scheme 3.7c). Similarly, compound 68 was first converted to compound 76 by treating
with NaH and BnBr, followed by isopropylidene removal and benzoylation to afford
compound 78. For benzoylation of compound 77, Lev protecting group was found to
migrate to the remaining free hydroxyl groups easily under heating conditions when we
performed the reaction at a relatively large scale (5 g). Subsequent Lev removal with
hydrazine afforded compound 72 in 87% yield. Due to the migration problem during
benzoylation, this route was also abandoned.
a)

O

HO
HO

OH

65

b) HO

O

c) HO

O

a
78%

O

HO
O

STol

O
68
O

b
PMBO
O
85%

O

STol

O
69

c
98%

O
d PMBO
e
HO
STol
STol
BzO
BzO
98%
81%
OBz
OBz
OH
71
72
O
O
O
BnO
BnO
g
h
STol
STol f
STol
O
HO
O
90%
O
92%
OH
95%
73
68
74
O

PMBO
HO
70

BnO
BzO

STol

STol

O

O
HO
STol
BzO
OBz
72
75
O
O
O
LevO
k LevO
STol
STol j
STol
O
HO
O
88%
O
90%
OH
76
68
77
STol i
60%
OBz

O

LevO
BzO
78

STol m
87%
OBz

HO
BzO
72

l
73%

O

STol
OBz

Scheme 3.7. Synthesis of xylose building block 72. Reagents and conditions: (a) 2o

methoxypropene, CSA, DMF, 60 C; (b) NaH, PMBCl, DMF; (c) CSA, DCM/MeOH; (d)
BzCl, DMAP, DCM, reflux; (e) DDQ, DCM/H2O; (f) NaH, BnBr, DMF; (g) CSA,
DCM/MeOH; (h) BzCl, DMAP, DCM, reflux; (i) DDQ, DCM/H2O, reflux; (j) LevOH, EDCHCl, DMAP, DCM; (k) CSA, DCM/MeOH; (l) BzCl, DMAP, DCM, reflux; (m) NH2NH2,
HOAc, DCM/MeOH;
200

3.3.5. Idose building block
Idose building blocks 79 and 80 (Figure 3.13) were synthesized by Dr. Mohamad
34

El-Dakdouki and Dr. Hang Dai according to literature procedure.

Figure 3.13. Idose building blocks 79 and 80.
3.4. Evaluation of “1 + 1” glycosylation
To figure out the best protecting group patterns for the above five
monosaccharide building blocks, we evaluated single step “1+1” glycosylation. Since
our group has extensive experience in HP/HS building block design and synthesis,

34,59

our evaluation started from the linker region.
3.4.1. “Gal + Gal” coupling
Since 4, 6-dibenzyl protected galactose is more stable compared to 4, 6benzylidene protected galactose, the first donor-acceptor pairs we screened are donors
81, 82 and acceptor 57. Synthesis of donor 81 was achieved by silylation of acceptor 57
with TBSOTf under low temperature while EDC-HCl mediated coupling of LevOH to
acceptor 57 afforded donor 82. Unfortunately, either donor 81 or 82 failed to glycosylate
acceptor 57 (Table 3.1, Entries 1 and 2). Both donor and acceptor decomposed based
on TLC analysis. Coupling of donor 82 to acceptor 61 only gave desired product 84 in
60

3% yield along with α-isomer (3%) and aglycon transfer product

(24%) (Table 3.1,

Entry 3). Changing the ratio of donor and acceptor did not improve the yield. Coupling in

201

the presence or absence of tri-tert-butylpyrimidine did not make any difference in the
outcome of the reaction. When we use 4, 6-benzylidene protected galactose 83 or 60 as
the donor, coupling to either acceptor 58 or 61 led to productive glycosylation (Table 3.1,
Entries 4, 5 and 6), with entry 4 giving the best coupling yields. We were able to
synthesize 8 g disaccharide 87 in 85% yield by coupling donor 83 to acceptor 61.

Table 3.1. Screening donor-acceptor pairs for Gal-Gal disaccharide synthesis.
o

o

Reagents and conditions: (a) TBSOTf, 2, 6-lutidine, DCM, -40 C-0 C; (b) LevOH,
EDC-HCl, DMAP, DCM; (c) LevOH, EDC-HCl, DMAP, DCM.

202

3.4.2. “Xyl + serine” coupling
Next we tested the coupling reaction between xylose donor and serine acceptor.
Serine acceptor 91 was synthesized following literature procedure from the
commercially available serine 90, for which carboxylate was protected as benzy ester
61

using BnBr and KHCO3.

Coupling of donor 75 to acceptor 91 yielded glycoside 92,

which was treated with DDQ in DCM/H2O under reflux condition to give compound 93 in
61% yield. Even large excess amount of DDQ was not able to push the reaction to
completion and 10-20% starting material was recovered. Therefore donor 75 was not
applied for large scale synthesis of compound 93. Coupling of donor 71 to acceptor 91
led to the formation of compound 94, which was treated with DDQ in DCM/H2O under
room temperature to yield compound 93 smoothly. Also, 4-OH in compound 72 can be
protected as silyl ether to give compound 95, which was coupled to acceptor 91 to yield
compound 96. We tried different condition to remove silyl ether protecting group as
shown in Scheme 3.8 with all these different conditions converting compound 96 to 93
in good yields.

203

Scheme 3.8. Screening donor-acceptor pairs for Xyl-Serine synthesis. Reagents and
o

conditions: (a) KHCO3, BnBr, Bu4NI, DMSO; (b) AgOTf, p-TolSCl, DCM, -78 C, then
o

91, TTBP, -78 C-r.t.; (c) DDQ, DCM/H2O, reflux; (d) DDQ, DCM/H2O; (e) TBSOTf, 2,
6-lutidine, -40

o

C-0

o

C; (f) p-TsOH, THF/H2O, 70% or HF/Pyridine, 72% or Tf2O,

THF/H2O, 80%.
3.4.3. “Gal + Xyl” coupling
To find the best donor-acceptor pair for this coupling, the first pair we tried is
donor 60 and acceptor 72 (Scheme 3.9). Pre-activation of donor 60 resulted in
complete consumption of this donor. However, upon addition of acceptor 72, donor 60
was converted to several compounds as indicated by TLC analysis. After silica gel
column purification, we were able to isolate six major compounds. Besides the desired
product 97, α-isomer 98 was isolated in 14% yield. Donor 60 was isolated in 15% with
60

its α-isomer 99 (77%). These two compounds were generated by aglycon transfer

204

due to the low nucleophilicity of 4-OH in acceptor 72. Furthermore, we recovered 8%
acceptor along with 5% glycal 100.

Scheme 3.9. Synthesis of disaccharide 97. Reagents and conditions: (a) AgOTf, po

o

TolSCl, DCM, -78 C, then 72, TTBP, -78 C-r.t..
Acceptor 72 has two electron-withdrawing benzoyl groups, which significantly
reduced the nucleophilicity of 4-OH. To increase its nucleophilicity, we tested compound
68 as acceptor for coupling to donor 60 (Scheme 3.10). Upon addition of acceptor 68,
o

TLC shows one major spot under -70 C. However, this spot was converted to another
spot along with some decomposition as the reaction was warmed up to room
temperature. This later spot turned out to be disaccharide 102 after column purification,
which showed the instability of isopropylidene protecting group during glycosylation
reaction. To solve this problem, we quenched the reaction by Et3N at -70

o

C. As a

result, we were able to able isolate disaccharide 101 in 65% yield. We further tried
coupling disaccharide 89 to acceptor 68, which was quenched by Et3N under -70

o

C.

However, we were only able to isolate trisaccharide 103 in 10-20% yield in the presence
205

or absence of TTBP (Scheme 3.11). To further test how stable isopropylidene group
was toward glycosylation reaction, we coupled disaccharide 101 to serine acceptor 91
(Scheme 3.12). However, this reaction failed to yield any desired product 104, with
donor and acceptor decomposed based on TLC analysis.

Scheme 3.10. Synthesis of disaccharide 101. Reagents and conditions: condition (a)
o

o

AgOTf, p-TolSCl, DCM, -78 C, then 68, TTBP, -78 C-r.t.; condition (b) AgOTf, po

o

o

TolSCl, DCM, -78 C, then 68, TTBP, -78 C--70 C.

Scheme 3.11. Synthesis of trisaccharide 103. Reagents and conditions: (a) AgOTf, po

o

o

TolSCl, DCM, -78 C, then 68, TTBP, -78 C- -70 C.

Scheme 3.12. Synthesis of trisaccharide 104. Reagents and conditions: (a) AgOTf, po

o

o

TolSCl, DCM, -78 C, then 91, TTBP, -78 C- -70 C.
206

To solve the aglycon transfer problem and improve the yield for “Gal + Xyl”
coupling, we decided to use compound 93 as the acceptor since this compound does
not have STol at the reducing end. Coupling of donor 60 to acceptor 93 gave compound
105 in 81% yield. Lev removal followed by another round of coupling to donor 60 by
mixing donor and acceptor together led to trisaccharide 107 in 83% yield, which was
also produced by coupling of disaccharide donor 89 to acceptor 93. Trisaccharide 107
was treated with NH2NH2/HOAc to yield compound 108 (Scheme 3.13). All the
glycosylation reactions shown in Scheme 3.13 were high yielding with good
reproducibility. This demonstrated the importance of having a non-activatable anomeric
leaving group in the acceptor whose hydroxyl group has low nucleophilicity.

Scheme 3.13. Synthesis of trisaccharide 108. Reagents and conditions: (a) AgOTf, pTolSCl, DCM, -78

o

C, then 93, TTBP, -78

207

o

C-0

o

C; (b) NH2NH2-H2O, HOAc,

o

o

DCM/MeOH; (c) AgOTf, p-TolSCl, DCM, 60, TTBP, -78 C-0 C; (d) NH2NH2-H2O,
HOAc, DCM/MeOH.
3.4.4. “Ido + GluN + Glu” coupling
Next we moved on to test the coupling reaction in HP/HS part to see whether our
protecting group patterns were suitable for glycosylation. Idose 79 was first converted to
compound 109 in 92% yield. Pre-activation of 109 formed a single spot on TLC, which
was followed by the addition of acceptor 40. However, the spot prior to addition of 40
was found unchanged after aqueous workup, which was isolated after column
purification and assigned as compound 111 based on NMR analysis. Acceptor 40 was
also recovered in 63% yield. To explain this result, we proposed a possible pathway for
the formation of 111 (Scheme 3.14). Pre-activation of 109 by AgOTf/p-TolSCl gave
oxacarbenium ion 112. Intramolecular nucleophilic attack formed intermediate 113,
which was further converted to product 111, which cannot be coupled to acceptor 40.
To avoid this cyclization problem, we treated compound 109 with DDQ to expose the
primary hydroxyl group, which was protected by ClAc group to give donor 115. Coupling
of 115 to acceptor 40 produced disaccharide 116. Although the glycosylation reaction
was clean based on TLC, 116 was isolated only in 48% yield presumably due to the low
stability of ClAc group. Coupling of disaccharide donor 116 to glucose acceptor 48 gave
trisacchride 117 only in 36% yield along with 10% cyclized product 118 (Scheme 3.15).
The latter was generated presumably by activating product 117 followed by cyclization
as the pathway shown in Scheme 3.14.

208

Scheme 3.14. Outcome of the coupling between donor 109 and acceptor 40. Reagents
o

o

and conditions: (a) TBSOTf, 2, 6-lutidine, -40 C-0 C; (b) AgOTf, p-TolSCl, DCM, -78
o

o

o

C, then 40, TTBP, -78 C-0 C.

Scheme 3.15. Synthesis of trisaccharide 117. Reagents and conditions: (a) DDQ,
o

o

DCM/H2O; (b) ClAcCl, Pyridine, DCM, 0 C-r.t.; (c) AgOTf, p-TolSCl, DCM, -78 C, then
o

o

o

o

o

40, TTBP, -78 C-0 C; (d) AgOTf, p-TolSCl, DCM, -78 C, then 48, TTBP, -78 C-0 C.
3.5. Synthetic design – Second Generation
Based on the single step glycosylation evaluated as discussed in Section 3.4.,
we designed a second generation retrosynthetic route (Scheme 3.16). Compound 121
209

would be used as a single amino acid building block for peptide coupling. This route
would potentially allow the solid phase synthesis of our final synthetic target 1. Since
ClAc was not quite stable during glycosylation and column purification, we would use
TBS and Lev as protecting groups to mask the primary hydroxyl groups which would be
oxidized to carboxylic acid later. For the two hydroxyl groups in A and B sugar units,
they would be protected by fluorenylmethyloxycarbonyl (Fmoc) group which could be
selectively removed for subsequent sulfation. For Gal-Gal disaccharide, 4, 6benzylidene protected monosaccharide building blocks were used to afford the F-G
sugar units.

210

Figure 3.14. Retrosynthetic analysis of glycopeptide 24.
3.6. Synthesis of building blocks
3.6.1. Synthesis of trisaccharide 124

211

Primary hydroxyl group in compound 114 was first protected by Lev to give donor
126 which glycosylated acceptor 40 to produce disaccharide 127 in 60% yield. This
disaccharide was used as a donor to couple to glucose 48 to yield 128. Disappointingly,
trisaccharide 128 was isolated in 34% yield, along with hemiacetal 129, cyclized
trisaccharide 130 and 18% recovered acceptor (Scheme 3.16).

Scheme 3.16. Synthesis of trisaccharide 128. Reagents and conditions: (a) LevOH,
o

o

o

EDC-HCl, DMAP, DCM; (b) AgOTf, p-TolSCl, DCM, -78 C, then 40, TTBP, -78 C-0 C;
o

o

o

(c) AgOTf, p-TolSCl, DCM, -78 C, then 48, TTBP, -78 C-0 C.
To improve the yield for trisaccharide 128 synthesis, we decided to make
disaccharide 131 first (Scheme 3.17). Coupling of donor 41 to acceptor 48 produced
disaccharide 131 in 62% yield along with 7% β-isomer 132 and 9% cyclized product 133.
Disaccharide 131 was converted to acceptor 134 in the presence of HF/Pyridine.
Glycosylation of donor 126 to acceptor 134 led to trisaccharide 128 in 50% yield
together with 20% cyclized product 130.

212

Scheme 3.17. Synthesis of trisaccharide 128. Reagents and conditions: (a) AgOTf, po

o

o

TolSCl, DCM, -78 C, then 48, TTBP, -78 C-0 C; (b) HF/Pyridine, Pyridine; (c) AgOTf,
o

o

o

p-TolSCl, DCM, -78 C, then 134, TTBP, -78 C-0 C.
To minimize the formation of cyclized product, we decided to convert PMB to Lev
at the disaccharide stage (Scheme 3.18). Compound 131 was first converted to
compound 136 by PMB removal and installation of Lev at primary hydroxyl group in
glucose unit. Compound 136 was treated with HF/Pyridine to remove the silyl ether
protecting group to afford disaccharide 137. Coupling of donor 126 to acceptor 137
produced trisaccharide 124 in 85% yield without any cyclization product being detected.
Alternatively, trisaccharide 128 synthesized in Scheme 3.17 was be converted to 124
by PMB removal and installation of Lev at the primary hydroxyl group.

213

OBn
O

OBn
O

OBn
O

b TBSO
OH 90% BnO
N3
O
O
136 BnO
STol
BzO

a TBSO
BnO
OPMB 71%
N3
N3
135 O
O
131 O
BnO
STol
BnO
BzO
OBn
O
HO
c
BnO
OLev
N3
93%
O
O
137 BnO
STol
BzO

TBSO
BnO

OBn
OLev
O
O
OBn BnO
O
N3
TBSO
128
O
BzO
BnO

OPMB
O STol
OBz

LevO BnO
O
TBSO

STol

OLev
O
BzO

STol

OBz

126
d 85%

OBn
OLev
O
O
OBn BnO
O
124 N3
TBSO
O
BzO
BnO

a, b
56%

OLev
O STol
OBz

Scheme 3.18. Synthesis of trisaccharide 124. Reagents and conditions: (a) DDQ,
DCM/H2O; (b) EDC-HCl, LevOH, DMAP, DCM; (c) HF/Pyridine, Pyridine; (d) AgOTf, po

o

o

TolSCl, DCM, -78 C, then 137, TTBP, -78 C-0 C.
3.6.2. Screening different conditions for the synthesis of disaccharide 122
Synthesis of disaccharide 122 started from the preparation of monosaccharide
building blocks. 4, 6-Benzylidene acetal in azidoglucoside 39 was removed under acidic
condition to give diol 138. Selective protection of primary hydroxyl group was achieved
by treating compound 138 with slightly excess amount of acetyl chloride under low
temperature. Primary hydroxyl group in the iodoside 80 was protected as a silyl ether
producing donor 140, which was subjected to glycosylation with acceptor 139.
Disappointingly, instead of getting the desired product 141, we isolated cyclized donor
142 and recovered 80% acceptor 139. The pathway for the formation of 142 was similar
to 111 as shown in Scheme 3.14. To solve this cyclization problem, we decided to
protect the primary hydroxyl group by TBDPSCl, which is sterically more hindred

214

compared to TBS protecting group. We were satisfied to find that coupling of donor 143
to acceptor 139 produced disaccharide 144 in 61% yield along with 7% isomer 145
(Scheme 3.18).

Scheme 3.18. Synthesis of disaccharide 144. Reagents and conditions: (a) p-TsOH,
o

o

DCM/MeOH; (b) AcCl, 2, 4, 6-collidine, -40 C-0 C, DCM; (c) TBSOTf, 2, 6-lutidine, -40
o

o

o

o

C-r.t., DCM; (d) AgOTf, p-TolSCl, DCM, -78 C, then 139, TTBP, -78 C-0 C; (e)

TBDPSCl, imidazole, DCM.
With disaccharide 144 in hand, we first treated this compound with NaOMe to
afford diol 146, which was further treated with FmocCl in order to give disaccharide 147.
However, this reaction turned out to be problematic, we were only able to isolate monoprotected disaccharide 148 with recovery of some starting material (Scheme 3.19). To
push the reaction to completion, we used up to 100 equiv. FmocCl and heated the
reaction to reflux, more disaccharide 148 was obtained but without the isolation of any
215

desired product 147. This is probably due to the low reactivity of secondary hydroxyl
group in 148 toward FmocCl. Instead of using FmocCl, we next tried to protect the diol
by Lev protecting group. By treating compound 146 with large excess of reagents (100
equiv. EDC-HCl and LevOH), we were able to get compound 149 in 60% yield along
with 20% mono-protected compound 150.

Scheme 3.19. Synthesis of disaccharide 149. Reagents and conditions: (a) NaOMe,
DCM/MeOH; (b) FmocCl, DMAP, DCM; (c) LevOH, EDC-HCl, DMAP, DCM.
3.7. Synthetic design – Third Generation
Based on the results of disaccharide 122 and trisaccharide 124 synthesis, we
have to revise our synthetic plan. Our third generation is shown in Figure 3.15. Since
we have used Lev to mask the hydroxyl group which will be sulfated later, we cannot
use Lev to mask the primary hydroxyl groups to be oxidized to carboxylic acids in
pentasaccharide 153. Instead, we will convert these hydroxyl groups to benzyl ester at
the pentasaccharide stage.

216

Figure 3.15. Retrosynthetic analysis of glycopeptide 24.
3.8. Synthesis of fully protected octasaccharide
3.8.1. Synthesis of pentasaccharide

217

Synthesis of disaccharide 125 was achieved by deprotecting 87 with
NH2NH2/HOAc. The “3 + 2” coupling of trisaccharide 124 with disaccharide 125
afforded the desired pentasaccharide 154 in 65% yield. Reductive acetylation of 154
with HSAc in pyridine

62

followed by Lev removal afforded pentasaccharide 156 in good

yields.
Ph
O
O

Ph
O
O
O

O
OLev
87

LevO

OLev
OBn O
b
O BnO
65% TBSO
BzO

124

Ph
O
O

Ph
O
O

a
O STol 89%
HO
OBz
OBn
O

O
OH

125

O STol
OBz

Ph
O
O

Ph
O
O

OLev
N3
O
O
O
BnO
OBz
154
c 79%

O

O
OH

O

O STol
OBz

OBn
Ph
Ph
OLev
O
OBn O
O
O
OLev
O
O
O BnO
AcHN
O
O
TBSO
O
STol
O
O
O
BzO
BnO
OBz
OH
OBz
155
d 95%
OH
TBSO

OBn O
O BnO

BzO

OBn
O

Ph
O
O

Ph
O
O

OH
AcHN
O
O
O
BnO
OBz
156

O
OH

O

O

STol
OBz

Scheme 3.20. Synthesis of pentasaccharide 157. Reagents and conditions: (a)
o

NH2NH2-H2O, HOAc, DCM/MeOH; (b) AgOTf, p-TolSCl, DCM, -78 C, then 125, TTBP,
o

o

-78 C-0 C; (c) HSAc, pyridine; (d) NH2NH2-H2O, HOAc, DCM/MeOH.
With compound 156 in hand, the next step was oxidation and benzylation. In
2006, our group developed a facile method for oxidation of primary alcohols using
218

TEMPO/NaOCl followed by Pinnick oxidation in one-pot
method for divergent HP/HS synthesis.

59

63

and we have applied this

For pentasaccharide 156, TLC showed one

major spot after the reaction. However, we found this spot to be compound 157
according to mass spectra analysis where besides oxidation of primary hydroxyl groups,
STol was also cleaved under this condition. This was presumably due to oxidation of the
thio ether moiety due to the excess NaOCl added first followed by hydrolysis to afford
the hemiacetal.

Scheme 3.21. Oxidation of pentasaccharide 156. Reagents and conditions: (a) TEMPO,
NaBr, n-Bu4NBr, NaOCl, NaHCO3, then NaClO2, 2-methyl-2-butene.
Next we tried TEMPO/BAIB mediated oxidation.

64

Using this condition, we were

able to oxidize the primary hydroxyl groups to carboxylic acids smoothly, which was
confirmed by mass spectra. Without purification, the crude compound 158 was directly
used for benzylation, which was carried out with phenyldiazomethane

65

to afford

compound 159. To remove the silyl ether protecting group, we first treated compound
159 with HF in pyridine. However, this reaction was found to be extremely slow. To

219

o

accelerate the reaction, we heated the reaction to 50 C and we were able to isolate
compound 160 in 63% yield after overnight reaction.

Scheme 3.22. Synthesis of pentasaccharide 160. Reagents and conditions: (a) TEMPO,
o

BAIB, t-BuOH, DCM, H2O; (b) PhCHN2 in ether, DCM; (c) HF/Pyridine, 50 C.
3.8.2.
Investigation of “2 + 5” coupling
With disaccharide 149 and pentasaccharide 160 in hand, we tried “2 + 5”
coupling to make heptasaccharide 161 (Scheme 3.23). However, this reaction did not
form any desired product 161. Instead, we recovered acceptor 160 (45%) and donor
149 (40%) which was regenerated due to aglycon transfer. For disaccharide donor 149,

220

we removed silyl ether protecting group and performed oxidation and benzyl ester
formation to get disaccharide 163 (Scheme 3.24). Disappointingly, coupling of donor
163 to acceptor 160 failed to yield desired product 164.

Scheme 3.23. Synthesis of heptasaccharide 161. Reagents and conditions: (a) AgOTf,
o

o

o

p-TolSCl, DCM, -78 C, then 160, TTBP, -78 C-0 C.

Scheme 3.24. Synthesis of heptasaccharide 164. Reagents and conditions: (a)
HF/Pyridine; (b) TEMPO, BAIB, t-BuOH, DCM, H2O; (c) PhCHN2 in ether, DCM; (d)
o

o

o

AgOTf, p-TolSCl, DCM, -78 C, then 160, TTBP, -78 C-0 C.
221

The failure of the “2 + 5” coupling shown in Schemes 3.23 and 3.24 might be a
result of low reactivity of the axial hydroxyl group in acceptor 160. In this acceptor, there
are a lot of electron-withdrawing groups especially the benzyl ester group next to the
axial hydroxyl group, which greatly reduced its nucleophilicity. Another possible
explanation is that N-acetyl group present in acceptor 160 has an inhibitory effect
toward glycosylation.

66

To figure out the reason for the failure of this coupling, we

carried out some model studies. Trisaccharide 124 was first treated with HF/Pyridine to
afford acceptor 165, in which there are no strong electron-withdrawing groups and Nacetyl group. However, coupling of donor 163 to acceptor 165 failed to give any desired
product 166. Again, we recovered 42% donor 163 and 35% acceptor 165. This result
excludes the above discussed reasons for the failure of the coupling.

Scheme 3.25. Synthesis of pentasaccharide 166. Reagents and conditions: (a)
o

o

o

o

HF/Pyridine, 50 C; (b) AgOTf, p-TolSCl, DCM, -78 C, then 165, TTBP, -78 C-0 C.
3.8.3. Investigation of “2 + 6” coupling
To avoid the aglycon transfer problem encountered in “2 + 5” coupling, we
decided to used pentasaccharide as the donor which can be coupled to xylose acceptor
93. Lev protection of pentasaccharide 160 afforded donor 167, which was coupled to

222

xylose acceptor 93. After the coupling, we were able to isolate 30% desired
hexasaccharide 168 along with 30% side product 169 and 42% acceptor 93 (Scheme
3.26). The formation of 169 was probably due to the participation of CH3CN during
glycosylation (Scheme 3.27). Upon pre-activation, donor 167 was converted to
oxacarbenium ion 170, which underwent nucleophilic attack by CH3CN (used for
dissolving AgOTf) to form 171. This intermediate was so stable that the anomeric
leaving group cannot be substituted by the nucleophile. During aqueous workup, this
intermediate was rearranged to give side product 169.

Scheme 3.26. Synthesis of hexasaccharide 168. Reagents and conditions: (a) LevOH,
o

o

o

EDC-HCl, DMAP, DCM; (b) AgOTf, p-TolSCl, DCM, -78 C, then 93, TTBP, -78 C-0 C.
223

Scheme 3.27. Possible pathway for the formation of 169.
With hexasaccharide 168 in hand, we removed Lev to expose the hydroxyl group
to afford acceptor 172. Then we performed “2 + 6” coupling by mixing donor 149 and
acceptor 172 together. However, this reaction failed with donor decomposed and
recovery of 63% acceptor 172 after column purification (Scheme 3.28). The low yield
for “5 + 1” coupling and the failure of “2 + 6” coupling might be due to the presence of Nacetyl group as mentioned before. So we did a control experiment as shown in Scheme
3.29. Donor 154 without N-acetyl group was coupled to acceptor 93. However, we only
isolated hexasaccharide 174 in 33% with 51% acceptor 93 recovered.

224

Scheme 3.28. Synthesis of octasaccharide 173. Reagents and conditions: (a) NH2NH2o

o

H2O, HOAc, DCM, MeOH; (b) AgOTf, p-TolSCl, 149, DCM, TTBP, -78 C-0 C.

Scheme 3.29. Synthesis of hexasaccharide 174. Reagents and conditions: (a) AgOTf,
o

o

p-TolSCl, 93, DCM, TTBP, -78 C-0 C.
3.9. Synthetic design – Fourth Generation
225

Since it is difficult to build up the α-linkage between B and C sugar units and the
β-linkage between G and H sugar units, we decided to revise our synthetic design
(Scheme 3.16). In this version, all the primary hydroxyl groups to be oxidized to
carboxylic acid will be masked by the TBDPS group. Octasaccharide 176 will be
assembled via “3 + 2 + 3” coupling. The α-linkage between B and C sugar units will be
synthesized first during the assembly of trisaccharide 177 and the β-linkage between G
and H sugar units will be synthesized by coupling donor 87 to acceptor acceptor 93.

226

Figure 3.16. Retrosynthetic analysis of glycopeptide 24.
3.10. Synthesis of glycopeptide 175
3.10.1. Synthesis of trisaccharide 177

227

As discussed earlier, we will build up the α-linkage first. Compound 139 was
converted to donor 273, which was glycosylated to acceptor 79 to afford 274 in 80%
yield. Selective removal of Ac in the presence of Bz was achieved by treating 274 with
Mg(OMe)2 under low temperature.

67

Subsequent Lev protection followed by PMB and

TBS removal afforded diol 185. Selective protection of primary hydroxyl group was
achieved by treating disaccharide 185 with TBDPSCl. Compound 186 will be use as the
acceptor for synthesizing trisaccharide 177. Synthesis of donor 188 started from
compound 143, which was treated with NaOMe in DCM/MeOH to afford compound 187.
Benzoylation of compound 187 produced donor 188 in 97% yield. Glycosylation of
donor 188 to acceptor 186 produced trisaccharide 177 in 52% yield. Besides the
desired product, we also isolated hemiacetal (11%) resulting from the hydrolysis of
activated donor and 22% acceptor 186.

228

Scheme 3.30. Synthesis of hexasaccharide 177. Reagents and conditions: (a) TBSOTf,
o

o

o

2, 6-lutidine, DCM, -40 C-0 C; (b) AgOTf, p-TolSCl, DCM, -78 C, then 79, TTBP, -78
o

o

o

o

C-0 C; (c) Mg(OMe)2, DCM, -20 C-0 C; (d) LevOH, EDC-HCl, DMAP, DCM; (e)

DDQ, DCM/H2O; (f) HF/Pyridine; (g) TBDPSCl, imidazole, DCM; (h) NaOMe,
o

DCM/MeOH; (i) LevOH, EDC-HCl, DMAP, DCM; (j) AgOTf, p-TolSCl, DCM, -78 C,
o

o

then 186, TTBP, -78 C-0 C.
3.10.2. Synthesis of disaccharide 178

229

Disaccharide 178 synthesis started from disaccharide 135. TBS removal followed
by selective protection of primary hydroxyl group furnished disaccharide 178, which will
be used for “3 + 2” coupling (Scheme 3.31).

Scheme 3.31. Synthesis of disaccharide 178. Reagents and conditions: (a) HF/Pyridine;
(b) TBDPSCl, imidazole, DCM.
3.10.3. Synthesis of trisaccharide 179
Synthesis of trisaccharide 179 was achieved by coupling donor 87 to acceptor 93.
TLC showed we got two spots (1:1) upon addition of acceptor and after column
purification, we were able to isolate the desired product 179 in 43% (Scheme 3.32). The
other spot was assigned to be α-isomer based on NMR analysis. To suppress the
formation of α-isomer, we tried different reaction conditions: pre-activation/nonpreactivation, with/without TTBP, different donor/acceptor ratios. However, all these
conditions led to the formation 1:1 anomeric mixture. In the donor, we have benzoyl
group as participating group to assist 1, 2-trans linkage formation as in the case of
donor 86/acceptor 93 coupling. The formation of anomeric mixture for this specific case
awaits further explanation.

230

Scheme 3.32. Synthesis of trisaccharide 179. Reagents and conditions: (a) AgOTf, po

o

TolSCl, DCM, TTBP, -78 C-0 C;
3.10.4. Synthesis of octasaccharide 176
Deprotection of trisaccharide 179 afforded acceptor 181 in 72% yield. “3 + 2”
coupling afforded pentasaccharide donor 182 by the pre-activation protocol smoothly,
which was further coupled to acceptor 181 to produce octasaccharide 176 in good
yields (Scheme 3.33). The success of these two couplings highlights the advantage of
our “3 + 2 + 3” coupling strategy. Identity and purity of compound 176 were confirmed
by NMR (δ: 0.92, 1.00, 1.03, 27 H, s, C(CH3)3 of TBDPS; 1.99, 2.03, 6 H, s, CH3 of Lev;
5.32, 2 H, s, PhCH) and mass spectra analysis.

231

Scheme 3.33. Synthesis of octasaccharide 176. Reagents and conditions: (a) NH2NH2o

o

H2O, HOAc, DCM/MeOH; (b) AgOTf, p-TolSCl, DCM, -78 C, then 178, TTBP, -78 C-0
o

o

o

C; (c) AgOTf, p-TolSCl, DCM, 181, TTBP, -78 C-0 C.

3.10.5. Synthesis of glycopeptide 175
To synthesize glycopeptide 175, we first prepared dipeptide 120 building block
which can be coupled to glycosyl amino acid. Synthesis of dipeptide 120 started from
BOP mediated coupling of serine 267 to glycine 268 to afford protected dipeptide 269 in
82% yield, followed by removal of tert-butyl group to produce 120 in 83% yield (Scheme
3.34). To prepare dipeptide 269, we first tried the pre-activation strategy. We first
232

dissolved serine 267 in DCM/THF, then added BOP and DIPEA as the base to the
reaction mixture. After 10 minutes, glycine 268 was added. However, we isolated
compound 190 as the major product (20%) together with trace amount of desired
product. The possible pathway for the formation of compound 190 is shown in Scheme
3.35. Pre-activation of 267 formed activated species 270, which underwent β-elimination
to afford 271 and 272. The latter acted as a nucleophile to react with 270 forming the
side product 190.

Scheme 3.34. Synthesis of dipeptide 120. Reagents and conditions: (a) BOP, DIPEA,
DCM/THF; (b) TFA, DCM.

BnO
OH

AcHN
267

O

DIPEA

(Me2N)3P
O N N
O
269 N

BnO
AcHN
268

O

AcHN
O

P(NMe2)3 + BnO
AcHN
271
272

AcHN

O
H O

P(NMe2)3
270
N

BnO
O

BnO

BnO
O
O

P(NMe2)3
270

AcHN
190 O

OBn

Scheme 3.35. Possible pathway for the formation of side product 190.
Octasaccharide 176 was treated with HF/Pyridine to afford triol 192 in 90% yield,
which was oxidized to carboxylic acid 193 by TEMPO/BAIB, followed by methyl ester
formation to produce 194 in 93% over two steps (Scheme 3.36). We protected the
carboxylic acid in the form of methyl ester instead of benzyl ester to achieve
orthogonality since we can selectively remove benzyl ester in the presence of methyl
ester, which is crucial for glycopeptide synthesis as discussed below.
233

OTBDPS
OBnO
BnO
O
PMBO
OLev

OLev

OLev
HO
PMBO

OBnO
BnO
O
OLev

OBn
Ph
Ph
O OTBDPS
O
O
O
O
N3
O BzO
O
O
O
O
O
O
BnO
OBz
OH
OBz
a 90%

OBz
O
O

FmocHN

CO2Bn

OTBDPS
OBn O
O BnO
N3
176
O
BzO
O

O
N3
O

OBn
O OH

OH

OBn O
O BnO
192 N3O
BzO
BnO
b

Ph
O
O

Ph
O
O
O

O
OBz

O
OH

O

OBz
O
O

O BzO
O
OBz

FmocHN
Ph
Ph
OBn O
O
O
O
O BnO
COOH O
193 N3O
O BzO
O
O
O
O
O
BzO
BnO
OBz
OH
OBz
c 93% over two steps

CO2Bn

OLev
OBn
Ph FmocHN
Ph
COOMe
O
O
O
O
OBn
O
O
MeOOC OBn BnO
O
O BnO
COOMe O
O
N3
194 N3O
O BzO
O
O
PMBO
O
O
O
O
BzO
BnO
OLev
OBz
OH
OBz

CO2Bn

FmocHN

CO2Bn

O
HOOC OBn BnO
O
PMBO
OLev

OLev
O COOH
N3
O

OBn
O

OBz
O
O

OBz
O
O

Scheme 3.36. Synthesis of octasaccharide 194. Reagents and conditions: (a)
HF/Pyridine; (b) TEMPO, BAIB, DCM, H2O, t-BuOH; (c) MeI, K2CO3, DMF.
To protect the remaining free hydroxyl group in octasaccharide 194, we treated
this compound with 10 equiv. Ac2O in pyridine. However, this reaction did not work. We
found that this protection could be achieved by using more Ac2O (100 equiv.) and
o

increasing the reaction temperature to 50 C. Removal of Fmoc in 195 was done by
piperidine in DCM. For this reaction, it is important to perform aqueous workup after the
234

reaction was complete. Concentration of the reaction mixture without aqueous workup
partially epimerized the serine residue and we were able to isolate the epimerized
product (5%) after column purification. Compound 196 was coupled to dipeptide 120 in
the presence of HATU) and DIPEA in 77% yield (Scheme 3.37).
OLev
OBn
Ph
COOMe
O
O
O
OBn O
O
MeOOC OBn BnO
O BnO
COOMe O
O
N3
N3
194
O
O
PMBO
O
O
BzO
BnO
OLev
OBz
a 88%
OLev
OBn
Ph
COOMe
O
O
O
O
OBn
O
MeOOC OBn BnO
O BnO
COOMe O
O
N3
195 N3
O
O
PMBO
O
O
BzO
BnO
OLev
OBz
b 70%
OLev
OBn
Ph
COOMe
O
O
O
OBn O
O
MeOOC OBn BnO
O BnO
COOMe O
O
N3
N3
196
O
O
PMBO
O
O
BzO
BnO
OLev
OBz
c 77%
OLev
OBn
Ph
COOMe
O
O
O
OBn O
O
MeOOC OBn BnO
O BnO
COOMe O
O
N3
N3
175
O
O
PMBO
O
O
BzO
BnO
OLev
OBz

Ph
O
O
O
OH

O BzO
O
OBz

O

Ph FmocHN
O
O
O BzO
O
O
O
OBz
OAc

Ph FmocHN
O
O
O BzO
O
O
O
OBz
OAc

Ph
O
O

H2N
O BzO
O
OBz

O

O
OAc

BnO

H
N

AcHN
O

OBz
O
O

CO2Bn
OBz
O
O

CO2Bn
OBz
O
O

CO2Bn
OBz
O
O

O
N
H

CO2Bn

Scheme 3.37. Synthesis of glycopeptide 175. Reagents and conditions: (a) Ac2O,
o

pyridine, 50 C; (b) piperidine, DCM; (c) HATU, DIPEA, 120, DMF.
3.11. Deprotection of glycopeptide 175

235

With the fully protected glycopeptide 175 in hand, our next goal is to remove all
the protecting groups to get fully deprotected target compound 24. To do this, we first
deprotected octasaccharide 195 as a model study.
3.11.1. Deprotection of octasaccharide 195
In general, six steps are required to achieve deprotection and they are: Lev
removal, azide reduction, O-sulfation, N-acetylation, ester hydrolysis, hydrogenation. To
achieve deprotection, we have to figure out the right reaction sequence (Figure 3.17).

Figure 3.17. Steps required for deprotection of octasaccharide 195.
Our trial started from Lev removal as the first step. Compound 195 was
converted to diol 198, followed by O-sulfation to give compound 199. For O-sulfation, it
is important that we keep the reaction system as dry as possible. Since we are doing
small scale reaction (< 10 mg), trace amount of water can kill the reaction. Therefore we
pre-dried DMF by molecular sieves prior to use and dried starting material through
azotropic distillation with toluene. Usually the reaction was quenched by Et3N and
MeOH after the reaction was complete.

34,37

In our case, when we added Et3N after the

236

reaction was complete, however, we found that small amount of Fmoc was cleaved and
trace amount of β-elimination product was also found based on mass spectra analysis.
To avoid this problem, we quenched the reaction with an aqueous solution of NaHCO3
or diluted the reaction with DCM/MeOH, then loaded the reaction mixture onto a LH-20
column. To reduce azide to free amine, we first tried Zn/HOAc condition.

68

However,

this condition did not work and we recovered the starting material. Then we tried the
Zn/HCOONH4 condition,

69

which failed again with recovery of the starting material. The

we tried Staudinger reduction condition, due to the backbone cleavage reaction caused
by Me3P during Staudinger reduction,

59

we decided to use Ph3P
P

41

which is much

bulkier. However, this reaction led to the formation of side product 201 which was the
intermediate of Staudinger reduction. To convert this intermediate to our target product,
o

we dissolved this compound in THF/H2O, heated this reaction under 65 C overnight.
Disappointingly, this intermediate decomposed under this condition. Due to the failure of
azide reduction on compound 199, we decided to reduce azide first and then perform Osulfation (Scheme 3.39). Zn/HCOONH4 successfully furnished compound 202. Then
we performed catalytic hydrogenation on this compound, however, this reaction failed to
give any desired product. We used HPLC to separate each and tried to determine the
molecular weight to determine each peak. Although we were able to see several peaks
after HPLC separation, we could not get any useful information from mass spectra. We
also tried other hydrogenation conditions (with/without HOAc, Pd(OH)2). But all these
conditions failed to give the desired product.
237

OLev
COOMe
O
OBn O
OBnO
MeOOC
BnO
O BnO
O
N3
195
PMBO
O
BzO
OLev

OBn
O

Ph
Ph
O
O
O
COOMe O
N3
O BzO
O
O
O
O
O
O
BnO
OBz
OAc
OBz

OBz
O
O

a 88%
OH

OBn
O

Ph FmocHN
Ph
O
O
O
COOMe O
N3
O BzO
O
O
O
O
O
O
BnO
OBz
OAc
OBz

CO2Bn

OBn
O

Ph FmocHN
Ph
O
O
O
COOMe O
N3
O BzO
O
O
O
O
O
O
BnO
OBz
OAc
OBz

CO2Bn

FmocHN
OBn
Ph
Ph
COOMe
O
O
O
OBn O
O
O
MeOOC OBn BnO
BnO
O
O
COOMe O
O
H2N
H2N
O BzO
O
200
O
O
PMBO
O
O
O
O
BzO
BnO
OSO3
OBz
OAc
OBz

CO2Bn

FmocHN

CO2Bn

O
MeOOC OBn BnO
O
PMBO
OH

COOMe
OBn O
O BnO
N3
198
O
BzO
O

b 82%
OSO3
O
MeOOC OBn BnO
O
PMBO
OSO3

COOMe
OBn O
O BnO
N3
199
O
BzO
c
O

OSO3

OBz
O
O

OBz
O
O

OBz
O
O

Scheme 3.38. Synthesis of octasaccharide 200. Reagents and conditions: (a) NH2NH2o

H2O, HOAc, DCM/MeOH; (b) SO3-Et3N, DMF, 55 C; (c) Zn, HOAc, DCM/MeOH or Zn,
HCOONH4, DCM/MeOH or PPh3, silica gel, DCM/MeOH.
OSO3

OBn
Ph
COOMe
O
O
O
O
OBn
O
OBn BnO
MeOOC
O BnO
COOMe O
O
N
N
O
Ph3P O
PMBO
Ph3P O
O
BzO
BnO
OSO3
OBz
201

Ph
O
O
O

O
OAc

O BzO
O
OBz
FmocHN

Figure 3.18. Side product 201 generated from Staudinger reaction.
238

OBz
O
O
CO2B
n

OH
O
MeOOC OBn BnO
O
PMBO
OH

COOMe
OBn O
O BnO
N3
198
O
BzO
O

a 73%

OBn
O

Ph
Ph
O
O
O
COOMe O
N3
O BzO
O
O
O
O
O
O
BnO
OBz
OAc
OBz

OH
OBn
Ph FmocHN
Ph
COOMe
O
O
O
O
OBn
O
O
MeOOC OBn BnO
O
O BnO
COOMe O
O
H2N
H2N
202
O BzO
O
O
O
PMBO
O
O
O
O
BzO
BnO
OH
OBz
OAc
OBz
b 85%
OSO3

OBz
O
O

CO2Bn
OBz
O
O

FmocHN
OBn
Ph
Ph
COOMe
O
O
OBn O
O
O
MeOOC OBn BnO
O
O BnO
COOMe O
O
H2N
H2N
O BzO
O
203
O
O
PMBO
O
O
O
O
BzO
BnO
OSO3
OBz
OAc
OBz
c

CO2Bn

FmocHN

CO2Bn

O

OBz
O
O

Scheme 3.39. Synthesis of octasaccharide 204. Reagents and conditions: (a) Zn,
o

HCOONH4, DCM/MeOH; (b) SO3-Et3N, DMF, 55 C; (c) H2, Pd/C, DCM/MeOH.
The difficulty of this hydrogenation reaction might be due to the presence of free
amine in this molecule

70

and this problem was also reported by Boons and
239

coworkers.

71

Thus we decided to try hydrolysis first and we used compound 206 as a

model compound. This compound was prepared from octasaccharide 194, which was
first treated with NH2NH2/HOAc to afford compound 205, followed by O-sulfation to
produce compound 206. The pH of the reaction has to be well controlled to minimize βelimination, which is a general problem for deprotection of glycopeptides.

72-79

To

hydrolyze compound 206, we controlled pH around 9 by LiOH/H2O2. However, instead
of getting the desired product 207, mass spectra analysis shows the formation of
several products and the possible structures are shown in Figure 3.19. Mass spectra
shows only some Bz got cleaved together with cleavage of serine residue from the
octasaccharide backbone.

240

Scheme 3.40. Synthesis of octasaccharide 207. Reagents and conditions: (a) NH2NH2o

H2O, HOAc, DCM/MeOH; (b) SO3-Et3N, DMF, 55 C; (c) LiOH, THF/H2O, pH = 9.

241

Figure 3.19. Possible structures of hydrolysis products.
To figure out the best pH for hydrolysis of esters without cleaving serine residue,
we did some model studies using compound 94 as the starting material. We prepared
Na2CO3/NaHCO3 with different pH. We found that under lower pH, hydrolysis was too
slow and never went to completion. While under higher pH, removal of ester was always
accompanied by β-elimination products such as compounds 217, 218 and 219.
Hydrolysis by LiOH (pH = 9.5) was also accompanied by β-elimination (Scheme 3.41).
However, we were excited to find that hydrolysis of compound 220 was successful and
we did not observe any β-elimination product based on mass spectra analysis (Scheme
3.42). Due to the absence of benzyl ester in the serine carboxylate terminal, βelimination is less likely to happen. This phenomenon is consistent with the literature
242

report. Kihlberg and coworkers found that deprotected glycopeptide is more stable
against β-elimination compared to a fully protected glycopeptide or a partially
deprotected one.

Scheme

3.41.

80

Hydrolysis

of

compound

94.

Reagents

and

conditions:

(a)

Na2CO3/NaHCO3 buffer, THF (1) pH = 9.2, (2) pH = 9.5, (3) pH = 10.1 (4) pH = 10.5; (b)
LiOH, MeOH/H2O, pH = 9.5.

Scheme 3.42. Hydrolysis of compound 220. Reagents and conditions: (a) LiOH,
MeOH/H2O, pH = 9.5.
Based on the results from these model studies, we decided to block all the free
amino group with Ac and hydrolysis would be carried out as the last step to minimize βelimination. Our new deprotection sequence started from acetylation of compound 196.
The two azido groups in 221 were converted to N-acetyl groups in a one-pot manner
following a literature procedure.

81

Subsequent Lev removal and O-sulfation furnished

compound 224 in good yields.
243

OLev
OBn
Ph
Ph
COOMe
O
O
O
O
OBn
O
O
MeOOC OBn BnO
O
O BnO
COOMe O
O
N3
N3
196
O BzO
O
O
O
PMBO
O
O
O
O
BzO
BnO
OLev
OBz
OAc
OBz
a 77%
OLev
OBn
Ph
Ph
COOMe
H2N
O
O
O
O
OBn
O
O
OBn BnO
MeOOC
O
O BnO
COOMe O
O
N3
221 N3O
O BzO
O
O
PMBO
O
O
O
O
BzO
BnO
OLev
OBz
OAc
OBz
b 72%
OLev
OBn
Ph
AcHN
Ph
COOMe
O
O
O
O
OBn
O
O
MeOOC OBn BnO
O
O BnO
COOMe O
O
AcHN
AcHN
O BzO
O
O
O
222
PMBO
O
O
O
O
BzO
BnO
OLev
OBz
OAc
OBz
c 88%
OH

OBn
AcHN
Ph
Ph
COOMe
O
O
O
OBn
O
O
MeOOC OBn BnO
O
O BnO
COOMe O
O
AcHN
AcHN
O BzO
O
O
O
223
PMBO
O
O
O
O
BzO
BnO
OH
OBz
OAc
OBz
d 92%
O

OSO3
OBn
Ph
Ph
COOMe
AcHN
O
O
O
OBn
O
O
OBnO
MeOOC
BnO
BnO
O
O
COOMe O
O
AcHN
AcHN
O BzO
O
O
O
PMBO
224
O
O
O
O
BzO
BnO
OSO3
OBz
OAc
OBz

AcHN

OBz
O
O

CO2Bn
OBz
O
O

CO2Bn
OBz
O
O

CO2Bn
OBz
O
O

CO2Bn
OBz
O
O

CO2Bn

Scheme 3.43. Synthesis of octasaccharide 224. Reagents and conditions: (a) Ac2O,
pyridine; (b) Zn, CuSO4 (sat.), Ac2O/THF/HOAc; (c) NH2NH2-H2O, HOAc, DCM/MeOH;
o

(d) SO3-Et3N, DMF, 55 C.
With compound 224 in hand, our last two steps will be catalytic hydrogenation
and ester hydrolysis. Hydrogenation was carried out in the presence of Pd/C and
244

hydrogen balloon. Mass spectra shows the reaction was complete after 24 hours. After
filtration and concentration, the crude product was subjected to hydrolysis by aqueous
NaOH solution (pH = 9.5). The progress of the reaction was monitored by mass spectra
and the reaction went to completion after 6 hours. Ogawa and coworkers reported a
two-step procedure for deprotecting glycosyl serine during the synthesis of partial
82-84

structure of chondroitin sulfate proteoglycan (CSPG).

The first step is deprotecting

methyl ester using LiOH and the second step is to remove Ac and Bz with NaOH (pH =
9.5). Following this protocol, we were able to get a much better mass spectrum with
high signal to noise ratio after LH-20 column purification. The identity of compound 226
1

was also confirmed by H NMR.

Scheme 3.44. Synthesis of octasaccharide 226. Reagents and conditions: (a) H2, Pd/C,
245

DCM/MeOH; (b) NaOH, THF/MeOH/H2O, pH = 9.5, yield 76%; (c) LiOH 1.25 M,
o

THF/H2O, 0 C, 1h, then NaOH, pH = 9.5, MeOH/H2O, yield 80%.
3.11.2. Deprotection of glycopeptide 175
Inspired by the successful synthesis of compound 226, we moved on to
deprotect glycopeptide 175. This compound was first converted to compound 227 under
reductive acetylation condition applied for compound 221. Subsequent Lev removal and
O-sulfation afforded compound 229 smoothly (Scheme 3.45). For O-sulfation, when a
large excess reagent was used, compound 228 was oversulfated. To better control the
result of this reaction, the amount of SO3-Et3N should be controlled. The oversulfation
problem probably arises from the PMB protecting group, on which Friedel-Crafts
reaction might happen with SO3. Fortunately, the oversulfated compound can be
converted to the desired product cleanly upon hydrogenation. Hydrogenation of
compound 229 afforded compound 230, which was subjected to hydrolysis under
Ogawa’s condition (Scheme 3.46). Our target glycopeptide 24 was obtained after G-15
column and lyophilization.

246

OLev
OBn
Ph
COOMe
O
O
O
OBn O
O
MeOOC OBn BnO
O BnO
COOMe O
O
N3
N3
175
O
O
PMBO
O
O
BzO
BnO
OLev
OBz

Ph
O
O
O BzO
O
OBz

O

O
OAc

BnO

a 76%

H
N

AcHN
OLev
OBn
Ph
COOMe
O
O
O
O
OBn
O
MeOOC OBn BnO
O BnO
COOMe O
O
AcHN
AcHN
O
O
227
PMBO
O
O
BzO
BnO
OLev
OBz

O
OAc

Ph
COOMe
O
O
OBn O
O
MeOOC OBn BnO
O BnO
COOMe O
O
AcHN
AcHN
O
O
PMBO
228
O
O
BzO
BnO
OH
OBz

O BzO
O
OBz

O

O
OAc

BnO

c 90%

H
N

AcHN
OSO3
OBn
Ph
COOMe
O
O
O
OBn
O
OBnO
MeOOC
BnO
O BnO
COOMe O
O
AcHN
AcHN
O
O
PMBO
229 BnO
O
O
BzO
OSO3
OBz

N
H

O
Ph
O
O

OBn
O

N
H

O BzO
O
OBz

O
OAc

BnO

H
N

AcHN
O

OBz
O
O

CO2Bn

OBz
O
O

O

O
Ph
O
O
O

CO2Bn

O

H
N

AcHN
OH

N
H

O BzO
O
OBz

BnO

b 84%

O

O
Ph
O
O
O

OBz
O
O

CO2Bn

OBz
O
O

O
N
H

CO2Bn

Scheme 3.45. Synthesis of glycopeptide 229. Reagents and conditions: (a) Zn, CuSO4
(sat.), Ac2O/THF/HOAc; (b) NH2NH2-H2O, HOAc, DCM/MeOH; (c) SO3-Et3N, DMF, 55
o

C.

247

OSO3
OBn
Ph
Ph
COOMe
O
O
O
O
OBn
O
O
MeOOC OBn BnO
O
O BnO
COOMe O
O
AcHN
AcHN
O
O
O
PMBO
O
229 BnO
O
O
BzO
OSO3
OAc
OBz
BnO

a 95%

O BzO
O
OBz
H
N

O

N
AcHN
H
OSO3
O
OH
COOMe
O
O
OH
O
OH O
OH
HO
MeOOC OH HO
HO
HO
O
O
COOMe
AcHN
AcHN
O BzO
O
O
O
HO
O
230
O
O
O
BzO
HO
OSO3
OBz
OAc
OBz
HO

b 72%

H
N

H
N

AcHN
O

CO2Bn

OBz
O
O

O

N
AcHN
OSO3
H
OH
O
COO
O
O
OH
O
OH O
OH
HO
OOC OH HO
HO
HO
O
O
COO
AcHN
AcHN
O HO
O
O
O
HO
O
24
O
O
O
BzO
HO
OSO3
OH
OH
OH
HO

OBz
O
O

CO2

OH
O

O

O
N
H

CO2

Scheme 3.46. Synthesis of glycopeptide 24. Reagents and conditions: (a) H2, Pd/C,
o

DCM/MeOH; (b) LiOH 1.25 M, THF/H2O, 0 C, 1 h, then NaOH, pH = 9.5, MeOH/H2O.
3.12. N-Sulfation
As mentioned in Section 3.1., HSPG contains various HS structures and amino
group in HS can be either sulfated or acetylated. We tested the possibility of installing
sulfates on octasaccharide 203. We tried standard conditions for N-sulfation (SO3Py),

85

but were not able to get desired product 231. Instead, we recovered starting
o

material. Heating the reaction to 55 C did not improve the situation. When we added
248

Et3N to the reaction, we were able to sulfate the free amino group. But this condition
resulted in β-elimination product 232 as indicated by mass spectra. Future work in this
part will focus on testing different N-sulfation conditions.
OSO3

OBn
Ph
COOMe
O
O
O
OBn O
O
MeOOC OBn BnO
O BnO
COOMe O
O
H2N
H2N
203
O
O
PMBO
O
O
BzO
BnO
OSO3
OBz
a or b or c

Ph
O
O
O
OAc

OSO3

OBn
Ph
COOMe
O
O
O
O
OBn
O
MeOOC OBn BnO
O BnO
COOMe O
O
O3SHN
O3SHN
O
O
PMBO
231
O
O
BzO
BnO
OSO3
OBz

OSO3

Ph
O
O

FmocHN

O

O
OAc

O BzO
O
OBz

FmocHN

OBn
O

Ph
COOMe
O
O
OBn O
O
MeOOC OBn BnO
O BnO
COOMe O
O
O3SHN
O3SHN
O
O
PMBO
232
O
O
BzO
BnO
OSO3
OBz

O BzO
O
OBz

O

Ph
O
O
O

O
OAc

O BzO
O
OBz

OBz
O
O

CO2Bn
OBz
O
O

CO2Bn
OBz
O
SO3

Scheme 3.47. N-Sulfation of octasaccharide 203. Reagents and conditions: (a) SO3-Py,
o

pyridine; (b) SO3-Py, pyridine, 55 C; (c) SO3-Py, Et3N, pyridine.
3.13. Synthesis of glycopeptide 233
Our next synthetic target is glycopeptide 233 bearing a longer peptide chain
which is synthetically more challenging compared to glycopeptide 24. We propose that it
could be accessed from fully protected glycopeptide 234, which could be assembled by
solid phase synthesis (SPS) using glycosyl serine as building block (Figure 20).

249

Figure 3.20. Retrosynthetic analysis of glycopeptide 233.
To test the possibility of using SPS to build compound 234, we first did a model
study. Selective hydrogenation of benzyl ester in compound 94 afforded carboxylic acid
220 within 6 hours. This reaction had to be closely monitored by TLC to avoid the
removal of PMB group. Resin bound peptide 236 was synthesized using SPS approach
by Suttipun Sungsuwan and Dr. Zhaojun Yin.

86

Upon cleavage of Fmoc, compound 220

was added to the resin. After the reaction, the peptide was cleaved off the resin by
250

cleavage cocktail. Mass spectra shows compound 238 was the product, in which PMB
was cleaved by the cocktail. Another model reaction we did is shown in Scheme 3.49.
Here we used trisaccharide 239 as the model compound, which was accessed from
selective removal of benzyl ester in compound 179. This transformation was achieved in
the presence of NH4OAc, which can inhibit the hydrogenation of ether type protecting
group.

87

The same SPS protocol was applied, however, after cocktail cleavage, we got

peptide 242 instead of glycopeptide 241.The generation of compound 242 was due to
the failure of coupling of 239 to the resin. Upon Ac2O capping and cocktail cleavage,
peptide 242 was generated. The failure of this coupling might be due to the presence of
Lev groups which interfered with peptide coupling.

251

Scheme 3.48. Solid phase synthesis of glycopeptide 238. Reagents and conditions: (a)
Pd/C, H2, DCM/MeOH; (b) HATU, DIPEA, 220, DMF; (c) TFA, phenol, TIPS, H2O.

252

Ph
O
O

Ph
O
O
O

O
OLev

LevO

O
FmocHN

N
H

Ph
Ph
a
O
O
78%
O
O
O O
O
O O
O
O
O LevO
O
O
BzO
BzO
OBz
OBz
OLev
OBz
OBz
179
239
FmocHN COOBn
FmocHN COOH
OBn
H O
H O
H O
H O
O
N
N
N
N
N
N
N
N
H O
resin
H O
H O
O
O
236
O OBn

BzO
O

HN

N
H

O

HN

LevO
OLev
O
O
O
O
O
O
Ph
Ph
O
H O
H
N
N
N
N
H O
O
240

O

H
N

O

solid phase
peptide synthesis
with 239
and 120

BzO
OBz
O O

O

b

N
H

O

O

O

O

N
H

resin

O

O OBn

c

O

OBn
H
N

O

H
N

NHAc

BnO

BzO
O

O

H
N

HN
HN
BnO

O
N
H

O

O

LevO
OLev
O
O
O
O
O
O
Ph
Ph
O
H O
H
N
N
N
N
H O
O
241

BzO
OBz
O O

AcHN

O
N
H

O

OBn
H
N

O
N
H

O

OH
O

O OBn

O

NHAc

H
N

O
N
H

H
N
O

O
N
H

H
N

O

H
N

N

O

O

O

N
H

OBn
H
N
O

O
N
H

OH
O

242
O OBn

Scheme 3.49. Solid phase synthesis of glycopeptide 241. Reagents and conditions: (a)

253

Pd/C, H2, NH4OAc, DCM/MeOH; (b) HATU, DIPEA, 239, DMF then HATU, DIPEA, 120,
DMF; (c) TFA, phenol, TIPS, H2O.
As there are multiple acid sensitive protecting groups such as 4, 6 bezylidene
acetal and Lev group in glycopeptide 234, we decided to synthesize this compound in
solution. This compound can be obtained via the coupling of 234 to peptide 244.
Compound 234 can be synthesized by selective hydrogenation of glycopeptide 175
while peptide 244 can be accessed from resin bound peptide 236.
Ph

Ph
OLev
OBn
COOMe
O
O
O
O
OBnO
MeOOC OBn BnO
COOMe O
BnO
O
O
AcHN
AcHN O
O
O
PMBO
BnO
O BzO
LevO
OBz
234
R=
BnO

H
N

AcHN

O
N
H

O

O

H
N

N
H

O

O

H
N

H
N

N
H

O

O
O
O
OAc

O

H
N

N

O

O
N
H

O

O

OBz
OR
O

O BzO
O
OBz
OBn
H O
N
O

N
H

OBn
O

O OBn
OLev
COOMe
O
OBnO
O BnO
N3
O
BzO
243

O
MeOOC OBn BnO
O
PMBO
LevO

O
COOMe O
N3O
O
O
BnO
OBz

+
O
H2N

N
H

H
N
O

O
N
H

H
N
O

244

O

H
N

N

Ph

Ph

OBn
O

O
N
H

O

O
O
O

OAc
BnO

Figure 3.21. Retrosynthetic analysis of glycopeptide 234.

OBz

O BzO
O
OBz
H
N

AcHN
OBn
O
H O
OBn
N
N
H O
O

O OBn

254

O

O

O

O
N
H

OH
O

To achieve selective hydrogenation on glycopeptide 175, we added NH4OAc to
the reaction mixture. The reaction was found to go to completion in 24 hours based on
TLC analysis (Scheme 3.50).
OLev
OBn
Ph
COOMe
O
O
O
O
OBn
O
MeOOC OBn BnO
BnO
O
COOMe O
O
N3
N3
175
O
O
PMBO
O
O
BzO
BnO
OLev
OBz
a 82%

OLev
OBn
Ph
COOMe
O
O
O
OBn O
O
MeOOC OBn BnO
BnO
O
COOMe O
O
N3
N3
243
O
O
PMBO
O
O
BzO
BnO
OLev
OBz

Ph
O
O
O BzO
O
OBz

O

O
OAc

BnO

H
N

AcHN

O
N
H

Ph O
O
O

O BzO
O
OBz

O

O
OAc

BnO

H
N

AcHN
O

OBz
O
O

CO2Bn

OBz
O
O

O
N
H

CO2H

Scheme 3.50. Synthesis of glycopeptide 243. Reagents and conditions: (a) Pd/C, H2,
NH4OAc, DCM/MeOH.
Resin bound peptide 236 was treated with the cleavage cocktail to yield the free
peptide 244. The purity and identity of this crude peptide was analyzed by HPLC and
mass spectra. Since this peptide also has free carboxylic acid, which will affect the
peptide coupling reaction, we decided to protect this acid as a methyl ester using
K2CO3 promoted methylation.

88-89

However, upon methylation, mass spectra shows

the formation of several compounds (245, 246, 247 and 248) (Scheme 3.51). The
formation of these side products was due to the basic nature of K2CO3, which cleaved
Fmoc and benzyl ester.
255

O
FmocHN

O

H
N

N
H

N
H

O

O

H
N

N

FmocHN

O

H
N

N
H

N
H

O

O

H
N

N

FmocHN

N
H

O

O

H
N

N

N
H

O

O

O OBn
O
H 2N

O

H
N

N
H

N
H

O

O

H
N

FmocHN

N
H

O
H2N

N
H

H
N

N

N
H

O

O

N
H

O

H
N
O

O

O
N
H

H
N

O

H
N

H
N

N

O
N

O

O
OH

N
H

O
244

OBn
H
N

O

N
H

O

OBn
H
N

O
247
18%

OMe

N
H

O
245
15%

O
246
22%

O

O

O

OBn
H
N

OBn
H
N

O

H
N

O OBn
O

resin

O

O OBn

O

H
N

N
H

N
H

O

O

O

H
N

O

N
H

O
236

O

H
N

b
O

O

O OBn

a
O

N
H

O

O

OBn
H
N

O

H
N

O

O
OMe

N
H

O

O

O OMe
OBn
H O
H O
N
N
N
N
H
H O
O
248
16%
O OMe

N
H

OMe
O

OMe
O

Scheme 3.51. Synthesis of peptide 245. Reagents and conditions: (a) TFA, phenol,
TIPS, H2O; (b) K2CO3, MeI, DMF
Due to the low yielding of methyl ester formation, we decided to use benzyl ester
to protect the peptide through benzylation with benzyl bromide.

61

This reaction gave

peptide 249 in 60% yield after column purification. Cleavage of Fmoc was achieved by
256

piperdine in DMF. Peptide 250 was precipitated from ether, air dried and directly used
for the next step without further purification. Coupling of peptide 250 to glycopeptide
243 was effected by HATU and DIPEA (Scheme 3.52). Besides product 251, we also
obtained β-elimination product due to the effect of base additive, which is consistent
with our model study (Scheme 3.53). Alternatively, we tried this coupling without any
base which was reported before to inhibit β-elimination.

90

As we expected, we isolated

glycopeptide 251 in 75% without any elimination product detected.

Scheme 3.52. Synthesis of glycopeptide 251. Reagents and conditions: (a)

257

BnBr,NaHCO3, DIPEA, DMF; (b) piepridine, DMF; (c) HATU, 243, DIPEA, DMF, yield
53%; (d) HATU, 243, DMF, yield 75%.
Trisaccharide 180 was treated with piperidine in DCM to afford compound 252,
which was coupled to dipeptide 120 to yield compound 253. Selective hydrogenation of
compound 253 led to compound 254 over 24 hours, which was subjected to the
coupling with peptide 250. Glycopeptide 255 was obtained in 52% yield together with
20% β-elimination product.

Scheme 3.53. Synthesis of glycopeptide 255. Reagents and conditions: (a) piperidine,
DCM; (b) HATU, 120, DIPEA, DMF; (c) H2, Pd/C, NH4OAc, DCM/MeOH; (d) HATU, 250,
DIPEA, DMF, yield 75%.
258

Deprotection of glycopeptide 251 started from Lev removal, which afforded
compound 256, followed by O-sulfation and azide reduction to afford compound 258.

Scheme 3.54. Synthesis of glycopeptide 258. Reagents and conditions: (a) NH2NH2o

H2O, HOAc, DCM/MeOH; (b) SO3-Et3N, DMF, 55 C; (c) Zn, HOAc, DCM/MeOH.

259

With compound 258 in hand, we tried N-sulfation. However, similar to the
reaction shown in Scheme 3.47, none of the conditions we tried gave the desired
product (Scheme 3.55).

Scheme 3.55. N-Sulfation of glycopeptide 258. Reagents and conditions: (a) SO3-Py,
o

pyridine; (b) SO3-Py, pyridine, 55 C; (c) SO3-Py, Et3N, pyridine.
Hydrogenation on either glycopeptide 257 or 258 failed due to the presence of
free amino group as discussed earlier. Ester hydrolysis on glycopeptide 257 also failed
due to β-elimination. As a result, we decided to convert azide to N-acetyl group as in the
case of octasaccharide deprotection. Subsequent Lev removal and O-sulfation afforded
glycopeptide 262.

260

OLev
OBn
Ph
COOMe
O
O
O
OBn
O
O
BnO
MeOOC OBn BnO
O
COOMe O
O
N3
N3
O
O
O
PMBO
O BzO
251 BnO
OLev
OBz
a 62%
OLev
OBn
Ph
COOMe
O
O
O
OBn
O
O
BnO
MeOOC OBn BnO
O
COOMe O
AcHN
O
AcHN
O
O
O
PMBO
O BzO
BnO
260
OLev
OBz

Ph
O
O
O

O
OAc

O BzO
O
OBz

OBz
O

OR

Ph
O
O
O

O
OAc

O BzO
O
OBz

OBz
OR
O

b 85%
OH

OBn
Ph
COOMe
O
O
O
OBn
O
O
BnO
MeOOC OBn BnO
O
COOMe O
AcHN
O
AcHN
O
O
O
PMBO
BnO
261
O BzO
OH
OBz

Ph
O
O
O

O
OAc

O BzO
O
OBz

OBz
OR
O

c 80%
OSO3
OBn
Ph
Ph
COOMe
O
O
O
OBn O
O
O
MeOOC OBn BnO
BnO
O
O
COOMe O
O
AcHN
AcHN
O BzO
O
O
O
PMBO
O
O
O
O BzO
BnO
262
OSO3
OBz
OAc
OBz
R=
OBn
BnO
H O
H O
H O
O
H O
H O
H
N
N
N
N
N
N
N
N
N
N
N
N
H
AcHN
H O
H O
H O
O
H O
O

OBz
OR
O

OBn
O

O OBn

Scheme 3.56. Synthesis of glycopeptide 262. Reagents and conditions: (a) Zn, CuSO4
(sat.), Ac2O/THF/HOAc; (b) NH2NH2-H2O, HOAc, DCM/MeOH; (c) SO3-Et3N, DMF, 55
o

C.
Hydrogenation of glycopeptide 262 afforded compound 263 in 95% yield. After

filtration and concentration, the crude product was subjected to Ogawa’s hydrolysis
261

condition. However, this condition failed to give the desired product. Instead, we got βelimination product based on mass spectra analysis. We also tried using NaOH or
NaOMe as base under different pH, none of which gave the right product (Scheme
3.57).

Scheme 3.57. Synthesis of glycopeptide 233. Reagents and conditions: (a) H2, Pd/C,
DCM/MeOH; (b) LiOH, 1.25 M, THF/H2O, then LH-20, NaOH, pH = 9.5, MeOH/H2O; (c)
NaOH, pH = 8-10, THF/MeOH/H2O; (d) NaOMe, pH = 8-10, MeOH.

262

At this point, we have to design an alternative route for accessing fully
deprotected glycopeptide 233. We propose that this compound can be accessed via
hydrogenation and ester hydrolysis of partially deprotected glycopeptide 264, which can
be synthesized by coupling glycopeptide 265 to peptide 250.

263

SO3

OH
COO
O
O
O
O
OH
OH
OH
OOC OH HO
HO
HO
HO
O
COO
O
OH
AcHN
AcHN
O HO
O
OR1
O
O
HO
O
O
O
O
O
HO
HO
OSO3
233
OH
OH
OH
R1 =
OH
HO
H O
H O
H O
O
H O
H O
O
H
N
N
N
N
N
N
N
N
N
N
N
N
H O
AcHN
H O
H O
H O
O
H O
O
O O
OSO3
OH
COOMe
O
O
O
O
OH
OH
OH
MeOOC OH HO
HO
HO
O
COOMe HO
OH
O
AcHN
AcHN
O HO
OR2
O
O
O
HO
O
O
O
O
264
O
HO
HO
OSO3
OH
OH
OH
R2 =
OBn
HO
H O
H O
H O
O
H O
H O
OBn
H
N
N
N
N
N
N
N
N
N
N
N
N
H O
AcHN
H O
H O
H O
O
H O
O
O OBn
OSO3
OH
COOMe
O
O
O
O
OH
OH
OH
MeOOC OH HO
HO
HO
O
COOMe HO
O
AcHN
AcHN
O HO
O
O
O
HO
O
O
O
O
HO 265
HO
OSO3
OH
OH
OH
HO

+

H
N

AcHN
O
H2N

N
H

H
N
O

O
N
H

H
N

O

H
N

N

O

O

O

250

N
H

OBn
H
N
O

O

O
N
H

OH
O

O

O
N
H

O
O

OBn
O

O OBn

Figure 3.21. Retrosynthetic analysis of glycopeptide 233.
To synthesize glycopeptide 265, we first did a model study (Scheme 3.58). We
tried to selectively remove Ac and Bz in the presence of methyl ester. After extensive

264

screening, we found using NaOMe in MeOH (pH 9.5) the reaction went to completion
after 32 hours. The progress of the reaction was monitored by mass spectroscopy.

Scheme 3.58. Synthesis of octasaccharide 266. Reagents and conditions: (a) NaOMe,
MeOH, pH = 9.5.
Encouraged by this result, we then tried synthesizing compound 266 under the
same condition. As we expected, partially deprotected glycopeptide 266 was obtained in
78% yield after 40 hours.

265

Scheme 3.59. Synthesis of Glycopeptide 265. Reagents and conditions: (a) NaOMe,
MeOH, pH = 9.5.
With glycopeptide 266 in hand, the next step was peptide coupling with
compound 250. It has been shown in literature that sulfates present in glycopeptide do
not affect peptide coupling reactions.

90

As we expected, this coupling produced

glycopeptide 264 in 60% yield. Subsequent hydrogenation and hydrolysis afforded
product 233. The pH of the hydrolysis was maintained around 9.5 by LiOH solution and
the progress of the reaction was monitored by mass spectroscopy.

266

OSO3
OH
COOMe
O
O
OH
O
O
OH
OH
HO
MeOOC OH HO
HO
HO
O
O
COOMe
AcHN
AcHN
O HO
O
O
O
HO
O
266
O
O
O
BzO
HO
OSO3
OH
OH
OH
HO

a 60%

H
N

OH
O

O

O

N CO2
AcHN
OSO3
H
OH
O
COOMe
O
O
O
OH O
OH
OH
MeOOC OH HO
HO
HO
O
COOMe HO
OH
O
AcHN
AcHN
O HO
O
OR1
O
O
HO
O
O
O
O
264
O
HO
HO
OSO3
OH
OH
OH
R1 =
OBn
HO
H O
O
H O
H O
O
H O
H
OBn
H
N
N
N
N
N
N
N
N
N
N
N
N
H O
AcHN
H O
H O
H O
O
H O
O
b
O OBn
OSO3
OH
COOMe
O
O
O
O
OH
OH
OH
MeOOC OH HO
HO
HO
O
COOMe HO
O
AcHN
AcHN
O HO
O
O
O
HO
O
O
O
275
O
HO
HO
OSO3
OH
OH
OH

OH
O

OR2

c 52% over two steps
OSO3
OH
COO
O
O
O
OH O
OH
OH
OOC OH HO
HO
HO
HO
O
COO
O
OH
AcHN
AcHN
O HO
O
OR2
O
O
HO
O
O
O
O
O
HO
233
HO
OSO3
OH
OH
OH
R2 =
OH
HO
H O
H O
H O
O
H O
H O
O
H
N
N
N
N
N
N
N
N
N
N
N
N
H O
AcHN
H O
H O
H O
O
H O
O
O O

Scheme 3.60. Synthesis of glycopeptide 233. Reagents and conditions: (a) HATU, 250,
2, 4, 6-collidine, DMF; (b) H2, Pd/C, DCM/MeOH; (c) LiOH, MeOH/H2O, pH = 9.5.
3.14. Conclusions

267

For the first time we chemically synthesized HS octasaccharide 226 and HS
glycopeptide 24, which are partial structures of HSPG. Synthesis of glycopeptide 233 is
even more challenging and the synthetic route used for compounds 226 and 24 cannot
be simply extended to the assembly of 233. Alternative route has been developed for
assembling the glycopeptide 233. Our success in making these glycopeptides lays the
foundation for synthesizing our final target HSPG 1.
3.15. Experimental Section
3.15.1. General experimental procedures
All reactions were carried out under nitrogen with anhydrous solvents in flame-dried
glassware, unless otherwise noted.

Glycosylation reactions were performed in the

presence of molecular sieves, which were flame–dried right before the reaction under
high vaccum. Glycosylation solvents were dried using a solvent purification system and
used directly without further drying. Chemicals used were reagent grade as supplied
except where noted. Compounds were visualized by UV light (254 nm) and by staining
with a yellow solution containing Ce(NH4)2(NO3)6 (0.5 g) and (NH4)6Mo7O24 4H2O
(24.0 g) in 6% H2SO4 (500mL). Flash column chromatography was performed on silica
1

gel 60 (230-400 Mesh). NMR spectra were referenced using residual CHCl3 (δ H-NMR
7.26 ppm,
1

13

C-NMR 77.0 ppm). Peak and coupling constants assignments are based

1

1

1

13

1

13

on H-NMR, H- H gCOSY and (or) H- C gHMQC and H- C gHMBC experiments.
o

All optical rotations were measured at 25 C using the sodium D line.
3.15.2. Characterization of anomeric stereochemistry

268

The stereochemistries of the newly formed glycosidic linkages were determined
3

1

by JH1,H1 through H-NMR and/or

1

1

JC1,H1 through gHMQC 2-D NMR (without H
3

decoupling). Smaller coupling constants of JH1,H2 (around 3 Hz) indicate α linkages
3

and larger coupling constants JH1,H1 (7.2 Hz or larger) indicate β linkages.
around 170 Hz suggests α linkages and 160 Hz suggests β linkages.

1

JC1,H1

91

3.15.3. General procedure for pre-activation based single-step glycosylation.
A solution of donor (60 μmol) and freshly activated molecular sieve MS 4 Å
(200 mg) in DCM (2 mL) was stirred at room temperature for 30 minutes, and cooled to
−78 °C, which was followed by addition of AgOTf (47 mg, 180 μmol) dissolved in Et2O
(1 mL) without touching the wall of the flask. After 5 minutes, orange colored p-TolSCl
(9.5 μL, 60 μmol) was added to the solution through a microsyringe. Since the reaction
temperature was lower than the freezing point of p-TolSCl, p-TolSCl was added directly
into the reaction mixture to prevent it from freezing on the flask wall. The characteristic
yellow color of p-TolSCl in the reaction solution dissipated rapidly within a few seconds
indicating depletion of p-TolSCl. After the donor was completely consumed according
to TLC analysis (about 5 minutes at −78 °C), a solution of acceptor (54 μmol) in DCM
(0.2 mL) was slowly added dropwise via a syringe together with one equivalent of TTBP.
The reaction mixture was warmed to 0 °C under stirring in 2 h. Then the mixture was
diluted with DCM (20 mL) and filtered over Celite. The Celite was further washed with
DCM until no organic compounds were observed in the filtrate by TLC. All DCM
solutions were combined and washed twice with a saturated aqueous solution of
269

NaHCO3 (20 mL) and twice with water (10 mL). The organic layer was collected and
dried over Na2SO4. After removal of the solvent, the desired oligosaccharide was
purified from the reaction mixture via silica gel flash chromatography.
3.15.4. General procedure for protection of 6-OH with Lev
The compound containing 6-OH (1 equiv.) was dissolved in DCM (for 0.5 g of
compound, 5 mL), followed by addition of levulinoyl acid (1.4 equiv.), EDC-HCl (1.6
equiv.) and DMAP (0.1 equiv.). The mixture was stirred at room temperature overnight
and then was diluted with DCM (100 mL). The organic phase was washed with
saturated NaHCO3 and then dried over Na2SO4. The solvent was concentrated in
vacuo and the compound was purified by silica gel column chromatography
3.15.5. General procedure for deprotection of Lev
The Lev-protected compound (1 equiv.) was dissolved in DCM/MeOH (for 150
mg of compound, 2.4 mL, 1:1) and acetic acid (0.2 mL). The mixture was cooled to 0
o

C, followed by addition hydrazine monohydrate (5 equiv. for each Lev). The mixture

was stirred at 0 °C for 2h and then was quenched by acetone (0.28 mL). The mixture
was stirred at room temperature for another 1h and the acetone was evaporated under
vaccum. The residue was diluted with EtOAc (50 mL) and washed with saturated
NaHCO3, 10% HCl and water and the organic phase was dried over Na2SO4. The
solvent was concentrated in vacuo and the compound was purified by silica gel column
chromatography.
3.15.6. General procedure for O-sulfation

270

The mixture of OH-containing compound (for 20 mg of compound, 1 equiv.),
DMF (1 mL) and SO3-NEt3 (20 equiv. per OH) was stirred at 55 °C for 24 h. The
mixture was cooled to room temperature and then diluted with DCM/MeOH (1mL/1mL).
The resulting solution was layered on the top of Sephadex LH-20 chromatography
column that was eluted with DCM/MeOH (1/1, v/v). The fractions containing product
were combined and evaporated to dryness under vacuo without further purification.
3.15.7. General procedure for global debenzylation
The mixture of the Bn/PMB-containing compound (for 3 mg of compound, 1
equiv.), DCM/MeOH (1 mL/1 mL) and Pd/C (15 mg) was stirred under H2 at room
temperature overnight and then filtered via PTFE membrane (pore size 0.22 μm). The
filtrate was concentrated to dryness under vacuum and then diluted with H2O (15 mL).
The aqueous phase was further washed with DCM (5 mL × 3) and MeOH (5 mL×3) and
then the aqueous phase was dried under vacuum. The crude product was further
purified by Sephadex LH-20 chromatography column. The fractions containing product
were combined and evaporated to dryness under vacuo without further purification.
3.15.8. General procedure for solid phase peptide synthesis
Amino acids were purchased from Chem-impex. H-Gly-2-CTrt Resin was purchased
from Advanced ChemTech (loading level: 0.6 mmol/g). Reaction vessels (10 mL,
disposable) and the Domino Block Synthesizer were purchased from Torvig.
(1) Pause Point:
If it is necessary to pause the synthesis, after each coupling-washing procedure, the
resin is washed with DCM five times and dried with nitrogen gas at room temperature.

271

The syringe was closed with a plunger and a cap, and stored at < 4 °C. Before
resuming the synthesis, the sample was allowed to reach room temperature, and the
dry resin was swelled as described in the following.
(2) Select the right syringe:
The optimal available volume for 6 mL syringe is < 4.8 mL;
The optimal available volume for 12 mL syringe is < 8.0 mL;
The concentration of amino acid used for coupling typically is 0.2 ~ 0.4 M;
The final volume of swelling resin, solvent, coupling reagent need to be considered
before choosig the right syringe. As an example for 200mg H-Gly-2-ClTrt-Resin with 10
eq Amino acid, 9.9 eq HBTU, 20 eq DIPEA, the final reaction volume is about 6 ~ 7 mL.
Therefore, the 12 mL syringe should be used.
(3) Reagent:
HBTU: MW 379.24
223 mg (4.9 eq) for 200 mg resin (0.6 mmol/g);
451 mg (9.9 eq) for 200 mg resin (0.6 mmol/g);
DIPEA: MW 129.24

d 0.742

0.21 mL (10 eq) for 200 mg resin (0.6 mmol/g);
0.42 mL (20 eq) for 200 mg resin (0.6 mmol/g).
DIPEA usually is 2 eq;
20% Piperidine in DMF:
20 mL piperidine + 80 mL DMF

10 mL/ g resin

Capping Reagent:
10 mL Ac2O + 10 mL DIPEA + 80 mL DMF

272

Kaiser Reagent:
80% Phenol in EtOH (W/V);
2 mL 0.001M KCN + 98 mL Pyridine;
5% Ninhydrin in EtOH (W/V).
(4) Standard Washing Procedure:
This procedure is performed when Fmoc is removed or the coupling reaction is finished.
(a) Push the plunger to remove the reaction mixture, and then pull it back to right
position;
(b) Put the syringe in the plate;
(c) Fill the syringe with DMF;
(d) Take the plate out of the shaker, carefully wash the plunger and edge of the
syringe with DMF, shake for 10s, and remove the solvent by filtration;
(e) Repeat steps 1 and 2 three times with DMF;
(f) Repeat steps 1 and 2 three times with DCM;
(g) Repeat steps 1 and 2 three times with DMF.
Section 1: Resin Swelling
(a) Place the dry resin 200mg in the syringe;
(b) Fill the reactor with DCM until all the resin beads are immersed;
(c) Shake for 30 min;
(d) Remove DCM by filtration by vacuum.
Section 2: Removal of Fmoc
(a) The swelled resin is washed once with DMF;
(b) Fill the syringe with 20% piperidine in DMF about 3 ml and shake for 30 min;

273

(c) Remove the DMF/piperidine solution by pushing the plunger;
(d) Repeat steps 2 and 3 once;
(e) Repeat the standard washing procedure once;
Section 3: Coupling Reaction
(a) Mix the Fmoc protected amino acid (5 eqiv.), DIPEA (10 eqiv.), and HBTU (4.9
eqiv.) with DMF in a dry vial. The reaction mixture is then transferred to syringe
when all the compounds are completely dissolved;
(b) Shake the reaction for 2 h;
(c) Repeat standard washing procedure;
(d) Check the resin with Kaiser Method to make sure no free amine is left; Repeat
steps 1 and 2 if necessary.
Section 4: Capping
(a) Fill the syringe with the capping reagent, and shake for 15 min;
(b) Remove the capping solution by pushing the plunger and repeat step 1 for 15
min;
(c) Repeat the standard washing procedure;
Section 5: Final Cleavage
(a) Weigh the resin and place it in a round bottom flask. When the resin is dry, swell
it as described in section 1;
(b) Add 10 mL of cleavage cocktail (TFA/H2O/Phenol/TIPS 8.5/0.5/0.5/0.5) per
100mg of resin, stir gently for 2h;
(c) Filter the resin and wash it twice with fresh cleavage cocktail. Recover the filtrate
in a round-bottom flask;
274

(d) Concentrate the cleavage cocktail in vacuum to approximately ¼ of its original
volume;
(e) Under vigorous stirring, add cold MTBE to precipitate the peptide. At least 10
times the initial TFA volume of MTBE should be added to precipitate the
unprotected peptide. When the peptide does not precipitate, concentrate the
solution in vacuum and go directly to step 8;
(f) Filter out the precipitate;
(g) Triturate and wash by filtration the precipitated peptide three times with MBTE;
(h) Solubilize the peptide in CH3CN/H2O/TFA 50/50/0.1 and lyophilize. Solvent used
for this step can be changed to increase solubility. The crude peptide is used for
HPLC analysis.
3.15.9. General procedure for HPLC analysis
(1) Preparation of Sample:
(a) Transfer a sample containing ~ 1- 2 mg dry peptide-resin to a small syringe (2
ml);
(b) Add 300 μl of the cleavage cocktail to the dried peptide resin, stir for 3h;
(c) Collect the solution in a small HPLC vial, dilute with 400 μl ACN/H2O 1/1 and mix;
(d) At this point, the solution can be analyzed in an analytical HPLC system (inject
20 μl) and /or further diluted (1/10) to be injected (2 μl) in liquid chromatographmass spectrometer.
(2) Preparation of HPLC Solvent:
(a) Eluent A: weak mobile phase solvent.
Case A: 0.1% TFA in H2O (1 ml TFA + 1 L H2O)
275

Case B: 0.12 % TFA in H2O (1.2 ml TFA + 1 L H2O)
(b) Eluent B: strong mobile phase solvent.
Case A: 80% ACN / 0.1% TFA
or 0.085% TFA/ACN (v/v)
Case B: 1.0 mL TFA + 700 ml ACN + 300 ml H2O
(3) Choice of HPLC Column:
(a) low picomole amount of peptide: 0.21 cm × 25 cm , 0.3 ml/min
(b) < 1 mg peptide: 0.46 cm × 25 cm , 1.0-1.5 ml/min
(c) 1.0-10.0 mg peptide: 1.0 cm × 25 cm , 2.0 ml/min
(d) >10 mg peptide: 2.2 cm × 25 cm , 6.0-10.0 ml/min
(4) Column Preparation:
(a) Eluent B, 2 ml/min, 20 min;
(b) Decrease Eluent B to 0% over 10 min using a linear gradient;
(c) Increase Eluent B to 100% over 10 min using a linear gradient;
(d) Repeat 2;
(e) Equilibrate the column with Eluent A for 20 min.
(5) Detector: 214 nm
3.15.10 Detailed experimental procedures and NMR, MS data
p-Tolyl 2-azido-3, 6-di-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-1-thio-βD-glucopyranoside (41).

34

Trichloroethoxycarbonyl chloride (7.7 mL, 57.2 mmol) was

added dropwise over a period of 1 hour at room temperature to a vigorously stirred
solution of D-glucosamine hydrochloride 32 (10 g, 46.3 mmol) and NaHCO3 (11.8 g,
276

139.9 mmol) in water (92 mL). The mixture was stirred for another 2 hours and then
filtered to give a yellowish solid, which was dried under vacuum. The obtained crude
solid 33 (17.76 g) was dissolved in pyridine (60 mL) and then acetic anhydride (30 mL)
was added at 0

°C

over a period of 30 minutes. The mixture was stirred at room

temperature under N2 overnight and then quenched with ethanol (20 mL) at 0 °C. The
mixture was concentrated and the resulting residue was diluted with ethyl acetate and
washed with saturated aqueous solution of NaHCO3, 10 % HCl, water, and brine. The
organic phase was dried over Na2SO4, filtered and concentrated. Without separation,
the obtained crude solid 34 (78% over two steps) and p-toluenethiol (6.34 g, 51 mmol)
were dissolved in DCM (50 mL) and the solution was cooled to 0 °C. Tin chloride (21.8
mL, 185 mmol) was added dropwise at 0 °C and the mixture was stirred under N2 at
room temperature overnight. The mixture was diluted with DCM (400 mL) and washed
with saturated aqueous solution of NaHCO3 until the pH is 7 and then dried over
Na2SO4, filtered and concentrated. The obtained crude product was recrystallized from
EtOAc/hexanes

to

afford

p-tolyl

3,

4,

6-tri-O-acetyl-2-deoxy-2-N-

trichloroethoxycarbonylamino-1-thio-D-glucopyranoside 35 as white solid (16.6 g, 81%).
1

H-NMR (500 MHz, CDCl3): δ 2.00-2.10 (m, 10 H), 2.20 (s, 1 H), 2.34 (s, 3 H), 3.63-

3.71 (m, 2 H), 4.00-4.34 (m, 3 H), 4.60-4.84 (m, 4 H), 5.01 (t, 1 H, J = 10 Hz), 5.19-5.29
(m, 2 H), 7.10-7.13 (m, 2 H), 7.40-7.42 (m, 2 H). Compound 35 (10 g, 17 mmol) was
dissolved in THF (40 mL), H2O (40 mL). NaOH powder (8.2 g, 205 mmol) was added

277

slowly at 0 °C and the mixture was stirred under N2 at room temperature overnight. The
mixture was neutralized by 10% HCl and concentrated to dryness. Silica gel column
chromatography (DCM/MeOH) purification of the resulting residue afforded p-tolyl 2amino-2-deoxy-1-thio-β-D-glucopyranoside 36 as white solid (3.9 g, 80%).

1

H-NMR

(500 MHz, CD3OD): δ 2.26 (s, 3 H, SPhCH3), 2.67 (t, 1 H, J = 9.6 Hz, H-2), 3.20-3.34
(m, 2 H), 3.59-3.63 (m, 1 H), 3.78-3.82 (dd, 1 H, J = 2.0 Hz, J = 12.0 Hz), 4.15-4.18 (m,
1 H), 4.53 (d, 1 H, J = 10 Hz, H-1), 7.08-7.11 (m, 2 H), 7.42-7.44 (m, 2 H). Compound
36, K2CO3 (5 g, 36.2 mmol) and catalytic amount of ZnCl2 (40 mg, 0.8 mmol) were
dissolved in MeOH (40 mL) and H2O (10 mL). Freshly prepared TfN3 (60 mL in DCM,
51 mmol) was added and the mixture was stirred at room temperature overnight. The
solvent was evaporated and the resulting residue was diluted with EtOAc (300 mL).
The mixture was neutralized by conc. HCl until the pH value is 6-7 and then
1

concentrated to dryness to afford the crude solid 37. H-NMR (500 MHz, CD3OD): δ
2.31 (s, 3 H, SPhCH3), 3.11-3.15 (m, 1 H), 3.30-3.34 (m, 2 H), 3.40-3.44 (m, 1 H), 3.693.73 (m, 1 H), 3.75-3.79 (m, 1 H), 3.86-3.89 (m, 1 H), 4.46-4.48 (d, 1 H, J = 10 Hz, H-1),
7.13-7.15 (m, 2 H), 7.46-7.48 (m, 2 H). Compound 37, camphorsulfonic acid (0.54 g, 3.4
mmol) and benzaldehyde dimethylacetal (1.92 mL, 18.7 mmol) in acetonitrile (30 mL)
was stirred at room temperature overnight. The reaction was quenched by addition of
Et3N and then diluted with EtOAc (200 mL). The organic phase was washed with
saturated aqueous solution of NaHCO3, water and then dried over Na2SO4, filtered and

278

concentrated. Silica gel column chromatography (2:1 Hexanes–EtOAc) afforded p-tolyl
2-azido-4,6-O-benzylidene-2-deoxy-1-thio-β-D-glucopyranoside 38 as white solid (3.6 g,
73% for two steps).

1

H-NMR (500 MHz, CDCl3): δ 2.35 (s, 3 H, SPhCH3), 3.28-3.32 (m,

1 H), 3.40-3.46 (m, 1 H), 3.72-3.76 (m, 2 H), 4.33-4.37 (m, 1 H), 4.46 (d, 1 H, J = 10 Hz,
H-1), 5.50 (s, 1H, CHPh), 7.13-7.16 (m, 2 H), 7.33-7.46 (m, 9 H). Compound 38 (3.5 g,
8.76 mmol) was dissolved in DMF (50 mL) and the solution was cooled to 0 °C. NaH
(0.46 g, 60% NaH in mineral oil, 11.4 mmol) was added in portions, followed by addition
of benzyl bromide (1.36 mL, 11.4 mmol). The mixture was stirred at room temperature
under N2 for 2 h and then diluted with EtOAc (200 mL). The mixture was washed with
saturated NaHCO3, water and then dried over Na2SO4, filtered and concentrated.
Silica gel column chromatography (2:1 Hexanes–EtOAc) afforded p-tolyl 2-azido-3-Obenzyl-4,6-O-benzylidene-2-deoxy-1-thio-β-D-glucopyranoside 39 as white solid (4.3 g,
92%).

1

H-NMR (500 MHz, CDCl3): δ 2.34 (s, 3H, SPhCH3), 3.28-3.32 (m, 1 H), 3.40-

3.45 (m, 1 H, H-3), 3.56-3.65 (m, 2 H, H-6a, H-6b), 3.76 (t, 1 H, J = 10 Hz, H-2), 4.354.37 (m, 1 H, H-4), 4.41 (d, 1 H, J = 10.5 Hz, H-1), 4.76 (d, 1 H, J = 11 Hz, PhCH2),
4.89 (d, 1 H, J = 11 Hz, PhCH2), 5.54 (s, 1 H, CHPh), 7.12-7.14 (m, 2 H), 7.26-7.39 (m,
8 H), 7.42-7.46 (m, 4 H). Compound 39 (4 g, 8.2 mmol) and Et3SiH (13.3 mL, 82 mmol)
in DCM (40 mL) together with MS AW 300 were added TFA (6.6 mL, 82 mmol). The
o

reaction mixture was stirred under N2 at 0 C for 1 hour and quenched with NaHCO3.
The product was extracted with DCM (x2) and the organic phase was washed with brine,

279

dried

(Na2SO4)

and

concentrated

for

flash

column

chromatography

(hexane/DCM/EtOAc). Compound 40 was obtained as a colorless viscous oil (4.1 g,
1

98%). H-NMR (500 MHz, CDCl3): δ 2.33 (s, 3H, SPhCH3), 2.83 (d, 1 H, J = 3 Hz, OH),
3.28 (t, 1 H, J = 9.5 Hz,H-6a), 3.36 (t, 1 H, J = 9.5 Hz, H-6b), 3.41-3.45 (m, 1 H), 3.583.61(m, 1 H), 3.73-3.80 (m, 2 H), 4.37 (d, 1 H, J = 10 Hz, H-1), 4.54-4.61 (m, 2 H,
PhCH2O), 4.81-4.90 (m, 2 H, PhCH2O), 7.07-7.10 (m, 2 H), 7.29-7.40 (m, 9 H), 7.477.50 (m, 2 H). Compound 40 (4 g, 8.15 mmol) in DCM (50 mL) was cooled down to –40

°C,

followed by sequential addition of 2, 6-lutidine (1.9 mL, 16.3 mmol) and TBSOTf

(2.8 mL, 12.2 mmol).

The resulting solution was warmed up very slowly to room

temperature. The mixture was quenched by Et3N and then diluted with DCM (100 mL).
The organic phase was washed with saturated NaHCO3 and then dried over Na2SO4,
filtered and the solvents were removed in vacuo. Silica gel column chromatography
(Hexanes–EtOAc) afforded compound 41 as white solid (4.2 g, 85%).

1

H-NMR

(500 MHz, CDCl3): δ 0.85 (s, 9 H, C(CH3)3), 2.30 (s, 3H, SPhCH3), 3.23-3.30 (m, 2 H),
3.37-3.41 (m, 1 H), 3.54-3.63 (m, 2 H), 3.72-3.75 (dd, 1 H, J = 2.5 Hz, J = 11 Hz), 4.394.41 (m, 1 H), 4.50-4.61 (m, 2 H, PhCH2O), 4.73-4.87 (m, 2 H, PhCH2O), 7.00-7.02 (m,
2 H), 7.24-7.36 (m, 10 H), 7.46-7.49 (m, 2 H).
2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-1-thio-β-D-

p-Tolyl
34

glucopyranoside (48).

β-D-Glucopyranosyl pentaacetate 42 (10 g, 25.64 mol), p-

toluenethiol (3.62 g, 29 mmol) were dissolved in DCM (100 mL) and boron trifluoride
280

etherate (10.1 mL, 75 mmol) was added dropwise at room temperature. The mixture
was stirred under N2 at room temperature for 20 hours and then diluted with DCM (200
mL). The organic phase was washed with saturated aqueous solution of NaHCO3 until
the pH is 7 and then dried over Na2SO4, filtered and concentrated to afford crude
1

product 43, which was recrystalized from hexane/EtOAc. H-NMR (500 MHz, CDCl3): δ
1.94 (s, 3 H, COCH3), 1.97 (s, 3 H, COCH3), 2.04 (s, 3 H, COCH3), 2.05 (s, 3 H,
COCH3), 2.31 (s, 3H, SPhCH3), 3.64-3.68 (m, 1 H), 4.12-4.20 (m, 2 H), 4.60 (m, 1 H, J
= 10 Hz, H-1), 4.87-4.91 (m, 1 H), 4.96-5.00 (m, 1 H), 5.17 (t, 1 H, J = 9 Hz), 7.07-7.09
(m, 2 H), 7.34-7.36 (m, 2 H). Compound 43 (10.7g, 92%) was dissolved in MeOH (50
mL) and DCM (50 mL). 5.4 M NaOMe (19 mL, 0.1 mol) was added and the mixture was
stirred at room temperature overnight. The mixture was neutralized by conc. HCl until
the pH is around 7 and then concentrated and dried under vacuum. Silica gel column
chromatography (9:1 DCM–MeOH) afforded p-tolyl 1-thio-β-D-glucopyranoside 44 as
white solid (6.75 g, quantitative).

1

H-NMR (500 MHz, CD3OD): δ 2.21 (s, 3 H, SPhCH3),

3.04-3.08 (m, 1 H), 3.13-3.18 (m, 2 H), 3.24-3.27 (m, 2 H), 3.53-3.57 (m, 1 H), 3.73-3.76
(m, 1 H), 4.40 (d, 1 H, J1,2 = 10 Hz, H-1), 7.01-7.03 (m, 2 H), 7.34-7.36 (m, 2 H). The
mixture of compound 44 (6.75 g, 23.6 mmol), p-anisaldehyde dimethylacetal (10.8 mL,
28.3 mmol) and camphorsulfonic acid (2.4 g, 10.6 mmol) in anhydrous DMF (80 mL)
was swirled on a rotary evaporator under aspirator pressure at 50 °C for 3 h, then was
warmed up to 70 °C to remove methanol generated until the reaction was completed
281

judging by TLC. The resulting solution was diluted with diethyl ether (200 mL) followed
by washing with saturated aqueous solution of NaHCO3, water and then dried over
Na2SO4, filtered and concentrated. Silica gel column chromatography (1:1 Hexanes–
EtOAc) afforded p-tolyl 4,6-O-p-methoxybenzylidene-1-thio-β-D-glucopyranoside 45 as
white solid (6.86 g, 72%).

1

H-NMR (500 MHz, CDCl3): δ 2.34 (s, 3H, SPhCH3), 2.60 (d,

1 H, J = 2.0 Hz, OH), 2.71 (d, 1 H, J = 2.0 Hz, OH), 3.38-3.48 (m, 3 H), 3.71-3.82 (m, 5
H), 4.32-4.35 (m, 1 H), 4.54 (d, 1 H, J = 9.5 Hz, H-1), 5.46 (s, 1 H, CH3OPhCH), 6.856.87 (m, 2 H), 7.12-7.14 (m, 2 H), 7.36-7.42 (m, 4 H). Compound 45 (6.86 g, 16.98
mmol) was heated under reflux with dibutyltin oxide (4.24 g, 16.98 mmol) in a flask
equipped with a Dear-Stark device in anhydrous toluene (200 mL) for 3 h and then
concentrated to approximate 100 mL. After cooling the reaction mixture down to room
temperature, anhydrous DMF (100 mL) was added followed by addition of CsF (2.85 g,
18.75 mmol) and BnBr (2.24 mL, 18.75 mmol). The mixture was stirred for 4 h at 140

°C.

After the reaction was complete, DMF was removed under reduced pressure. The

residue was dissolved in DCM and extracted by 1 M aqueous solution of KF. The
organic phase was dried over Na2SO4, filtered and concentrated. Silica gel column
chromatography (5:1:1 hexane–EtOAc–DCM) afforded p-tolyl 3-O-benzyl-4,6-O-pmethoxybenzylidene-1-thio-β-D-glucopyranoside 46 as white solid (6.54 g, 78%).

1

H-

NMR (500 MHz, CDCl3): δ 2.32 (s, 3 H, SPhCH3), 2.50 (d, 1 H, OH), 3.43-3.48 (m, 2 H,
H-2, H-5), 3.57-3.67 (m, 2 H, H-6a, H-6b), 3.72-3.76 (m, 1 H, H-4), 3.79 (s, 3 H, OCH3),

282

4.33 (dd, 1 H, J = 5 Hz, J = 10.5 Hz, H-3), 4.54 (d, 1 H, J1,2 = 9.5 Hz, H-1), 4.76 (d, 1 H,
J = 12 Hz, CH2Ph), 4.91 (d, 1 H, J = 11.5 Hz, CH2Ph), 5.50 (s, 1 H, CHPh), 6.86-6.89
(m, 2 H), 7.09-7.11 (m, 2 H), 7.24-7.42 (m, 9 H). Compound 46 (6.54 g, 13.24 mmol)
was treated with benzoyl chloride (1.85 mL, 15.8 mmol), DMAP (3.83 g, 31.7 mmol) in
anhydrous DCM (50 mL) overnight at room temperature. The reaction mixture was
washed with saturated aqueous solution of NaHCO3, water and then dried over
Na2SO4, filtered and concentrated. Silica gel column chromatography (4:1:1 Hexanes–
EtOAc–DCM) afforded p-tolyl 2-O-benzoyl-3-O-benzyl-4,6-O-p-methoxybenzylidene-11

thio-β-D-glucopyranoside 47 as white solid (6.34 g, 80%). H-NMR (500 MHz, CDCl3): δ
2.30 (s, 3 H, SPhCH3), 3.54 (dt, 1 H, J = 5 Hz, J = 10 Hz, H-5), 3.74-3.86 (m, 3 H, H-3,
H-6a. H-6b), 3.80 (s, 3 H, OCH3), 4.37 (dd, 1 H, J = 4.5 Hz, 10.5 Hz, H-4), 4.63 (d, 1 H,
J = 12.0 Hz, OCH2Ph), 4.76 (d, 1 H, J1,2 = 10 Hz, H-1), 4.77 (d, 1 H, J = 12.0 Hz,
OCH2Ph), 5.21-5.25 (t, 1 H, H-2), 5.54 (s, 1 H, CHPh), 6.88-6.91 (m, 2 H), 7.02-7.11 (m,
7 H), 7.30-7.32 (m, 2 H), 7.38-7.41 (m, 2 H), 7.43-7.47 (m, 2 H), 7.57-7.60 (m, 1 H),
7.99-8.01 (m, 2 H). Compound 47 (6.34 g, 10.6 mmol) in anhydrous DMF (72 mL) was
cooled down to 0 °C, followed by sequential addition of solid NaCNBH3 (5.32 g, 85.01
mmol) and TFA (7.88 mL, 106 mmol). The resulting suspension was stirred for 48 h
until no starting material was left. After neutralization by solid NaHCO3, the solution
was filterer and diluted with EtOAc, followed by washing with saturated aqueous
solution of NaHCO3, water and then dried over Na2SO4, filtered and concentrated.
283

Silica gel column chromatography (3:1 Hexanes–EtOAc) afforded p-tolyl 2-O-benzoyl-3O-benzyl-6-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside 48 as white solid (5.4 g,
85%).

1

H-NMR (500 MHz, CDCl3): δ 2.29 (s, 3 H, SPhCH3), 2.92 (d, 1 H, J = 2.5 Hz,

OH), 3.54-3.57 (m, 1 H, H-6b), 3.66-3.70 (m, 1 H, H-3), 3.73-3.80 (m, 6 H, H-4, H-5, H-6,
OCH3), 4.48-4.53 (m, 2 H, CH2Ph), 4.68-4.71 (m, 2 H, CH2Ph), 4.74 (d, 1 H, J1,2 = 10
Hz, H-1), 5.24 (t, 1 H, J = 9.5 Hz, H-2), 6.88-6.90 (m, 2 H), 7.01-7.03 (m, 2 H), 7.13-7.16
(m, 5 H), 7.24-7.27 (m, 2 H), 7.36-7.38 (m, 2 H), 7.44-7.48 (m, 2 H), 7.57-7.61 (m, 1 H),
8.06-8.08 (m, 2 H).
p-Tolyl 2-O-benzoyl-4, 6-di-O-benzyl-1-thio-β-D-galactopyranoside (57). β-DGalactopyranosyl pentaacetate 49 (10 g, 25.64 mol), p-toluenethiol (3.62 g, 29 mmol)
were dissolved in DCM (100 mL) and boron trifluoride etherate (10.1 mL, 75 mmol) was
added dropwise at room temperature.

The mixture was stirred under N2 at room

temperature for 20 hours and then diluted with DCM (200 mL). The organic phase was
washed with saturated aqueous solution of NaHCO3 until the pH is 7 and then dried
over Na2SO4, filtered and concentrated to afford crude product 50, which was
1

recrystalized from hexane/EtOAc. H-NMR (500 MHz, CDCl3): δ 1.95 (s, 3 H, COCH3),
2.02 (s, 3 H, COCH3), 2.07 (s, 3 H, COCH3), 2.09 (s, 3 H, COCH3), 2.32 (S, 3 H,
SPhCH3), 3.86-3.90 (m, 1 H), 4.07-4.18 (m, 2 H), 4.62 (d, 1 H, J1,2 = 10 Hz, H-1), 5.005.03 (m, 1 H), 5.19 (t, 1 H, J1,2 = 10 Hz ), 5.38 (dd, 1 H, J = 1 Hz, J = 3.5 Hz), 7.09-7.11
(m, 2 H), 7.38-7.40 (m, 2 H). Compound 50 (9.89 g, 85%) was dissolved in MeOH (50
mL) and DCM (50 mL). 5.4 M NaOMe (19 mL, 0.1 mol) was added and the mixture was
284

stirred at room temperature overnight. The mixture was neutralized by conc. HCl until
the pH is around 7 and then concentrated and dried under vacuum. Silica gel column
chromatography (9:1 DCM–MeOH) afforded p-tolyl 1-thio-β-D-galactopyranoside 51 as
1

white solid (5.86 g, 94%). H-NMR (500 MHz, CD3OD): δ 2.29 (s, 3 H, SPhCH3), 3.453.48 (m, 1 H), 3.51-3.53 (m, 1 H), 3.56 (t, 1 H, J = 4.5 Hz), 3.67-3.76 (m, 2 H), 3.87 (dd,
1 H, J = 1 Hz, J = 3.5 Hz), 4.49 (d, 1 H, J1,2 = 9.5 Hz ), 7.09-7.11 (m, 2 H), 7.43-7.45 (m,
2 H). Compound 51 with dibutyltin oxide (6.12 g, 24.6 mmol) in a flask equipped with a
Dear-Stark device in anhydrous toluene and THF (200 mL) for 3 h and then
concentrated. After cooling the reaction mixture down to room temperature, anhydrous
THF (100 mL) was added followed by addition of Bu4NBr (3.42 g, 22.5 mmol) and AllBr
(2.69 mL, 22.5 mmol). The mixture was stirred for 4 h under reflux. After the reaction
was complete, THF was removed under reduced pressure. The resulting residue was
purified by silica gel column (1:1, hexane-EtOAc) to afford p-tolyl 3-O-allyl-1-thio-β-D1

galactopyranoside 52 (4.67 g, 70%). H-NMR (500 MHz, CD3OD): δ 2.30 (s, 3 H,
SPhCH3), 3.13-3.34 (m, 1 H), 3.65-3.78 (m, 3 H, H-2, H-6a, H-6b), 4.06-4.07 (m, 1 H,
H-4), 4.13-4.25 (m, 2 H, CH2CHCH2O), 4.52 (d, 1 H, J1,2 = 9.5 Hz, H-1), 5.14-5.17 (m,
1 H, CH2CHCH2O), 5.30-5.35 (m, 1 H, CH2CHCH2O), 5.96-6.01 (m, 1 H,
CH2CHCH2O), 7.10-7.12 (m, 2 H), 7.44-7.46 (m, 2 H).

13

C-NMR (125 MHz, CD3OD): δ

13.8, 20.8, 21.1, 26.8, 54.1, 62.6, 67.3, 70.0, 71.8, 80.3, 83.5, 90.5, 117.3, 130.5, 131.8,
+

133.0, 136.4, 138.4. HRMS: C16H22O5S [M+NH4] calcd: 344.1532, obsd: 344.1542.
Compound 52 (4.67 g, 14.34 mmol) was dissolved in CH3CN (100 mL) followed by
285

addition of camphorsulfonic acid (999 mg, 4.302 mmol) and benzaldehyde dimethyl
acetal (3.24 mL, 21.51 mmol). The resulting mixture was stirred under room
temperature overnight. After the reaction was complete, it was quenched by Et3N and
diluted with EtOAc (100 mL) and the organic phase was extracted by sat. NaHCO3
solution and dried over Na2SO4. After concentration, the residue was purified by silica
gel column (5:1:1, hexanes-DCM-EtOAc) to afford p-tolyl 3-O-allyl-4, 6-O-benzylidene1

1-thio-β-D-galactopyranoside 53 (4.87 g, 82%). H-NMR (500 MHz, CD3Cl): δ 2.31 (s,
3 H, SPhCH3), 2.43 (d, 1 H, J = 1.5 Hz, OH), 3.44-3.47 (m, 2 H, H-3, H-5), 3.81 (dt, 1 H,
J = 1.5 Hz, J = 9.5 Hz, H-2), 3.98-4.01 (m, 1 H, H-6a), 4.13-4.21 (m, 3 H, H-4,
CH2CHCH2O), 4.34-4.37 (m, 1 H, H-6b), 4.56 (d, 1 H, J = 9.5 Hz, H-1), 5.15-5.18 (m, 1
H, CH2CHCH2O), 5.25-5.29 (m, 1 H, CH2CHCH2O), 5.47 (s, 1 H, PhCH), 5.86-5.93 (m,
1 H, CH2CHCH2O), 7.02-7.04 (m, 2 H), 7.29-7.38 (m, 5 H), 7.55-7.57 (m, 2 H).

13

C-

NMR (125 MHz, CD3Cl): δ 21.4, 67.3, 69.7, 70.3, 71.1, 73.6, 80.3, 87.4, 101.4, 118.0,
126.8, 126.9, 128.2, 129.2, 129.9, 134.6, 135.0, 138.1, 138.6. HRMS: C23H26O5S
+

[M+NH4] calcd: 432.1845, obsd: 432.1831. Compound 53 (4.87 g, 11.76 mmol) was
dissolved in dry pyridine (100 mL) followed by addition of DMAP (143 mg, 1.176 mmol)
o

and benzoyl chloride (2.05, 17.64 mmol). The resulting mixture was stirred under 50 C
overnight. After cooling down to room temperature, the reaction mixture was diluted with
DCM and extracted with 10% HCl solution. The combined organic phase was washed

286

with sat. NaHCO3 solution and dried over Na2SO4. After concentration, the residue was
purified by silica gel column (5:1:1, hexane-DCM-EtOAc) to afford p-tolyl 2-O-benzoyl-3O-allyl-4, 6-O-benzylidene-1-thio-β-D-galactopyranoside 54 (5.48 g, 90%).

1

H-NMR

(500 MHz, CD3Cl): δ 2.30 (s, 3 H, SPhCH3), 3.52-3.54 (m, 1 H, H-5), 3.75 (dd, 1 H, J =
3.5 Hz, J = 9.5 Hz, H-3), 3.97-4.08 (m, 3 H, H-6a, CH2CHCH2O), 4.28 (dd, 1 H, J = 1
Hz, J = 3.5 Hz, H-4), 4.37-4.40 (m, 1 H, H-6b), 4.78 (d, 1 H, J = 9.5 Hz, H-1), 5.02-5.05
(m, 1 H, CH2CHCH2O), 5.11-5.15 (m, 1 H, CH2CHCH2O), 5.43 (t, 1 H, J = 9.5 Hz, H-2),
5.67-5.76 (m, 1 H, CH2CHCH2O), 7.01-7.03 (m, 2 H), 7.30-7.34 (m, 3 H), 7.39-7.48 (m,
6 H), 7.55-7.59 (m, 1 H), 8.04-8.06 (m, 2H).

13

C-NMR (125 MHz, CD3Cl): δ 21.2, 69.3,

69.3, 70.1, 70.7, 73.8, 78.4, 85.5, 101.2, 117.4, 126.6, 127.5, 128.0, 128.3, 128.9, 129.4,
129.7, 130.3, 132.9, 134.4, 134.6, 137.6, 138.1, 164.8. HRMS: C30H30O6S [M+NH4]

+

calcd: 536.2107, obsd: 530.2036. Compound 54 (5.48 g, 10.58 mmol) was dissolved in
DCM/MeOH (1:1. 100 mL) followed by addition of p-TsOH (685 mg, 3.98 mmol). The
reaction mixture was kept under room temperature overnight and quenched with Et3N.
After concentration, the resulting residue was purified by silica gel column (2:1:2,
hexane-DCM-EtOAc)

to

afford

p-tolyl

2-O-benzoyl-3-O-allyl-1-thio-β-D-

1

galactopyranoside 55 (3.64 g, 80%). H-NMR (500 MHz, CD3Cl): δ 2.10-2.13 (m, 1 H,
OH), 2.29 (s, 3 H, SPhCH3), 2.60 (br, 1 H, OH), 3.59-3.65 (m, 2 H, H-3, H-5), 3.79-3.85
(m, 1 H, H-6a), 3.96-4.03 (m, 2 H, CH2CHCH2O), 4.07-4.11 (m, 2 H, H-4, H-6b), 4.72 (d,

287

1 H, J = 10 Hz, H-1), 5.06-5.09 (m, 1 H, CH2CHCH2O), 5.13-5.17 (m, 1 H,
CH2CHCH2O), 5.40 (t, 1 H, J = 10 Hz, H-2), 5.66-5.73 (m, 1 H, CH2CHCH2O), 7.047.06 (m, 2 H), 7.32-7.34 (m, 2 H), 7.43-7.47 (m, 2 H), 7.56-7.59 (m, 1 H), 8.04-8.06 (m,
2 H).

13

C-NMR (125 MHz, CD3Cl): δ 21.2, 62.6, 67.2, 69.3, 69.8, 71.0, 78.4, 79.5, 86.8,

118.1, 128.4, 129.0, 129.6, 129.8, 129.9, 132.9, 133.1, 133.9, 138.1, 165.2. HRMS:
+

C23H26O6S [M+H] calcd: 431.1528, obsd: 431.1508. Freshly activated MS AW 300 (2
g) was mixed with compound 55 (3.64 g, 8.47 mmol) in dry DMF. Under N2, the
resulting mixture was stirred under room temperature for 30 minutes, followed by
addition of NaH (485 mg, 20.38 mmol) and BnBr (4.03 mL, 33.88 mmol). After the
reaction was complete, it was quenched by 10% HCl and diluted with DCM. The organic
phase was extracted with sat. NaHCO3 and dried over Na2SO4. After concentration, the
residue was purified by silica gel column (4:1, hexane-EtOAc) to afford p-tolyl 2-O1

benzoyl-3-O-allyl-4, 6-di-O-benzyl-1-thio-β-D-galactopyranoside 56 (4.33 g, 84%). HNMR (500 MHz, CD3Cl): δ 2.26 (s, 3 H, SPhCH3), 3.62-3.69 (m, 4 H, H-3, H-5, H-6a,
H-6b), 3.94-3.99 (m, 2 H, CH2CHCH2O, H-4), 4.05-4.10 (m, 1 H, CH2CHCH2O), 4.404.47 (m, 2 H, CH2Ph), 4.56-4.59 (m, 1 H, CH2Ph), 4.73 (d, 1 H, J = 10 Hz, H-1), 4.954.97 (m, 1 H, CH2Ph), 5.03-5.05 (m, 1 H, CH2CHCH2O), 5.13-5.17 (m, 1 H,
CH2CHCH2O), 5.59 (t, 1 H, J = 10 Hz, H-2), 5.67-5.72 (m, 1 H, CH2CHCH2O), 6.976.99 (m, 2 H), 7.24-7.34 (m, 12 H), 7.43-7.46 (m, 2 H), 7.54-7.58 (m, 1 H), 8.05-8.07 (m,

288

2 H).

13

C-NMR (125 MHz, CD3Cl): δ 21.2, 68.8, 70.6, 71.3, 73.1, 73.6, 74.3, 77.7, 81.4,

87.2, 117.2, 127.4, 127.9, 128.0, 128.1, 128.3, 128.4, 129.4, 129.7, 130.2, 132.6, 132.9,
+

134.3, 137.5, 137.9, 138.5, 165.2. HRMS: C37H38O6S [M+NH4] calcd: 628.2733, obsd:
628.2721. Compound 56 (4.33 g, 7.11 mmol) wais dissolved in THF (50 mL), followed
by addition of 222 mg [Ir(COD)(Ph2MeP)2]PF6. The resulting mixture was stirred under
H2 for 3 h, followed by addition of H2O (60 mL) and I2 (3.5 g). After the reaction was
complete, the mixture was diluted with EtOAc and extracted with H2O. The combined
organic phase was dried over Na2SO4. After concentration, the residue was purified by
silica gel column (4:1, hexane-EtOAc) to afford p-tolyl 2-O-benzoyl-4, 6-di-O-benzyl-11

thio-β-D-galactopyranoside 57 (3 g, 74%). H-NMR (500 MHz, CD3Cl): δ 2.29 (s, 3 H,
SPhCH3), 2.47 (d, 1 H, J = 9.5 Hz, OH), 3.68-3.81 (m, 4 H, H-3, H-5, H-6a, H-6b), 3.953.96 (m, 1 H, H-4), 4.46-4.53 (m, 2 H, CH2Ph), 4.68-4.73 (m, 3 H, CH2Ph, H-1, J = 9.5
Hz), 5.20 (t, 1 H, J = 9.5 Hz, H-2), 7.00-7.02 (m, 2 H), 7.26-7.34 (m, 12 H), 7.42-7.45 (m,
2 H), 7.54-7.58 (m, 1 H), 8.03-8.05 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 21.4, 68.6,

72.5, 73.8, 74.6, 75.5, 77.7, 86.5, 127.8, 128.0, 128.1, 128.6, 128.7, 128.7, 129.1, 129.8,
130.0, 130.2, 133.3, 133.4, 137.9, 138.2, 138.4, 165.5. HRMS: C34H34O6S [M+NH4]

+

calcd: 588.2420, obsd: 588.2412.
p-Tolyl 2-O-benzoyl-4, 6-di-O-benzylidene -1-thio-β-D-galactopyranoside
57

(61).

p-Tolyl 1-thio-β-D-galactopyranoside 51 (5 g, 17.48 mmol) was dissolved in

289

CH3CN (100 mL) followed by addition of camphorsulfonic acid (1.21 g, 5.24 mmol) and
benzaldehyde dimethyl acetal (3.95 mL, 26.22 mmol). The resulting mixture was stirred
under room temperature overnight. After the reaction was complete, it was quenched by
Et3N and diluted with EtOAc (100 mL) and the organic phase was extracted by sat.
NaHCO3 solution and dried over Na2SO4. After concentration, the residue was
recrystalized

from

EtOH

to

afford

galactopyranoside 58 (5.3 g, 81%).

p-tolyl

4,

6-O-benzylidene-1-thio-β-D-

1

H-NMR (500 MHz, CD3Cl): δ

2.34 (s, 3 H,

SPhCH3), 2.45-2.49 (m, 2 H), 3.53 (br, 1 H), 3.59-3.69 (m, 2 H), 3.99-4.02 (m, 2 H,
CH2Ph), 4.18-4.19 (m, 1 H), 4.35-4.37 (m, 1 H), 4.44 (d, 1 H, J = 9 Hz, H-1), 5.48 (s, 1
H, PhCH), 7.08-7.10 (m, 2 H), 7.33-7.38 (m, 5 H), 7.55-7.57 (m, 2 H). Compound 58
(5.3 g, 14.16 mmol) was dissolved in dry DCM (100 mL), followed by addition of
dicyclohexylcarbodiimide (DCC) (4.53 g, 21.24 mmol), DMAP (878 mg, 7.1 mmol) and
LevOH (1.98 g, 17 mmol). The resulting mixture was stirred under room temperature for
3 h. The reaction was diluted with DCM and washed with 10% HCl solution, sat.
NaHCO3 solution sequentially. The combined organic phase was dried over Na2SO4
and purified by silica gel column (3:1. hexane-EtOAc) to afford p-tolyl 3-O-levulinoyl-4,
6-O-benzylidene-1-thio-β-D-galactopyranoside 59 (3.74 g, 56%).

1

H-NMR (500 MHz,

CD3Cl): δ 2.04 (s, 3 H, CH3COCH2CH2O), 2.32 (s, 3 H, SPhCH3), 2.47 (d, 1 H, OH),
2.55-2.57 (t, 2 H, J = 6.5 Hz, CH3COCH2CH2O), 2.68-2.71 (t, 2 H, J = 6.5 Hz,
CH3COCH2CH2O), 3.56 (br, 1 H), 3.85-3.91 (m, 1 H), 3.97-4.00 (m, 1 H), 4.28-4.36 (m,
290

2 H), 4.52 (d, 1 H, J = 9.5 Hz, H-1), 4.92 (dd, 1 H, J = 3.5 Hz, J = 10 Hz), 5.44 (s, 1 H,
PhCH), 7.05-7.07 (m, 3 H), 7.32-7.38 (m, 5 H), 7.55-7.57 (m, 2 H). Compound 59 (3.74
g, 7.93 mmol) was dissolved in dry DCM (100 mL) followed by addition of DMAP (97 mg,
0.793 mmol) and benzoyl chloride (1.38, 11.9 mmol). The resulting mixture was stirred
o

under 50 C overnight. After cooling down to room temperature, the reaction mixture
was diluted with DCM and extracted with 10% HCl solution. The combined organic
phase was washed with sat. NaHCO3 solution and dried over Na2SO4. After
concentration, the residue was purified by silica gel column (5:1:1, hexane-DCM-EtOAc)
to

afford

p-tolyl

2-O-benzoyl-3-O-levulinoyl-4,

galactopyranoside 60 (3.74 g, 82%).

1

6-O-benzylidene-1-thio-β-D-

H-NMR (500 MHz, CD3Cl): δ

1.84 (s, 3 H,

CH3COCH2CH2O), 2.31 (s, 3 H, SPhCH3), 2.38-2.55 (m, 4 H, CH3COCH2CH2O), 3.62
(br, 1 H), 4.01-4.04 (m, 1 H), 4.36-4.39 (m, 2 H), 4.79 (d, 1 H, J = 9.5 Hz, H-1), 5.15 (dd,
1 H, J = 3.5 Hz, J = 10 Hz), 5.46 (s, 1 H, PhCH), 5.15 (t, 1 H, J = 9.5 Hz, H-2), 7.03-7.05
(m, 2 H), 7.32-7.46 (m, 9 H), 7.55-7.58 (m, 1 H), 8.00-8.02 (m, 2 H). Compound 60
(3.74 g, 6.5 mmol) was dissolved in DCM/MeOH (1:1, 100 mL), followed by addition of
HOAc (30 mL) and NH2NH2-H2O (4 mL). The resulting reaction mixture was stirred
under room temperature overnight and quenched by acetone, diluted with DCM. The
organic phase was extracted with sat. NaHCO3 solution. After drying over Na2SO4 and
concentration, the resulting rsidue was purified by silica gel column (4:1, hexane-EtOAc)
to afford p-tolyl 2-O-benzoyl-4, 6-O-benzylidene-1-thio-β-D-galactopyranoside 61 (2.8 g,
90%). Compound 54 (1 g, 1.93 mmol) wais dissolved in THF (30 mL), followed by

291

addition of 60 mg [Ir(COD)(Ph2MeP)2]PF6. The resulting mixture was stirred under H2
for 3 h, followed by addition of H2O (15 mL) and I2 (948 mg). After the reaction was
complete, the mixture was diluted with EtOAc and extracted with H2O. The combined
organic phase was dried over Na2SO4. After concentration, the residue was purified by
silica gel column (4:1, hexane-EtOAc) to afford p-tolyl 2-O-benzoyl-4, 6-di-O-benzyl-11

thio-β-D-galactopyranoside 61 (710 mg, 77%). H-NMR (500 MHz, CD3Cl): δ 2.32 (s, 3
H, SPhCH3), 2.54-2.56 (m, 1 H, OH), 3.59 (br, 1 H, H-5), 3.84-3.89 (m, 1 H, H-3), 4.034.06 (m, 1 H, H-6a), 4.23-4.25 (m, 1 H, H-4), 4.39-4.42 (m, 1 H, H-6b), 4.75 (d, 1 H, J
=10 Hz, H-1), 5.17 (t, 1 H, J = 9.5 Hz, H-2), 5.51 (s, 1 H, PhCH), 7.05-7.07 (m, 2 H),
7.35-7.46 (m, 9 H), 7.55-7.59 (m, 1 H), 8.05-8.07 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl):

δ 21.3, 69.1, 69.9, 70.7, 73.0, 75.7, 84.8, 101.5, 126.6, 126.9, 128.1, 128.3, 129.3,
129.5, 129.9, 133.1, 134.6, 137.4, 138.4, 165.5.
p-Tolyl 2, 3-di-O-benzoyl-4-chloroacetyl-1-thio-β-D-xylopyranose (67). A
solution of D-xylose 62 (10 g, 66.67 mmol) in dry pyridine (50 mL) was added Ac2O (44
mL, 457.6 mmol). The resulting mixture was stirred under room temperature overnight.
After the reaction was complete, it was diluted by DCM and washed with 10% HCl
solution. The combined organic phase was further extracted with sat. NaHCO3 and
dried over Na2SO4. After concentration, the crude product 63 (19.7 g, 93%) was directly
used for next step without further purification. D-Xylopyranosyl tetraacetate 63 (19.7 g,
62 mol), p-toluenethiol (8.75 g, 70.4 mmol) were dissolved in DCM (100 mL) and boron
292

trifluoride etherate (24.5 mL) was added dropwise at room temperature. The mixture
was stirred under N2 at room temperature for 20 hours and then diluted with DCM (200
mL). The organic phase was washed with saturated aqueous solution of NaHCO3 until
the pH is 7 and then dried over Na2SO4, filtered and concentrated to afford crude
product 64 (14.7 g, 62%), which was recrystalized from hexane/EtOAc.

1

H-NMR

(500 MHz, CD3Cl): δ 2.04 (s, 3 H, COCH3), 2.05 (s, 3 H, COCH3), 2.11 (s, 3 H,
COCH3), 2.36 (s, 3 H, SPhCH3), 3.40 (dd, 1 H, J = 9 Hz, J = 11.5 Hz, H-5a), 4.27 (dd, 1
H, J = 5 Hz, J = 7 Hz, H-5b), 4.73 (d, 1 H, J = 8.5 Hz, H-1), 4.90-4.95 (m, 2 H, H-2, H-4),
5.18 (t, 1 H, J = 8.5 Hz, H-3), 7.13-7.15 (m, 2 H), 7.37-7.39 (m, 2 H).

13

C-NMR (125

MHz, CD3Cl): δ 20.6, 20.6, 20.7, 21.1, 65.3, 68.4, 69.8, 72.2, 86.4, 128.1, 129.7, 133.4,
138.5, 169.2, 169.7, 169.9. HRMS: C18H22O7S [M+NH4]

+

calcd: 400.1430, obsd:

400.1442. Compound 64 (14.7 g, 38.44 mmol) was dissolved in MeOH (50 mL) and
DCM (50 mL). Freshly prepared NaOMe solution in MeOH was added to maintain pH
above 12 and the mixture was stirred at room temperature overnight. The mixture was
neutralized by conc. HCl until the pH is around 7 and then concentrated and dried under
vacuum to afford p-tolyl 1-thio-β-D-xylopyranose 65 as white solid (8.86 g, 90%) which
1

was directly used without further purification. H-NMR (500 MHz, CD3OD): δ 2.38 (s, 3
H, SPhCH3), 3.15-3.22 (m, 2 H, H-2, H-5a), 3.32-3.35 (m, 1 H, H-5b), 3.42-3.47 (m, 1 H,
H-4), 3.91-3.94 (dd, 1 H, J = 5 Hz, J = 11.5 Hz, H-3), 4.46 (d, 1 H, J = 9 Hz, H-1), 7.127.14 (m, 2 H), 7.40-7.42 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 20.8, 70.4, 70.9, 73.6,
293

+

79.2, 90.3, 130.5, 130.8, 133.9, 139.0. HRMS: C12H16O4S [M+H] calcd: 257.0848,
obsd: 257.0857. Compound 65 (1 g, 3.91 mmol) was dissolved dixoane, followed by
addition of Bu2SnO (1.4 g, 5.62 mmol). The resulting mixture was boiled under reflux for
2 h and then was evaporated to dryness. The resulting residue was dissolved in 40 mL
DCM, and a solution of chloroacetyl chloride (ClAcCl) (336 μL, 4.15 mmol) in DCM (4
mL) was added while stirring. After 80 minutes, the reaction was diluted with DCM and
washed with sat. NaHCO3 and dried over Na2SO4. After, concentration, the residue
was purified by silica gel column (1:1, hexane-EtOAc) to afford p-tolyl 4-chloroacetyl-11

thio-β-D-xylopyranose 66 (882 mg, 68%). H-NMR (500 MHz, CDCl3): δ 2.33 (s, 3 H,
SPhCH3), 2.74-2.94 (m, 2 H, OH), 3.27-3.35 (m, 2 H, H-2, H-5a), 3.72 (dd, 1 H, J = 9
Hz, H-3), 4.03-4.16 (m, H-5b, ClCH2CO), 4.41 (d, 1 H, J = 9 Hz, H-1), 4.82-4.87 (m, 1 H,
H-4), 7.11-7.13 (m, 2 H), 7.39-7.41 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 21.1, 40.6,

66.1, 71.9, 72.3, 74.9, 88.5, 126.9, 129.9, 133.7, 138.9, 166.8. HRMS: C14H17ClO5S
+

[M+Na] calcd: 355.0383, obsd: 355.0309. Compound 66 (882 mg, 2.66 mmol) was
dissolved in DCM (20 mL), followed by addition of DMAP (32 mg, 0.266 mmol) and
benzoyl chloride (464 μL, 3.99 mmol). The resulting mixture was stirred under room
temperature overnight and diluted with DCM. After extraction with 10% HCl solution, the
combined organic phase was further washed with sat. NaHCO3 solution and dried over
Na2SO4. Silica gel column (4:1, hexane/EtOAc) purification afforded p-Tolyl 2, 3-di-Obenzoyl-4-chloroacetyl-1-thio-β-D-xylopyranose 67 (72 mg, 5%).
294

1

H-NMR (500 MHz,

CDCl3): δ 2.36 (s, 3 H, SPhCH3), 3.69-3.73 (m, 2 H, H-5a, H-5b), 3.97-4.05 (m, 2 H,
ClCH2CO), 4.12-4.16 (m, 1 H, H-4), 5.10 (d, 1 H, J = 6.5 Hz, H-1), 5.39 (t, 1 H, J = 7 Hz,
H-2), 5.60 (t, 1 H, J = 7 Hz, H-3), 7.13-7.15 (m, 2 H), 7.40-7.44 (m, 6 H), 7.55-7.58 (m, 3
H), 8.01-8.03 (m, 3 H).
p-Tolyl 2, 3-di-O-benzoyl-4-p-methoxybenzyl-1-thio-β-D-xylopyranose (71).
A solution of p-tolyl 1-thio-β-D-xylopyranose 65 (7 g, 27.34 mmol) in dry DMF (50 mL)
was added camphorsulfonic acid (953 mg, 4.1 mmol). The resulting mixture was stirred
o

under 60 C. 2-Methoxy propene (7.85 mL, 82.02 mmol) was added into the reaction
mixture in portions. The reaction was stirred for another 2 h. After the reaction was
complete, it was cooled back to room temperature and quenched with Et3N. The
resulting mixture was concentrated and purified by silica gel column (4:1:1, hexaneDCM-EtOAc) to afford p-tolyl 2, 3-isopropylidene-1-thio-β-D-xylopyranose 68 (6.3 g,
1

78%). H-NMR (500 MHz, CD3Cl): δ 1.41 (s, 3 H, C(CH3)2), 1.46 (s, 3 H, C(CH3)2),
2.31 (s, 3 H, SPhCH3), 2.53 (d, 1 H, J = 4 Hz, OH), 3.16-3.20 (m, 2 H, H-2, H-5a), 3.48
(t, 1 H, J = 9 Hz, H-3), 3.89-4.62 (m, 1 H, H-4), 4.06-4.10 (m, 1 H, H-5b), 4.69 (d, 1 H, J
= 9.5 Hz, H-1), 7.09-7.11 (m, 2 H), 7.42-7.44 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ

21.1, 26.4, 26.6, 68.9, 69.8, 75.0, 82.8, 85.5, 111.1, 127.7, 129.6, 133.6, 138.4. HRMS:
+

C15H20O4S [M+H]

calcd: 297.1161, obsd: 297.1160. Compound 68 (6.3 g, 21.33

mmol) was dissolved in 40 mL DMF, followed by addition of NaH (1 g, 25.6 mmol) and
PMBCl (3.76 mL, 27.73 mmol). After stirring under room temperature overnight, the
reaction was quenched by 10% HCl solution and diluted with DCM. The organic phase
295

was extracted with sat. NaHCO3 and dried over Na2SO4. Silica gel column (4:1,
hexane/EtOAc) purification afforded p-Tolyl 2, 3-isopropylidene-4-p-methoxybenzyl-11

thio-β-D-xylopyranose 69 (7.54 g, 85%). H-NMR (500 MHz, CD3Cl): δ 1.43 (s, 3 H,
C(CH3)2), 1.47 (s, 3 H, C(CH3)2), 2.31 (s, 3 H, SPhCH3), 3.16-3.21 (m, 2 H, H-2, H-5a),
3.59 (t, 1 H, J = 9 Hz, H-3), 3.67-3.72 (m, 1 H, H-4), 3.77 (s, 3 H, CH3OPhCH2O), 4.014.04 (m, 1 H, H-5b), 4.47-4.49 (m, 1 H, CH3OPhCH2O), 4.69 (d, 1 H, J = 9.5 Hz, H-1),
4.69-4.71 (m, 1 H, CH3OPhCH2O), 6.83-6.86 (m, 2 H), 7.08-7.10 (m, 2 H), 7.22-7.24 (m,
2 H), 7.42-7.44 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 21.1, 26.5, 26.7, 55.2, 68.4,

71.8, 75.2, 75.3, 82.4, 85.4, 111.0, 113.8, 127.9, 129.4, 129.5, 130.0, 133.5, 138.3,
159.3. HRMS: C23H28O5S [M+H]

+

calcd: 417.1736, obsd: 417.1725. Compound 69

(7.54 g, 18.13 mmol) was dissolve in DCM/MeOH (1:1, 60 mL), followed by addition of
camphorsulfonic acid (4.23 g, 18.13 mmol). The resulting mixture was stirred under
room temperature overnight. After the reaction was complete, it was quenched by Et3N
and concentrated. The residue was purified by silica gel column (1:1, hexane-EtOAc) to
afford p-tolyl 4-p-methoxybenzyl-1-thio-β-D-xylopyranose 70 (6.68 g, 98%).

1

H-NMR

(500 MHz, CD3Cl): δ 2.32 (s, 3 H, SPhCH3), 2.64 (br, 2 H, OH), 3.19-3.24 (m, 1 H, H5a), 3.05-3.35 (m, 1 H, H-2), 3.39-3.44 (m, 1 H, H-4), 3.60-3.65 (m, 1 H, H-3), 3.78 (s, 3
H, CH3OPhCH2O), 4.01-4.04 (m, 1 H, H-5b), 4.44 (d, 1 H, J = 9 Hz, H-1), 4.53-4.59 (m,
2 H, CH3OPhCH2O), 6.83-6.86 (m, 2 H), 7.09-7.11 (m, 2 H), 7.21-7.23 (m, 2 H), 7.38-

296

7.40 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 21.1, 55.2, 67.1, 71.8, 72.6, 88.8, 113.9,
+

127.9, 129.4, 129.7, 129.9, 133.2, 138.4, 159.4. HRMS: C20H24O5S [M+NH4] calcd:
394.1688, obsd: 394.1669. Compound 70 (6.68 g, 17.77 mmol) and DMAP (2.17 g,
17.77 mmol) were dissolved in DCM (100 mL). Benzoyl chloride (4.95 mL, 42.65 mmol)
was added into the reaction mixture while stirring and the reaction was left under reflux
overnight. After the reaction was complete, it was diluted with DCM and washed with
10% HCl solution. The combined organic phase was extracted with sat. NaHCO3 and
dried over Na2SO4. Silica gel column (2:1, hexane/EtOAc) purification afforded p-Tolyl 2,
1

3-di-O-benzoyl-4-p-methoxybenzyl-1-thio-β-D-xylopyranose 71 (10.2 g, 98%). H-NMR
(500 MHz, CD3Cl): δ 2.30 (s, 3 H, SPhCH3), 3.48-3.52 (m, 1 H, H-5a), 3.70-3.74 (m, 1
H, H-4), 3.73 (s, 3 H, CH3OPhCH2O), 4.21-4.25 (m, 1 H, H-5b), 4.47-4.52 (m, 2 H,
CH3OPhCH2O), 4.91 (d, 1 H, J = 8 Hz, H-1), 5.28 (t, 1 H, J = 8 Hz, H-2), 5.55 (t, 1 H, J
= 8 Hz, H-3), 6.68-6.71 (m, 2 H), 7.06-7.11 (m, 4 H), 7.32-7.39 (m, 6 H), 7.47-7.52 (m, 2
H), 7.92-7.96 (m, 4 H).

13

C-NMR (125 MHz, CD3Cl): δ 21.1, 55.1, 66.4, 70.5, 72.3, 73.7,

73.9, 87.1, 113.8, 128.3, 128.3, 129.1, 129.4, 129.4, 129.6, 129.7, 129.8, 129.9, 133.0,
+

133.1, 138.2, 159.3, 165.2, 165.5. HRMS: C34H32O7S [M+NH4] calcd: 602.2212, obsd:
602.2203.
p-Tolyl 2, 3-di-O-benzoyl-4-O-benzyl-1-thio-β-D-xylopyranose (75). p-Tolyl 2,
3-isopropylidene-1-thio-β-D-xylopyranose 68 (600 mg, 2.03 mmol) was dissolved in 10
mL DMF, followed by addition of NaH (95 mg, 2.44 mmol) and BnBr (314 μL, 2.64
297

mmol). After stirring under room temperature overnight, the reaction was quenched by
10% HCl solution and diluted with DCM. The organic phase was extracted with sat.
NaHCO3 and dried over Na2SO4. Silica gel column (4:1, hexane/EtOAc) purification
afforded p-Tolyl 2, 3-isopropylidene-4-benzyl-1-thio-β-D-xylopyranose 73 (703 mg,
1

90%). H-NMR (500 MHz, CD3Cl): δ 1.48 (s, 3 H, C(CH3)2), 1.53 (s, 3 H, C(CH3)2),
2.34 (s, 3 H, SPhCH3), 3.22-3.29 (m, 2 H, H-2, H-4), 3.67 (t, 1 H, J = 9 Hz, H-3), 3.723.78 (m, 1 H, H-5a), 4.09-4.12 (m, 1 H, H-5b), 4.57-4.60 (m, 1 H, PhCH2O), 4.75 (d, 1 H,
J = 9.5 Hz, H-1), 4.80-4.83 (m, 1 H, PhCH2O), 7.11-7.13 (m, 2 H), 7.26-7.35 (m, 5 H),
7.49-7.51 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 20.8, 26.3, 26.5, 68.0, 71.7, 75.0,

75.3, 82.2, 85.0, 113.2, 127.4, 127.4, 127.6, 129.3, 133.3, 137.7, 138.0. HRMS:
+

C22H26O4S [M+Na] calcd: 410.1528, obsd: 410.1547. Compound 73 (703 mg, 1.82
mmol) was dissolve in DCM/MeOH (1:1, 10 mL), followed by addition of
camphorsulfonic acid (423 mg, 1.82 mmol). The resulting mixture was stirred under
room temperature overnight. After the reaction was complete, it was quenched by Et3N
and concentrated. The residue was purified by silica gel column (1:1, hexane-EtOAc) to
1

afford p-tolyl 4-benzyl-1-thio-β-D-xylopyranose 74 (581 mg, 92%). H-NMR (500 MHz,
CD3Cl): δ 2.34 (s, 3 H, SPhCH3), 3.22-3.27 (m, 1 H, H-5a), 3.42-3.53 (m, 2 H, H-2, H4), 3.71-3.75 (m, 1 H, H-3), 3.97-3.99 (m, 1 H, OH), 4.03-4.06 (m, 1 H, H-5b), 4.18 (br, 1
H, OH), 4.54 (d, 1 H, J = 9 Hz, H-1), 4.61-4.76 (m, 2 H, PhCH2), 7.11-7.13 (m, 2 H),
7.29-7.36 (m, 5 H), 7.46-7.48 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 21.4, 67.1, 72.1,
298

73.0, 77.4, 88.8, 127.8, 127.9, 128.4, 128.6, 129.7, 133.0, 138.0. HRMS: C19H22O4S
+

[M+NH4] calcd: 364.1583, obsd: 364.1582. Compound 74 (581 mg, 1.68 mmol) and
DMAP (205 mg, 1.68 mmol) were dissolved in DCM (10 mL). Benzoyl chloride (468 μL,
4.03 mmol) was added into the reaction mixture while stirring and the reaction was left
under reflux overnight. After the reaction was complete, it was diluted with DCM and
washed with 10% HCl solution. The combined organic phase was extracted with sat.
NaHCO3 and dried over Na2SO4. Silica gel column (2:1, hexane/EtOAc) purification
afforded p-Tolyl 2, 3-di-O-benzoyl-4-p-methoxybenzyl-1-thio-β-D-xylopyranose 75 (881
1

mg, 95%). H-NMR (500 MHz, CD3Cl): δ 2.31 (s, 3 H, SPhCH3), 3.53-3.57 (m, 1 H, H5a), 3.74-3.78 (m, 1 H, H-4), 4.27-4.31 (m, 1 H, H-5b), 4.56-4.60 (m, 2 H, PhCH2), 4.95
(d, 1 H, J = 9 Hz, H-1), 5.28-5.32 (m, 1 H, H-2), 5.58-5.62 (m, 1 H, H-3), 7.07-7.09 (m, 2
H), 7.19-7.24 (m, 5 H), 7.32-7.40 (m, 6 H), 7.47-7.53 (m, 2 H), 7.93-7.98 (m, 4 H).
+

HRMS: C33H30O6S [M+NH4] calcd: 572.2107, obsd: 572.2096.
p-Tolyl 2, 3-di-O-benzoyl-4-O-levulinoyl-1-thio-β-D-xylopyranose (78). pTolyl 2, 3-isopropylidene-1-thio-β-D-xylopyranose 68 (500 mg, 1.69 mmol) was
dissolved in 10 mL DMF, followed by addition of LevOH (205 μL, 2.03 mmol), DMAP
(206 mg, 1.69 mmol) and EDC-HCl (389 mg, 2.03 mmol). After stirring under room
temperature overnight, the reaction was quenched by 10% HCl solution and diluted with
DCM. The organic phase was extracted with sat. NaHCO3 solution and dried over
Na2SO4. Silica gel column (3:1, hexanes/EtOAc) purification afforded p-Tolyl 2, 3-

299

isopropylidene-4-levulinoyl-1-thio-β-D-xylopyranose 76

(586 mg, 88%).

1

H-NMR

(500 MHz, CD3Cl): δ 1.40 (s, 3 H, C(CH3)2), 1.46 (s, 3 H, C(CH3)2), 2.15 (s, 3 H,
CH3COCH2CH2CO), 2.32 (s, 3 H, SPhCH3), 2.51-2.81 (m, 4 H, CH3COCH2CH2CO),
3.21-3.28 (m, 2 H, H-2, H-5a), 3.69-3.73 (m, 1 H, H-3), 4.20-4.24 (m, 1 H, H-5b), 4.76 (d,
1 H, J = 9.5 Hz, H-1), 4.91-4.96 (m, 1 H, H-4), 7.09-7.11 (m, 2 H), 7.42-7.44 (m, 2 H).
+

HRMS: C20H26O6S [M+NH4] calcd: 412.2522, obsd: 412.2529. Compound 76 (586
mg, 1.49 mmol) was dissolved in DCM/MeOH (1:1, 8 mL), followed by addition of
camphorsulfonic acid (346 mg, 1.49 mmol). The resulting mixture was stirred under
room temperature overnight. After the reaction was complete, it was quenched by Et3N
and concentrated. The residue was purified by silica gel column (1:1, hexane-EtOAc) to
1

afford p-tolyl 4-benzyl-1-thio-β-D-xylopyranose 77 (475 mg, 90%). H-NMR (500 MHz,
CD3Cl): δ 2.16 (s, 3 H, CH3COCH2CH2CO), 2.31 (s, 3 H, SPhCH3), 2.53-2.57 (m, 3 H,
OH, CH3COCH2CH2CO), 2.75-2.78 (m, 2 H, CH3COCH2CH2CO), 2.87 (br, 1 H, OH),
3.26-3.37 (m, 2 H, H-2, H-5a), 3.68-3.72 (m, 1 H, H-3), 4.08-4.12 (m, 1 H, H-5b), 4.42 (d,
1 H, J = 9.5 Hz, H-1), 4.77-4.82 (m, 1 H, H-4), 7.10-7.13 (m, 2 H), 7.40-7.42 (m, 2 H).
+

HRMS: C17H22O6S [M+NH4] calcd: 372.1481, obsd: 372.1489. Compound 77 (475
mg, 1.34 mmol) and DMAP (163 mg, 1.34 mmol) were dissolved in DCM (8 mL).
Benzoyl chloride (373 μL, 3.21 mmol) was added into the reaction mixture while stirring
and the reaction was left under reflux overnight. After the reaction was complete, it was
diluted with DCM and washed with 10% HCl solution. The combined organic phase was

300

extracted with sat. NaHCO3 and dried over Na2SO4. Silica gel column (3:1,
hexane/EtOAc) purification afforded p-Tolyl 2, 3-di-O-benzoyl-4-O-levulinoyl-1-thio-β-Dxylopyranose 78 (550 mg, 73%).

1

H-NMR (500 MHz, CD3Cl): δ

2.04 (s, 3 H,

CH3COCH2CH2CO), 2.31 (s, 3 H, SPhCH3), 2.41-2.67 (m, 4 H, CH3COCH2CH2CO),
3.59-3.63 (m, 1 H, H-5a), 4.43-4.46 (m, 1 H, H-5b), 5.03 (d, 1 H, J = 6 Hz, H-1), 5.085.11 (m, 1 H, H-4), 5.34 (t, 1 H, J = 6.5 Hz, H-2), 5.57 (t, 1 H, J = 6.5 Hz, H-3), 7.08-7.10
(m, 2 H), 7.36-7.39 (m, 6 H), 7.49-7.53 (m, 2 H), 7.97-7.99 (m, 2 H).

13

C-NMR (125

MHz, CD3Cl): δ 14.1, 20.9, 21.0, 27.7, 29.5, 37.6, 60.2, 64.3, 68.4, 69.9, 71.5, 86.6,
128.3, 128.3, 128.6, 128.8, 129.2, 129.6, 129.7, 129.8, 133.2, 133.2, 133.3, 138.3,
164.9, 165.2, 171.5, 205.8. HRMS: C31H30O8S [M+NH4]

+

calcd: 580.2005, obsd:

580.2008.
p-Tolyl 2, 3-di-O-benzoyl-1-thio-β-D-xylopyranose (72). p-Tolyl 2, 3-di-Obenzoyl-4-O-p-methoxybenzyl-1-thio-β-D-xylopyranose 71 (10.2 g, 17.41 mmol) was
dissolved in DCM/H2O (10:1, 50 mL), followed by addition of DDQ (5.9 g, 26.11 mmol).
The resulting mixture was stirred under room temperature overnight. After the reaction
was complete, it was diluted with DCM, washed with sat. NaHCO3, dried over Na2SO4.
Silica gel column (4:1, hexane/EtOAc) purification afforded p-Tolyl 2, 3-di-O-benzoyl-1thio-β-D-xylopyranose 72 (6.54 g, 81%). p-Tolyl 2, 3-di-O-benzoyl-4-O-benzyl-1-thio-βD-xylopyranose 75 (881 mg, 1.59 mmol) was dissolved in DCM/H2O (10:1, 10 mL),
followed by addition of DDQ (543 mg, 2.39 mmol). The resulting mixture was stirred
301

under reflux overnight. After the reaction was complete, it was diluted with DCM,
washed with sat. NaHCO3, dried over Na2SO4. Silica gel column (4:1, hexane/EtOAc)
purification afforded p-Tolyl 2, 3-di-O-benzoyl-1-thio-β-D-xylopyranose 72 (444 mg,
60%). p-Tolyl 2, 3-di-O-benzoyl-4-O-benzyl-1-thio-β-D-xylopyranose 78 (550 mg, 0.98
mmol) was dissolved in DCM/MeOH (1:1, 8 mL), followed by addition of HOAc (6 mL)
and NH2NH2-H2O (570 μL). The resulting mixture was stirred under reflux overnight.
After the reaction was complete, it was diluted with DCM, washed with sat. NaHCO3,
dried over Na2SO4. Silica gel column (4:1, hexane/EtOAc) purification afforded p-Tolyl 2,
1

3-di-O-benzoyl-1-thio-β-D-xylopyranose 72 (396 mg, 87%). H-NMR (500 MHz, CD3Cl):

δ 2.32 (s, 3 H, SPhCH3), 3.02 (br, 1 H, OH), 3.53-3.58 (m, 1 H, H-5a), 3.94-3.98 (m, 1
H, H-4), 4.38-4.41 (m, 1 H, H-5b), 4.98 (d, 1 H, J = 7 Hz, H-1), 5.29 (t, 1 H, J = 7.5 Hz,
H-3), 5.38 (t, 1 H, J = 7.5 Hz, H-2), 7.09-7.11 (m, 2 H), 7.36-7.40 (m, 6 H), 7.51-7.54 (m,
2 H), 7.96-8.02 (m, 4 H).

13

C-NMR (125 MHz, CD3Cl): δ 21.2, 67.6, 68.2, 70.1, 76.0,

86.8, 128.4, 128.4, 128.7, 128.8, 129.2, 129.7, 129.7, 129.9, 133.2, 133.3, 133.5, 138.3,
+

165.0, 166.8. HRMS: C26H24O6S [M+NH4] calcd: 482.1637, obsd: 482.1657.
p-Tolyl 2-O-benzoyl-3-O-tert-butyl-dimethylsilyl-4, 6-di-O-benzyl-1-thio-β-Dgalactopyranoside (81). Compound 57 (500 mg, 0.87 mmol) in DCM (5 mL) was
cooled down to –40 °C, followed by sequential addition of 2, 6-lutidine (203 μL, 1.74
mmol) and TBSOTf (299 μL, 1.3 mmol). The resulting solution was warmed up very
slowly to room temperature. The mixture was quenched by Et3N and then diluted with
302

DCM (100 mL). The organic phase was washed with saturated NaHCO3 and then dried
over Na2SO4, filtered and the solvents were removed in vacuo.

Silica gel column

chromatography (Hexanes–EtOAc) afforded compound 81 as white solid (468 mg, 78%).
1

H-NMR (500 MHz, CDCl3): δ -0.13 (s, 3 H, Si(CH3)2C(CH3)3), 0.07 (s, 3 H,

Si(CH3)2C(CH3)3), 0.74 (s, 3 H, Si(CH3)2C(CH3)3), 2.26 (s, 3H, SPhCH3), 3.62-3.74 (m,
3 H, H-5, H-6a, H-6b), 3.81-3.82 (m, 1 H, H-4), 3.92-3.96 (m, 1 H, H-3), 4.42-4.48 (m, 2
H, PhCH2), 4.48-4.51 (m, 1 H, PhCH2), 4.66-4.70 (d, 1 H, J = 10 Hz, H-1), 5.04-5.06 (m,
1 H, PhCH2), 5.56-5.61 (m, 1 H, H-2), 6.95-6.97 (m, 2 H), 7.25-7.33 (m, 12 H), 7.417.45 (m, 2 H), 7.53-7.57 (m, 1 H), 8.02-8.04 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ -

4.8, -3.8, 14.4, 18.0, 21.2, 21.3, 25.7, 25.8, 60.6, 69.2, 73.8, 75.2, 76.0, 77.9, 87.5,
127.6, 127.9, 127.9, 128.1, 128.4, 128.5, 128.6, 129.6, 130.0, 130.1, 130.6, 132.7,
137.7, 138.2, 139.0, 165.4. HRMS: C40H48O6SSi [M+NH4]

+

calcd: 702.3285, obsd:

702.3273.
p-Tolyl

2-O-benzoyl-3-O-levulinoyl-4,

6-di-O-benzyl-1-thio-β-D-

galactopyranoside (82). Compound 58 (600 mg, 1.05 mmol) was dissolved in dry
DCM (8 mL), followed by addition of EDC-HCl (336 mg, 1.57 mmol), DMAP (65 mg,
0.53 mmol) and LevOH (147 mg, 1.26 mmol). The resulting mixture was stirred under
room temperature for overnight. The reaction was diluted with DCM and washed with
10% HCl solution, sat. NaHCO3 solution sequentially. The combined organic phase was
dried over Na2SO4 and purified by silica gel column (3:1. hexane-EtOAc) to afford p303

tolyl 2-O-benzoyl-3-O-levulinoyl-4, 6-di-O-benzyl-1-thio-β-D-galactopyranoside 82 (611
1

mg, 87%). H-NMR (600 MHz, CDCl3): δ 1.96 (s, 3 H, CH3COCH2CH2), 2.28 (s, 3H,
SPhCH3), 2.25-2.58 (m, 4 H, CH3COCH2CH2), 3.60-3.69 (m, 2 H, H-6a, H-6b), 3.793.81 (m, 1 H, H-5), 4.05-4.06 (m, 1 H, H-4), 4.41-4.48 (m, 2 H, PhCH2), 4.54-4.56 (m, 1
H, PhCH2), 4.75-4.77 (m, 1 H, PhCH2), 4.77 (d, 1 H, J = 8 Hz, H-1), 5.17-5.19 (m, 1 H,
H-3), 5.61-5.64 (m, 1 H, H-2), 6.99-7.01 (m, 2 H), 7.26-7.35 (m, 12 H), 7.42-7.45 (m, 2
H), 7.55-7.57 (m, 1 H), 8.00-8.02 (m, 2 H).

13

C-NMR (150 MHz, CD3Cl): δ 21.1, 27.9,

29.4, 37.6, 68.3, 68.7, 73.4, 74.3, 74.7, 75.1, 77.4, 86.9, 127.5, 127.7, 127.7, 127.8,
128.2, 128.3, 129.1, 129.5, 129.6, 129.8, 132.8, 133.1, 137.7, 137.8, 138.1, 165.1,
+

171.9, 205.9. HRMS: C39H40O8S [M+NH4] calcd: 686.2788, obsd: 686.2770.
p-Tolyl 2, 3-di-O-levulinoyl-4, 6-O-benzylidene-1-thio-β-D-galactopyranoside
57

(83).

Compound 58 (5 g, 13.36 mmol) was dissolved in dry DCM (100 mL), followed

by addition of EDC-HCl (7.1 g, 33.4 mmol), DMAP (325 mg, 2.67 mmol) and LevOH
(3.9 g, 33.4 mmol). The resulting mixture was stirred under room temperature for
overnight. The reaction was diluted with DCM and washed with 10% HCl solution, sat.
NaHCO3 solution sequentially. The combined organic phase was dried over Na2SO4
and purified by silica gel column (3:1. hexane-EtOAc) to afford p-tolyl 2, 3-di-Olevulinoyl-4, 6-O-benzylidene-1-thio-β-D-galactopyranoside 83 (7 g, 92%).

1

H-NMR

(500 MHz, CD3Cl): δ 1.98 (s, 3 H, CH3COCH2CH2), 2.14 (s, 3 H, CH3COCH2CH2),
2.30 (s, 3 H, SPhCH3), 2.47-2.85 (m, 8 H, CH3COCH2CH2), 3.51-3.53 (m, 1 H), 3.95304

3.98 (m, 1 H), 4.24-4.25 (m, 1 H), 4.30-4.33 (m, 1 H), 4.61 (d, 1 H, J = 9.5 Hz, H-1),
5.01 (dd, 1 H, J = 3 Hz, J = 10 Hz), 5.23-5.27 (m, 1 H), 5.41 (s, 1 H, PhCH), 7.03-7.05
(m, 2 H), 7.30-7.36 (m, 5 H), 7.45-7.47 (m, 2 H).
p-Tolyl 2-O-benzoyl-3-O-levulinoyl-4, 6-di-O-benzyl -β-D-galactopyranosyl(1→3)-2-O-benzoyl-4,

6-O-benzylidene-1-thio-β-D-galactopyranoside

(84).

Compound 84 was synthesized from donor 82 and acceptor 61 in 3% yield following the
1

general procedure of single step glycosylation. H-NMR (500 MHz, CD3Cl): δ 1.94 (s, 3
H, CH3COCH2CH2), 2.28 (s, 3 H, SPhCH3), 2.15-2.51 (m, 4 H, CH3COCH2CH2), 3.433.51 (m, 3 H), 3.69-3.72 (m, 1 H), 3.90-3.98 (m, 2 H), 4.12-4.18 (m, 1 H), 4.30-4.40 (m,
3 H), 4.50-4.53 (m, 1 H, PhCH2), 4.73 (d, 1 H, J = 8 Hz), 4.73-4.76 (m, 1 H, PhCH2),
4.80 (d, 1 H, J = 8 Hz), 4.96 (dd, 1 H, J = 3 Hz, J = 10 Hz), 5.36-5.42 (m, 2 H, PhCH2),
5.54-5.58 (m, 1 H), 6.96-6.98 (m, 2 H), 7.19-7.52 (m, 23 H), 7.63-7.65 (m, 2 H), 7.807.82 (m, 2 H).
p-Tolyl 2, 3-di-O-levulinoyl-4, 6-O-benzylidene-β-D-galactopyranosyl-(1→3)2-O-benzoyl-4, 6-O-benzylidene-1-thio-β-D-galactopyranoside (87). Compound 87
was synthesized from donor 83 and acceptor 61 in 85% yield following the general
1

procedure of single step glycosylation. H-NMR (500 MHz, CD3Cl): δ 1.95 (s, 3 H,
CH3COCH2CH2), 2.00 (s, 3 H, CH3COCH2CH2), 2.28 (s, 3 H, SPhCH3), 2.10-2.69 (m,
8 H, CH3COCH2CH2), 3.35 (br, 1 H), 3.52 (br, 1 H), 3.95-4.03 (m, 2 H), 4.13-4.19 (m, 3
H), 4.33-4.36 (m, 1 H), 4.43 (d, 1 H, J = 3 Hz), 4.65-4.68 (m, 2 H), 4.72 (d, 1 H, J = 9.5
Hz), 5.24-5.28 (m, 1 H), 5.42 (s, 1 H, PhCH), 5.48-5.53 (m, 1 H), 5.54 (s, 1 H, PhCH),
305

6.98-7.00 (m, 2 H), 7.29-7.34 (m, 6 H), 7.41-7.48 (m, 8 H), 7.57-7.61 (m, 1 H), 8.01-8.04
(m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 21.1, 27.5, 28.1, 29.5, 29.5, 37.7, 37.7, 66.4,

68.2, 68.7, 69.0, 69.4, 70.3, 71.7, 73.1, 75.9, 86.1, 100.7, 100.8, 100.9, 126.3, 126.5,
127.9, 128.1, 128.2, 128.5, 128.7, 129.0, 129.4, 129.6, 130.0, 133.2, 133.7, 137.4,
137.8, 137.9, 164.7, 171.2, 172.1, 206.5, 206.6.
p-Tolyl

6-O-benzylidene-β-D-

2-O-benzoyl-3-O-levulinoyl-4,

galactopyranosyl-(1→3)-4,

6-O-benzylidene-1-thio-β-D-galactopyranoside

(88).

Compound 88 was synthesized from donor 60 and acceptor 58 in 60% yield following
the general procedure of single step glycosylation.
p-Tolyl

6-O-benzylidene-β-D-

2-O-benzoyl-3-O-levulinoyl-4,

6-O-benzylidene-1-thio-β-D-

galactopyranosyl-(1→3)-2-O-benzoyl-4,

galactopyranoside (89). Compound 89 was synthesized from donor 60 and acceptor
1

61 in 62% yield following the general procedure of single step glycosylation. H-NMR
(500 MHz, CD3Cl): δ 1.83 (s, 3 H, CH3COCH2CH2), 2.25 (s, 3 H, SPhCH3), 2.34-2.56
(m, 4 H, CH3COCH2CH2), 3.41 (br, 1 H), 3.49 (br, 1 H), 3.91-4.16 (m, 3 H), 4.27-4.34
(m, 3 H), 4.40-4.41 (m, 1 H), 4.75 (d, 1 H, J = 9.5 Hz), 4.90-4.94 (m, 2 H), 5.36 (s, 1 H,
PhCH), 5.40-5.52 (m, 1 H), 5.45 (s, 1 H, PhCH), 5.53-5.57 (m, 1 H), 6.94-6.96 (m, 2 H),
7.19-7.53 (m, 18 H), 7.69-7.71 (m, 2 H), 7.88-7.90 (m, 2 H).

13

C-NMR (125 MHz,

CD3Cl): δ 21.4, 28.4, 29.6, 37.9, 66.7, 68.9, 69.1, 69.2, 69.4, 70.4, 72.4, 73.4, 76.1,
76.5, 86.3, 100.2, 101.0, 101.2, 126.6, 128.1, 128.2, 128.4, 128.5, 128.8, 129.3, 129.5,

306

129.6, 129.9, 129.9, 130.4, 133.1, 133.1, 134.0, 137.8, 137.9, 138.1, 164.8, 165.3,
+

172.2, 206.2. HRMS: C52H50O14S [M+NH4] calcd: 948.3265, obsd: 948.3286.
61

Fmoc-Ser-OBn (91).

Dry DMSO (6 mL) was added to a 25 mL flask under

nitrogen containing compound 90 (0.962 g, 2.94 mmol), KHCO3 (0.442 g, 4.40 mmol),
and tetrabutylammonium iodide (0.1086 g, 0.294 mmol). The resulting white suspension
was stirred under room temperature until a homogeneous solution was obtained. BnBr
(1.048 mL, 8.80 mmol) was added to the reaction mixture and the reaction was kept for
8 h under room temperature. After the reaction was complete indicated by TLC, it was
quenched by water and the mixture was extracted by EtOAc. The combined organic
layer was washed by sat. NaHCO3, sat. Na2S2O3 and brine sequentially. After drying
over Na2SO4, the solution was concentrated to give yellow oil. The oil was cooled to -78
o

C and triturated with hexanes. The obtained yellow solid was filtered and washed with
1

hexanes under suction until a white solid was obtained (1 g, 82%). H-NMR (500 MHz,
CD3Cl): δ 1.95 (br, 1 H), 3.91-4.03 (m, 2 H), 4.20 (t, 1 H, J = 7 Hz), 4.39-4.49 (m, 3 H),
5.17-5.25 (m, 2 H), 5.64-5.68 (m, 1 H), 7.26-7.39 (m, 9 H), 7.57-7.59 (m, 2 H), 7.74-7.76
(m, 2 H).
N-Fluorenylmethyloxycarbonyl-O-(2,

3-di-O-benzoyl-4-O-benzyl

-β-D-

xylopyranosyl)-L-serine benzyl ester (92). Compound 92 was synthesized from donor
75 and acceptor 91 in 83% yield following the general procedure of single step
1

glycosylation. H-NMR (500 MHz, CD3Cl): δ 3.37-3.42 (m, 1 H, H-5a), 3.64-3.70 (m, 1 H,

307

H-4), 3.79-3.82 (m, 1 H, OCH2CH), 3.89-3.92 (m, 1 H, H-5b), 4.11-4.14 (m, 1 H), 4.194.23 (m, 1 H), 4.28-4.35 (m, 2 H), 4.50-4.55 (m, 1 H, OCH2CH), 4.59 (br, 2 H, PhCH2),
4.64 (d, 1 H, J = 5.5 Hz, H-1), 5.08-5.22 (m, 3 H, H-2, PhCH2), 5.55-5.59 (m, 2 H), 7.187.29 (m, 14 H), 7.35-7.42 (m, 5 H), 7.51-7.55 (m, 3 H), 7.74-7.76 (m, 2 H), 7.92-7.97 (m,
4 H).

13

C-NMR (150 MHz, CD3Cl): δ 47.3, 54.5, 60.6, 62.6, 67.4, 67.6, 69.2, 71.0, 72.1,

72.7, 74.0, 76.1, 101.2, 120.1, 125.4, 127.3, 127.9, 127.9, 128.0, 128.1, 128.5, 128.5,
128.6, 128.6, 128.7, 129.4, 129.6, 130.0, 130.1, 133.4, 133.5, 135.4, 137.6, 141.4,
143.9, 144.1, 156.1, 165.5, 165.7, 169.7. HRMS: C51H45NO11 [M+NH4]

+

calcd:

865.3336, obsd: 865.3331.
N-Fluorenylmethyloxycarbonyl-O-(2, 3-di-O-benzoyl-4-O-p-methoxybenzyl -

β-D-xylopyranosyl)-L-serine benzyl ester (94). Compound 94 was synthesized from
donor 71 and acceptor 91 in 76% yield following the general procedure of single step
1

glycosylation. H-NMR (500 MHz, CD3Cl): δ 3.32-3.28 (m, 1 H), 3.62-3.67 (m, 1 H),
3.73 (s, 1 H, CH3OPh), 3.79-3.82 (m, 1 H), 3.86-3.91 (m, 1 H), 4.10-4.14 (m, 1 H), 4.184.22 (m, 1 H), 4.28-4.34 (m, 2 H), 4.49-4.54 (m, 2 H), 4.61 (d, 1 H, J = 6 Hz, H-1), 5.085.21 (m, 3 H), 5.52-5.59 (m, 2 H), 6.71-6.73 (m, 2 H), 7.11-7.14 (m, 2 H), 7.23-7.28 (m,
8 H), 7.35-7.42 (m, 6 H), 7.50-7.55 (m, 3 H), 7.74-7.76 (m, 2 H), 7.90-7.96 (m, 4 H).
+

HRMS: C52H47NO12 [M+NH4] calcd: 895.3442, obsd: 895.3451.
N-Fluorenylmethyloxycarbonyl-O-(2, 3-di-O-benzoyl-4-O-p-methoxybenzyl -

β-D-xylopyranosyl)-L-serine (220). Compound 94 (261 mg, 0.298 mmol) was
dissolved in DCM/MeOH (1:1, 8 mL), followed by addition of Pd/C (25 mg) and NH4OAc
308

(80 mg, 1.041 mmol). The resulting mixture was stirred under H2 atomosphere. The
reaction was carefully monitored by TLC. After the complete disappearance of starting
material, the reaction was diluted with DCM and filtered. After concentration, the residue
1

was purified by silica gel column to afford compound 220 (199 mg, 85%). H-NMR
(500 MHz, CD3OD): δ 3.45-3.50 (m, 1 H), 3.65 (s, 3 H, CH3OPh), 3.73-3.78 (m, 1 H),
3.87 (dd, 1 H, J = 3.5 Hz, J = 5.5 Hz), 4.03-4.23 (m, 5 H), 4.33-4.35 (m, 1 H), 4.41-4.52
(m, 2 H), 4.70 (d, 1 H, J = 6.5 Hz, H-1), 5.17 (dd, 1 H, J = 7 Hz, J = 8.5 Hz), 5.52 (t, 1 H,
J = 8.5 Hz), 6.63-6.66 (m, 2 H), 7.05-7.12 (m, 2 H), 7.16-7.27 (m, 5 H), 7.32-7.38 (m, 5
H), 7.48-7.56 (m, 2 H), 7.83-7.88 (m, 4 H).

13

C-NMR (125 MHz, CD3Cl): δ 21.5, 55.3,

55.5, 55.5, 63.8, 67.7, 69.7, 72.6, 73.1, 74.1, 75.2, 78.6, 78.8, 79.1, 101.7, 114.5, 120.7,
120.7, 126.0, 127.9, 127.9, 128.6, 128.6, 129.0, 129.2, 129.3, 129.7, 130.2, 130.4,
130.5, 130.5, 130.7, 134.1, 134.2, 142.2, 142.3, 144.7, 144.9, 157.7, 160.5, 166.6,
-

166.9, 172.8. ESI-MS: C45H41NO12 [M-1] calcd: 786.26, obsd: 786.26.
N-Fluorenylmethyloxycarbonyl-O-(2,

3-di-O-benzoyl-4-O-tert-

butyldimethylsilyl-β-D-xylopyranosyl)-L-serine benzyl ester (96). Compound 72
(400 mg, 0.86 mmol) in DCM (5 mL) was cooled down to –40 °C, followed by sequential
addition of 2, 6-lutidine (203 μL, 1.74 mmol) and TBSOTf (299 μL, 1.3 mmol). The
resulting solution was warmed up very slowly to room temperature. The mixture was
quenched by Et3N and then diluted with DCM (100 mL).

The organic phase was

washed with saturated NaHCO3 and then dried over Na2SO4, filtered and the solvents
were removed in vacuo. Silica gel column chromatography (Hexanes–EtOAc) afforded
309

p-Tolyl 2, 3-di-O-benzoyl-4-O-tert-butyldimethylsilyl-1-thio-β-D-xylopyranoside 95 as
white solid (448 mg, 90%) (HRMS: C32H38O6SSi [M+NH4]

+

calcd: 596.2502, obsd:

596.2480) which was used as donor to couple to acceptor 91 to produce compound 96
in 83% yield following the general procedure of single step glycosylation.

1

H-NMR

(600 MHz, CD3Cl): δ -0.12 (s, 3 H, Si(CH3)2C(CH3)3), 0.02 (s, 3 H, Si(CH3)2C(CH3)3),
0.76 (s, 9 H, Si(CH3)2C(CH3)3), 3.31-3.35 (m, 1 H), 3.83-3.96 (m, 3 H), 4.10-4.13 (m, 1
H), 4.18-4.20 (m, 1 H), 4.30-4.34 (m, 2 H), 4.49-4.52 (m, 1 H), 4.57 (d, 1 H, J = 6.5 Hz,
H-1), 5.11-5.17 (m, 2 H, PhCH2), 5.22-5.25 (m, 1 H), 5.46-5.49 (m, 1 H), 5.54-5.56 (m,
1 H), 7.22-7.40 (m, 14 H), 7.47-7.55 (m, 3 H), 7.75-7.77 (m, 2 H), 7.90-7.95 (m, 4 H).
13

C-NMR (150 MHz, CD3Cl): δ -5.0, -4.7, 17.7, 25.4, 47.0, 54.3, 65.9, 67.1, 67.3, 68.9,

69.1, 71.5, 74.7, 101.6, 119.9, 119.9, 125.1, 127.0, 127.6, 127.7, 128.1, 128.3, 128.3,
128.5, 129.2, 129.5, 129.7, 129.7, 133.0, 133.1, 135.2, 141.2, 141.2, 143.7, 143.8,
+

155.9, 165.3, 165.5, 169.4. HRMS: C50H53NO11Si [M+NH4] calcd: 889.3732, obsd:
889.3686.
N-Fluorenylmethyloxycarbonyl-O-(2,

3-di-O-benzoyl-β-D-xylopyranosyl)-L-

serine benzyl ester (93). Compound 92 (300 mg, 0.354 mmol) was dissolved in 6 mL
o

DCM/H2O (10:1) and cooled down to 0 C, followed by addition of 804 mg DDQ. The
resulting mixture was stirred under reflux overnight. After cooling down to room
temperature, the reaction mixture was diluted with DCM and washed with sat. NaHCO3.
The combined organic phase was dried over Na2SO4 and concentrated. Silica gel
310

column purification afforded compound 93 (163 mg, 61%). Compound 94 (3 g, 3.42
o

mmol) was dissolved in 50 mL DCM/H2O (10:1) and cooled down to 0 C, followed by
addition of 1.55 g DDQ. The resulting mixture was stirred under room temperature
overnight. After cooling down to room temperature, the reaction mixture was diluted with
DCM and washed with sat. NaHCO3. The combined organic phase was dried over
Na2SO4 and concentrated. Silica gel column purification afforded compound 93 (2.2 g,
85%). Compound 96 (1 g, 1.15 mmol) was dissolved in 20 mL DCM/H2O (10:1) and
o

cooled down to 0 C, followed by dropwise addition of 290 μL Tf2O. The resulting
mixture was stirred under room temperature for another 2 h. After the reaction was
complete, it was diluted with DCM and washed with sat. NaHCO3. The combined
organic phase was dried over Na2SO4 and concentrated. Silica gel column purification
1

afforded compound 93 (696 mg, 80%). H-NMR (500 MHz, CD3Cl): δ 3.05 (d, 1 H, J = 5
Hz, OH), 3.38-3.45 (m, 4 H), 3.83 (dd, 1 H, J = 2.5 Hz, J = 8.5 Hz), 3.88-3.93 (m, 1 H),
4.05-4.14 (m, 2 H), 4.22-4.26 (m, 1 H), 4.30-4.35 (m, 2 H), 4.53-4.55 (m, 1 H), 4.65 (d, 1
H, J = 4.5 Hz, H-1), 5.08-5.18 (m, 2 H, PhCH2), 5.24-5.32 (m, 2 H), 5.57 (d, 1 H, J = 7
Hz), 7.25-7.46 (m, 14 H), 7.51-7.55 (m, 3 H), 7.73-7.76 (m, 2 H), 7.94-7.99 (m, 4 H).
13

C-NMR (125 MHz, CD3Cl): δ 47.0, 54.2, 54.3, 64.0, 64.1, 67.1, 67.3, 67.4, 67.6, 68.3,

68.4, 69.0, 69.1, 70.2, 70.3, 75.0, 75.1, 100.6, 100.7, 119.9, 120.0, 124.7, 125.1, 125.1,
127.1, 127.7, 128.2, 128.4, 128.5, 128.5, 128.8, 129.0, 129.7, 129.8, 129.9, 130.0,

311

132.6, 133.4, 133.4, 133.6, 133.7, 135.1, 141.2, 143.7, 143.8, 155.9, 165.1, 165.5,
+

167.0, 169.5. HRMS: C44H39NO11 [M+NH4] calcd: 775.2867, obsd: 775.2867.
p-Tolyl

2-O-benzoyl-3-O-levulinoyl-4,

galactopyranosyl-(1→4)-2,

6-O-benzylidene

3-di-O-benzoyl-1-thio-β-D-xylopyranoside

-β-D(97).

Compound 97 was synthesized from donor 60 and acceptor 72 in 17% yield following
1

the general procedure of single step glycosylation. H-NMR (500 MHz, CD3Cl): δ 1.86
(s, 3 H, CH3COCH2CH2), 2.28 (s, 3 H, SPhCH3), 2.40-2.60 (m, 4 H, CH3COCH2CH2),
3.18 (br, 1 H), 3.41-3.45 (m, 1 H), 3.66-3.77 (m, 2 H), 3.98-4.04 (m, 1 H), 4.08-4.13 (m,
1 H), 4.21-4.22 (m, 1 H), 4.74 (d, 1 H, J = 8 Hz), 4.89 (d, 1 H, J = 7.5 Hz), 5.03 (dd, 1 H,
J = 3.5 Hz, J = 10.5 Hz), 5.24 (t, 1 H, J = 8 Hz), 5.32 (m, 1 H, PhCH), 5.53 (dd, 1 H, J =
8 Hz, J = 10.5 Hz), 5.63 (t, 1 H, J = 8 Hz), 7.03-7.05 (m, 3 H), 7.27-7.58 (m, 17 H), 7.947.99 (m, 4 H).

13

C-NMR (125 MHz, CD3Cl): δ 21.1, 28.2, 29.4, 37.7, 66.0, 66.6, 68.2,

69.3, 70.5, 71.9, 73.1, 73.3, 76.1, 76.4, 86.8, 100.9, 102.1, 126.5, 128.0, 128.3, 128.5,
128.9, 129.3, 129.3, 129.6, 129.7, 129.7, 129.9, 133.0, 133.1, 133.3, 137.4, 138.2,
164.8, 165.0, 165.3, 172.0.
p-Tolyl

2-O-benzoyl-3-O-levulinoyl-4,

galactopyranosyl-(1→4)-2,

6-O-benzylidene

3-di-O-benzoyl-1-thio-β-D-xylopyranoside

-α-D(98).

1

Compound 98 is a side product during the synthesis of 97. H-NMR (500 MHz, CD3Cl):

δ 1.88 (s, 3 H, CH3COCH2CH2), 2.31 (s, 3 H, SPhCH3), 2.39-2.58 (m, 4 H,
CH3COCH2CH2), 3.58-3.64 (m, 1 H), 3.83 (br, 1 H), 4.04-4.10 (m, 2 H), 4.25-4.36 (m, 2
H), 4.43 (d, 1 H, J = 3.5 Hz), 4.87-4.89 (m, 1 H), 5.19 (t, 1 H, J = 8.5 Hz), 5.42-5.58 (d, 5
312

H), 7.08-7.19 (m, 6 H), 7.32-7.54 (m, 14 H), 7.60-7.62 (m, 2 H), 7.87-7.89 (m, 2 H).

13

C-

NMR (125 MHz, CD3Cl): δ 21.1, 28.1, 29.4, 37.7, 62.9, 67.8, 68.3, 68.9, 70.7, 73.7,
74.1, 74.7, 87.0, 98.6, 100.9, 126.2, 128.0, 128.1, 128.1, 128.3, 128.6, 129.0, 129.5,
129.6, 129.7, 129.8, 132.8, 133.0, 133.1, 133.5, 137.3, 165.4, 171.9, 165.3, 205.9.
1

Glycal (100). Compound 100 is a side product during the synthesis of 97. HNMR (500 MHz, CD3Cl): δ 3.55-3.58 (m, 1 H), 4.06-4.08 (m, 1 H), 5.16-5.17 (m, 1 H),
5.21-5.28 (m, 2 H), 5.67 (br, 1 H), 7.42-7.48 (m, 4 H), 7.55-7.61 (m, 2 H), 8.04-8.06 (m,
2 H).
p-Tolyl

2-O-benzoyl-3-O-levulinoyl-4,

galactopyranosyl-(1→4)-2,

6-O-benzylidene

3-O-isopropylidene-1-thio-β-D-xylopyranoside

-β-D(101).

Compound 101 was synthesized from donor 60 and acceptor 68 in 65% yield following
the general procedure of single step glycosylation and the reaction was quenched by
o

1

Et3N under -70 C. H-NMR (600 MHz, CD3Cl): δ 1.36 (s, 6 H, C(CH3)2), 1.88 (s, 3 H,
CH3COCH2CH2), 2.30 (s, 3 H, SPhCH3), 2.43-2.63 (m, 4 H, CH3COCH2CH2), 3.09 (t,
1 H, J = 7.5 Hz), 3.18 (dd, 1 H, J = 7.5 Hz, J = 10 Hz), 3.54 (br, 1 H), 3.69 (t, 1 H, J =
7.5 Hz), 3.88-3.92 (m, 1 H), 3.98-4.01 (m, 1 H), 4.05-4.09 (m, 1 H), 4.27-4.30 (m, 1 H),
4.36-4.37 (m, 1 H), 4.61 (d, 1 H, J = 7.5 Hz), 4.78 (d, 1 H, J = 6.5 Hz), 5.11 (dd, 1 H, J =
3.5 Hz, J = 9 Hz), 5.50 (s, 1 H, PhCH), 5.56-5.59 (m, 1 H), 7.06-7.08 (m, 2 H), 7.33-7.44
(m, 7 H), 7.50-7.58 (m, 3 H), 7.96-7.98 (m, 2 H).

13

C-NMR (150 MHz, CD3Cl): δ 14.1,

21.0, 21.1, 26.4, 26.6, 28.2, 29.4, 37.7, 60.3, 66.7, 67.8, 68.8, 68.9, 72.1, 73.4, 75.0,
75.9, 80.8, 85.0, 99.9, 101.0, 110.7, 126.3, 127.4, 128.1, 128.4, 129.0, 129.5, 129.6,
313

+

133.2, 133.7, 137.4, 138.4, 165.0, 172.0, 206.0. HRMS: C40H44O12S [M+NH4] calcd:
766.2897, obsd: 766.2872.
p-Tolyl

2-O-benzoyl-3-O-levulinoyl-4,

galactopyranosyl-(1→4)-1-thio-β-D-xylopyranoside

6-O-benzylidene

-β-D-

Compound

101 was

(102).

synthesized from donor 60 and acceptor 68 in 40% yield following the general
procedure of single step glycosylation and the reaction was quenched by Et3N under 0
o

1

H-NMR (500 MHz, CD3Cl): δ 1.90 (s, 3 H, CH3COCH2CH2), 2.29 (s, 3 H,

C.

SPhCH3), 2.44-2.64 (m, 4 H, CH3COCH2CH2), 3.11-3.16 (m, 1 H), 3.27-3.33 (m, 1 H),
3.38-3.76 (m, 4 H), 3.94 (br, 1 H), 4.07-4.09 (m, 2 H), 4.28-4.30 (m, 1 H), 4.34 (d, 1 H, J
= 9.5 Hz), 4.40 (d, 1 H, J = 3.5 Hz), 4.65 (d, 1 H, J = 8 Hz), 5.13 (dd, 1 H, J = 3.5 Hz, J =
10.5 Hz), 5.51 (s, 1 H, PhCH), 5.57-5.61 (m, 1 H), 7.05-7.07 (m, 2 H), 7.34-7.60 (m, 10
H), 7.94-7.97 (m, 2 H). ESI-MS: C37H40O12SNa calcd 731.22, obsd 731.30.
p-Tolyl

2-O-benzoyl-3-O-levulinoyl-4,

6-O-benzylidene

-β-D-

galactopyranosyl-(1→3)-2-O-benzoyl-4, 6-O-benzylidene -β-D-galactopyranosyl(1→4)-2, 3-O-isopropylidene-1-thio-β-D-xylopyranoside (103). Compound 103 was
synthesized from donor 89 and acceptor 68 in 10-20% yield following the general
procedure of single step glycosylation and the reaction was quenched by Et3N under o

1

70 C. H-NMR (500 MHz, CD3Cl): δ 1.34 (s, 3 H, C(CH3)2), 1.35 (s, 3 H, C(CH3)2),
1.84 (s, 3 H, CH3COCH2CH2), 2.29 (s, 3 H, SPhCH3), 2.36-2.57 (m, 4 H,
CH3COCH2CH2), 3.03-3.11 (m, 2 H), 3.43 (br, 1 H), 3.63 (t, 1 H, J = 9 Hz), 3.78-3.84

314

(m, 1 H), 3.89-3.92 (m, 1 H), 3.93-3.96 (m, 1 H), 3.99-4.02 (m, 1 H), 4.09-4.12 (m, 1 H),
4.22-4.26 (m, 2 H), 4.28-4.29 (m, 1 H), 4.36-4.37 (m, 1 H), 4.56 (d, 1 H, J = 9.5 Hz),
4.69 (d, 1 H, J = 8.5 Hz), 4.93-4.96 (m, 1 H), 4.97 (d, 1 H, J = 8.5 Hz), 5.39 (s, 1 H,
PhCH), 5.43-5.47 (m, 1 H), 5.46 (s, 1 H, PhCH), 5.57 (dd, 1 H, J = 8 Hz, J = 10.5 Hz),
7.04-7.06 (m, 2 H), 7.19-7.25 (m, 5 H), 7.32-7.38 (m, 3 H), 7.41-7.55 (m, 4 H), 7.71-7.73
(m, 2 H), 7.85-7.87 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 21.1, 26.4, 26.6, 28.1, 29.3,

37.7, 66.5, 67.0, 67.8, 68.6, 68.8, 68.9, 70.6, 72.1, 73.2, 75.0, 75.1, 75.8, 75.9, 80.8,
85.0, 100.0, 100.3, 100.6, 100.9, 110.6, 126.1, 126.4, 127.5, 127.9, 128.2, 128.3, 128.5,
129.1, 129.2, 129.5, 129.6, 129.9, 132.9, 133.6, 137.5, 137.5, 138.3, 164.6, 165.1,
165.5, 171.9, 206.0. HRMS: C60H62O18S [M+NH4]

+

calcd: 1120.4001, obsd:

1120.4078.
N-Fluorenylmethyloxycarbonyl-O-[2-O-benzoyl-3-O-levulinoyl-4,

6-O-

benzylidene -β-D-galactopyranosyl-(1→4)-2, 3-di-O-benzoyl-β-D-xylopyranosyl]-Lserine benzyl ester (105). Compound 105 was synthesized from donor 60 and
acceptor 93 in 81% yield following the general procedure of single step glycosylation.
1

H-NMR (500 MHz, CD3Cl): δ 1.87 (s, 3 H, CH3COCH2CH2), 2.41-2.61 (m, 4 H,

CH3COCH2CH2), 3.23-3.31 (m, 2 H), 3.71-3.83 (m, 4 H), 3.90-3.95 (m, 1 H), 4.09-4.13
(m, 1 H), 4.20-4.24 (m, 3 H), 4.29-4.33 (m, 1 H), 4.45-4.51 (m, 1 H), 4.55 (d, 1 H, J = 6
Hz), 4.76 (d, 1 H, J = 8 Hz), 5.00-5.17 (m, 4 H), 5.35 (s, 1 H, PhCH), 5.53-5.62 (m, 3 H),
7.20-7.48 (m, 21 H), 7.51-7.56 (m, 3 H), 7.74-7.76 (m, 2 H), 7.94-7.98 (m, 6 H).

13

C-

NMR (150 MHz, CD3Cl): δ 28.4, 29.6, 37.9, 38.1, 47.3, 54.5, 62.5, 66.9, 67.4, 67.5,

315

68.5, 69.2, 69.3, 69.6, 71.0, 72.0, 72.1, 73.4, 76.2, 91.5, 100.8, 101.2, 102.3, 120.2,
125.4, 126.5, 126.7, 127.3, 127.3, 127.9, 127.9, 128.2, 128.3, 128.4, 128.5, 128.5,
128.6, 128.6, 128.7, 128.8, 129.1, 129.3, 129.6, 129.7, 129.8, 130.0, 130.1, 130.1,
133.3, 133.6, 135.3, 137.7, 141.4, 143.9, 144.0, 156.1, 165.1, 165.3, 165.7, 169.6,
+

172.3, 206.3. HRMS: C69H63NO19 [M+NH4] calcd: 1227.4338, obsd: 1227.3872.
N-Fluorenylmethyloxycarbonyl-O-[2-O-benzoyl-4,
galactopyranosyl-(1→4)-2,

6-O-benzylidene-β-D-

3-di-O-benzoyl-β-D-xylopyranosyl]-L-serine

benzyl

ester (106). Compound 106 was synthesized from compound 105 in 90% yield
1

following the general procedure of Lev deprotection. H-NMR (600 MHz, CD3Cl): δ 2.60
(d, 1 H, J = 9.5 Hz, OH), 3.26-3.34 (m, 2 H), 3.74-3.86 (m, 5 H), 3.93-3.97 (m, 1 H),
4.09-4.13 (m, 2 H), 4.21-4.33 (m, 2 H), 4.48-4.50 (m, 1 H), 4.57 (d, 1 H, J = 5 Hz), 4.71
(d, 1 H, J = 7 Hz), 5.02-5.10 (m, 2 H, COOCH2Ph), 5.16-5.19 (m, 1 H), 5.27-5.30 (m, 1
H), 5.40 (s, 1 H, PhCH), 5.56 (d, 1 H, J = 7.5 Hz), 5.61 (t, 1 H, J = 6 Hz), 7.21-7.47 (m,
23 H), 7.52-7.54 (m, 3 H), 7.74-7.76 (m, 2 H), 7.95-8.01 (m, 5 H).

13

C-NMR (150 MHz,

CD3Cl): δ 47.0, 54.2, 62.2, 66.8, 67.2, 67.3, 68.3, 69.0, 70.7, 71.6, 71.8, 73.0, 75.3,
75.7, 100.5, 101.4, 101.7, 119.9, 125.1, 126.2, 126.5, 127.0, 127.7, 127.7, 127.9, 128.1,
128.2, 128.3, 128.3, 128.4, 128.4, 128.4, 128.5, 129.1, 129.1, 129.5, 129.6, 129.8,
129.9, 133.1, 133.2, 135.1, 137.2, 141.2, 143.7, 143.8, 155.9, 165.1, 165.5, 165.9,
+

169.4. HRMS: C64H57NO17 [M+NH4] calcd: 1129.3970, obsd: 1129.3920.
N-Fluorenylmethyloxycarbonyl-O-[2-O-benzoyl-3-O-levulinoyl-4,
benzylidene

-β-D-galactopyranosyl-(1→3)-2-O-benzoyl-4,

316

6-O-

6-O-benzylidene-β-D-

galactopyranosyl-(1→4)-2,

3-di-O-benzoyl-β-D-xylopyranosyl]-L-serine

benzyl

ester (107). Compound 107 was synthesized from donor 60 and acceptor 106 or donor
86 and acceptor 93 in 82% yield following the general procedure of single step
1

glycosylation. H-NMR (600 MHz, CD3Cl): δ 1.83 (s, 3 H, CH3COCH2CH2), 2.36-2.56
(m, 4 H, CH3COCH2CH2), 3.14-3.23 (m, 2 H), 3.42 (br, 1 H), 3.65-3.77 (m, 4 H), 3.823.87 (m, 1 H), 3.93-3.95 (m, 1 H), 4.09-4.11 (m, 2 H), 4.18-4.32 (m, 6 H), 4.43-4.47 (m,
1 H), 4.49 (d, 1 H, J = 5 Hz), 4.69 (d, 1 H, J = 6.5 Hz), 4.93-5.12 (m, 5 H), 5.24 (s, 1 H,
PhCH), 5.42-5.44 (m, 1 H), 5.45 (s, 1 H, PhCH), 5.50-5.57 (m, 3 H), 7.14-7.27 (m, 20 H),
7.29-7.37 (m, 9 H), 7.41-7.52 (m, 6 H), 7.70-7.75 (m, 3 H), 7.86-7.88 (m, 2 H), 7.91-7.94
(m, 3 H).

13

C-NMR (150 MHz, CD3Cl): δ 28.1, 29.3, 29.7, 37.2, 37.7, 47.0, 54.2, 66.5,

67.0, 67.2, 67.2, 68.2, 68.6, 68.9, 69.0, 71.0, 72.1, 73.2, 75.7, 75.8, 99.9, 100.5, 100.9,
102.4, 119.9, 125.1, 126.2, 126.4, 127.0, 127.6, 127.7, 128.2, 128.2, 128.3, 128.3,
128.4, 128.4, 128.5, 129.1, 129.3, 129.5, 129.6, 129.7, 129.8, 129.9, 132.9, 133.0,
+

137.5, 141.2, 165.1, 165.5, 165.9, 169.4, 206.0. MALDI-MS: C89H81NO25 [M+Na]
calcd: 1587.59, obsd: 1588.20.
N-Fluorenylmethyloxycarbonyl-O-[2-O-benzoyl-4,

6-O-benzylidene

-β-D-

6-O-benzylidene-β-D-galactopyranosyl-

galactopyranosyl-(1→3)-2-O-benzoyl-4,

(1→4)-2, 3-di-O-benzoyl-β-D-xylopyranosyl]-L-serine benzyl ester (108). Compound
108 was synthesized from compound 107 in 86% yield following the general procedure
1

of Lev deprotection. H-NMR (600 MHz, CD3Cl): δ 2.51 (d, 1 H, J = 9.5 Hz, OH), 3.16
(br, 1 H), 3.22-3.25 (m, 1 H), 3.40 (br, 1 H), 3.63-3.77 (m, 5 H), 3.85-3.89 (m, 1 H), 3.953.97 (m, 1 H), 4.09-4.22 (m, 6 H), 4.28-4.33 (m, 1 H), 4.45-4.48 (m, 1 H), 4.51 (d, 1 H, J
317

= 5 Hz), 4.72 (d, 1 H, J = 7 Hz), 4.92 (d, 1 H, J = 7 Hz), 4.98-5.06 (m, 2 H, COOCH2Ph),
5.10-5.13 (m, 1 H), 5.22 (s, 1 H, PhCH), 5.26-5.29 (m, 1 H), 5.50 (s, 1 H, PhCH), 5.465.58 (m, 3 H), 7.14-7.37 (m, 28 H), 7.42-7.46 (m, 4 H), 7.49-7.52 (m, 2 H), 7.73-7.75 (m,
2 H), 7.77-7.79 (m, 2 H), 7.92-7.96 (m, 5 H).

13

C-NMR (150 MHz, CD3Cl): δ 29.6, 47.0,

54.2, 62.2, 66.7, 67.0, 67.2, 67.2, 68.2, 68.7, 69.0, 70.7, 71.1, 71.6, 72.0, 72.5, 74.7,
75.5, 75.8, 99.5, 100.4, 100.6, 101.3, 102.5, 114.7, 119.9, 125.1, 126.2, 126.3, 127.0,
127.0, 127.6, 127.7, 127.8, 128.1, 128.2, 128.3, 128.3, 128.3, 128.4, 129.1, 129.3,
129.4, 129.5, 129.7, 129.7, 129.9, 129.9, 132.9, 133.1, 135.1, 137.3, 137.5, 141.2,
143.7, 143.8, 155.9, 164.6, 165.0, 165.5, 166.4, 169.4. MALDI-MS: C84H75NO23
+

[M+Na] calcd: 1488.47, obsd: 1488.65.
p-Tolyl

2-O-benzoyl-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-6-O-p34

methoxybenzyl-1-thio-α-L-idopyranoside (109). Compound 79

(500 mg, 0.83 mmol)

in DCM (5 mL) was cooled down to –40 °C, followed by sequential addition of 2, 6lutidine (203 μL, 1.74 mmol) and TBSOTf (299 μL, 1.3 mmol). The resulting solution
was warmed up very slowly to room temperature. The mixture was quenched by Et3N
and then diluted with DCM (100 mL). The organic phase was washed with saturated
NaHCO3 and then dried over Na2SO4, filtered and the solvents were removed in vacuo.
Silica gel column chromatography (Hexanes–EtOAc) afforded compound 109 (547 mg,
1

92%). H-NMR (600 MHz, CD3Cl): δ 2.28 (s, 3 H, SPhCH3), 3.67-3.70 (m, 1 H, H-3),
3.76-3.79 (m, 3 H, H-4, H-6a, H-6b), 3.80 (s, 3 H, CH3OPhCH2O), 4.48 (br, 2 H,
318

CH3OPhCH2O), 4.66-4.68 (m, 1 H, OCH2Ph), 4.78-4.82 (m, 1 H), 4.88-4.90 (m, 1 H,
OCH2Ph), 5.36-5.37 (m, 1 H, H-2), 5.52 (d, 1 H, J = 2 Hz, H-1), 6.86-6.88 (m, 2 H),
6.98-7.00 (m, 2 H), 7.24-7.27 (m, 3 H), 7.29-7.32 (m, 2 H), 7.36-7.42 (m, 4 H), 7.45-7.46
(m, 2 H), 7.51-7.54 (m, 1 H), 8.02-8.04 (m, 2 H).

13

C-NMR (150 MHz, CD3Cl): δ 17.9,

21.0, 25.6, 55.2, 68.7, 69.4, 69.9, 72.4, 72.8, 74.7, 86.1, 113.6, 127.8, 128.1, 128.1,
128.3, 129.2, 129.5, 129.7, 130.0, 130.3, 131.9, 132.3, 133.1, 137.3, 137.5, 159.0,
+

165.6. HRMS: C41H50O7SSi [M+NH4] calcd: 732.3390, obsd: 732.3377.
p-Tolyl

2-O-benzoyl-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-6-O-p-

methoxybenzyl-1-thio-α-L-idopyranosyl-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-βD-glucopyranoside (110). Synthesis of compound 110 from donor 109 and acceptor
40 following the general procedure of single step glycosylation failed. Compound 111
(60%) and acceptor 40 (40%) were isolated.
p-Tolyl

2-O-benzoyl-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-6-O-

chloroacetyl-1-thio-α-L-idopyranoside (115). Compound 109 (1 g, 1.4 mmol) was
dissolved in DCM/H2O (20 mL, 10:1), followed by addition of DDQ (476 mg). The
resulting mixture was stirred under room temperature overnight. After the reaction was
complete, it was diluted with DCM, washed with sat. NaHCO3. The combined organic
phase was dried over Na2SO4 and concentrated. Column purification afforded p-tolyl 2O-benzoyl-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-1-thio-α-L-idopyranoside 114 (682 mg,
1

82%). H-NMR (500 MHz, CD3Cl): δ -0.28 (br, 3 H, Si(CH3)2), -0.10 (br, 3 H, Si(CH3)2),
0.67 (s, 9 H, C(CH3)3), 1.82 (dd, 1 H, J = 3 Hz, J = 10 Hz, H-4), 2.30 (s, 3 H, SPhCH3),
319

3.67-3.75 (m, 3 H, H-3, H-6a, OH), 3.90-3.95 (m, 1 H, H-5), 4.65-4.68 (m, 2 H, H-6b,
CH2Ph), 4.90-4.93 (m, 1 H, CH2Ph), 5.39-5.41 (m, 1 H, H-2), 5.53 (br, 1 H, H-1), 7.097.11 (m, 2 H), 7.26-7.45 (m, 9 H), 7.51-7.54 (m, 1 H), 8.03-8.06 (m, 2 H).

13

C-NMR (125

MHz, CD3Cl): δ -5.48, -4.66, 17.9, 21.0, 25.6, 62.7, 68.9, 69.5, 69.6, 72.3, 74.2, 86.2,
127.9, 128.2, 128.4, 129.6, 129.7, 130.0, 131.5, 132.6, 133.2, 137.4, 137.8, 165.5.
+

HRMS: C33H42O6SSi [M+NH4] calcd: 612.2815, obsd: 612.2813. Compound 114 (682
mg, 1.14 mmol) was dissolved in DCM (10 mL), followed by addition of ClAcCl (108 μL,
o

1.368 mmol) and pyridine (110 μL, 1.368 mmol) under 0 C. The resulting mixture was
stirred under room temperature for 2 h. After the reaction was complete, it was diluted
with DCM, washed with 10% HCl and sat. NaHCO3. The combined organic phase was
dried over Na2SO4 and concentrated. Column purification afforded compound 115 (653
mg, 85%).

1

H-NMR (600 MHz, CD3Cl): δ -0.30 (s, 3 H, Si(CH3)2), -0.10 (s, 3 H,

Si(CH3)2), 0.68 (s, 9 H, C(CH3)3), 2.31 (s, 3 H, SPhCH3), 3.72-3.75 (m, 2 H, H-3, H-4),
3.98 (s, 2 H, ClCH2CO), 4.29-4.32 (m, 1 H, H-6b), 4.47-4.50 (m, 1 H, H-6b), 4.65-4.67
(m, 1 H, CH2Ph), 4.85-4.87 (m, 1 H, H-5), 4.92-4.94 (m, 1 H, CH2Ph), 5.38 (br, H-2),
5.53 (br, H-1), 7.10-7.11 (m, 2 H), 7.27-7.30 (m, 1 H), 7.33-7.39 (m, 4 H), 7.43-7.46 (m,
4 H), 7.51-7.54 (m, 1 H), 8.03-8.05 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 17.9, 21.0,

25.5, 40.6, 65.4, 66.9, 68.2, 69.2, 72.3, 73.7, 86.1, 128.0, 128.2, 128.2, 128.4, 129.5,

320

129.6, 130.0, 131.8, 133.2, 137.2, 137.6, 165.5, 165.7. HRMS: C35H43ClO7SSi
+

[M+NH4] calcd: 688.2531, obsd: 688.2548.
p-Tolyl

2-O-benzoyl-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-6-O-

chloroacetyl-α-L-idopyranosyl-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-1-thio-β-Dglucopyranoside (116). Compound 116 was synthesized from donor 115 and acceptor
1

40 in 48% yield following the general procedure of single step glycosylation. H-NMR
(600 MHz, CD3Cl): δ -0.15 (s, 3 H, Si(CH3)2), -0.05 (s, 3 H, Si(CH3)2), 0.78 (s, 9 H,
C(CH3)3), 2.28 (s, 3 H, SPhCH3), 3.20-3.24 (m, 1 H), 3.28-3.32 (m, 1 H), 3.36-3.39 (m,
1 H), 3.64-3.85 (m, 8 H), 3.96-4.02 (m, 1 H), 4.24-4.37 (m, 4 H), 4.45-4.52 (m, 2 H,
CH2Ph), 4.65-4.80 (m, 3 H), 4.88-4.92 (m, 1 H), 5.14-5.17 (m, 2 H), 7.00-7.12 (m, 2 H),
7.18-7.43 (m, 19 H), 7.53-7.56 (m, 1 H), 7.95-7.98 (m, 2 H).

13

C-NMR (125 MHz,

CD3Cl): δ -5.2, -4.5, 17.9, 21.1, 25.7, 40.5, 40.6, 64.0, 64.3, 64.9, 68.2, 68.6, 68.8, 69.2,
70.3, 70.7, 71.8, 73.0, 73.2, 73.3, 74.3, 75.1, 75.3, 75.5, 79.5, 80.1, 83.2, 85.7, 97.0,
126.6, 127.4, 127.5, 127.6, 127.8, 127.9, 128.0, 128.1, 128.2, 128.2, 128.2, 128.3,
128.3, 128.3, 129.5, 129.7, 129.7, 129.8, 129.9, 132.4, 133.3, 134.5, 137.5, 137.9,
+

138.1, 138.8, 165.4, 166.7. HRMS: C55H64ClN3O11SSi [M+NH4] calcd: 1055.4063,
obsd: 1055.4050.
p-Tolyl

2-O-benzoyl-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-6-O-

chloroacetyl-α-L-idopyranosyl-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-β-Dglucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-1-thio-β-D-

321

glucopyranoside (117). Compound 117 was synthesized from donor 116 and acceptor
1

48 in 36% yield following the general procedure of single step glycosylation. H-NMR
(500 MHz, CD3Cl): δ -0.16 (s, 3 H, Si(CH3)2), -0.07 (s, 3 H, Si(CH3)2), 0.77 (s, 9 H,
C(CH3)3), 2.25 (s, 3 H, SPhCH3), 3.22-3.27 (m, 2 H), 3.49-3.77 (m, 14 H), 3.94-4.03 (m,
4 H), 4.14-4.37 (m, 7 H), 4.61-4.75 (m, 6 H), 4.95 (d, 1 H, J = 11 Hz), 5.07-5.29 (m, 3 H),
5.56 (d, 1 H, J = 3.5 Hz), 6.79-6.81 (m, 2 H), 6.96-7.58 (m, 32 H), 7.91-7.93 (m, 2 H),
8.05-8.07 (m, 2 H). ESI-MS: C83H96ClN4O18SSi calcd: 1531.56, obsd: 1531.66.
+

MALDI-MS: [M+Na] calcd: 1538.23, obsd: 1538.21.
p-Tolyl

2-O-benzoyl-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-6-O-levulinoyl-

1-thio-α-L-idopyranoside (126). Compound 114 (650 mg, 1.09 mmol) was dissolved in
dry DCM (10 mL), followed by addition of EDC-HCl (579 mg, 2.7 mmol), DMAP (26 mg,
0.27 mmol) and LevOH (316 mg, 2.7 mmol). The resulting mixture was stirred under
room temperature for overnight. The reaction was diluted with DCM and washed with
10% HCl solution, sat. NaHCO3 solution sequentially. The combined organic phase was
dried over Na2SO4 and purified by silica gel column (3:1. hexane-EtOAc) to compound
1

126 (674 mg, 89%). H-NMR (500 MHz, CD3Cl): δ -0.30 (s, 3 H, Si(CH3)2), -0.10 (s, 3
H, Si(CH3)2), 0.68 (s, 9 H, C(CH3)3), 2.13 (s, 3 H, CH3COCH2CH2), 2.29 (s, 3 H,
SPhCH3), 2.55-2.71 (m, 4 H, CH3COCH2CH2), 3.71-3.76 (m, 2 H, H-3, H-4), 4.22-4.25
(m, 1 H, H-6a), 4.34-4.38 (m, 1 H, H-6b), 4.65-4.68 (m, 1 H, PhCH2), 4.83-4.86 (m, 1 H,
H-5), 4.91-4.94 (m, 1 H, PhCH2), 5.37-5.39 (m, 1 H, H-2), 5.32 (br, 1 H, H-1), 7.08-7.10
322

(m, 2 H), 7.25-7.29 (m, 1 H), 7.32-7.38 (m, 4 H), 7.43-7.52 (m, 5 H), 8.03-8.05 (m, 2 H).
13

C-NMR (125 MHz, CD3Cl): δ -5.7, -4.6, 17.8, 20.9, 25.5, 27.6, 29.6, 37.7, 63.9, 66.9,

68.1, 69.2, 72.1, 73.6, 86.0, 127.9, 128.0, 128.1, 128.3, 129.5, 129.9, 132.0, 133.1,
137.2, 137.3, 165.5, 172.3, 206.1.
p-Tolyl

2-O-benzoyl-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-6-O-levulinoyl-

α-L-idopyranosyl-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-1-thio-β-Dglucopyranoside (127). Compound 127 was synthesized from donor 126 and acceptor
1

40 in 60% yield following the general procedure of single step glycosylation. H-NMR
(600 MHz, CD3Cl): δ -0.16 (s, 3 H, Si(CH3)2), -0.05 (s, 3 H, Si(CH3)2), 0.77 (s, 9 H,
C(CH3)3), 2.07 (s, 3 H, CH3COCH2CH2), 2.26 (s, 3 H, SPhCH3), 2.32-2.55 (m, 4 H,
CH3COCH2CH2), 3.20-3.23 (m, 1 H), 3.29-3.33 (m, 1 H), 3.39-3.42 (m, 1 H), 3.63-3.71
(m, 3 H), 3.77-3.79 (m, 1 H), 3.82 (t, 1 H, J = 7.5 Hz), 4.02-4.05 (m, 1 H), 4.24-4.30 (m,
3 H), 4.43-4.49 (m, 2 H, CH2Ph), 4.65-4.72 (m, 3 H), 4.96 (d, 1 H, J = 9 Hz), 5.12-5.16
(m, 2 H), 6.97-6.99 (m, 2 H), 7.17-7.29 (m, 13 H), 7.32-7.42 (m, 6 H), 7.52-7.56 (m, 1 H),
7.94-7.96 (m, 2 H).

13

C-NMR (150 MHz, CD3Cl): δ -5.7, -5.1, 17.9, 20.0, 25.7, 27.7,

29.7, 37.7, 62.4, 65.1, 68.3, 68.9, 69.2, 70.8, 73.1, 73.3, 74.6, 75.4, 76.7, 79.3, 83.3,
85.9, 97.2, 127.0, 127.4, 127.5, 127.8, 128.0, 128.1, 128.2, 128.3, 128.3, 129.5, 129.6,
129.8, 133.2, 134.1, 137.6, 138.0, 138.1, 138.5, 165.4, 172.2, 206.3. HRMS:
+

C58H69N3O12SSi [M+NH4] calcd: 1077.4715, obsd: 1077.4650.
p-Tolyl

2-O-benzoyl-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-6-O-levulinoyl-

α-L-idopyranosyl-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl323

(1→4)-2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside
(128). Compound 128 was synthesized from donor 127 and acceptor 48 in 34% or
donor 126 and acceptor 134 in 50% yield following the general procedure of single step
1

glycosylation. H-NMR (600 MHz, CD3Cl): δ -0.12 (s, 3 H, Si(CH3)2), -0.02 (s, 3 H,
Si(CH3)2), 0.81 (s, 9 H, C(CH3)3), 2.08 (s, 3 H, CH3COCH2CH2), 2.28 (s, 3 H,
SPhCH3), 2.38-2.60 (m, 4 H, CH3COCH2CH2), 3.21-3.23 (dd, 1 H, J = 3.5 Hz, J = 9 Hz),
3.25-3.28 (m, 1 H), 3.52-3.66 (m, 6 H), 3.77-3.80 (m, 4 H), 3.96-3.99 (m, 3 H), 4.04-4.14
(m, 3 H), 4.21-4.29 (m, 4 H), 4.39-4.41 (m, 1 H), 4.64-4.75 (m, 5 H), 5.04-5.08 (m, 2 H),
5.17 (t, 1 H, J = 4.5 Hz), 5.26-5.31 (m, 1 H), 5.59 (d, 1 H, J = 3 Hz), 6.81-6.83 (m, 2 H),
6.98-6.99 (m, 2 H), 7.07-7.31 (m, 20 H), 7.34-7.38 (m, 6 H), 7.44-7.47 (m, 2 H), 7.527.59 (m, 2 H), 7.91-7.93 (m, 2 H), 8.08-8.10 (m, 2 H).

13

C-NMR (150 MHz, CD3Cl): δ -

5.2, -4.6, 14.0, 14.1, 17.9, 21.0, 21.0, 22.6, 25.2, 25.7, 27.7, 29.7, 31.5, 34.6, 37.7, 55.1,
60.3, 62.0, 63.1, 67.8, 68.6, 68.9, 69.5, 71.1, 71.5, 72.1, 72.8, 73.0, 73.3, 73.4, 74.6,
74.9, 74.9, 78.2, 78.9, 85.1, 85.8, 96.8, 97.2, 113.5, 127.4, 127.5, 127.6, 127.6, 127.7,
127.8, 128.0, 128.0, 128.0, 128.1, 128.2, 128.2, 128.2, 128.3, 128.4, 129.2, 129.5,
129.7, 129.8, 130.2, 133.2, 133.6, 137.2, 137.6, 137.9, 138.1, 138.3, 159.0, 165.0,
+

165.3, 172.2, 206.3. MALDI-MS: C86H97N3O19SSi [M+Na]

calcd: 1559.85, obsd:

1560.67.
Trisaccharide (130). Compound 130 was a side product generated during the
1

synthesis of 128. H-NMR (600 MHz, CD3Cl): δ -0.15 (s, 3 H, Si(CH3)2), -0.04 (s, 3 H,
Si(CH3)2), 0.80 (s, 9 H, C(CH3)3), 2.08 (s, 3 H, CH3COCH2CH2), 2.39-2.59 (m, 4 H,
324

CH3COCH2CH2), 3.20-3.23 (m, 1 H), 3.52 (br, 1 H), 3.57-3.80 (m, 9 H), 3.90 (t, 1 H, J =
8.5 Hz), 3.98-4.11 (m, 3 H), 4.27-4.32 (m, 3 H), 4.37-4.47 (m, 2 H, CH2Ph), 4.64-4.77
(m, 6 H), 4.85-4.88 (m, 2 H), 4.93 (br, 1 H), 5.10 (d, 1 H, J = 3.5 Hz), 5.18 (t, 1 H, J =
3.5 Hz), 5.54 (br, 1 H), 7.19-7.38 (m, 22 H), 7.44-7.54 (m, 4 H), 7.93-7.95 (m, 2 H),
8.13-8.15 (m, 2 H).

13

C-NMR (150 MHz, CD3Cl): δ -5.0, -4.3, 18.2, 26.0, 28.0, 30.0,

38.0, 62.3, 63.7, 65.1, 68.7, 69.1, 69.4, 71.3, 71.6, 72.5, 73.5, 73.6, 74.7, 75.3, 75.6,
75.7, 78.7, 97.3, 98.5, 99.8, 127.6, 127.8, 127.8, 128.0, 128.1, 128.2, 128.2, 128.3,
128.5, 128.5, 128.6, 128.6, 128.8, 129.8, 129.9, 130.0, 130.1, 133.5, 133.5, 137.8,
137.9, 138.1, 138.4, 165.7, 165.9, 172.5, 206.6. MALDI-MS: C71H81N3O18Si [M+Na]

+

calcd: 1314.53, obsd: 1314.85.
p-Tolyl

2-azido-3,6-di-O-benzyl-4-O-tert-butyl-dimethylsilyl-2-deoxy-α-D-

glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-1-thio-β-Dglucopyranoside (131). Compound 131 was synthesized from donor 41 and acceptor
1

48 in 62% yield following the general procedure of single step glycosylation. H-NMR
(500 MHz, CD3Cl): δ -0.04 (s, 3 H, Si(CH3)2), -0.01 (s, 3 H, Si(CH3)2), 0.80 (s, 9 H,
C(CH3)3), 2.28 (s, 3 H, SPhCH3), 3.20 (dd, 1 H, J = 4 Hz, J = 5 Hz), 3.40-3.42 (m, 1 H),
3.50-3.53 (m, 1 H), 3.65-3.79 (m, 10 H), 3.85-3.88 (m, 1 H), 4.01-4.03 (m, 2 H), 4.344.37 (m, 1 H), 4.43-4.49 (m, 4 H), 4.66-4.84 (m, 6 H), 5.10-5.15 (m, 1 H), 5.62 (d, 1 H, J
= 4 Hz), 6.85-6.88 (m, 2 H), 7.00-7.02 (m, 2 H), 7.12-7.46 (m, 21 H), 7.55-7.58 (m, 1 H),
8.07-8.09 (m, 2 H).

325

2-azido-3,6-di-O-benzyl-4-O-tert-butyl-dimethylsilyl-2-deoxy-β-D-

p-Tolyl

glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-1-thio-β-Dglucopyranoside (132). Compound 132 was a side product generated during the
1

synthesis of 131. H-NMR (500 MHz, CD3Cl): δ -0.00 (s, 3 H, Si(CH3)2), -0.01 (s, 3 H,
Si(CH3)2), 0.84 (s, 9 H, C(CH3)3), 2.29 (s, 3 H, SPhCH3), 3.18 (t, 1 H, J = 7.5 Hz), 3.343.42 (m, 2 H), 3.55-3.62 (m, 3 H), 3.68-3.82 (m, 7 H), 4.15-4.18 (m, 1 H), 4.42 (d, 1 H, J
= 7 Hz), 4.51-4.56 (m, 1 H), 4.61-4.65 (m, 2 H), 4.70-4.77 (m, 4 H), 4.92 (d, 1 H, J =9.5
Hz), 5.24 (t, 1 H, J = 8 Hz), 6.83-6.85 (m, 2 H), 7.05-7.19 (m, 9 H), 7.25-7.45 (m, 14 H),
7.56-7.59 (m, 1 H), 8.02-8.04 (m, 2 H).
Anhydrosugar (133). Compound 133 was a side product generated during the
1

synthesis of 131. H-NMR (600 MHz, CD3Cl): δ -0.03 (s, 3 H, Si(CH3)2), -0.00 (s, 3 H,
Si(CH3)2), 0.88 (s, 9 H, C(CH3)3), 3.20 (dd, 1 H, J = 3 Hz, J = 8.5 Hz), 3.56-3.74 (m, 6
H), 3.80-3.82 (m, 1 H), 4.10-4.14 (m, 2 H), 4.48-4.52 (m, 2 H), 4.57-4.59 (m, 2 H), 4.664.69 (m, 1 H), 4.76 (d, 1 H, J = 3 Hz), 4.83-4.85 (m, 1 H), 4.89-4.91 (m, 1 H), 4.96 (br, 1
H), 5.62 (br, 1 H), 7.22-7.43 (m, 17 H), 7.49-7.52 (m, 1 H), 8.15-8.17 (m, 2 H).

13

C-NMR

(150 MHz, CD3Cl): δ -4.7, -3.7, 17.9, 25.9, 63.8, 64.9, 69.1, 69.2, 71.4, 72.3, 72.4, 73.2,
74.6, 74.7, 75.6, 77.7, 80.3, 98.8, 99.5, 127.0, 127.3, 127.3, 127.5, 127.7, 127.9, 128.0,
128.2, 128.2, 128.3, 128.3, 129.8, 133.1, 137.6, 137.9, 137.9, 165.6. HRMS:
+

C46H55N3O10Si [M+NH4] calcd: 855.4000, obsd: 855.3960.
p-Tolyl

2-azido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl-(1→4)-2-O-

benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside
326

(134).

Compound 131 (820 mg, 0.758 mmol) was dissolved in pyridine (6 mL) in a plastic flask
o

followed by addition of 65-70% HF-pyridine solution (6 mL) under 0 C. The solution
was stirred overnight until complete disappearance of starting material as judged by
TLC analysis. The reaction mixture was quenched by solid NaHCO3 and diluted with
DCM. The aqueous phase was extracted with DCM twice. The combined organic phase
was further washed with sat. NaHCO3 and dried over Na2SO4. Column purification
1

afforded compound 134 (608 mg, 83%). H-NMR (600 MHz, CD3Cl): δ 2.31 (s, 3 H,
SPhCH3), 2.72 (d, 1 H, J = 1.5 Hz, OH), 3.20 (dd, 1 H, J = 3.5 Hz, J = 8.5 Hz), 3.40 (dd,
1 H, J = 3.5 Hz, J = 8.5 Hz), 3.56 (dd, 1 H, J = 2.5 Hz, J = 8.5 Hz), 3.65-3.67 (m, 1 H),
3.70-3.83 (m, 8 H), 4.01-4.06 (m, 2 H), 4.34-4.59 (m, 2 H, CH2Ph), 4.50 (s, 2 H), 4.694.71 (m, 1 H), 4.76-4.79 (m, 2 H), 4.88 (s, 2 H), 5.32-5.36 (m, 1 H), 5.63 (d, 1 H, J = 3.5
Hz), 6.87-6.89 (m, 2 H), 7.03-7.05 (m, 2 H), 7.16-7.43 (m, 19 H), 7.47-7.50 (m, 2 H),
7.59-7.61 (m, 1 H), 8.12-8.14 (m, 2 H).

13

C-NMR (150 MHz, CD3Cl): δ 14.1, 20.9, 21.0,

55.1, 60.3, 62.4, 68.8, 69.8, 70.3, 72.1, 72.6, 72.7, 73.2, 73.5, 74.5, 74.9, 78.9, 79.3,
85.2, 85.9, 97.3, 113.6, 127.5, 127.6, 127.7, 127.8, 127.8, 127.9, 128.1, 128.2, 128.3,
128.4, 128.4, 129.1, 129.5, 129.7, 129.8, 130.3, 133.2, 133.6, 137.2, 137.6, 138.0,
138.2, 159.0, 165.0. HRMS: C55H57N3O11S [M+NH4]

+

calcd: 985.4058, obsd:

985.4047.
p-Tolyl

2-azido-3,6-di-O-benzyl-4-O-tert-butyl-dimethylsilyl-2-deoxy-β-D-

glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-1-thio-β-D-glucopyranoside

(135).

Compound 131 (300 mg, 0.277 mmol) was dissolved in DCM/H2O (10:1, 5 mL),
327

followed by addition of DDQ (95 mg, 0.42 mmol). The resulting mixture was stirred
under room temperature overnight. After the reaction was complete, it was diluted with
DCM, washed with sat. NaHCO3, dried over Na2SO4. Silica gel column purification
1

afforded compound 135 (189 mg, 71%). H-NMR (500 MHz, CD3Cl): δ -0.10 (s, 3 H,
Si(CH3)2), -0.08 (s, 3 H, Si(CH3)2), 0.78 (s, 9 H, C(CH3)3), 2.30 (s, 3 H, SPhCH3), 3.04
(d, 1 H, J = 6 Hz), 3.15 (dd, 1 H, J = 5.5 Hz, J = 8.5 Hz), 3.40 (dd, 1 H, J = 5.5 Hz, J =
8.5 Hz), 3.46-3.52 (m, 2 H), 3.61-3.65 (m, 2 H), 3.70-3.72 (m, 1 H), 3.88-3.94 (m, 2 H),
4.02 (t, 1 H, J = 7.5 Hz), 4.11 (t, 1 H, J = 7.5 Hz), 4.45-4.47 (m, 1 H), 4.62-4.81 (m, 6 H),
5.31 (t, 1 H, J = 8 Hz), 5.61 (d, 1 H, J = 3.5 Hz), 7.06-7.08 (m, 2 H), 7.11-7.18 (m, 5 H),
7.23-7.35 (m, 12 H), 7.54-7.57 (m, 1 H), 8.05-8.07 (m, 2 H).

13

C-NMR (125 MHz,

CD3Cl): δ -4.8, -3.9, 14.0, 17.8, 21.0, 21.0, 25.7, 60.2, 61.3, 63.2, 68.5, 71.3, 72.6, 73.0,
73.1, 73.3, 74.4, 74.8, 79.1, 80.2, 84.9, 86.5, 97.9, 114.2, 127.1, 127.3, 127.4, 127.5,
127.7, 127.8, 128.1, 128.1, 128.3, 128.3, 128.8, 129.5, 129.6, 129.7, 133.1, 133.1,
+

137.2, 137.3, 137.7, 138.0, 164.9. HRMS: C53H63N3O10SSi [M+NH4] calcd: 979.4347,
obsd: 979.4371.
p-Tolyl

2-azido-3,6-di-O-benzyl-4-O-tert-butyl-dimethylsilyl-2-deoxy-β-D-

glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-1-thio-β-Dglucopyranoside (136). Compound 135 (189 mg, 0.197 mmol) was dissolved in dry
DCM (100 mL), followed by addition of EDC-HCl (105 mg, 0.49 mmol), DMAP (15 mg,
0.04 mmol) and LevOH (57 mg, 0.49 mmol). The resulting mixture was stirred under
room temperature for overnight. The reaction was diluted with DCM and washed with

328

10% HCl solution, sat. NaHCO3 solution sequentially. The combined organic phase was
dried over Na2SO4 and purified by silica gel column (3:1. hexane-EtOAc) to afford
1

compound 136 (187 mg, 90%). H-NMR (500 MHz, CD3Cl): δ -0.01 (s, 3 H, Si(CH3)2),
0.02 (s, 3 H, Si(CH3)2), 0.87 (s, 9 H, C(CH3)3), 2.19 (s, 3 H, CH3COCH2CH2), 2.33 (s,
3 H, SPhCH3), 2.54-2.73 (m, 4 H, CH3COCH2CH2), 3.27 (dd, 1 H, J = 4 Hz, J = 10.5
Hz), 3.58-3.77 (m, 6 H), 3.94-3.98 (m, 2 H), 4.04-4.08 (m, 1 H), 4.24-4.28 (m, 1 H),
4.47-4.50 (m, 1 H), 4.60-4.64 (m, 2 H), 4.70-4.72 (m, 1 H), 4.77-4.88 (m, 4 H), 5.30-5.34
(m, 1 H), 5.62 (d, 1 H, J = 4 Hz), 7.09-7.11 (m, 2 H), 7.15-7.21 (m, 5 H), 7.25-7.29 (m, 2
H), 7.32-7.39 (m, 10 H), 7.45-7.48 (m, 2 H), 7.56-7.61 (m, 1 H), 8.09-8.12 (m, 2 H).
+

HRMS: C58H69N3O12SSi [M+NH4] calcd: 1077.4715, obsd: 1077.4690.
p-Tolyl

2-azido-3,6-di-O-benzyl-2-deoxy-β-D-glucopyranosyl-(1→4)-2-O-

benzoyl-3-O-benzyl-6-O-levulinoyl-1-thio-β-D-glucopyranoside

(137).

Compound

136 (187 mg, 0.177 mmol) was dissolved in pyridine (2 mL) in a plastic flask followed by
o

addition of 65-70% HF-pyridine solution (2 mL) under 0 C. The solution was stirred
overnight until complete disappearance of starting material as judged by TLC analysis.
The reaction mixture was quenched by solid NaHCO3 and diluted with DCM. The
aqueous phase was extracted with DCM twice. The combined organic phase was
further washed with sat. NaHCO3 and dried over Na2SO4. Column purification afforded
compound 137 (155 mg, 93%).

1

H-NMR (500 MHz, CD3Cl): δ 2.16 (s, 3 H,

CH3COCH2CH2), 2.32 (s, 3 H, SPhCH3), 2.56-2.72 (m, 4 H, CH3COCH2CH2), 2.82 (d,
329

1 H, J = 3 Hz), 3.23 (dd, 1 H, J = 4 Hz, J = 10 Hz), 3.60-3.62 (m, 1 H), 3.68-3.80 (m, 5
H), 3.90 (t, 1 H, J = 9.5 Hz), 4.02 (t, 1 H, J = 9 Hz), 4.18 (dd, 1 H, J = 5.5 Hz, J = 12
Hz), 4.52-4.59 (m, 3 H), 4.68-4.70 (m, 1 H), 4.75-4.79 (m, 2 H), 4.85-4.91 (m, 2 H,
CH2Ph), 5.30 (t, 1 H, J = 4.5 Hz), 5.57 (t, 1 H, J = 4 Hz), 7.08-7.10 (m, 2 H), 7.14-7.24
(m, 5 H), 7.26-7.48 (m, 14 H), 7.57-7.60 (m, 1 H), 8.09-8.11 (m, 2 H).
p-Tolyl

2-O-benzoyl-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-6-O-levulinoyl-

α-L-idopyranosyl-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl(1→4)-2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-1-thio-β-D-glucopyranoside

(124).

Compound 128 (110 mg, 0.071 mmol) was dissolved in DCM/H2O (10:1, 3 mL),
followed by addition of DDQ (24 mg, 0.108 mmol). The resulting mixture was stirred
under room temperature overnight. After the reaction was complete, it was diluted with
DCM, washed with sat. NaHCO3, dried over Na2SO4. Silica gel column purification
1

afforded intermediate. H-NMR (500 MHz, CD3Cl): δ -0.15 (s, 3 H, Si(CH3)2), -0.03 (s, 3
H, Si(CH3)2), 0.80 (s, 9 H, C(CH3)3), 2.08 (s, 3 H, CH3COCH2CH2), 2.32 (s, 3 H,
SPhCH3), 2.34-2.61 (m, 4 H, CH3COCH2CH2), 3.24 (dd, 1 H, J = 3.5 Hz, J = 10 Hz),
3.50-3.89 (m, 10 H), 3.99-4.02 (m, 3 H), 4.25-4.29 (m, 12 H), 4.34-4.37 (m, 1 H, PhCH2),
4.48-4.51 (m, 1 H, PhCH2), 4.65-4.80 (m, 6 H), 5.02 (d, 1 H, J = 10.5 Hz), 5.06-5.07 (m,
1 H), 5.13-5.15 (m, 1 H), 5.27-5.31 (m, 1 H), 5.57 (d, 1 H, J = 4 Hz), 7.08-7.47 (m, 28 H),
7.55-7.60 (m, 2 H), 7.95-7.98 (m, 2 H), 8.07-8.09 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl):

δ -5.3, -4.5, 14.1, 17.9, 21.0, 21.1, 25.7, 27.7, 29.7, 37.7, 60.3, 61.5, 62.2, 63.0, 68.0,

330

68.7, 69.0, 70.6, 71.8, 72.3, 72.9, 73.0, 73.4, 74.7, 74.9, 75.1, 76.4, 78.3, 78.9, 84.9,
86.5, 97.1, 97.6, 127.4, 127.6, 127.6, 127.7, 127.8, 128.0, 128.0, 128.1, 128.2, 128.3,
128.3, 128.4, 128.7, 129.4, 129.6, 129.8, 129.8, 133.0, 133.3, 137.3, 137.4, 137.5,
+

138.1, 138.2, 165.0, 165.5, 172.3, 206.3. MALDI-MS: C78H89N3O18SSi [M+Na] calcd:
1439.70, obsd: 1439.38. The intermediate was dissolved in dry DCM (5 mL), followed
by addition of EDC-HCl (27 mg, 0.125 mmol), DMAP (3.8 mg, 0.01 mmol) and LevOH
(15 mg, 0.126 mmol). The resulting mixture was stirred under room temperature for
overnight. The reaction was diluted with DCM and washed with 10% HCl solution, sat.
NaHCO3 solution sequentially. The combined organic phase was dried over Na2SO4
and purified by silica gel column to afford compound 124 (61 mg, 56%). Alternatively,
compound 124 was synthesized from donor 126 and acceptor 137 in 85% yield
1

following the general procedure of single step glycosylation. H-NMR (600 MHz, CD3Cl):

δ -0.19 (s, 3 H, Si(CH3)2), -0.07 (s, 3 H, Si(CH3)2), 0.76 (s, 9 H, C(CH3)3), 2.08 (s, 3 H,
CH3COCH2CH2), 2.13 (s, 3 H, CH3COCH2CH2), 2.30 (s, 3 H, SPhCH3), 2.38-2.64 (m,
8 H, CH3COCH2CH2), 3.26 (dd, 1 H, J = 3 Hz, J = 8.5 Hz), 3.46-3.48 (m, 1 H), 3.643.72 (m, 5 H), 3.76-3.79 (m, 1 H), 3.82-3.85 (m, 1 H), 3.95-4.01 (m, 3 H), 4.06-4.11 (m,
1 H), 4.18-4.22 (m, 1 H), 4.28-4.32 (m, 1 H), 4.40-4.48 (m, 3 H), 4.63-4.78 (m, 6 H),
5.00 (d, 1 H, J = 8.5 Hz), 5.11-5.15 (m, 2 H), 5.22-5.26 (m, 1 H), 5.51 (d, 1 H, J = 3.5
Hz), 7.05-7.29 (m, 19 H), 7.32-7.38 (m, 7 H), 7.43-7.46 (m, 2 H), 7.51-7.58 (m, 2 H),
7.92-7.94 (m, 2H), 8.06-8.08 (m, 2 H).

13

C-NMR (150 MHz, CD3Cl): δ -5.2, -4.6, 17.9,

21.1, 25.7, 27.7, 27.7, 29.7, 29.8, 37.7, 37.8, 62.5, 63.2, 63.3, 67.7, 68.1, 68.9, 70.3,
331

71.9, 72.7, 72.9, 73.3, 74.2, 74.5, 74.6, 75.0, 76.2, 78.3, 84.5, 86.0, 97.0, 97.9, 127.4,
127.5, 127.6, 127.6, 127.7, 127.8, 128.0, 128.2, 128.2, 128.3, 128.3, 128.4, 128.5,
129.5, 129.5, 129.6, 129.8, 133.2, 133.3, 133.4, 137.2, 137.6, 137.7, 138.1, 138.2,
+

165.1, 165.5, 171.9, 172.2, 206.1, 206.3. MALDI-MS: C83H95N3O20SSi [M+Na] calcd:
1537.80, obsd: 1537.16.
p-Tolyl

2-azido-3-O-benzyl-2-deoxy-1-thio-β-D-glucopyranoside

(138).

Compound 39 (1.1 g, 2.25 mmol) was dissolved in DCM/MeOH (1:1, 5 mL), followed by
addition of p-TsOH (641 mg, 3.37 mmol). The resulting reaction mixture was stirred
under room temperature overnight and diluted with DCM. The organic phase was
extracted with sat. NaHCO3 solution, dried over Na2SO4 and purified by silica gel
column to afford compound 138 (829 mg, 92%). The identity of the compound was
confirmed by comparison with literature data.
p-Tolyl

34

2-azido-3-O-benzyl-6-O-acetyl-2-deoxy-1-thio-β-D-glucopyranoside

(139). Compound 18 (829 mg, 2.07 mmol) was dissolved in 2, 4, 6-collidine (4 mL).
Acetyl chloride (0.147 mL, 2.07 mmol) was added dropwise at –40 °C under N2. The
mixture was stirred at –40 °C for 6 h and then the temperature was warmed up slowly to
room temperature. The mixture was poured into EtOAc and washed with 10% HCl,
brine and saturated NaHCO3. The organic phase was dried over Na2SO4, filtered and
the solvents were removed in vacuo. Silica gel column chromatography (3:1 Hexanes–
EtOAc) afforded compound 139 as white solid (779 mg, 85%). The identity of the
compound was confirmed by comparison with literature data.
332

34

p-Tolyl

2-O-benzoyl-3-O-benzyl-4-O-p-methoxybenzyl-6-O-tert-butyl-

dimethylsilyl-1-thio-α-L-idopyranoside (140). Compound 80 (420 mg, 0.7 mmol) in
DCM (5 mL) was cooled down to –40 °C, followed by sequential addition of 2, 6-lutidine
(165 μL, 1.4 mmol) and TBSOTf (242 μL, 1.05 mmol).

The resulting solution was

warmed up very slowly to room temperature. The mixture was quenched by Et3N and
then diluted with DCM (10 mL).

The organic phase was washed with saturated

NaHCO3 and then dried over Na2SO4, filtered and the solvents were removed in vacuo.
Silica gel column chromatography (Hexanes–EtOAc) afforded compound 140 (440 mg,
1

88%). H-NMR (500 MHz, CD3Cl): δ 0.06 (s, 3 H, Si(CH3)2), 0.08 (s, 3 H, Si(CH3)2),
0.90 (s, 9 H, C(CH3)3), 2.29 (s, 3 H, SPhCH3), 3.61 (br, 1 H), 3.76 (s, 3 H, CH3OPh),
3.86-3.90 (m, 2 H), 3.94-3.98 (m, 1 H), 4.34-4.42 (m, 2 H, PhCH2), 4.58-4.62 (m, 1 H,
PhCH2), 4.63-4.66 (m, 1 H, PhCH2), 4.84-4.87 (m, 1 H), 5.39-5.43 (m, 1 H, H-2), 5.53
(br, 1 H, H-1), 6.71-6.73 (m, 2 H), 7.05-7.08 (m, 4 H), 7.27-7.51 (m, 10 H), 7.95-7.97 (m,
2 H).

13

C-NMR (125 MHz, CD3Cl): δ -5.4, -5.3, 18.2, 21.0, 25.9, 55.1, 62.0, 69.4, 70.0,

72.2, 72.5, 73.5, 86.1, 127.7, 127.8, 128.2, 128.3, 129.3, 129.5, 129.7, 129.9, 130.1,
+

131.8, 132.3, 132.9, 137.1, 137.6, 159.5, 165.8. HRMS: C41H50O7SSi [M+NH4] calcd:
732.3390, obsd: 732.3326.
p-Tolyl

2-O-benzoyl-3-O-benzyl-4-O-p-methoxybenzyl-6-O-tert-

butyldimethylsilyl-α-L-idopyranosyl-(1→4)-2-azido-3-O-benzyl-6-O-acetyl-2-deoxy-

333

1-thio-β-D-glucopyranoside (141). Synthesis of compound 141 from donor 140 and
acceptor 139 following the general procedure of single step glycosylation failed.
Anhydrosugar (142). Compound 142 was a side product during the synthesis of
compound 141.

1

H-NMR (600 MHz, CD3Cl): δ 3.67-3.70 (m, 1 H), 3.78 (s, 3 H,

CH3OPh), 3.80-3.83 (m, 1 H), 3.93 (t, 1 H, J = 7 Hz), 4.15 (t, 1 H, J = 6.5 Hz), 4.42 (t, 1
H, J = 4 Hz), 4.56-4.58 (m, 1 H, PhCH2), 4.65-4.77 (m, 3 H, PhCH2), 4.99 (dd, 1 H, J =
1.5 Hz, J = 7 Hz), 5.48 (d, 1 H, J = 1.5 Hz), 6.84-6.86 (m, 2 H), 7.17-7.24 (m, 7 H), 7.397.42 (m, 2 H), 7.53-7.55 (m, 1 H), 7.99-8.01 (m, 2 H).

13

C-NMR (150 MHz, CD3Cl): δ

55.2, 65.5, 72.9, 73.3, 74.8, 79.0, 79.2, 99.2, 113.9, 127.5, 127.7, 128.2, 128.3, 129.4,
129.5, 129.8, 129.9, 133.2, 138.1, 159.4, 165.7. ESI-MS: C28H28O7Na calcd: 499.18,
+

obsd: 499.19. HRMS: C28H28O7 [M+NH4] calcd: 494.2179, obsd: 494.2161.
p-Tolyl

2-O-benzoyl-3-O-benzyl-4-O-p-methoxybenzyl-6-O-tert-butyl-

diphenylsilyl-1-thio-α-L-idopyranoside (143). Compound 80 (1.3 g, 2.16 mmol) was
dissolved in 10 mL DCM, followed by addition of imidazole (176 mg, 2.59 mmol) and
TBDPSCl (840 μL, 3.24 mmol). The resulting mixture was stirred under room
temperature overnight and diluted with DCM. The solution was washed with 10% HCl
solution, sat. NaHCO3 solution and dried over Na2SO4. After concentration, column
1

purification afforded compound 143 (1.45 g, 80%). H-NMR (500 MHz, CD3Cl): δ 1.13
(s, 9 H, C(CH3)3), 2.31 (s, 3 H, SPhCH3), 3.74-3.76 (m, 1 H), 3.79 (s, 3 H, CH3OPh),
3.96-4.05 (m, 2 H, H-6a, H-6b), 4.10-4.12 (m, 1 H, H-3), 4.36-4.48 (m, 2 H, PhCH2),
334

4.73-4.76 (m, 2 H, H-5, PhCH2), 4.92-4.95 (m, 1 H, PhCH2), 5.48-5.49 (m, 1 H, H-2),
5.60 (dd, 1 H, J = 2.5 Hz, H-1), 6.72-6.74 (m, 2 H), 7.02-7.05 (m, 4 H), 7.32-7.53 (m, 16
H), 7.72-7.78 (m, 4 H), 8.02-8.04 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 19.1, 21.0,

26.8, 55.1, 63.0, 69.4, 70.1, 72.2, 72.4, 72.6, 73.7, 86.2, 113.5, 127.6, 127.7, 127.7,
128.1, 128.3, 129.3, 129.4, 129.6, 129.7, 129.9, 129.9, 131.8, 132.4, 132.9, 133.1,
+

133.2, 135.5, 135.6, 137.2, 137.6, 159.0, 165.5. HRMS: C51H54O7SSi [M+NH4] calcd:
856.3703, obsd: 856.3743.
p-Tolyl

2-O-benzoyl-3-O-benzyl-4-O-p-methoxybenzyl-6-O-tert-

butyldiphenylsilyl-α-L-idopyranosyl-(1→4)-2-azido-3-O-benzyl-6-O-acetyl-2-deoxy1-thio-β-D-glucopyranoside (144). Compound 144 was synthesized from donor 143
and acceptor 139 in 61% yield following the general procedure of single step
glycosylation.

1

H-NMR (500 MHz, CD3Cl): δ 1.05 (s, 9 H, C(CH3)3), 1.86 (s, 3 H,

COCH3), 2.29 (s, 3 H, SPhCH3), 3.12-3.17 (m, 1 H), 3.35-3.39 (m, 2 H), 3.59-3.63 (m, 1
H), 3.71-3.73 (m, 1 H), 3.76 (s, 3 H, CH3OPh), 3.77-3.82 (m, 1 H), 3.88-3.92 (m, 1 H),
4.06-4.09 (m, 1 H), 4.12-4.16 (m, 3 H), 4.22 (d, 1 H, J = 10 Hz), 4.30-4.34 (m, 2 H),
4.44-4.47 (m, 1 H, PhCH2), 4.59-4.62 (m, 1 H, PhCH2), 4.66 (s, 1 H), 4.91-4.93 (m, 1 H,
PhCH2), 5.15 (t, 1 H, J = 5.5 Hz), 5.21-5.22 (m, 1 H), 6.71-6.74 (m, 2 H), 7.02-7.07 (m,
4 H), 7.16-7.19 (m, 8 H), 7.25-7.44 (m, 12 H), 7.51-7.55 (m, 1 H), 7.62-7.66 (m, 4 H),
7.93-7.95 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 19.0, 20.6, 21.1, 26.9, 55.2, 62.7,

62.9, 64.6, 70.8, 71.5, 72.4, 72.6, 73.5, 75.2, 75.6, 75.8, 83.4, 85.6, 98.6, 113.7, 126.7,

335

127.5, 127.7, 127.7, 127.7, 127.9, 127.9, 128.1, 128.2, 128.2, 128.3, 128.4, 128.5,
128.5, 129.3, 129.4, 129.4, 129.6, 129.6, 129.7, 129.8, 129.8, 129.9, 129.9, 130.1,
132.8, 132.9, 133.1, 134.3, 135.4, 135.5, 135.6, 135.7, 137.7, 137.8, 138.7, 159.2,
+

165.3, 170.3. HRMS: C66H71N3O12SSi [M+NH4] calcd: 1175.4871, obsd: 1175.4918.
p-Tolyl

2-O-benzoyl-3-O-benzyl-4-O-p-methoxybenzyl-6-O-tert-

butyldiphenylsilyl-β-L-idopyranosyl-(1→4)-2-azido-3-O-benzyl-6-O-acetyl-2-deoxy1-thio-β-D-glucopyranoside (145). Compound 145 was a side product generated
1

during the synthesis of 144. H-NMR (500 MHz, CD3Cl): δ 1.06 (s, 9 H, C(CH3)3), 1.72
(s, 3 H, COCH3), 2.31 (s, 3 H, SPhCH3), 3.16-3.20 (m, 1 H), 3.29-3.34 (m, 1 H), 3.383.42 (m, 1 H), 3.53 (s, 1 H), 3.65 (t, 1 H, J = 9.5 Hz), 3.70-3.72 (m, 1 H), 3.72 (s, 3 H,
CH3OPh), 3.93-3.97 (m, 2 H), 4.00-4.05 (m, 1 H), 4.09-4.13 (m, 1 H), 4.22-4.28 (m, 3 H),
4.37-4.40 (m, 1 H), 4.47-4.63 (m, 2 H, PhCH2), 4.81 (br, 2 H), 5.17 (br, 1 H), 5.25 (br, 1
H), 6.57-6.59 (m, 2 H), 6.88-6.90 (m, 2 H), 7.03-7.05 (m, 2 H), 7.16-7.52 (m, 21 H),
7.63-7.66 (m, 4 H), 7.99-8.02 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 19.1, 20.5, 21.1,

26.8, 55.1, 61.5, 62.8, 64.6, 68.0, 71.2, 72.1, 72.4, 72.8, 74.5, 74.6, 76.1, 84.4, 85.5,
98.1, 113.4, 126.7, 127.7, 127.7, 127.7, 127.9, 128.0, 128.2, 128.4, 128.5, 128.5, 129.3,
129.6, 129.7, 129.7, 129.8, 129.9, 130.0, 132.8, 133.1, 133.3, 134.2, 135.4, 135.5,
+

137.0, 137.3, 138.6, 158.9, 166.1, 170.2. HRMS: C66H71N3O12SSi [M+NH4] calcd:
1175.4871, obsd: 1175.4896.
p-Tolyl

3-O-benzyl-4-O-p-methoxybenzyl-6-O-tert-butyldiphenylsilyl-α-L-

idopyranosyl-(1→4)-2-azido-3-O-benzyl-2-deoxy-1-thio-β-D-glucopyranoside (146).
336

Compound 144 (60 mg, 0.052 mmol) in 3 mL DCM was added freshly prepared NaOMe
in MeOH (1 M) dropwisely. The pH was maintained above 12. After the reaction was
complete indicated by TLC analysis, it was netralized by 1 M HOAc to pH around 6 and
diluted with DCM. The solution was washed with sat. NaHCO3 solution and dried over
Na2SO4. After concentration, column purification afforded compound 146 (50 mg, 95%).
1

H-NMR (500 MHz, CD3Cl): δ 1.00 (s, 9 H, C(CH3)3), 2.30 (s, 3 H, SPhCH3), 2.41 (br, 1

H), 3.15-3.19 (m, 1 H), 3.21 (br, 1 H), 3.29-3.32 (m, 1 H), 3.33-3.37 (m, 1 H), 3.58-3.68
(m, 4 H), 3.75-3.77 (m, 5 H), 3.82-3.88 (m, 2 H), 4.15-4.19 (m, 1 H), 4.30-4.32 (m, 2 H),
4.43-4.45 (m, 1 H), 4.56-4.64 (m, 3 H), 4.84-4.86 (m, 1 H), 5.10 (d, 1 H, J = 3 Hz), 5.27
(d, 1 H, J = 1 Hz), 6.77-6.77 (m, 2 H), 7.03-7.05 (m, 2 H), 7.08-7.10 (m, 2 H), 7.18-7.19
(m, 3 H), 7.22-7.24 (m, 3 H), 7.27-7.32 (m, 8 H), 7.37-7.41 (m, 3 H), 7.58-7.62 (m, 3 H).
13

C-NMR (125 MHz, CD3Cl): δ 14.1, 19.0, 21.1, 26.8, 55.1, 60.3, 62.4, 62.6, 65.1, 69.3,

69.7, 72.6, 73.0, 74.3, 75.0, 75.3, 76.4, 79.6, 83.5, 86.0, 100.9, 113.7, 126.6, 127.5,
127.6, 127.6, 127.9, 128.0, 128.1, 128.2, 128.4, 129.2, 129.6, 129.8, 132.9, 133.0,
134.2, 135.5, 135.6, 137.6, 137.8, 138.8, 159.3. ESI-MS: C57H65N3O10SSi [M+2Na]

+

calcd: 1057.42, obsd: 1057.34.
p-Tolyl

2-O-fluorenylmethyloxycarbonyl-3-O-benzyl-4-O-p-methoxybenzyl-

6-O-tert-butyldiphenylsilyl-α-L-idopyranosyl-(1→4)-2-azido-3-O-benzyl-6-Ofluorenylmethyloxycarbonyl-2-deoxy-1-thio-β-D-glucopyranoside (147). Compound
146 (50 mg, 0.0492 mmol) was dissolved in DCM (3 mL), followed by addition of DMAP
(60 mg, 0.49 mmol) and FmocCl (126 mg, 0.49 mmol). The resulting reaction mixture

337

was stirred under room temperature overnight. This reaction failed to give the desired
product 147.
p-Tolyl

2-O-levulinoyl-3-O-benzyl-4-O-p-methoxybenzyl-6-O-tert-

butyldiphenylsilyl-α-L-idopyranosyl-(1→4)-2-azido-3-O-benzyl-6-O-levulinoyl-2deoxy-1-thio-β-D-glucopyranoside (149). Compound 146 (210 mg, 0.207 mmol) was
dissolved in dry DCM (5 mL), followed by addition of EDC-HCl (1.1 g, 5.1 mmol), DMAP
(160 mg, 0.42 mmol) and LevOH (598 mg, 5.14 mmol). The resulting mixture was
stirred under room temperature for overnight. The reaction was diluted with DCM and
washed with 10% HCl solution, sat. NaHCO3 solution sequentially. The combined
organic phase was dried over Na2SO4 and purified by silica gel column (hexane-EtOAc)
1

to afford compound 149 (151 mg, 60%). H-NMR (500 MHz, CD3Cl): δ 0.97 (s, 9 H,
C(CH3)3), 2.10 (s, 3 H, CH3COCH2CH2), 2.12 (s, 3 H, CH3COCH2CH2), 2.31 (s, 3 H,
SPhCH3), 2.41-2.79 (m, 8 H, CH3COCH2CH2), 3.15 (t, 1 H, J = 9.5 Hz), 3.35 (t, 1 H, J
= 9.5 Hz), 3.45-3.50 (m, 1 H), 3.58-3.63 (m, 2 H), 3.73-3.75 (m, 1 H), 3.75 (s, 3 H,
CH3OPh), 3.84-3.89 (m, 1 H), 3.94 (t, 1 H, J = 5.5 Hz), 4.04-4.11 (m, 2 H), 4.24-4.27 (m,
2 H), 4.41-4.43 (m, 1 H), 4.46-4.49 (m, 1 H), 4.55-4.64 (m, 3 H), 4.86-4.89 (m, 2 H),
5.02 (d, 1 H, J = 4.5 Hz), 6.73-6.75 (m, 2 H), 7.01-7.03 (m, 2 H), 7.07-7.09 (m, 2 H),
7.18-7.20 (m, 3 H), 7.25-7.31 (m, 12 H), 7.36-7.42 (m, 3 H), 7.56-7.61 (m, 4 H).

13

C-

NMR (125 MHz, CD3Cl): δ 19.0, 21.1, 26.8, 27.8, 27.8, 29.7, 29.8, 37.6, 37.8, 55.2,
62.6, 62.9, 64.7, 70.4, 71.0, 72.3, 73.5, 74.2, 75.3, 75.6, 75.7, 83.4, 85.6, 98.3, 113.6,
126.7, 127.5, 127.6, 127.7, 128.0, 128.1, 128.3, 128.4, 129.5, 129.7, 129.7, 129.8,
338

132.9, 133.0, 134.3, 135.6, 135.6, 137.8, 137.9, 138.7, 159.2, 171.8, 172.2, 206.2,
206.3. ESI-MS: C61H77N3O14SSi [M+NH4]

+

calcd: 1225.49, obsd: 1126.30. HRMS:

calcd: 1225.5318, obsd: 1225.6290.
p-Tolyl

3-O-benzyl-4-O-p-methoxybenzyl-6-O-tert-butyldiphenylsilyl-α-L-

idopyranosyl-(1→4)-2-azido-3-O-benzyl-6-O-levulinoyl-2-deoxy-1-thio-β-Dglucopyranoside (150). Compound 150 was a side product generated during the
1

synthesis of 149. H-NMR (500 MHz, CD3Cl): δ 0.98 (s, 9 H, C(CH3)3), 2.13 (s, 3 H,
CH3COCH2CH2), 2.30 (s, 3 H, SPhCH3), 2.52-2.72 (m, 4 H, CH3COCH2CH2), 3.15 (t,
1 H, J = 10 Hz), 3.29-3.34 (m, 2 H), 3.41-3.44 (m, 1 H), 3.56-3.61 (m, 3 H), 3.64-3.68 (m,
1 H), 3.72-3.76 (m, 1 H), 3.75 (s, 3 H, CH3OPh), 3.82-3.84 (m, 1 H), 4.16 (dd, 1 H, J = 5
Hz, J = 12 Hz), 4.19-4.24 (m, 1 H), 4.26-4.30 (m, 2 H), 4.42-4.57 (m, 4 H), 4.62-4.65 (m,
1 H), 4.81-4.84 (m, 1 H), 4.98-4.99 (m, 1 H), 6.74-6.76 (m, 2 H), 7.02-7.04 (m, 2 H),
7.07-7.09

(m,

2

H),

7.17-7.42

C62H71N3O12SSiNa [M+NH4]

+

(m,

18

H),

7.55-7.59

(m,

4

H).

ESI-MS:

calcd: 1127.45, obsd: 1128.30. HRMS: calcd:

1127.4871, obsd: 1127.4825.
p-Tolyl 4, 6-O-benzylidene-β-D-galactopyranosyl-(1→3)-2-O-benzoyl-4, 6-Obenzylidene-1-thio-β-D-galactopyranoside (125). Compound 125 was synthesized
from compound 87 in 89% yield following the general procedure of Lev deprotection.
1

H-NMR (500 MHz, (CD3)2CO): δ 2.31 (s, 3 H, SPhCH3), 3.32-3.37 (m, 1 H), 3.45-3.54

(m, 3 H), 3.68-3.70 (m, 1 H), 3.82 (br, 1 H), 4.09-4.30 (m, 6 H), 4.46 (d, 1 H, J = 8 Hz),
4.59 (d, 1 H, J = 3 Hz), 5.00 (d, 1 H, J = 10 Hz), 5.05 (t, 1 H, J = 9.5 Hz), 5.56 (s, 1 H,

339

PhCH), 5.64 (s, 1 H, PhCH), 7.07-7.09 (m, 2 H), 7.29-7.36 (m, 6 H), 7.44-7.51 (m, 8 H),
7.60-7.63 (m, 1 H), 8.02-8.04 (m, 2 H).

13

C-NMR (150 MHz, (CD3)2CO): δ 21.1, 67.5,

69.6, 69.7, 70.2, 70.9, 71.5, 73.1, 76.7, 77.2, 79.9, 86.1, 101.2, 101.5, 106.0, 127.2,
127.6, 128.6, 129.1, 129.2, 129.4, 129.8, 130.2, 130.4, 131.5, 133.7, 133.7, 138.3,
+

139.7, 139.7, 166.0. HRMS: C40H40O11S [M+NH4] calcd: 746.2635, obsd: 746.2517.
p-Tolyl

2-O-benzoyl-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-6-O-levulinoyl-

α-L-idopyranosyl-(1→4)-2-azido-3,

6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-

(1→4)-2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-β-D-glucopyranosyl-(1→3)-4,
benzylidene-β-D-galactopyranosyl-(1→3)-2-O-benzoyl-4,
galactopyranosyl-1-thio-β-D-galactopyranoside

(154).

6-O-

6-O-benzylidene-β-DCompound

164

was

synthesized from donor 124 and acceptor 125 in 65% yield following the general
1

procedure of single step glycosylation. H-NMR (500 MHz, CD3Cl): δ -0.17 (s, 3 H,
Si(CH3)2), -0.04 (s, 3 H, Si(CH3)2), 0.78 (s, 9 H, C(CH3)3), 1.99 (s, 3 H,
CH3COCH2CH2), 2.09 (s, 3 H, CH3COCH2CH2), 2.33 (s, 3 H, SPhCH3), 2.25-2.60 (m,
8 H, CH3COCH2CH2), 3.25-3.29 (m, 2 H), 3.37-3.40 (m, 1 H), 3.45-3.53 (m, 3 H), 3.643.76 (m, 5 H), 3.78-3.83 (m, 2 H), 3.87-3.92 (m, 1 H), 3.94-4.02 (m, 7 H), 4.15-4.35 (m,
6 H), 4.40-4.48 (m, 3 H), 4.62-4.82 (m, 6 H), 4.98 (d, 1 H, J = 8 Hz), 5.04 (d, 1 H, J =
10.5 Hz), 5.06-5.14 (m, 2 H), 5.21-5.25 (m, 1 H), 5.39 (s, 1 H), 5.44-5.48 (m, 2 H), 5.555.57 (m, 1 H), 7.04-7.06 (m, 2 H), 7.11-7.62 (m, 41 H), 7.91-7.96 (m, 4 H), 8.01-8.04 (m,
2 H).

13

C-NMR (125 MHz, CD3Cl): δ -5.4, -4.6, 14.1, 17.8, 20.9, 21.1, 25.6, 27.6, 29.5,

29.7, 37.6, 37.6, 60.2, 62.4, 63.1, 66.6, 68.0, 68.7, 68.9, 69.0, 69.1, 70.1, 71.8, 72.3,
340

72.8, 73.2, 73.7, 74.3, 74.4, 74.9, 75.6, 76.0, 76.0, 77.9, 78.2, 78.4, 83.0, 85.0, 96.9,
97.7, 100.0, 100.1, 101.0, 104.3, 125.8, 126.6, 127.1, 127.3, 127.4, 127.5, 127.6, 127.8,
127.9, 127.9, 127.9, 127.9, 128.1, 128.1, 128.2, 128.2, 128.3, 128.5, 128.9, 129.3,
129.4, 129.5, 129.6, 129.6, 129.7, 130.3, 132.8, 133.1, 134.3, 137.2, 137.5, 137.6,
137.7, 138.1, 138.1, 165.0, 165.2, 165.4, 172.1, 172.1, 206.3, 206.6. MALDI-MS:
+

C116H127N3O31SSi [M+Na] calcd: 2142.40, obsd: 2144.35.
p-Tolyl

2-O-benzoyl-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-6-O-levulinoyl-

α-L-idopyranosyl-(1→4)-2-N-acetyl-3, 6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl(1→4)-2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-β-D-glucopyranosyl-(1→3)-4,
benzylidene-β-D-galactopyranosyl-(1→3)-2-O-benzoyl-4,

6-O-

6-O-benzylidene-β-D-

galactopyranosyl-1-thio-β-D-galactopyranoside (155). Compound 154 (17 mg, 0.008
mmol) was dissolved in thioacetic acid (HSAc) and pyridine (1:1, 2 mL). The resulting
reaction was left under room temperature overnight. After the the reaction was complete,
it was diluted with DCM and washed with 10% HCl and sat. NaHCO3 sequentially.After
drying over Na2SO4, it was concentrated and purified by column (Hexanes–EtOAc, 1:3)
1

to afford compound 155 (13 mg, 79%). H-NMR (500 MHz, CD3Cl): δ -0.16 (s, 3 H,
Si(CH3)2), -0.05 (s, 3 H, Si(CH3)2), 0.77 (s, 9 H, C(CH3)3), 1.36 (3s, 3 H, NHCOCH3),
2.00 (s, 3 H, CH3COCH2CH2), 2.09 (s, 3 H, CH3COCH2CH2), 2.31 (s, 3 H, SPhCH3),
2.40-2.66 (m, 8 H, CH3COCH2CH2), 3.25 (br, 2 H), 3.34-3.38 (m, 1 H), 3.52 (s, 1 H),
3.55-3.68 (m, 6 H), 3.71-3.75 (m, 1H), 3.77-3.81 (m, 1 H), 3.84-3.88 (m, 1 H), 3.91-4.16
(m, 10 H), 4.22-4.54 (m, 11 H), 4.60-4.63 (m, 1 H), 4.68-4.77 (m, 4 H), 4.89-4.91 (m, 2
341

H), 5.10-5.12 (m, 1 H), 5.17-5.23 (m, 2 H), 5.37 (s, 1 H), 5.42-5.46 (m. 2 H), 6.23 (d, 1 H,
J = 9.5 Hz), 6.99-7.09 (m, 7 H), 7.14-7.55 (m, 36 H), 7.86-7.88 (m, 2 H), 7.96-7.99 (m, 4
H).

13

C-NMR (125 MHz, CD3Cl): δ -5.3, -4.6, 14.1, 17.8, 20.9, 21.2, 22.3, 25.6, 27.7,

27.8, 29.6, 29.7, 37.7, 60.3, 62.1, 66.6, 68.1, 68.8, 68.9, 69.2, 69.3, 70.1, 70.7, 73.0,
73.1, 73.2, 73.6, 74.5, 75.0, 75.5, 76.0, 76.5, 78.4, 78.5, 81.6, 85.1, 97.3, 100.0, 100.2,
101.1, 104.3, 125.8, 126.7, 127.1, 127.2, 127.4, 127.5, 127.6, 127.7, 127.9, 127.9,
128.0, 128.0, 128.1, 128.2, 128.2, 128.2, 128.3, 128.4, 128.5, 128.9, 129.4, 129.4,
129.5, 129.5, 129.6, 129.8, 130.3, 132.9, 133.0, 133.2, 134.3, 136.4, 137.5, 137.6,
137.7, 137.9, 138.2, 138.4, 165.1, 165.3, 165.5, 170.1, 171.0, 172.2, 172.4, 206.8,
+

206.8. MALDI-MS: C118H131NO32SSi [M+Na] calcd: 2158.44, obsd: 2158.67.
p-Tolyl

2-O-benzoyl-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-α-L-

idopyranosyl-(1→4)-2-N-acetyl-3,

6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-

(1→4)-2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyl-(1→3)-4, 6-O-benzylidene-β-Dgalactopyranosyl-(1→3)-2-O-benzoyl-4,

6-O-benzylidene-β-D-galactopyranosyl-1-

thio-β-D-galactopyranoside (156). Compound 156 was synthesized from compound
155 in 95% yield following the general procedure of Lev deprotection.

1

H-NMR

(500 MHz, CD3Cl): δ -0.14 (s, 3 H, Si(CH3)2), -0.04 (s, 3 H, Si(CH3)2), 0.77 (s, 9 H,
C(CH3)3), 1.24 (s, 3 H, NHCOCH3), 2.32 (s, 3 H, SPhCH3), 2.41-2.44 (m, 2 H), 2.572.61 (m, 1 H), 3.16-3.20 (m, 1 H), 3.23 (s, 1 H), 3.44-3.59 (m, 4 H), 3.63-3.86 (m, 10 H),
3.93-4.04 (m, 6 H), 4.14-4.16 (m, 1 H), 4.21 (s, 1 H, J = 8 Hz), 4.32-4.44 (m, 6 H), 4.544.81 (m, 6 H), 4.99-5.06 (m, 3 H), 5.17-5.24 (m, 2 H), 5.39-5.47 (m, 3 H), 6.34 (d, 1 H, J
= 9.5 Hz), 5.17-5.23 (m, 2 H), 5.37 (s, 1 H), 5.42-5.46 (m. 2 H), 6.23 (d, 1 H, J = 9.5 Hz),
342

7.03-7.06 (m, 4 H), 7.12-7.47 (m, 37 H), 7.52-7.59 (m, 2 H), 7.93-7.95 (m, 2 H), 8.008.04 (m, 4 H).

13

C-NMR (125 MHz, CD3Cl): δ -5.2, -4.4, 14.2, 17.8, 20.9, 21.2, 22.3,

25.6, 27.7, 27.9, 29.6, 29.7, 37.8, 61.0, 61.4, 66.8, 67.9, 68.7, 68.9, 69.3, 70.1, 70.1,
70.4, 70.5, 72.9, 73.1, 73.3, 73.8, 73.9, 74.8, 75.2, 75.4, 75.9, 76.0, 78.1, 78.5, 81.7,
85.0, 98.0, 100.0, 100.1, 101.0, 104.2, 125.2, 125.8, 126.7, 126.9, 127.6, 127.7, 127.7,
127.8, 127.9, 127.9, 128.0, 128.1, 128.1, 128.2, 128.3, 128.3, 128.4, 128.5, 128.9,
128.9, 129.4, 129.4, 129.5, 129.6, 129.7, 129.8, 130.2, 132.9, 133.0, 133.3, 134.4,
136.7, 137.5, 137.5, 137.6, 137.6, 137.8, 137.8, 138.3, 152.8, 165.2, 165.5, 165.6,
+

167.0, 170.3. MALDI-MS: C108H119NO28SSi [M+Na] calcd: 1962.24, obsd: 1963.33.
2-O-Benzoyl-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-α-L-idopyranosyluronic
acid-(1→4)-2-N-acetyl-3,

6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-

benzoyl-3-O-benzyl-β-D-glucopyranosyluronic acid-(1→3)-4, 6-O-benzylidene-β-Dgalactopyranosyl-(1→3)-2-O-benzoyl-4,

6-O-benzylidene-β-D-galactopyranosyl-1-

hydroxyl-D-galactopyranoside (157). Compound 156 (19 mg, 0.0098 mmol) was
o

dissolved in DCM (1 mL) and H2O (170 μL) under 0 C. An aqueous solution of NaBr (1
M, 5 μL), an aqueous solution of tetrabutylammonium bromide (1 M, 10 μL), TEMPO (1
mg) and a saturated solution of NaHCO3 (25 μL) were added to the above solution. The
resulting mixture was treated with an aqueous solution of NaOCl (30 μL) and
continuously stirred for 1 h under room temperature. It was later neutralized with 1 M
HCl (10 μL) to pH 6-7. After neutralization, tBuOH (0.7 mL), 2-methylbut-2-ene in THF
(2 M, 1.4 mL) and a solution of NaClO2 (10 mg, 0.044 mM) and NaH2PO4 (8 mg, 0.34
mM) in water (40 μL) were added. The reaction mixture was stirred under room
343

temperature for 1-2 h, diluted with saturated NaH2PO4 solution (5 mL), and extracted
with EtOAc. The organic layer was combined and dried over Na2SO4. After
concentration, the identity of the major spot was confirmed by ESI-MS. ESI-MS:
-

-

C101H107NO31Si [M-H] calcd: 1858.66, obsd: 1857.60, [M-2 H+2 Na] 1880.66, obsd:
1879.60, [M-2 H]

2-

calcd: 928.83, obsd: 927.90, [M-3 H+Na ]

2-

calcd: 939.83, obsd:

938.90.
p-Tolyl

benzyl

2-O-benzoyl-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-α-L-

idopyranosyluronate-(1→4)-2-N-acetyl-3,

2-O-benzoyl-3-O-benzyl-β-D-

glucopyranosyl-(1→4)-benzyl
glucopyranosyluronate-(1→3)-4,

6-di-O-benzyl-2-deoxy-α-D-

6-O-benzylidene-β-D-galactopyranosyl-(1→3)-2-

O-benzoyl-4, 6-O-benzylidene-β-D-galactopyranosyl-1-thio-β-D-galactopyranoside
(159). Compound 156 (132 mg, 0.07 mmol) was dissolved in DCM/tBuOH/H2O (4:4:1,
4.5 mL), followed by addition of TEMPO (6 mg) and BAIB (350 mg). The resulting
mixture was stirred under room temperature overnight. After the reaction was complete
indicated by TLC analysis, it was neutralized by 1 M HCl solution to adjust pH around 6.
The solution was first diluted with DCM, then extracted with H2O. The combined organic
phase was dried over Na2SO4. After concentration, the crude product was confirmed by
-

ESI-MS analysis. ESI-MS: C108H114NO30SSi calcd: [M-H] calcd: 1964.68, obsd:
2-

1966.40, [M-2 H]

calcd: 981.84, obsd: 982.30. The crude compound was dissolved in

DCM (1 mL) and was treated with phenyl diazomethane

344

65

solution in diethyl ether (500

μL). The reaction was stirred under room temperature for 3 h and concentrated to afford
1

compound 159 (106 mg, 63% over two steps). H-NMR (500 MHz, CD3Cl): δ -0.18 (s, 3
H, Si(CH3)2), -0.12 (s, 3 H, Si(CH3)2), 0.72 (s, 9 H, C(CH3)3), 1.30 (s, 3 H, NHCOCH3),
2.31 (s, 3 H, SPhCH3), 2.61 (s, 1 H), 3.11 (s, 1 H), 3.29-3.33 (m, 1 H), 3.39-3.43 (m, 1
H), 3.52-3.68 (m, 6 H), 3.72-3.76 (m, 3 H), 3.82-3.86 (m, 2 H), 3.92-4.15 (m, 9 H), 4.184.26 (m, 2 H), 4.30-4.36 (m, 2 H), 4.42-4.48 (m, 4 H), 4.58-4.63 (m, 2 H), 4.67 (s, 2H),
4.71-4.98 (m, 7 H), 5.16-5.19 (m, 1 H), 5.23-5.27 (m, 1 H), 5.30 (s, 1 H), 5.37-5.43 (m, 2
H), 5.58-5.65 (m, 2 H), 7.01-7.54 (m, 55 H), 7.88-7.97 (m, 4 H).

13

C-NMR (125 MHz,

CD3Cl): δ -5.8, -4.8, 14.4, 17.8, 20.9, 21.2, 22.3, 25.6, 27.7, 27.9, 29.6, 29.7, 31.2, 32.3,
34.2, 51.6, 63.9, 66.7, 66.8, 67.2, 67.5, 68.6, 68.9, 69.1, 69.3, 70.0, 70.2, 71.8, 72.2,
72.4, 72.6, 73.2, 73.2, 73.6, 74.3, 75.0, 75.0, 75.3, 75.9, 76.5, 77.7, 78.3, 81.2, 85.0,
98.3, 98.9, 100.0, 100.9, 101.0, 104.0, 114.0, 117.0, 125.2, 125.8, 125.9, 126.3, 126.7,
126.7, 126.9, 127.0, 127.3, 127.4, 127.4, 127.5, 127.6, 127.9, 127.9, 127.9, 128.0,
128.0, 128.1, 128.1, 128.1, 128.2, 128.2, 128.3, 128.4, 128.4, 128.4, 128.4, 128.5,
128.5, 128.9, 128.9, 129.0, 129.4, 129.5, 129.6, 129.7, 129.8, 130.2, 132.9, 133.0,
133.1, 133.1, 134.4, 135.0, 135.0, 136.6, 137.6, 137.6, 137.7, 137.8, 138.1, 138.3,
138.8, 164.9, 165.4, 165.6, 167.5, 169.2, 169.7. ESI-MS: C122H127NO30SSi [M+NH4]
calcd: 2163.79, obsd: 2165.10, [M+H+NH4]

2+

+

calcd: 1082.39, obsd: 1083.10. MALDI-

+

MS: [M+Na] calcd: 2170.45, obsd: 2171.79.
p-Tolyl benzyl 2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate-(1→4)-2-Nacetyl-3, 6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-benzyl 2-O-benzoyl-3345

O-benzyl-β-D-glucopyranosyluronate-(1→3)-4,
galactopyranosyl-(1→3)-2-O-benzoyl-4,

6-O-benzylidene-β-D-

6-O-benzylidene-β-D-galactopyranosyl-1-

thio-β-D-galactopyranoside (160). Compound 159 (31 mg, 0.0144 mmol) was
o

dissolved in 2 mL pyridine, followed by addition of 115 μL HF-Pyridine under 0 C. The
resulting mixture was stirred under room temperature overnight. After the reaction was
complete, it was quenched by solid NaHCO3 and diluted with DCM. The solution was
extracted with 10% HCl solution and sat. NaHCO3 solution. The combined organic
phase was dried over Na2SO4. After concentration, it was purified by silica gel column
(toluene-acetone, 2:3) to afford compound 160 (25 mg, 63%).

1

H-NMR (600 MHz,

CD3Cl): δ 1.25 (s, 3 H, NHCOCH3), 2.31 (s, 3 H, SPhCH3), 2.70-2.72 (m, 1 H), 3.12 (s,
1 H), 3.35-3.39 (m, 1 H), 3.52-3.56 (m, 1 H), 3.61-3.66 (m, 1 H), 3.71-3.88 (m, 4 H),
3.96-3.98 (m, 2 H), 4.03-4.17 (m, 4 H), 4.30-4.47 (m, 5 H), 4.57-4.66 (m, 3 H), 4.72-4.80
(m, 2 H), 4.89-5.07 (m, 5 H), 5.20 (br, 1 H), 5.28-5.44 (m, 4 H), 5.67-5.69 (m, 1 H), 7.017.58 (m, 55 H), 7.92-8.00 (m, 4 H).

13

C-NMR (125 MHz, CD3Cl): δ 21.0, 21.2, 22.3,

51.5, 66.6, 66.7, 67.4, 67.7, 68.1, 68.3, 68.5, 68,7, 69.0, 69.4, 70.2, 70.3, 71.8, 72.4,
72.5, 73.1, 73.2, 74.3, 74.5, 75.0, 75.3, 76.0, 78.1, 78.3, 81.1, 85.0, 98.1, 99.5, 100.1,
100.9, 101.1, 104.0, 114.7, 125.9, 126.7, 126.9, 127.0, 127.1, 127.4, 127.6, 127.9,
127.9, 127.9, 128.0, 128.1, 128.1, 128.2, 128.2, 128.3, 128.4, 128.4, 128.4, 128.4,
128.5, 128.5, 128.6, 128.9, 129.4, 129.5, 129.6, 129.7, 129.7, 130.2, 133.1, 133.2,
133.7, 134.4, 134.9, 135.4, 136.5, 137.5, 137.6, 137.6, 137.9, 138.3, 138.3, 164.9,

346

165.0, 165.6, 167.4, 169.0, 169.7. MALDI-MS: C116H113NO30SSi [M+Na]

+

calcd:

2056.19, obsd: 2056.43.
p-Tolyl

2-O-levulinoyl-3-O-benzyl-4-O-p-methoxybenzyl-6-O-tert-butyl-

diphenylsilyl-α-L-idopyranosyl-(1→4)-2-azido-3-O-benzyl-6-O-levulinoyl-α-Dglucopyranosyl-(1→4)-benzyl

2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate-

(1→4)-2-N-acetyl-3, 6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-benzyl 2-Obenzoyl-3-O-benzyl-β-D-glucopyranosyluronate-(1→3)-4,
galactopyranosyl-(1→3)-2-O-benzoyl-4,

6-O-benzylidene-β-D-

6-O-benzylidene-β-D-galactopyranosyl-1-

thio-β-D-galactopyranoside (161). Synthesis of compound 161 from donor 149 and
acceptor 160 following the general procedure of single step glycosylation failed.
p-Tolyl

2-O-levulinoyl-3-O-benzyl-4-O-p-methoxybenzyl-α-L-idopyranosyl-

(1→4)-2-azido-3-O-benzyl-6-O-levulinoyl-β-D-glucopyranoside

(162).

Compound

149 (32 mg, 0.0265 mmol) was dissolved in 2 mL pyridine, followed by addition of 300
o

μL HF-Pyridine under 0 C. The resulting mixture was stirred under room temperature
overnight. After the reaction was complete, it was quenched by solid NaHCO3 and
diluted with DCM. The solution was extracted with 10% HCl solution and sat. NaHCO3
solution. The combined organic phase was dried over Na2SO4. After concentration, it
was purified by silica gel column (hexane-EtOAc, 1:1) to afford compound 162 (25 mg,
63%).

1

H-NMR (500 MHz, CD3Cl): δ 2.13 (s, 3 H, CH3COCH2CH2), 2.16 (s, 3 H,

CH3COCH2CH2), 2.33 (s, 3 H, SPhCH3), 2.44-2.76 (m, 8 H, CH3COCH2CH2), 3.203.28 (m, 2 H), 3.33 (t, 1 H, J = 9.5 Hz), 3.39-3.42 (m, 1 H), 3.47-3.51 (m, 2 H), 3.72-3.83
347

(m, 5 H), 3.96-3.99 (m, 1 H), 4.13-4.17 (m, 1 H), 4.29-4.31 (m, 2 H), 4.50-4.53 (m, 2 H),
4.59-4.61 (m, 1 H), 4.66-4.72 (m, 3 H), 4.83-4.84 (m, 1 H), 4.88-4.90 (m, 1 H), 6.80-6.83
(m, 2 H), 7.10-7.13 (m, 4 H), 7.28-7.36 (m, 5 H), 7.42-7.44 (m, 2 H).

13

C-NMR (125

MHz, CD3Cl): δ 21.1, 27.8, 27.9, 29.7, 29.8, 37.6, 37.8, 55.2, 61.8, 62.4, 65.0, 68.9,
69.7, 72.1, 73.0, 73.2, 74.3, 74.6, 75.7, 83.4, 85.8, 97.5, 113.7, 126.4, 127.8, 127.9,
128.0, 128.3, 128.4, 129.6, 129.6, 129.7, 134.5, 137.5, 137.8, 138.9, 159.4, 171.9,
172.1, 206.6, 206.7.
p-Tolyl

benzyl

2-O-levulinoyl-3-O-benzyl-4-O-p-methoxybenzyl-α-L-

idopyranosyluronate-(1→4)-2-azido-3-O-benzyl-6-O-levulinoyl-β-Dglucopyranoside (163). Compoun 156 (20 mg, 0.0205 mmol) was dissolved in
DCM/tBuOH/H2O (4:4:1, 4.5 mL), followed by addition of TEMPO (2 mg) and BAIB (138
mg). The resulting mixture was stirred under room temperature overnight. After the
reaction was complete indicated by TLC analysis, it was neutralized by 1 M HCl solution
to adjust pH around 6. The solution was first diluted with DCM, then extracted with H2O.
The combined organic phase was dried over Na2SO4. After concentration, the crude
-

product (ESI-MS: C51H56N3O15S [M-H] calcd: 982.34, obsd: 982.10) was dissolved in
DCM (1 mL) and was treated with phenyl diazomethane solution in diethyl ether (100
μL). The reaction was stirred under room temperature for 3 h and concentrated to afford
compound 163 (15 mg, 63% over two steps) after column purification (hexane-EtOAc,
1:2).

1

H-NMR (600 MHz, CD3Cl): δ 2.11 (s, 3 H, CH3COCH2CH2), 2.14 (s, 3 H,

CH3COCH2CH2), 2.32 (s, 3 H, SPhCH3), 2.49-2.82 (m, 8 H, CH3COCH2CH2), 3.20 (t,
348

2 H, J = 8.5 Hz), 3.38 (t, 1 H, J = 8 Hz), 3.49-3.52 (m, 1 H), 3.74 (s, 3 H, CH3OPh),
3.78-3.82 (m, 3 H), 4.15 (dd, 1 H, J = 3.5 Hz, J = 10 Hz), 4.27-4.31 (m, 2 H), 4.38-4.40
(m, 1 H), 4.50 (dd, 1 H, J = 1.5 Hz, J = 10 Hz), 4.59-4.68 (m, 4 H), 4.83-4.87 (m, 2 H),
4.90-4.92 (m, 1 H), 5.24 (d, 1 H, J = 4.5 Hz), 6.71-6.74 (m, 2 H), 7.00-7.02 (m, 2 H),
7.09-7.11 (m, 2 H), 7.18-7.22 (m, 5 H), 7.24-7.31 (m, 10 H), 7.42-7.44 (m, 2 H).

13

C-

NMR (125 MHz, CD3Cl): δ 21.1, 27.8, 27.9, 29.7, 29.8, 37.6, 37.9, 55.2, 62.5, 64.6,
66.6, 70.9, 71.2, 72.4, 73.2, 75.0, 75.1, 75.1, 75.5, 82.9, 85.7, 97.7, 113.6, 126.6, 127.5,
127.7, 127.8, 128.1, 128.2, 128.3, 128.3, 128.3, 128.4, 129.3, 129.4, 129.7, 134.4,
135.2, 137.7, 137.7, 138.8, 159.3, 169.3, 171.8, 172.2, 206.2, 206.5. HRMS:
+

C58H63N3O15S [M+NH4] calcd: 1091.4324, obsd: 1091.4363.
p-Tolyl

benzyl

2-O-levulinoyl-3-O-benzyl-4-O-p-methoxybenzyl-α-L-

idopyranosyluronate-(1→4)-2-azido-3-O-benzyl-6-O-levulinoyl-2-deoxy-α-Dglucopyranosyl-(1→4)-benzyl

2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate-

(1→4)-2-N-acetyl-3, 6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-benzyl 2-Obenzoyl-3-O-benzyl-β-D-glucopyranosyluronate-(1→3)-4,
galactopyranosyl-(1→3)-2-O-benzoyl-4,

6-O-benzylidene-β-D-

6-O-benzylidene-β-D-galactopyranosyl-1-

thio-β-D-galactopyranoside (164). Synthesis of compound 164 from donor 163 and
acceptor 160 following the general procedure of single step glycosylation failed.
p-Tolyl

2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-α-L-idopyranosyl-(1→4)-2-

azido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl6-O-levulinoyl-1-thio-β-D-glucopyranoside (165). Compound 124 (51 mg, 0.0337
mmol) was dissolved in 2 mL pyridine, followed by addition of 400 μL HF-Pyridine under
349

o

o

0 C. The resulting mixture was stirred under 50 C overnight. After the reaction was
complete, it was quenched by solid NaHCO3 and diluted with DCM. The solution was
extracted with 10% HCl solution and sat. NaHCO3 solution. The combined organic
phase was dried over Na2SO4. After concentration, it was purified by silica gel column
1

(hexane-EtOAc, 2:1) to afford compound 165 (38 mg, 82%). H-NMR (500 MHz, CD3Cl):

δ 2.09 (s, 3 H, CH3COCH2CH2), 2.14 (s, 3 H, CH3COCH2CH2), 2.34 (s, 3 H, SPhCH3),
2.37-2.68 (m, 8 H, CH3COCH2CH2), 3.28 (dd, 1 H, J = 4.5 Hz, J = 10.5 Hz), 3.56-3.63
(m, 2 H), 3.69-3.95 (m, 8 H), 3.99-4.05 (m, 2 H), 4.11-4.14 (m, 1 H), 4.45-4.47 (m, 3 H),
4.57-4.67 (m, 4 H), 4.72-4.83 (m, 4 H), 5.13 (s, 1 H), 5.20 (br, 1 H), 5.25-5.29 (m, 1 H),
5.59 (d, 1 H, J = 4 Hz), 7.06-7.47 (m, 28 H), 7.55-7.59 (m, 2 H), 7.93-7.95 (m, 2 H),
8.07-8.09 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 21.1, 21.1, 27.7, 27.8, 29.7, 29.8,

29.8, 37.8, 37.8, 63.0, 63.2, 63.6, 65.2, 66.8, 67.6, 68.0, 72.1, 72.2, 72.7, 73.0, 73.3,
73.7, 74.6, 74.9, 75.0, 78.6, 84.6, 86.0, 97.0, 97.9, 127.4, 127.4, 127.5, 127.7, 127.7,
128.0, 128.2, 128.3, 128.3, 128.4, 128.5, 128.6, 129.0, 129.5, 129.6, 129.7, 129.8,
133.4, 133.6, 137.1, 137.4, 137.6, 137.8, 138.2, 165.1, 165.1, 172.1, 172.2, 206.1,
+

206.5. HRMS: C77H81N3O20S [M+NH4] calcd: 1417.5478, obsd: 1417.5514.
p-Tolyl

benzyl

2-O-levulinoyl-3-O-benzyl-4-O-p-methoxybenzyl-α-L-

idopyranosyluronate-(1→4)-2-azido-3-O-benzyl-6-O-levulinoyl-2-deoxy-α-Dglucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-α-L-idopyranosyl(1→4)-2-azido-3,

6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-benzyl

350

2-O-

benzoyl-3-O-benzyl-6-O-levulinoyl-1-thio-β-D-glucopyranoside (166). Synthesis of
compound 166 from donor 163 and acceptor 165 following the general procedure of
single step glycosylation failed.
p-Tolyl

benzyl

2-O-benzoyl-3-O-benzyl-4-O-levulinoyl-α-L-

idopyranosyluronate-(1→4)-2-N-acetyl-3,

2-O-benzoyl-3-O-benzyl-β-D-

glucopyranosyl-(1→4)-benzyl
glucopyranosyluronate-(1→3)-4,

6-di-O-benzyl-2-deoxy-α-D-

6-O-benzylidene-β-D-galactopyranosyl-(1→3)-2-

O-benzoyl-4, 6-O-benzylidene-β-D-galactopyranosyl-1-thio-β-D-galactopyranoside
(167). Compound 160 (18 mg, 0.00886 mmol), EDC-HCl (16 mg, 0.0177 mmol), DMAP
(10 mg, 0.0177 mmol) and LevOH (2 μL, 0.0177 mmol) were dissolved in 2 mL DCM.
The resulting mixture was stirred under room temperature overnight. After the the
reaction was complete, it was diluted with DCM and washed with 10% HCl and sat.
NaHCO3 sequentially.After drying over Na2SO4, it was concentrated and purified by
column (toluene–acetone, 4:1) to afford compound 167 (14 mg, 76%).

1

H-NMR

(500 MHz, CD3Cl): δ 1.19 (s, 3 H, NHCOCH3), 2.31 (s, 3 H, CH3COCH2CH2), 2.012.08 (m, 1 H), 2.33 (s, 3 H, SPhCH3), 2.26-2.46 (m, 4 H, CH3COCH2CH2), 2.86-2.93
(m, 1 H), 3.12 (s, 1 H), 3.34-3.37 (m, 1 H), 3.51-3.56 (m, 2 H), 3.61 (t, 2 H, J = 10 Hz),
3.70-3.89 (m, 6 H), 3.93-3.97 (m, 3 H), 4.05-4.16 (m, 4 H), 4.32-4.48 (m, 8 H), 4.56-4.59
(m, 1 H), 4.64-4.66 (m, 1 H), 4.70-4.78 (m, 3 H), 4.86-4.92 (m, 2 H), 4.96-5.04 (m, 3 H),
5.10-5.17 (m, 3 H), 5.28 (m, 2 H), 5.38-5.44 (m, 3 H), 5.70 (d, 1 H, J = 9.5 Hz), 7.01-

351

7.03 (m, 2 H), 7.07-7.56 (m, 51 H), 7.93-8.04 (m, 6 H). ESI-MS: C121H119NO32S
+

[M+Na] calcd: 2152.74, obsd: 2154.79.
N-Fluorenylmethyloxycarbonyl-O-[benzyl

2-O-benzoyl-3-O-benzyl-4-O-

levulinoyl-α-L-idopyranosyluronate-(1→4)-2-N-acetyl-3,

2-O-benzoyl-3-O-benzyl-β-D-

D-glucopyranosyl-(1→4)-benzyl
glucopyranosyluronate-(1→3)-4,

6-di-O-benzyl-2-deoxy-α-

6-O-benzylidene-β-D-galactopyranosyl-(1→3)-2-

O-benzoyl-4, 6-O-benzylidene-β-D-galactopyranosyl-(1→4)-2, 3-di-O-benzoyl-β-Dxylopyranosyl]-L-serine benzyl ester (168). Compound 168 was synthesized from
donor 167 and acceptor 93 in 30% yield following the general procedure of single step
1

glycosylation. H-NMR (600 MHz, CD3Cl): δ 1.20 (s, 3 H, NHCOCH3), 2.14 (s, 3 H,
CH3COCH2CH2), 2.28-2.45 (m, 4 H, CH3COCH2CH2), 2.61 (s, 1 H), 3.20 (s, 1 H),
3.40-3.65 (m, 6 H), 3.72-3.91 (m, 8 H), 3.96-4.02 (m, 3 H), 4.06-4.24 (m, 8 H), 4.26-4.35
(m, 4 H), 4.38-4.53 (m, 6 H), 4.56-4.63 (m, 3 H), 4.70-4.76 (m, 2 H, PhCH2), 4.91-4.99
(m, 4 H), 5.04-5.17 (m, 6 H), 5.26-5.30 (m, 1 H), 5.34-5.50 (m, 5 H), 5.52-5.60 (m, 2 H),
5.67 (d, 1 H, J = 8 Hz), 7.05-7.46 (m, 64 H), 7.50-7.57 (m, 4 H), 7.66-7.68 (m, 2 H),
7.73-7.76 (m, 2 H), 7.85-7.87 (m, 4 H), 7.98-8.00 (m, 2 H). MALDI-MS: C158H150N2O43
+

[M+Na] calcd: 2787.87, obsd: 2790.59.
N-Fluorenylmethyloxycarbonyl-O-[benzyl
idopyranosyluronate-(1→4)-2-N-acetyl-3,

6-di-O-benzyl-2-deoxy-α-D2-O-benzoyl-3-O-benzyl-β-D-

glucopyranosyl-(1→4)-benzyl
glucopyranosyluronate-(1→3)-4,

2-O-benzoyl-3-O-benzyl-α-L-

6-O-benzylidene-β-D-galactopyranosyl-(1→3)-2352

O-benzoyl-4, 6-O-benzylidene-β-D-galactopyranosyl-(1→4)-2, 3-di-O-benzoyl-β-Dxylopyranosyl]-L-serine benzyl ester (172). Compound 172 was synthesized from
1

compound 168 in 82% yield following the general procedure of Lev deprotection. HNMR (500 MHz, CD3Cl): δ 1.10 (s, 3 H, NHCOCH3), 2.71 (d, 1 H, J = 11 Hz), 3.21 (s, 1
H), 3.39-3.49 (m, 3 H), 3.55-3.64 (m, 4 H), 3.71-3.93 (m, 9 H), 3.99-4.36 (m, 16 H),
4.39-4.56 (m, 7 H), 4.60-4.66 (m, 4 H), 4.74-4.77 (m, 1 H), 4.91-4.94 (m, 3 H), 5.02-5.07
(m, 3 H), 5.09-5.20 (m, 4 H), 5.26-5.30 (m, 3 H), 5.35 (s, 2 H), 5.40-5.46 (m, 2 H), 5.49
(s, 1 H), 5.42-5.60 (m, 2 H), 5.67 (d, 1 H, J = 10 Hz), 7.07-7.46 (m, 65 H), 7.50-7.59 (m,
4 H), 7.65-7.67 (m, 2 H), 7.85-7.87 (m, 3 H), 7.91-7.93 (m, 2 H).
N-Fluorenylmethyloxycarbonyl-O-[2-O-levulinoyl-3-O-benzyl-4-O-pmethoxybenzyl-6-O-tert-butyldiphenylsilyl-α-L-idopyranosyl-(1→4)-2-azido-3-Obenzyl-6-O-levulinoyl-2-deoxy-1-thio-α-D-glucopyranosyl-(1→4)-benzyl

2-O-

benzoyl-3-O-benzyl-α-L-idopyranosyluronate-(1→4)-2-N-acetyl-3, 6-di-O-benzyl-2deoxy-α-D-glucopyranosyl-(1→4)-benzyl
glucopyranosyluronate-(1→3)-4,

2-O-benzoyl-3-O-benzyl-β-D-

6-O-benzylidene-β-D-galactopyranosyl-(1→3)-2-

O-benzoyl-4, 6-O-benzylidene-β-D-galactopyranosyl-(1→4)-2, 3-di-O-benzoyl-β-Dxylopyranosyl]-L-serine benzyl ester (173). Synthesis of compound 173 from donor
149 and acceptor 172 following the general procedure of single step glycosylation failed.
N-Fluorenylmethyloxycarbonyl-O-[2-O-benzoyl-3-O-benzyl-4-O-tertbutyldimeythylsilyl-6-O-levulinoyl-α-L-idopyranosyl-(1→4)-2-azido-3,

6-di-O-

benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-

β-D-glucopyranosyl-(1→3)-4,

6-O-benzylidene-β-D-galactopyranosyl-(1→3)-2-O353

benzoyl-4,

6-O-benzylidene-β-D-galactopyranosyl-(1→4)-2,

3-di-O-benzoyl-β-D-

xylopyranosyl]-L-serine benzyl ester (174). Compound 174 was synthesized from
donor 154 and acceptor 93 in 33% yield following the general procedure of single step
1

glycosylation. H-NMR (500 MHz, CD3Cl): δ -0.20 (s, 3 H, Si(CH3)2), -0.07 (s, 3 H,
Si(CH3)2), 0.78 (s, 9 H, C(CH3)3), 1.95 (s, 3 H, CH3COCH2CH2), 2.06 (s, 3 H,
CH3COCH2CH2), 2.15 (d, 1 H, J = 3 Hz), 2.31-2.61 (m, 8 H, CH3COCH2CH2), 3.203.24 (m, 1 H), 3.33 (s, 1 H), 3.42-4.78 (m, 36 H), 4.98-5.23 (m, 5 H), 5.36-5.60 (m, 5 H),
+

7.02-7.99 (m, 68 H). MALDI-MS: C153H158N4O42Si [M+Na]

calcd: 2775.98, obsd:

2777.54.
p-Tolyl

2-azido-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-6-O-acetyl-2-deoxy-

1-thio-β-D-glucopyranoside (273). Compound 139 (3.2 g, 7.12 mmol) in DCM (50 mL)
was cooled down to –40 °C, followed by sequential addition of 2, 6-lutidine (1.65 mL, 14
mmol) and TBSOTf (2.42 mL, 10.5 mmol). The resulting solution was warmed up very
slowly to room temperature. The mixture was quenched by Et3N and then diluted with
DCM (100 mL). The organic phase was washed with saturated NaHCO3 and then dried
over Na2SO4, filtered and the solvents were removed in vacuo.

Silica gel column

chromatography (Hexanes–EtOAc) afforded compound 273 (3.33 g, 84%). The identity
of the compound was confirmed by comparison with literature data.
p-Tolyl

59

2-azido-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-6-O-acetyl-2-deoxy-

α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-1-thio-α354

L-idopyranoside (274). Compound 274 was synthesized from donor 273 and acceptor
79 in 80% yield following the general procedure of single step glycosylation. The identity
of the compound was confirmed by comparison with literature data.
p-Tolyl

59

2-azido-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-2-deoxy-α-D-

glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-1-thio-α-Lidopyranoside (276). Compound 274 (2.2 g, 2.13 mmol) was dissolved in 50 mL dry
o

DCM and cooled down to -20 C. Fresh methanolic Mg(OMe)2 solution (8%) (21 mL)
was added to the reaction mixture. The resulting mixture was left under N2 and
monitored by TLC. After the reaction was complete, it was neutralized by 1 M HOAc to
pH 5 and diluted with DCM. After washing with sat. NaHCO3 solution and drying over
Na2SO4, the solution was concentrated and purified by silica gel column to afford
1

compound 276 (1.81 g, 86%). H-NMR (500 MHz, CD3Cl): δ -0.14 (s, 3 H, Si(CH3)2), 0.01 (s, 3 H, Si(CH3)2), 0.85 (s, 9 H, C(CH3)3), 1.70-1.73 (m, 1 H), 2.28 (s, 3 H,
SPhCH3), 3.18 (m, 1 H, J = 3.5 Hz, J = 10 Hz), 3.31-3.35 (m, 1 H), 3.44-3.48 (m, 1 H),
3.53-3.66 (m, 4 H), 3.73-3.75 (m, 2 H), 3.79 (s, 3 H, CH3OPh), 3.99-4.02 (m, 1 H,
CH2Ph), 4.12-4.14 (m, 1 H), 4.24-4.27 (m, 1 H), 4.49 (s, 2 H), 4.58 (d, 1 H, J = 4.0 Hz),
4.70-4.73 (m, 1 H, CH2Ph), 4.88-4.91 (m, 1 H), 4.92-4.94 (m, 1 H, CH2Ph), 5.31-5.33
(m, 1 H), 5.53 (br, 1 H), 6.85-6.87 (m, 2 H), 7.00-7.02 (m, 2 H), 7.08-7.10 (m, 2 H), 7.207.29 (m, 7 H), 7.32-7.36 (m, 4 H), 7.38-7.45 (m, 4 H), 8.08-8.10 (m, 2 H). HRMS:
+

C54H65N3O11SSi [M+NH4] calcd: 1009.4453, obsd: 1009.4430.
355

p-Tolyl

2-azido-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-6-O-levulinoyl-2-

deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-1thio-α-L-idopyranoside (183). Compound 183 was synthesized from compound 276 in
81% yield following the general procedure for protecting 6-OH with Lev.

1

H-NMR

(500 MHz, CD3Cl): δ -0.09 (s, 3 H, Si(CH3)2), -0.01 (s, 3 H, Si(CH3)2), 0.88 (s, 9 H,
C(CH3)3), 2.14 (s, 3 H, CH3COCH2CH2), 2.31 (s, 3 H, SPhCH3), 2.55-2.76 (m, 4 H,
CH3COCH2CH2), 3.27-3.29 (m, 1 H), 3.35-3.39 (m, 1 H), 3.50-3.54 (m, 1 H), 3.73-3.86
(m, 7 H), 4.02-4.15 (m, 2 H), 4.17 (br, 1 H), 4.24-4.27 (m, 1 H), 4.31-4.34 (m, 1 H), 4.504.56 (m, 2 H), 4.68-4.69 (m, 1 H), 4.75-4.77 (m, 1 H), 4.94-4.97 (m, 2 H), 5.37 (br, 1 H),
5.58 (br, 1 H), 6.88-6.90 (m, 2 H), 7.03-7.05 (m, 2 H), 7.14-7.16 (m, 2 H), 7.24-7.30 (m,
6 H), 7.34-7.39 (m, 4 H), 7.43-7.49 (m, 5 H), 8.12-8.14 (m, 2 H).

13

C-NMR (125 MHz,

CD3Cl): δ -4.9, -3.9, 14.1, 17.8, 21.0, 25.8, 27.6, 29.7, 37.6, 55.1, 60.2, 62.9, 64.4, 67.1,
69.1, 70.0, 70.8, 71.2, 71.7, 72.5, 72.8, 74.4, 74.7, 80.4, 86.3, 98.2, 113.6, 126.9, 127.2,
127.8, 127.9, 128.0, 128.3, 128.3, 129.1, 129.5, 129.7, 129.8, 130.0, 131.7, 132.2,
133.1, 137.3, 137.4, 137.6, 159.0, 165.5, 172.3, 206.0. HRMS: C59H71N3O13SSi
+

[M+NH4] calcd: 1107.4821, obsd: 1107.4768.
p-Tolyl

2-azido-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-6-O-levulinoyl-2-

deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-1-thio-α-Lidopyranoside (184). Compound 183 (1.6 g, 1.48 mmol) was dissolved in DCM/H2O
(10:1, 30 mL), followed by addition of DDQ (500 mg, 2.25 mmol). The resulting mixture

356

was stirred under room temperature overnight. After the reaction was complete, it was
diluted with DCM, washed with sat. NaHCO3 solution, dried over Na2SO4. Silica gel
1

column purification afforded compound 184 (1.29 g, 90%). H-NMR (500 MHz, CD3Cl):

δ -0.17 (s, 3 H, Si(CH3)2), -0.03 (s, 3 H, Si(CH3)2), 0.84 (s, 9 H, C(CH3)3), 2.14 (s, 3 H,
CH3COCH2CH2), 2.31 (s, 3 H, SPhCH3), 2.57-2.72 (m, 4 H, CH3COCH2CH2), 3.223.25 (m, 2 H), 3.35-3.39 (m, 1 H), 3.68 (br, 1 H), 3.76-3.98 (m, 6 H), 4.06-4.10 (m, 1 H),
4.15 (br, 1 H), 4.40 (br, 1 H, J = 10.5 Hz), 4.52 (s, 1 H), 4.73-4.75 (m, 1 H, CH2Ph),
4.82-4.85 (m, 1 H), 4.95-4.97 (m, 1 H, CH2Ph), 5.36 (s, 1 H), 5.56 (s, 1 H), 7.03-7.10 (m,
4 H), 7.18-7.48 (m, 13 H), 8.10-8.12 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ -4.8, -3.9,

14.0, 17.8, 21.0, 25.7, 27.5, 29.7, 37.6, 61.3, 63.3, 64.4, 67.8, 69.7, 71.0, 71.1, 71.6,
72.3, 74.2, 75.9, 80.3, 86.3, 99.2, 126.7, 127.1, 127.9, 128.1, 128.2, 128.4, 129.6, 129.6,
129.9, 131.6, 132.2, 133.1, 137.2, 137.5, 137.6, 165.4, 172.3, 206.4. HRMS:
+

C51H63N3O12SSi [M+NH4] calcd: 987.4245, obsd: 987.4199.
p-Tolyl

2-azido-3-O-benzyl-4-O-tert-butyl-dimethylsilyl-2-deoxy-α-D-

glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-1-thio-α-L-idopyranoside

(185).

Compound 184 (1.29 g, 1.33 mmol) was dissolved in pyridine (10 mL) in a plastic flask
o

followed by addition of 65-70% HF-pyridine solution (15 mL) under 0 C. The solution
was stirred overnight until complete disappearance of starting material as judged by
TLC analysis. The reaction mixture was quenched by solid NaHCO3 and diluted with
DCM. The aqueous phase was extracted with DCM twice. The combined organic phase
357

was further washed with sat. NaHCO3 and dried over Na2SO4. Column purification
1

afforded compound 185 (1.08 g, 95%). H-NMR (500 MHz, CD3Cl): δ 2.13 (s, 3 H,
CH3COCH2CH2), 2.26-2.29 (m, 1 H), 2.31 (s, 3 H, SPhCH3), 2.54-2.72 (m, 4 H,
CH3COCH2CH2), 2.92 (br, 1 H), 3.21 (dd, 1 H, J = 3.5 Hz, J = 10 Hz), 3.31-3.35 (m, 1
H), 3.39-3.44 (m, 1 H), 3.69-3.71 (m, 1 H), 3.75-3.79 (m, 1 H), 3.85-3.90 (m, 2 H), 4.044.07 (m, 1 H, CH2Ph), 4.12-4.14 (m, 1 H), 4.21 (dd, 1 H, J = 2 Hz, J = 12 Hz), 4.26-4.28
(m, 1 H, CH2Ph), 4.35 (dd, 1 H, J = 5.5 Hz, J = 12 Hz), 4.55 (d, 1 H, J = 4 Hz), 4.734.76 (m, 1 H, CH2Ph), 4.80-4.83 (m, 1 H), 4.95-4.98 (m, 1 H, CH2Ph), 5.38 (s, 1 H),
5.55 (s, 1 H), 7.10-7.16 (m, 4 H), 7.24-7.30 (m, 4 H), 7.34-7.47 (m, 9 H), 8.13-8.15 (m, 2
H).

13

C-NMR (125 MHz, CD3Cl): δ 14.1, 20.9, 21.0, 27.6, 29.7, 37.7, 60.3, 61.4, 63.2,

63.3, 68.0, 69.7, 70.5, 71.1, 71.2, 72.4, 74.8, 74.9, 80.2, 86.3, 98.6, 127.8, 127.9, 128.1,
128.3, 128.3, 128.4, 129.7, 129.9, 131.6, 132.4, 133.1, 137.2, 137.6, 137.7, 165.6,
+

173.1, 206.8. HRMS: C45H49N3O12S [M+NH4] calcd: 873.3381, obsd: 873.3345.
p-Tolyl

2-azido-3-O-benzyl-6-O-levulinoyl-2-deoxy-α-D-glucopyranosyl-

(1→4)-2-O-benzoyl-3-O-benzyl-6-O-tert-butyl-diphenylsilyl-1-thio-α-Lidopyranoside (186). Compound 185 (1.08 g, 1.26 mmol) was dissolved in 10 mL DCM,
followed by addition of imidazole (102 mg, 1.5 mmol) and TBDPSCl (487 μL, 1.88
mmol). The resulting mixture was stirred under room temperature overnight and diluted
with DCM. The solution was washed with 10% HCl solution, sat. NaHCO3 solution and
dried over Na2SO4. After concentration, column purification afforded compound 186
358

1

(1.28 g, 93%). H-NMR (500 MHz, CD3Cl): δ 1.08 (s, 9 H, C(CH3)3), 2.13 (s, 3 H,
CH3COCH2CH2), 2.29 (s, 3 H, SPhCH3), 2.47-2.69 (m, 4 H, CH3COCH2CH2), 2.76 (d,
1 H, J = 4.5 Hz), 3.24 (dd, 1 H, J = 3.5 Hz, J = 10 Hz), 3.31-3.36 (m, 1 H), 3.41-3.45 (m,
1 H), 3.56-3.61 (m, 2 H), 3.75 (br, 1 H), 3.89-3.97 (m, 2 H), 4.14-4.17 (m, 1 H, CH2Ph),
4.22-4.23 (m, 1 H), 4.33-4.36 (m, 1 H), 4.40-4.43 (m, 1 H, CH2Ph), 4.68 (d, 1 H, J = 3.5
Hz), 4.75-4.81 (m, 2 H), 4.95-4.97 (m, 1 H, CH2Ph), 5.39-5.41 (m, 1 H), 5.58-5.59 (m, 1
H), 6.99-7.01 (m, 2 H), 7.18-7.20 (m, 2 H), 7.26-7.50 (m, 19 H), 7.69-7.76 (m, 4 H),
8.12-8.15 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 14.1, 19.1, 21.0, 26.8, 27.6, 29.6,

37.8, 60.3, 62.5, 63.3, 63.5, 69.3, 69.4, 70.2, 70.3, 70.9, 72.5, 72.7, 74.9, 75.0, 79.9,
86.7, 98.5, 127.7, 127.7, 127.8, 127.9, 127.9, 128.3, 128.3, 128.4, 129.5, 129.7, 129.8,
129.9, 131.6, 132.7, 132.8, 132.9, 133.1, 135.5, 135.6, 137.5, 137.5, 137.8, 165.6,
+

173.4, 206.4. HRMS: C61H67N3O12SSi [M+NH4] calcd: 1111.4558, obsd: 1111.4517.
p-Tolyl 3-O-benzyl-4-O-p-methoxybenzyl-6-O-tert-butyl-diphenylsilyl-1-thioα-L-idopyranoside (187). Compound 143 (2.7 g, 3.22 mmol) was dissolved in
DCM/MeOH (1:1, 20 mL) and was added freshly prepared NaOMe in MeOH (5 M) to
maintain the pH above 12. After the reaction was complete, the reaction was diluted
with 10% HCl solution until the pH was around 6. The solution was washed with 10%
HCl solution, sat. NaHCO3 solution and the organic phase was dried over Na2SO4.
1

After concentration, column purification afforded compound 187 (2.1 g, 89%). H-NMR
(500 MHz, CD3Cl): δ 1.11 (s, 9 H, C(CH3)3), 2.30 (s, 3 H, SPhCH3), 3.71-3.73 (m, 1 H,

359

OH), 3.79 (s, 1 H, CH3OPh), 3.82-3.84 (m, 1 H), 3.85-3.87 (m, 2 H), 4.03-4.10 (m, 2 H),
4.39-4.42 (m, 1 H), 4.52-4.55 (m, 1 H), 4.57-4.60 (m, 1 H), 4.75-4.81 (m, 2 H), 5.39 (br,
1 H, H-1), 6.80-6.82 (m, 2 H), 7.01-7.03 (m, 2 H), 7.09-7.11 (m, 2 H), 7.34-7.47 (m, 13
13

C-NMR (125 MHz, CD3Cl): δ 19.1, 20.9, 26.8, 55.1, 62.6, 67.9,

H), 7.70-7.73 (m, 4 H).

69.4, 71.7, 71.9, 72.6, 73.4, 89.7, 113.8, 127.6, 127.6, 127.7, 128.4, 128.9, 129.4, 129.6,
129.6, 129.7, 131.9, 133.0, 133.1, 133.3, 135.5, 135.5, 136.9, 137.6, 159.4. HRMS:
+

C44H50O6SSi [M+NH4] calcd: 752.3441, obsd: 752.3485.
p-Tolyl

2-O-levulinoyl-3-O-benzyl-4-O-p-methoxybenzyl-6-O-tert-butyl-

diphenylsilyl-1-thio-α-L-idopyranoside (188). Compound 187 (2.1 g, 2.86 mmol) was
dissolved in 10 mL DCM, followed by addition of LevOH (347 μL, 3.43 mmol), DMAP
(348 mg, 2.86 mmol) and EDC-HCl (657 mg, 3.43 mmol). After stirring under room
temperature overnight, the reaction was quenched by 10% HCl solution and diluted with
DCM. The organic phase was extracted with sat. NaHCO3 solution and dried over
1

Na2SO4. Silica gel column purification afforded compound 188 (2.3 g, 97%). H-NMR
(500 MHz, CD3Cl): δ 1.05 (s, 9 H, C(CH3)3), 2.13 (s, 3 H, CH3COCH2CH2), 2.27 (s, 3 H,
SPhCH3), 2.52-2.75 (m, 4 H, CH3COCH2CH2), 3.59-3.61 (m, 1 H, H-4), 3.77 (s, 1 H,
CH3OPh), 3.88-3.91 (m, 3 H, H-3, H-6a, H-6b), 4.30-4.32 (m, 1 H), 4.45-4.47 (m, 1 H),
4.61-4.64 (m, 2 H), 4.82-4.85 (m, 1 H), 5.16-5.18 (m, 1 H, H-2), 5.42 (d, 1 H, J = 2.5 Hz,
H-1), 6.75-6.77 (m, 2 H), 6.97-6.99 (m, 2 H), 7.05-7.07 (m, 2 H), 7.31-7.43 (m, 13 H),
7.64-7.69 (m, 4 H).

13

C-NMR (125 MHz, CD3Cl): δ 14.1, 19.0, 20.9, 20.9, 26.7, 28.0,

360

29.7, 37.6, 55.1, 60.2, 62.9, 69.3, 70.0, 71.9, 72.5, 73.3, 86.0, 113.6, 127.6, 127.7,
127.8, 128.3, 129.4, 129.4, 129.5, 129.9, 131.8, 132.0, 133.1, 133.2, 135.5, 135.5,
137.1, 137.7, 159.1, 165.4, 171.7, 206.1. HRMS: C49H56O8SSi [M+NH4]

+

calcd:

850.3809, obsd: 850.3805.
p-Tolyl

2-O-levulinoyl-3-O-benzyl-4-O-p-methoxybenzyl-6-O-tert-butyl-

diphenylsilyl-α-L-idopyranosyl-(1→4)-2-azido-3-O-benzyl-6-O-levulinoyl-2-deoxyα-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-tert-butyl-diphenylsilyl-1thio-α-L-idopyranoside (177). Compound 177 was synthesized from donor 188 and
acceptor 186 in 52% yield following the general procedure of single step glycosylation.
1

H-NMR (500 MHz, CD3Cl): δ 1.03 (s, 9 H, C(CH3)3), 1.06 (s, 9 H, C(CH3)3), 2.01 (s, 3

H, CH3COCH2CH2), 2.02 (s, 3 H, CH3COCH2CH2), 2.27 (s, 3 H, SPhCH3), 2.31-2.68
(m, 8 H, CH3COCH2CH2), 3.23 (dd, 1 H, J = 4 Hz, J = 10.5 Hz), 3.41 (t, 1 H, J = 9 Hz),
3.53-3.55 (m, 1 H), 3.66-3.71 (m, 2 H), 3.77 (s, 1 H, CH3OPh), 3.82-3.95 (m, 7 H), 3.984.02 (m, 3 H), 4.15-4.17 (m, 1 H), 4.23 (d, 1 H, J = 10.5 Hz), 4.29-4.31 (m, 1 H), 4.334.36 (m, 1 H), 4.58-4.62 (m, 4 H), 4.78-4.83 (m, 2 H), 4.87-4.89 (m, 2 H), 4.93-4.95 (m,
1 H), 5.37 (t, 1 H, J = 4.5 Hz), 5.58 (d, 1 H, J = 3.5 Hz), 6.73-6.75 (m, 2 H), 6.96-6.99 (m,
4 H), 7.12-7.17 (m, 6 H), 7.22-7.40 (m, 23 H), 7.45-7.49 (m, 1 H), 7.56-7.61 (m, 4 H),
7.65-7.67 (m, 2 H), 7.72 (m 2 H), 8.06-8.08 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ

14.1, 19.1, 19.1, 21.0, 26.8, 26.9, 27.7, 27.9, 29.5, 29.6, 29.6, 34.6, 37.6, 55.2, 60.3,
62.9, 63.9, 64.0, 70.1, 70.4, 70.8, 71.4, 72.2, 73.0, 73.6, 74.3, 74.9, 75.2, 75.4, 75.6,
75.9, 78.9, 86.4, 97.9, 98.7, 113.6, 113.9, 127.1, 127.6, 127.6, 127.7, 127.7, 127.7,

361

127.8, 127.8, 128.0, 128.1, 128.2, 128.3, 128.6, 129.4, 129.4, 129.5, 129.6, 129.7,
129.7, 129.8, 129.8, 129.9, 131.3, 132.0, 132.8, 132.9, 133.1, 133.3, 135.6, 135.6,
135.6, 135.6, 137.5, 137.7, 137.9, 138.1, 159.2, 165.3, 171.7, 172.1, 206.0, 206.2.
+

MALDI-MS: C103H115N3O20SSi2 [M+Na] calcd: 1826.26, obsd: 1826.33.
p-Tolyl

2-azido-3,

6-di-O-benzyl-4-O-tert-butyl-dimethylsilyl-2-deoxy-α-D-

glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-1-thio-β-D-glucopyranoside

(191).

Compound 135 (1.3 g, 1.35 mmol) was dissolved in pyridine (10 mL) in a plastic flask
o

followed by addition of 65-70% HF-pyridine solution (15 mL) under 0 C. The solution
was stirred overnight until complete disappearance of starting material as judged by
TLC analysis. The reaction mixture was quenched by solid NaHCO3 and diluted with
DCM. The aqueous phase was extracted with DCM twice. The combined organic phase
was further washed with sat. NaHCO3 and dried over Na2SO4. Column purification
1

afforded compound 191 (800 mg, 70%). H-NMR (600 MHz, CD3Cl): δ 2.31 (s, 3 H,
SPhCH3), 2.43-2.46 (m, 1 H), 2.51-2.53 (m, 1 H), 3.16-3.19 (m, 1 H), 3.50-3.53 (m, 1 H),
3.58-3.66 (m, 3 H), 3.72-3.77 (m, 2 H), 3.79-3.83 (m, 1 H), 3.90-3.94 (m, 1 H), 4.00-4.06
(m, 2 H), 4.50-4.58 (m, 2 H, CH2Ph), 4.67-4.69 (m, 1 H, CH2Ph), 4.74-4.81 (m, 3 H),
4.88-4.90 (m, 1 H), 5.30 (t, 1 H, J = 7.5 Hz), 5.58-5.59 (m, 1 H), 7.07-7.09 (m, 2 H),
7.12-7.20 (m, 5 H), 7.26-7.39 (m, 12 H), 7.43-7.47 (m, 2 H), 7.56-7.59 (m, 1 H), 8.078.09 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 21.1, 21.1, 60.3, 61.7, 62.4, 69.4, 71.0,

72.0, 72.3, 73.0, 73.6, 74.6, 75.0, 78.9, 79.6, 84.9, 86.5, 97.8, 113.7, 127.6, 127.7,
127.8, 127.8, 128.0, 128.2, 128.4, 128.5, 128.6, 128.7, 129.6, 129.8, 133.1, 133.3,
362

137.3, 137.3, 137.9, 138.3, 165.1, 171.7, 172.1, 206.0, 206.2. HRMS: C47H49N3O10S
+

[M+NH4] calcd: 865.3482, obsd: 865.3478.
p-Tolyl

2-azido-3,

6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-

benzoyl-3-O-benzyl-6-O-tert-butyl-diphenylsilyl-1-thio-β-D-glucopyranoside (178).
Compound 191 (800 mg, 0.945 mmol) was dissolved in 10 mL DCM, followed by
addition of imidazole (102 mg, 1.5 mmol) and TBDPSCl (487 μL, 1.88 mmol). The
resulting mixture was stirred under room temperature overnight and diluted with DCM.
The solution was washed with 10% HCl solution, sat. NaHCO3 solution and dried over
Na2SO4. After concentration, column purification afforded compound 178 (930 mg,
1

92%). H-NMR (500 MHz, CD3Cl): δ 1.09 (s, 9 H, C(CH3)3), 2.28 (s, 3 H, SPhCH3),
2.31-2.32 (m, 1 H), 3.13-3.19 (m, 2 H), 3.25-3.28 (m, 1 H), 3.43-3.47 (m, 1 H), 3.60-3.67
(m, 3 H), 3.92-3.97 (m, 2 H), 4.00-4.04 (m, 2 H), 4.20-4.35 (m, 2 H, CH2Ph), 4.68-4.76
(m, 2 H, CH2Ph), 4.82-4.87 (m, 3 H), 5.34-5.38 (m, 1 H), 5.59 (d, 1 H, J = 3.5 Hz), 6.987.00 (m, 2 H), 7.12-7.20 (m, 7 H), 7.27-7.41 (m, 16 H), 7.44-7.47 (m, 2 H), 7.56-7.59 (m,
1 H), 7.70-7.72 (m , 4 H), 8.08-8.10 (m, 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 19.3, 21.0,

26.9, 62.4, 63.8, 69.0, 70.8, 72.0, 72.9, 73.1, 73.5, 74.4, 74.9, 79.2, 79.6, 85.0, 87.3,
97.6, 127.6, 127.7, 127.7, 127.8, 127.9, 127.9, 128.2, 128.3, 128.4, 128.5, 129.6, 129.6,
129.7, 129.8, 130.4, 131.9, 133.1, 133.3, 133.6, 135.5, 135.8, 137.3, 137.5, 137.5,
+

138.0, 165.2. HRMS: C63H67N3O10SSi [M+NH4] calcd: 1103.4660, obsd: 1103.4486.

363

N-Fluorenylmethyloxycarbonyl-O-[2, 3-di-O-levulinoyl-4, 6-O-benzylidene -

β-D-galactopyranosyl-(1→3)-2-O-benzoyl-4,
galactopyranosyl-(1→4)-2,

6-O-benzylidene-β-D-

3-di-O-benzoyl-β-D-xylopyranosyl]-L-serine

benzyl

ester (179). Compound 179 was synthesized from donor 87 and acceptor 93 in 43%
1

yield following the general procedure of single step glycosylation. H-NMR (600 MHz,
CD3Cl): δ 1.96 (s, 3 H, CH3COCH2CH2), 2.02 (s, 3 H, CH3COCH2CH2), 2.13-2.40 (m,
3 H, CH3COCH2CH2), 2.51-2.70 (m, 5 H, CH3COCH2CH2), 3.19 (s, 1 H), 3.23-3.27 (m,
1 H), 3.35 (s, 1 H), 3.70-3.76 (m, 3 H), 3.80-3.83 (m, 1 H, CH2Ph), 3.88-3.92 (m, 1 H),
3.95-3.98 (m, 1 H), 4.08-4.13 (m, 2 H), 4.16-4.24 (m, 4 H), 4.29-4.33 (m, 2 H), 4.46-4.48
(m, 1 H), 4.53 (d, 1 H, J = 6.0 Hz), 4.68-4.71 (m, 3 H), 4.99-5.08 (m, 2 H, CH2Ph), 5.125.15 (m, 1 H), 5.26-5.30 (m, 1 H), 5.52-5.44 (m, 2 H), 5.51-5.60 (m, 3 H), 7.15-7.47 (m,
27 H), 7.51-7.57 (m, 3 H), 7.74-7.76 (m, 2 H), 7.93-8.01 (m , 6 H).

13

C-NMR (125 MHz,

CD3Cl): δ 27.4, 28.1, 29.5, 29.6, 37.6, 37.7, 47.0, 54.2, 60.3, 62.3, 66.4, 67.1, 67.1,
67.2, 68.2, 68.2, 68.7, 69.0, 70.8, 71.2, 71.6, 71.6, 73.1, 75.5, 75.6, 75.7, 100.5, 100.6,
100.9, 102.2, 119.9, 125.1, 126.3, 126.3, 127.0, 127.0, 127.6, 127.7, 127.8, 128.1,
128.2, 128.3, 128.3, 128.3, 128.4, 128.5, 128.7, 129.0, 129.1, 129.4, 129.5, 129.7,
129.9, 133.0, 133.1, 133.4, 135.1, 137.4, 137.7, 141.2, 141.2, 143.6, 143.8, 155.8,
164.6, 165.1, 165.5, 169.4, 171.1, 172.1, 206.5, 206.5. HRMS: C87H83NO26 [M+NH4]

+

calcd: 1576.5625, obsd: 1576.5555.
N-Fluorenylmethyloxycarbonyl-O-[2, 3-di-O-levulinoyl-4, 6-O-benzylidene -

β-D-galactopyranosyl-(1→3)-2-O-benzoyl-4,
364

6-O-benzylidene-α-D-

galactopyranosyl-(1→4)-2,

3-di-O-benzoyl-β-D-xylopyranosyl]-L-serine

benzyl

ester (180). Compound 180 was a side product generated in 45% yield during the
synthesis of compound 179.

1

H-NMR (600 MHz, CD3Cl): δ 1.94 (s, 3 H,

CH3COCH2CH2), 1.95 (s, 3 H, CH3COCH2CH2), 1.96-2.15 (m, 3 H, CH3COCH2CH2),
2.41-2.66 (m, 5 H, CH3COCH2CH2), 3.44 (s, 1 H), 3.46-3.49 (m, 1 H), 3.77 (s, 1 H),
3.81-3.84 (m, 1 H), 3.96-4.12 (m, 6 H), 4.14-4.17 (m, 1 H), 4.23-4.36 (m, 6 H), 4.50-4.52
(m, 2 H), 4.61 (d, 1 H, J = 5.5 Hz), 4.68 (dd, 1 H, J = 3 Hz, J = 8.5 Hz), 4.73 (d, 1 H, J =
6.5 Hz), 5.10-5.17 (m, 3 H), 5.25-5.29 (m, 1 H), 5.40-5.41 (m, 1 H), 5.45-5.49 (m, 2 H),
5.54-5.61 (m, 3 H), 7.14-7.46 (m, 26 H), 7.50-7.55 (m, 6 H), 7.66-7.68 (m, 2 H), 7.737.76 (m , 2 H), 7.85-7.87 (m 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 14.1, 20.9, 27.1, 28.0,

29.5, 29.5, 37.5, 37.6, 47.0, 54.2, 60.3, 63.8, 64.0, 66.4, 67.2, 67.3, 68.3, 68.8, 68.9,
70.0, 71.6, 71.7, 72.0, 72.8, 73.2, 75.1, 76.2, 98.6, 100.4, 100.8, 101.2, 101.3, 119.9,
125.1, 126.1, 126.2, 127.0, 127.0, 127.6, 127.6, 128.0, 128.1, 128.1, 128.2, 128.2,
128.3, 128.3, 128.5, 128.6, 128.8, 128.9, 129.0, 129.4, 129.4, 129.6, 133.0, 133.1,
133.2, 135.1, 137.4, 137.6, 141.2, 141.2, 143.6, 143.7, 155.8, 165.1, 165.2, 165.3,
169.3, 170.9, 172.0, 206.2, 206.5. ESI-MS: C87H83NO26 calcd: [M+NH4]

+

calcd:

+

1575.52, obsd: 1575.90. HRMS: [M+NH4] calcd: 1575.5625, obsd: 1575.5486.
N-Fluorenylmethyloxycarbonyl-O-[4,

6-O-benzylidene

-β-D-

6-O-benzylidene-β-D-galactopyranosyl-

galactopyranosyl-(1→3)-2-O-benzoyl-4,

(1→4)-2, 3-di-O-benzoyl-β-D-xylopyranosyl]-L-serine benzyl ester (181). Compound
181 was synthesized from compound 179 in 72% yield following the general procedure
365

1

for Lev deprotection. H-NMR (500 MHz, CD3Cl): δ 2.53-2.55 (m, 1 H), 2.77 (s, 1 H),
3.25-3.36 (m, 4 H), 3.64-3.68 (m, 1 H), 3.73-3.76 (m, 2 H), 3.80-3.84 (m, 2 H), 3.95-4.06
(m, 4 H), 4.10-4.14 (m, 1 H), 4.17-4.34 (m, 6 H), 4.48-4.50 (m, 1 H), 4.56 (d, 1 H, J = 5.5
Hz), 4.78 (d, 1 H, J = 8 Hz), 5.01-5.09 (m, 2 H, CH2Ph), 5.15-5.18 (m, 1 H), 5.44-5.47
(m, 2 H), 5.54-5.63 (m, 3 H), 7.20-7.54 (m, 29 H), 7.73-7.76 (m, 2 H), 7.94-7.97 (m, 6 H),
8.33-8.37 (m 1 H).

13

C-NMR (125 MHz, CD3Cl): δ 47.0, 54.2, 62.2, 66.7, 67.0, 67.2,

67.3, 68.3, 69.0, 70.7, 71.0, 71.3, 71.5, 72.0, 75.0, 75.5, 75.9, 77.9, 100.4, 101.1, 101.2,
101.9, 104.1, 119.9, 125.1, 126.2, 126.6, 127.0, 127.0, 127.6, 127.7, 128.0, 128.2,
128.2, 128.3, 128.3, 128.4, 128.4, 128.5, 128.7, 128.8, 129.1, 129.2, 129.5, 129.5,
129.6, 129.8, 129.9, 130.8, 133.1, 133.1, 133.2, 135.1, 137.4, 137.6, 141.2, 141.2,
+

143.6, 143.8, 155.9, 165.1, 165.5, 165.6, 169.4. MALDI-MS: C77H71NO22 [M+Na]
calcd: 1385.38, obsd: 1385.57.
p-Tolyl

2-O-levulinoyl-3-O-benzyl-4-O-p-methoxybenzyl-6-O-tert-butyl-

diphenylsilyl-α-L-idopyranosyl-(1→4)-2-azido-3-O-benzyl-6-O-levulinoyl-2-deoxyα-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-tert-butyl-diphenylsilyl-αL-idopyranosyl-(1→4)-2-azido-3,

6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-

(1→4)-2-O-benzoyl-3-O-benzyl-6-O-tert-butyl-diphenylsilyl-1-thio-β-Dglucopyranoside (182). Compound 182 was synthesized from donor 177 and acceptor
1

178 in 93% yield following the general procedure of single step glycosylation. H-NMR
(600 MHz, CD3Cl): δ 0.99 (s, 9 H, C(CH3)3), 1.00 (s, 9 H, C(CH3)3), 1.04 (s, 9 H,
C(CH3)3), 2.00 (s, 3 H, CH3COCH2CH2), 2.01 (s, 3 H, CH3COCH2CH2), 2.25 (s, 3 H,
366

SPhCH3), 2.30-2.65 (m, 8 H, CH3COCH2CH2), 3.05-3.10 (m, 1 H), 3.13-3.15 (m, 2 H),
3.18-3.20 (m, 1 H), 3.40-3.44 (m, 2 H), 3.47-3.59 (m, 3 H), 3.64-3.69 (m, 2 H), 3.72-3.76
(m, 6 H), 3.82-4.03 (m, 10 H), 4.08-4.20 (m, 6 H), 4.28-4.31 (m, 2 H), 4.53-4.57 (m, 2 H),
4.60-4.64 (m, 2 H), 4.69-4.79 (m, 6 H), 4.84-4.89 (m, 2 H), 4.97 (d, 1 H, J = 9.5 Hz),
5.16-5.19 (m, 2 H), 5.27-5.31 (m, 1 H), 5.46-5.48 (m, 1 H), 6.71-6.73 (m, 2 H), 6.93-6.97
(m, 4 H), 7.02-7.38 (m, 52 H), 7.42-7.49 (m, 4 H), 7.53-7.76 (m, 12 H), 8.03-8.06 (m 4
H).

13

C-NMR (125 MHz, CD3Cl): δ 19.2, 19.2, 19.2, 21.0, 26.8, 26.9, 27.0, 27.7, 27.9,

29.6, 29.6, 37.6, 55.2, 62.2, 62.6, 63.0, 63.0, 63.7, 63.9, 68.1, 69.6, 69.9, 70.6, 70.9,
71.9, 72.0, 72.9, 73.2, 73.2, 74.2, 74.3, 74.5, 74.6, 74.7, 74.9, 74.9, 75.0, 78.4, 78.9,
79.7, 84.6, 87.0, 97.7, 97.7, 98.3, 113.6, 127.1, 127.3, 127.4, 127.5, 127.6, 127.6, 127.7,
127.7, 127.8, 127.8, 127.9, 128.0, 128.0, 128.1, 128.2, 128.2, 128.3, 128.3, 128.4,
128.6, 129.4, 129.6, 129.6, 129.6, 129.7, 129.7, 129.8, 129.8, 129.9, 130.3, 132.0,
132.9, 133.2, 133.3, 133.5, 135.6, 135.6, 135.6, 135.6, 135.7, 137.4, 137.5, 137.5,
137.8, 137.8, 138.1, 138.2, 159.2, 165.2, 165.3, 171.7, 172.1, 206.1, 206.1. MALDI-MS:
+

C159H174N6O30SSi3 [M+Na] calcd: 2768.43, obsd: 2790.93.
N-Fluorenylmethyloxycarbonyl-O-[2-O-levulinoyl-3-O-benzyl-4-O-pmethoxybenzyl-6-O-tert-butyl-diphenylsilyl-α-L-idopyranosyl-(1→4)-2-azido-3-Obenzyl-6-O-levulinoyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl6-O-tert-butyl-diphenylsilyl-α-L-idopyranosyl-(1→4)-2-azido-3,

6-di-O-benzyl-2-

deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-tert-butyldiphenylsilyl-β-D-glucopyranosyl-(1→4)-4, 6-O-benzylidene-β-D-galactopyranosyl(1→3)-2-O-benzoyl-4,

6-O-benzylidene-β-D-galactopyranosyl-(1→4)-2,
367

3-di-O-

benzoyl-β-D-xylopyranosyl]-L-serine benzyl ester (176). Compound 176 was
synthesized from donor 182 and acceptor 181 in 71% yield following the general
procedure of single step glycosylation.

1

H-NMR (600 MHz, CD3Cl): δ 0.92 (s, 9 H,

C(CH3)3), 1.00 (s, 9 H, C(CH3)3), 1.03 (s, 9 H, C(CH3)3), 1.99 (s, 3 H, CH3COCH2CH2),
2.00 (s, 3 H, CH3COCH2CH2), 2.20 (s, 1 H), 2.27-2.66 (m, 8 H, CH3COCH2CH2), 3.023.06 (m, 2 H), 3.09-3.15 (m, 2 H), 3.18-3.22 (m, 2 H), 3.30-3.46 (m, 5 H), 3.47-3.49 (m,
1 H), 3.53-3.57 (m, 1 H), 3.64-3.87 (m, 19 H), 3.94-4.33 (m, 18 H), 4.48-4.77 (m, 12 H),
4.84-4.88 (m, 2 H), 4.96-5.03 (m, 3 H), 5.14-5.18 (m, 3 H), 5.22-5.27 (m, 1 H), 5.31 (s, 2
H), 5.42-5.44 (m, 1 H), 5.47-5.51 (m, 1 H), 5.53-5.55 (m, 1 H), 5.58-5.62 (m, 1 H), 6.716.73 (m, 2 H), 6.94-6.96 (m, 4 H), 7.01-7.03 (m, 2 H), 7.04-7.07 (m, 3 H), 7.11-7.38 (m,
78 H), 7.43-7.64 (m, 18 H), 7.42-7.46 (m, 2 H), 7.94-8.02 (m 10 H).

13

C-NMR (125 MHz,

CD3Cl): δ 19.2, 19.2, 19.3, 26.7, 26.7, 26.9, 27.0, 27.6, 27.9, 29.6, 29.6, 29.6, 37.6,
47.0, 54.2, 55.2, 62.2, 62.3, 62.6, 62.9, 62.9, 63.7, 64.2, 66.8, 67.0, 67.2, 67.3, 68.0,
68.2, 69.0, 69.5, 68.7, 69.9, 70.6, 70.7, 71.3, 71.5, 71.8, 72.0, 73.2, 73.2, 73.3, 73.9,
74.1, 74.2, 74.3, 74.4, 74.7, 74.8, 74.9, 75.0, 75.0, 75.2, 75.5, 75.5, 75.8, 76.5, 78.1,
78.4, 78.9, 83.3, 97.6, 97.7, 98.3, 100.1, 100.5, 101.0, 101.4, 102.0, 104.0, 113.6, 119.9,
125.1, 126.0, 126.6, 127.0, 127.1, 127.3, 127.4, 127.5, 127.6, 127.7, 127.8, 127.8,
127.8, 127.9, 127.9, 127.9, 128.0, 128.0, 128.1, 128.2, 128.2, 128.3, 128.3, 128.3,
128.4, 128.5, 128.6, 128.7, 129.1, 129.4, 129.5, 129.6, 129.6, 129.7, 129.7, 129.9,
130.0, 132.9, 133.1, 133.1, 133.1, 133.2, 133.2, 133.3, 133.5, 135.1, 135.4, 135.4,
135.6, 135.6, 135.6, 137.5, 137.5, 137.6, 137.7, 138.1, 138.2, 141.2, 143.6, 143.8,

368

155.9, 159.1, 165.1, 165.2, 165.3, 165.3, 165.5, 169.4, 171.7, 172.1, 206.1, 206.2.
+

MALDI-MS: C222H231N7O25Si3: [M+NH4] calcd: 3931.48, obsd: 3930.60.
N-(Acetyl)-O-(benzyl)-L-serglycine-t-butyl-ester (269). Serine 267 (1.329 g,
5.6 mmol), glycine 268 (940 mg, 5.6 mmol) were dissolved in DCM/THF (1:1, 20 mL),
followed by addition of BOP (4.95 g, 11.2 mmol) and DIPEA (1.85 mL, 11.2 mol). The
resulting mixture was stirred under room temperature overnight. After the reaction was
complete, it was diluted with DCM. The solution was washed with 10% HCl solution, sat.
NaHCO3 solution and dried over Na2SO4. After concentration, column purification
1

afforded compound 269 (2.05 g, 82%). H-NMR (500 MHz, CD3Cl): δ 1.44 (s, 9 H,
C(CH3)3), 1.99 (s, 3 H, CH3CONH), 3.52 (dd, 1 H, J = 7 Hz, J = 9.5 Hz), 3.86-3.90 (m, 3
H), 4.51-4.60 (m, 3 H), 6.44-6.45 (m, 1 H, NH), 6.95-6.98 (m, 1 H, NH), 7.13-7.23 (m, 1
H), 7.24-7.33 (m, 4 H).

13

C-NMR (125 MHz, CD3Cl): δ 23.1, 27.9, 42.1, 52.3, 69.1, 73.4,

82.2, 125.2, 127.8, 127.9, 128.1, 128.4, 128.9, 137.3, 168.4, 170.0, 170.2. HRMS:
+

C18H26N2O5 [M+H] calcd: 351.1920, obsd: 351.1895.
N-(Acetyl)-O-(benzyl)-L-serglycine (120). Compound 269 (2.05 g, 1.86 mmol)
was dissolved in 4 mL DCM, followed by addition of TFA (4 mL). The resulting mixture
was stirred under room temperature until the reaction was complete indicated by TLC
analysis. The solution was concentrated to dryness to afford compound 120 which was
1

used directly for next step without further purification. H-NMR (500 MHz, CD3OD): δ
2.00 (s, 3 H, CH3CONH), 3.70-3.77 (m, 2 H), 3.91-3.93 (m, 2 H), 4.52-4.57 (m, 2 H),
6.63 (t, 1 H, J = 4.5 Hz), 7.24-7.27 (m, 1 H), 7.29-7.34 (m, 4 H), 8.21-8.24 (m, 1 H,
369

COOH).

13

C-NMR (125 MHz, CD3OD): δ 22.5, 41.9, 54.7, 70.7, 74.2, 128.7, 128.9,
+

128.9, 129.3, 139.2, 172.5, 173.4. HRMS: C14H18N2O5 [M+H] calcd: 295.1294, obsd:
295.1287.
N-Fluorenylmethyloxycarbonyl-O-[2-O-levulinoyl-3-O-benzyl-4-O-pmethoxybenzyl-α-L-idopyranosyl-(1→4)-2-azido-3-O-benzyl-6-O-levulinoyl-2deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-α-L-idopyranosyl(1→4)-2-azido-3, 6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3O-benzyl-β-D-glucopyranosyl-(1→4)-4,
(1→3)-2-O-benzoyl-4,

6-O-benzylidene-β-D-galactopyranosyl-

6-O-benzylidene-β-D-galactopyranosyl-(1→4)-2,

3-di-O-

benzoyl-β-D-xylopyranosyl]-L-serine benzyl ester (192). Compound 176 (715 mg,
0.178 mmol) was dissolved in pyridine (6 mL) in a plastic flask followed by addition of
o

65-70% HF-pyridine solution (4.2 mL) under 0 C. The solution was stirred overnight
until complete disappearance of starting material as judged by TLC analysis. The
reaction mixture was quenched by solid NaHCO3 and diluted with DCM. The aqueous
phase was extracted with DCM twice. The combined organic phase was further washed
with sat. NaHCO3 and dried over Na2SO4. Column purification afforded compound 192
1

(520 mg, 90%). H-NMR (500 MHz, CD3Cl): δ 2.11 (s, 3 H, CH3COCH2CH2), 2.14 (s, 3
H, CH3COCH2CH2), 2.43-2.78 (m, 8 H, CH3COCH2CH2), 3.20-3.32 (m, 7 H), 3.36-3.42
(m, 2 H), 3.46-3.53 (m, 3 H), 3.56-3.64 (m, 4 H), 3.68-4.35 (m, 41 H), 4.41-4.44 (m, 1 H),
4.47-4.53 (m, 4 H), 4.56-4.58 (m, 1 H), 4.63-4.65 (m, 1 H), 4.69-4.93 (m, 11 H), 5.025.11 (m, 4 H), 5.16-5.20 (m, 2 H), 5.25-5.29 (m, 1 H), 5.35-5.40 (m, 2 H), 5.52-5.64 (m,
370

4 H), 6.81-6.84 (m, 2 H), 7.12-7.47 (m, 66 H), 7.53-7.58 (m, 2 H), 7.96-8.01 (m, 8 H),
8.07-8.09 (m 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 14.1, 20.9, 27.7, 27.8, 29.6, 29.7,

37.6, 37.7, 46.9, 54.1, 55.1, 60.3, 60.8, 61.1, 61.6, 62.2, 62.3, 63.3, 63.6, 66.7, 66.9,
67.1, 67.2, 67.5, 68.0, 68.1, 68.7, 69.0, 69.2, 69.3, 69.8, 69.9, 70.0, 70.6, 71.3, 71.5,
71.8, 72.1, 72.3, 72.7, 72.8, 73.1, 73.4, 73.8, 74.1, 74.6, 74.8, 75.0, 75.1, 75.3, 75.4,
75.6, 76.5, 78.7, 78.8, 83.2, 97.0, 97.3, 97.5, 97.5, 100.1, 100.4, 100.9, 101.8, 103.8,
113.7, 119.9, 125.0, 125.8, 126.5, 127.0, 127.0, 127.5, 127.6, 127.6, 127.7, 127.7,
127.8, 127.8, 127.9, 128.0, 128.0, 128.1, 128.1, 128.2, 128.2, 128.2, 128.3, 128.3,
128.4, 128.4, 128.5, 128.7, 129.0, 129.4, 129.5, 129.5, 129.6, 129.7, 129.7, 129.8,
129.8, 133.0, 133.1, 133.1, 133.2, 135.0, 137.1, 137.4, 137.4, 137.5, 137.6, 137.8,
141.1, 141.1, 143.6, 143.7, 155.8, 159.3, 165.0, 165.1, 165.3, 165.4, 165.7, 169.4,
+

171.0, 171.8, 172.1, 206.7, 206.8. MALDI-MS: C181H183N7O52 [M+Na] calcd: 3311.41,
obsd: 3311.30.
N-Fluorenylmethyloxycarbonyl-O-[methyl 2-O-levulinoyl-3-O-benzyl-4-O-pmethoxybenzyl-α-L-idopyranosyluronate-(1→4)-2-azido-3-O-benzyl-6-O-levulinoyl2-deoxy-α-D-glucopyranosyl-(1→4)-methyl

2-O-benzoyl-3-O-benzyl-α-L-

idopyranosyluronate-(1→4)-2-azido-3, 6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl(1→4)-2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyl-(1→4)-4, 6-O-benzylidene-β-D6-O-benzylidene-β-D-galactopyranosyl-

galactopyranosyl-(1→3)-2-O-benzoyl-4,

(1→4)-2, 3-di-O-benzoyl-β-D-xylopyranosyl]-L-serine benzyl ester (194). Compound
192 (115 mg, 0.035 mmol) was dissolved in DCM/tBuOH/H2O (4:4:1, 4.5 mL), followed
by addition of TEMPO (4 mg) and BAIB (90 mg). The resulting mixture was stirred
371

under room temperature overnight. After the reaction was complete indicated by TLC
analysis, it was neutralized by 1 M HCl solution to adjust pH around 6. The solution was
first diluted with DCM, then extracted with H2O. The combined organic phase was dried
over Na2SO4. After concentration, the crude product was confirmed by MALDI-MS
+

analysis. MALDI-MS: C181H177N7O55 calcd: [M+Na] calcd: 3351.00, obsd: 3351.13.
The crude compound was dissolved in dry DMF (5 mL), to which was added MeI (17 μL,
0.263 mmol) and K2CO3 (73 mg, 0.525 mmol). The resulting mixture was stirred under
room temperature overnight. The reaction mixture was diluted with DCM and H2O. The
aqueous phase was extracted with DCM twice. The combined organic phase was
further washed with sat. NaHCO3 and dried over Na2SO4. Column purification afforded
compound 194 (110 mg, 93%).

1

H-NMR (500 MHz, CD3Cl): δ 2.04 (s, 3 H,

CH3COCH2CH2), 2.11 (s, 3 H, CH3COCH2CH2), 2.43-2.74 (m, 8 H, CH3COCH2CH2),
3.18-3.59 (m, 17 H), 3.62-3.99 (m, 19 H), 4.06-4.25 (m, 11 H), 4.29-4.78 (m, 19 H),
4.85-4.89 (m, 2 H), 4.93-4.95 (m, 1 H), 5.02-5.09 (m, 2 H), 5.13-5.17 (m, 2 H), 5.21-5.28
(m, 2 H), 5.33-5.38 (m, 2 H), 5.45-5.61 (m, 4 H), 6.76-6.81 (m, 1 H), 7.09-7.48 (m, 68 H),
7.51-7.56 (m, 2 H), 7.74-7.76 (m, 1 H), 7.85-7.89 (m, 2 H), 7.93-7.99 (m, 7 H), 8.07-8.09
(m 1 H).

13

C-NMR (125 MHz, CD3Cl): δ 14.0, 21.4, 22.6, 27.7, 27.8, 27.9, 29.3, 29.5,

29.6, 29.7, 31.8, 37.6, 37.9, 47.0, 51.7, 51.9, 52.1, 54.2, 55.2, 61.9, 62.2, 62.7, 63.0,
66.8, 66.9, 67.1, 67.3, 68.2, 68.7, 69.0, 69.8, 69.9, 70.2, 70.7, 70.8, 71.2, 71.3, 71.5,
72.3, 73.1, 73.4, 73.7, 73.8, 74.2, 74.4, 74.6, 75.0, 75.4, 75.4, 75.5, 75.7, 77.8, 77.9,

372

82.6, 97.2, 97.7, 97.9, 99.2, 100.0, 100.5, 100.6, 101.0, 101.8, 103.8, 113.7, 119.9,
125.1, 125.2, 125.8, 126.6, 127.0, 127.0, 127.3, 127.4, 127.6, 127.6, 127.7, 127.7,
127.8, 127.8, 127.9, 127.9, 128.0, 128.0, 128.1, 128.1, 128.2, 128.2, 128.3, 128.3,
128.3, 128.4, 128.6, 128.8, 129.0, 129.0, 129.2, 129.3, 129.4, 129.5, 129.6, 129.6,
129.7, 129.8, 133.1, 133.2, 133.4, 135.1, 137.2, 137.4, 137.4, 137.6, 137.6, 137.8,
137.9, 141.2, 143.6, 143.8, 155.8, 159.3, 164.9, 165.1, 165.1, 165.4, 165.5, 168.9,
+

169.4, 169.4, 169.9, 171.8, 172.3, 206.1, 206.5. MALDI-MS: C184H183N7O55 [M+Na]
calcd: 3395.44, obsd: 3395.40.

N-Fluorenylmethyloxycarbonyl-O-[methyl 2-O-levulinoyl-3-O-benzyl-4-O-pmethoxybenzyl-α-L-idopyranosyluronate-(1→4)-2-azido-3-O-benzyl-6-O-levulinoyl2-deoxy-α-D-glucopyranosyl-(1→4)-methyl

2-O-benzoyl-3-O-benzyl-α-L-

idopyranosyluronate-(1→4)-2-azido-3, 6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl(1→4)-2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyl-(1→4)-2-O-acetyl-4,
benzylidene-β-D-galactopyranosyl-(1→3)-2-O-benzoyl-4,
galactopyranosyl-(1→4)-2,

6-O-

6-O-benzylidene-β-D-

3-di-O-benzoyl-β-D-xylopyranosyl]-L-serine

benzyl

ester (195). Compound 194 (30 mg, 0.0089 mmol) was dissolved in 2 mL pyridine,
o

followed by addition of 1 mL Ac2O. The resulting mixture was stirred under 50 C
overnight. After cooling back to room temperature, it was diluted with DCM, washed with
10% HCl, sat. NaHCO3. The combined organic phase was dried over Na2SO4. Column
1

purification afforded compound 195 (27 mg, 88%). H-NMR (500 MHz, CD3Cl): δ 2.04
(s, 3 H, CH3COCH2CH2), 2.11 (s, 3 H, CH3COCH2CH2), 2.33 (s, 3 H, COCH3), 2.44-

373

2.75 (m, 8 H, CH3COCH2CH2), 3.10-3.36 (m, 9 H), 3.44-3.57 (m, 9 H), 3.65-3.97 (m, 18
H), 4.06-4.22 (m, 10 H), 4.27-4.42 (m, 4 H), 4.45-4.70 (m, 13 H), 4.74-4.79 (m, 3 H),
4.84-4.89 (m, 3 H), 4.99-5.07 (m, 2 H), 5.11-5.15 (m, 3 H), 5.17-5.22 (m, 2 H), 5.27-5.33
(m, 3 H), 5.36-5.38 (m, 1 H), 5.44-5.48 (m, 1 H), 5.52-5.58 (m, 3 H), 6.79-6.81 (m, 2 H),
7.07-7.55 (m, 69 H), 7.73-7.77 (m, 2 H), 7.88-8.00 (m, 9 H).

13

C-NMR (125 MHz,

CD3Cl): δ 20.1, 21.4, 27.8, 27.9, 29.5, 29.7, 37.6, 37.9, 47.0, 51.7, 51.9, 52.1, 54.2,
55.2, 62.3, 62.7, 63.0, 66.7, 67.0, 67.1, 67.2, 68.1, 69.0, 69.8, 70.8, 70.9, 71.2, 71.3,
71.3, 71.7, 72.3, 72.9, 73.4, 73.7, 73.8, 73.9, 74.5, 74.7, 75.0, 75.2, 75.4, 75.4, 75.7,
77.9, 82.6, 97.2, 97.7, 97.9, 99.2, 100.1, 100.4, 100.5, 102.2, 113.7, 119.9, 125.1, 125.2,
126.0, 126.4, 127.0, 127.0, 127.4, 127.6, 127.6, 127.6, 127.7, 127.7, 127.8, 127.8,
127.9, 127.9, 128.0, 128.0, 128.0, 128.1, 128.2, 128.2, 128.3, 128.3, 128.3, 128.4,
128.4, 128.5, 128.6, 128.7, 129.0, 129.1, 129.2, 129.3, 129.5, 129.5, 129.7, 129.9,
129.9, 133.1, 133.4, 135.1, 137.0, 137.3, 137.5, 137.7, 137.8, 137.8, 137.9, 141.2,
143.6, 143.8, 155.9, 159.3, 164.5, 164.6, 165.0, 165.1, 165.5, 168.8, 168.9, 169.4,
169.9, 171.8, 172.3, 206.1, 206.5. MALDI-MS: C186H185N7O56

[M+Na]

+

calcd:

3435.18, obsd: 3435.70.
N-(Acetyl)-O-(benzyl)-L-seryl-glycyl-O-[methyl 2-O-levulinoyl-3-O-benzyl-4O-p-methoxybenzyl-α-L-idopyranosyluronate-(1→4)-2-azido-3-O-benzyl-6-Olevulinoyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl 2-O-benzoyl-3-O-benzyl-α-Lidopyranosyluronate-(1→4)-2-azido-3, 6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl(1→4)-2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyl-(1→4)-2-O-acetyl-4,
benzylidene-β-D-galactopyranosyl-(1→3)-2-O-benzoyl-4,
374

6-O-

6-O-benzylidene-β-D-

galactopyranosyl-(1→4)-2,

3-di-O-benzoyl-β-D-xylopyranosyl]-L-serine

benzyl

ester (175). Compound 195 (31 mg, 0.009 mmol) was dissolved in DCM (0.6 mL),
followed by addition of 46 μL piperidine. After 3 h, the mixture was diluted with DCM and
extracted with H2O. The combined organic phase was dried over Na2SO4. Column
1

purification afforded compound 196 (20 mg, 70%). H-NMR (500 MHz, CD3Cl): δ 2.04
(s, 3 H, CH3COCH2CH2), 2.11 (s, 3 H, CH3COCH2CH2), 2.33 (s, 3 H, COCH3), 2.432.77 (m, 8 H, CH3COCH2CH2), 3.07 (br, 1 H), 3.16-3.19 (m, 2 H), 3.22-3.34 (m, 8 H),
3.45-3.58 (m, 10 H), 3.64-4.02 (m, 20 H), 4.04-4.20 (m, 8 H), 4.35-4.42 (m, 3 H), 4.454.78 (m, 16 H), 4.84-4.88 (m, 3 H), 4.98 (s, 2 H), 5.09-5.15 (m, 3 H), 5.17-5.22 (m, 2 H),
5.31 (d, 1 H, J = 11 Hz), 5.36-5.38 (m, 1 H), 5.41-5.46 (m, 1 H), 5.52-5.57 (m, 2 H),
6.79-6.81 (m, 2 H), 7.07-7.54 (m, 62 H), 7.88-7.98 (m, 10 H). Compound 196 (22 mg,
0.0069 mmol) and compound 120 (4 mg, 0.0138 mmol) were dissolved in 0.6 mL DMF,
followed by addition of HATU (5.2 mg, 0.0138 mmol) and DIPEA (4.8 μL, 0.0276 mmol).
The resulting mixture was stirred under room temperature overnight and diluted with
DCM. The solution was washed with 10% HCl, sat. NaHCO3. The combined organic
phase was dried over Na2SO4. Column purification afforded compound 175 (18 mg,
77%).

1

H-NMR (500 MHz, CD3Cl): δ 1.95 (s, 3 H, CH3COCH2CH2), 2.04 (s, 3 H,

CH3COCH2CH2), 2.33 (s, 3 H, COCH3), 2.37-2.73 (m, 8 H, CH3COCH2CH2), 2.782.93(m, 6 H), 3.12-3.38 (m, 8 H), 3.44-3.57 (m, 8 H), 3.69-3.97 (m, 18 H), 3.06-4.20 (m,
9 H), 4.38-4.89 (m, 23 H), 4.95-5.27 (m, 7 H), 5.30-5.55 (m, 6 H), 6.45-6.47 (m, 1 H),
6.67-6.71 (m, 1 H), 6.79-6.81 (m, 2 H), 6.93-6.96 (m, 1 H), 7.09-7.54 (m, 67 H), 7.88375

7.99 (m, 10 H).

13

C-NMR (125 MHz, CD3Cl): δ 20.1, 21.4, 23.0, 27.8, 27.9, 29.5, 29.7,

37.6, 37.9, 38.5, 42.8, 51.7, 51.9, 52.1, 52.5, 52.7, 55.2, 61.9, 62.2, 62.8, 63.1, 66.7,
67.1, 67.3, 68.3, 69.1, 69.1, 69.8, 70.8, 71.0, 71.2, 71.3, 71.5, 72.3, 72.9, 73.3, 73.4,
73.6, 73.7, 73.8, 74.5, 74.5, 74.6, 75.0, 75.3, 75.4, 75.4, 75.5, 75.5, 77.8, 77.9, 82.6,
97.2, 97.7, 98.0, 99.2, 100.1, 100.1, 100.4, 100.5, 102.2, 113.7, 125.2, 126.0, 126.3,
127.3, 127.4, 127.6, 127.6, 127.7, 127.7, 127.8, 127.8, 127.8, 127.9, 127.9, 127.9,
128.0, 128.0, 128.1, 128.2, 128.2, 128.2, 128.3, 128.3, 128.3, 128.4, 128.4, 128.5,
128.6, 128.7, 128.9, 129.0, 129.2, 129.3, 129.3, 129.5, 129.5, 129.5, 129.7, 129.7,
129.9, 130.0, 133.1, 133.1, 133.3, 133.4, 135.0, 137.0, 137.3, 137.4, 137.5, 137.7,
137.7, 137.8, 137.8, 137.8, 138.0, 159.4, 164.5, 164.6, 165.0, 165.1, 165.7, 168.3,
168.8, 169.0, 169.3, 169.9, 170.3, 170.4, 171.8, 172.3, 206.1, 206.5. MALDI-MS:
+

C185H191N9O58 [M+Na] calcd: 3489.23, obsd: 3489.22.
N-Fluorenylmethyloxycarbonyl-O-[methyl 3-O-benzyl-4-O-p-methoxybenzylα-L-idopyranosyluronate-(1→4)-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl(1→4)-methyl

2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate-(1→4)-2-azido-3,

6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-β-Dglucopyranosyl-(1→4)-2-O-acetyl-4, 6-O-benzylidene-β-D-galactopyranosyl-(1→3)2-O-benzoyl-4, 6-O-benzylidene-β-D-galactopyranosyl-(1→4)-2, 3-di-O-benzoyl-βD-xylopyranosyl]-L-serine benzyl ester (198). Compound 198 was synthesized from
1

compound 195 in 88% yield following the general procedure of Lev deprotection. HNMR (500 MHz, CD3Cl): δ 1.45 (s, 3 H), 3.10 (s, 1 H), 3.19-3.35 (m, 14 H), 3.38-3.41 (m,
7 H), 3.50-3.89 (m, 27 H), 3.92-3.99 (m, 4 H), 4.06-4.23 (m, 12 H), 4.27-4.60 (m, 17 H),
376

4.64-4.80 (m, 8 H), 4.84-4.89 (m, 2 H), 4.99-5.08 (m, 2 H), 5.11-5.22 (m, 6 H), 5.27-5.39
(m, 4 H), 5.44-5.58 (m, 4 H), 6.80-6.82 (m, 2 H), 7.08-7.54 (m, 58 H), 7.73-7.76 (m, 2 H),
+

7.88-8.00 (m, 10 H). MALDI-MS: C176H173N7O52 [M+Na]

calcd: 3241.27, obsd:

3241.03.
Octasaccharide 199. Compound 199 was synthesized from compound 198 in
2-

88% yield following the general procedure of O-sulfation. ESI-MS: C176H171N7O58S2
calcd: [M-2H]

2-

calcd: 1687.00, obsd: 1687.80.

Staudinger reaction intermediate 201. Compound 199 (2 mg, 0.006 mmol)
was dissolved in THF/H2O (9:1. 0.5 mL), followed by addition of PPh3 (2 mg) and silica
gel (1 mg). The resulting mixture was stirred under room temperature overnight. TLC
shows the formation of a major spot, which was identified as comound 201 based on
2-

mass spectra analysis. ESI-MS: C176H171N7O58S2

calcd: [M-2H]

2-

calcd: 1921.09,

obsd: 1921.60.
Octasaccharide 202. Compound 198 (2 mg) was dissolved in DCM/MeOH (1:1,
0.6 mL), followed by addition of Zn (2 mg) and HCOONH4 (2 mg). The resulting mixture
was stirred under room temperature overnight. After filtration, the reaction mixture was
loaded onto LH-20 column. The fractions containing octasaccharide was combined and
concentrated to afford compound 202 (1.4 mg, 73%). MALDI-MS: C176H177N3O52
+

calcd: [M+H] calcd: 3165.13, obsd: 3164.74.
N-Fluorenylmethyloxycarbonyl-O-[methyl 3-O-benzyl-4-O-p-methoxybenzylα-L-idopyranosyluronate-(1→4)-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl377

(1→4)-methyl

2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate-(1→4)-2-azido-3,

6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-β-Dglucopyranosyl-(1→4)-4,
benzoyl-4,

6-O-benzylidene-β-D-galactopyranosyl-(1→3)-2-O-

6-O-benzylidene-β-D-galactopyranosyl-(1→4)-2,

3-di-O-benzoyl-β-D-

xylopyranosyl]-L-serine benzyl ester (205). Compound 205 was synthesized from
1

compound 194 in 84% yield following the general procedure of Lev deprotection. HNMR (500 MHz, CD3Cl): δ 3.22-3.35 (m, 8 H), 3.39-3.49 (m, 7 H), 3.54-3.58 (m, 1 H),
3.61-4.29 (m, 31 H), 4.33-4.38 (m, 1 H), 4.40-4.82 (m, 16 H), 4.89-4.93 (m, 2 H), 4.98 (d,
1 H, J = 7.5 Hz), 5.05-5.13 (m, 2 H), 5.16-5.31 (m, 4 H), 5.36-5.42 (m, 2 H), 5.50-5.65
(m, 4 H), 6.83-6.85 (m, 2 H), 7.12-7.50 (m, 59 H), 7.55-7.60 (m, 2 H), 7.77-7.80 (m, 2 H),
+

7.92-8.01 (m, 8 H). MALDI-MS: C174H171N7O51 [M+H] calcd: 3177.24, obsd: 3177.50.
Octasaccharide 206. Compound 206 was synthesized from compound 205 in
2-

77% yield following the general procedure of O-sulfation. ESI-MS: C174H169N7O57S2
2-

[M-2H]

calcd: 1666.00, obsd: 1666.53.

N-Acetyl-O-[methyl

2-O-levulinoyl-3-O-benzyl-4-O-p-methoxybenzyl-α-L-

idopyranosyluronate-(1→4)-2-azido-3-O-benzyl-6-O-levulinoyl-2-deoxy-α-Dglucopyranosyl-(1→4)-methyl

2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate-

(1→4)-2-azido-3, 6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3O-benzyl-β-D-glucopyranosyl-(1→4)-2-O-acetyl-4,

6-O-benzylidene-β-D-

6-O-benzylidene-β-D-galactopyranosyl-

galactopyranosyl-(1→3)-2-O-benzoyl-4,

(1→4)-2, 3-di-O-benzoyl-β-D-xylopyranosyl]-L-serine benzyl ester (221). Compound
378

196 (9 mg, 0.003 mmol) was dissolved in 0.5 mL pyridine, followed by addition of 10 μL
Ac2O. The resulting mixture was stirred under room temperature overnight. After
cooling back to room temperature, it was diluted with DCM, washed with 10% HCl, sat.
NaHCO3. The combined organic phase was dried over Na2SO4. Column purification
1

afforded compound 221 (7 mg, 77%). H-NMR (500 MHz, CD3Cl): δ 1.22 (s, 3 H), 1.42
(s, 3 H), 2.03 (s, 3 H, CH3COCH2CH2), 2.10 (s, 3 H, CH3COCH2CH2), 2.42-2.75 (m, 8
H, CH3COCH2CH2), 3.08 (m, 1 H), 3.16-3.32 (m, 10 H), 3.39-3.55 (m, 11 H), 3.61-3.98
(m, 21 H), 4.04-4.09 (m, 4 H), 4.12-4.21 (m, 7 H), 4.34-4.40 (m, 3 H), 4.44-4.53 (m, 6 H),
4.56-4.76 (m, 13 H), 4.83-4.88 (m, 3 H), 5.01-5.12 (m, 6 H), 5.16-5.21 (m, 2 H), 5.275.32 (m, 3 H), 5.35-5.37 (m, 1 H), 5.41-5.45 (m, 1 H), 5.51-5.55 (m, 2 H), 6.27-6.29 (m,
1 H), 6.78-6.81 (m, 2 H), 7.06-7.54 (m, 62 H), 7.86-7.97 (m, 10 H).

13

C-NMR (125 MHz,

CD3Cl): δ 14.1, 20.1, 22.6, 27.8, 27.9, 28.9, 29.1, 29.3, 29.5, 29.6, 29.7, 31.0, 31.9,
33.8, 37.6, 37.9, 51.7, 51.9, 52.1, 52.2, 55.2, 62.5, 62.7, 63.0, 63.9, 66.7, 67.0, 67.2,
68.1, 69.1, 69.8, 70.5, 70.9, 71.3, 71.8, 72.3, 72.9, 73.4, 73.7, 74.4, 75.0, 75.2, 75.4,
75.6, 77.9, 82.6, 97.2, 97.7, 97.9, 99.2, 100.0, 100.3, 100.5, 100.6, 102.1, 113.7, 125.2,
125.9, 126.0, 126.3, 127.3, 127.6, 127.7, 127.8, 127.8, 127.9, 127.9, 128.0, 128.1,
128.2, 128.3, 128.3, 128.4, 128.4, 128.6, 129.0, 129.0, 129.2, 129.3, 129.5, 129.5,
129.6, 129.7, 129.8, 129.9, 132.9, 133.1, 133.3, 133.4, 135.1, 137.0, 137.3, 137.5,
137.6, 137.7, 137.8, 137.8, 137.9, 159.3, 164.5, 164.6, 165.0, 165.1, 165.4, 168.8,
168.9, 169.4, 169.4, 169.8, 169.9, 171.8, 172.3, 206.1, 206.1. MALDI-MS:
+

C173H177N7O55 [M+Na] calcd: 3257.27, obsd: 3257.83.
379

N-Acetyl-O-[methyl

2-O-levulinoyl-3-O-benzyl-4-O-p-methoxybenzyl-α-L-

idopyranosyluronate-(1→4)-2-N-acetyl-3-O-benzyl-6-O-levulinoyl-2-deoxy-α-Dglucopyranosyl-(1→4)-methyl

2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate-

(1→4)-2-N-acetyl-3, 6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl3-O-benzyl-β-D-glucopyranosyl-(1→4)-2-O-acetyl-4,

6-O-benzylidene-β-D-

6-O-benzylidene-β-D-galactopyranosyl-

galactopyranosyl-(1→3)-2-O-benzoyl-4,

(1→4)-2, 3-di-O-benzoyl-β-D-xylopyranosyl]-L-serine benzyl ester (222). Compoud
221 (30 mg, 0.0093 mmol) was dissolved in THF/Ac2O/HOAc (3:2:1, 6 mL), followed by
addition of Zn (480 mg), CuSO4 (saturated solution, 30 μL). The resulting mixture was
stirred under room temperature overnight. After filtration, the mixture was diluted with
DCM and washed with sat. NaHCO3. The combined organic phase was dried over
1

Na2SO4 and purified by silica gel column to afford compound 222 (22 mg, 72%). HNMR (500 MHz, CD3Cl): δ 1.28 (s, 3 H), 1.29 (s, 3 H), 1.40 (s, 3 H), 2.08 (s, 3 H,
CH3COCH2CH2), 2.13 (s, 3 H, CH3COCH2CH2), 2.33 (s, 3 H), 2.45-2.77 (m, 8 H,
CH3COCH2CH2), 3.08 (s, 1 H), 3.18 (s, 1 H), 3.22-3.27 (m, 2 H), 3.33-3.41 (m, 8 H),
3.48-3.58 (m, 7 H), 3.61-3.94 (m, 19 H), 4.01-4.28 (m, 15 H), 4.31-4.70 (m, 22 H), 4.754.77 (m, 1 H), 4.81-4.83 (m, 1 H), 4.89-5.21 (m, 13 H), 5.28-5.33 (m, 4 H), 5.36-5.38 (m,
1 H), 5.42-5.46 (m, 1 H), 5.52-5.57 (m, 2 H), 6.09-6.12 (m, 1 H), 6.77-6.79 (m, 2 H),
+

6.99-7.55 (m, 61 H), 7.87-8.03 (m, 11 H). MALDI-MS: C177H185N3O57 [M+Na] calcd:
3289.35, obsd: 3289.72.

380

N-Acetyl-O-[methyl

3-O-benzyl-4-O-p-methoxybenzyl-α-L-

idopyranosyluronate-(1→4)-2-N-acetyl-3-O-benzyl-2-deoxy-α-D-glucopyranosyl(1→4)-methyl 2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate-(1→4)-2-N-acetyl-3,
6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-β-Dglucopyranosyl-(1→4)-2-O-acetyl-4, 6-O-benzylidene-β-D-galactopyranosyl-(1→3)2-O-benzoyl-4, 6-O-benzylidene-β-D-galactopyranosyl-(1→4)-2, 3-di-O-benzoyl-βD-xylopyranosyl]-L-serine benzyl ester (223). Compound 223 was synthesized from
1

compound 222 in 88% yield following the general procedure of Lev deprotection. HNMR (500 MHz, CD3Cl): δ 1.20 (s, 3 H), 1.27 (s, 3 H), 1.40 (s, 3 H), 2.33 (s, 3 H), 3.08
(s, 1 H), 3.18-3.41 (m, 17 H), 3.48-3.89 (m, 28 H), 3.97-4.11 (m, 8 H), 4.18-4.26 (m, 6
H), 4.35-4.69 (m, 21 H), 4.75-4.77 (m, 2 H), 4.85-5.14 (m, 9 H), 5.18-5.22 (m, 3 H),
5.28-5.36 (m, 5 H), 5.42-5.46 (m, 1 H), 5.53-5.57 (m, 2 H), 6.09-6.11 (m, 1 H), 6.79-6.81
(m, 2 H), 6.99-7.55 (m, 61 H), 7.87-8.03 (m, 11 H).

13

C-NMR (125 MHz, CD3Cl): δ 14.1,

18.4, 20.0, 20.8, 22.4, 22.5, 22.7, 23.2, 28.9, 29.3, 29.4, 29.5, 29.6, 30.8, 31.0, 31.9,
46.3, 51.5, 51.8, 52.0, 52.3, 52.6, 55.2, 59.3, 61.5, 62.5, 66.6, 67.0, 67.2, 68.1, 69.1,
70.2, 70.9, 71.8, 72.3, 72.6, 72.8, 73.3, 73.4, 74.2, 74.5, 74.6, 74.7, 75.2, 75.4, 75.7,
77.8, 78.4, 81.5, 98.2, 100.3, 100.5, 100.6, 101.1, 102.1, 113.8, 125.9, 126.0, 126.4,
126.7, 126.9, 127.0, 127.1, 127.5, 127.6, 127.8, 127.8, 127.9, 128.0, 128.1, 128.1,
128.2, 128.2, 128.3, 128.3, 128.4, 128.6, 128.7, 128.8, 128.9, 129.1, 129.5, 129.6,
129.6, 129.7, 129.8, 129.8, 129.9, 133.1, 133.3, 133.8, 135.1, 136.2, 136.9, 137.5,
137.7, 137.9, 138.1, 138.3, 159.5, 164.5, 164.6, 165.0, 165.4, 165.5, 168.2, 168.9,
+

169.4, 169.8. MALDI-MS: C167H173N3O53 [M+Na] calcd: 3093.15, obsd: 3093.15.
381

Octasaccharide 224. Compound 224 was synthesized from compound 223 in
2-

92% yield following the general procedure of O-sulfation. ESI-MS: C167H171N3O59S2
2-

[M-2H]

calcd: 1612.99, obsd: 1613.52.

Octasaccharide 225. Compound 225 was synthesized from compound 224 in
98% yield following the general procedure of global debenzylation. ESI-MS:
3-

C96H112N3O58S2

2-

[M-2H]

3-

calcd: 1149.77, obsd: 1150.06, [M-3H]

calcd: 766.18,

obsd: 766.41.
Octasaccharide 226. Compound 225 (2 mg, 0.0087 mmol) was dissolved in
o

THF/H2O (5:1, 300 μL) and cooled to 0 C. 17μL LiOH (1.25 M) solution was added to
o

the reaction mixture and the reaction was kept under 0 C for 1 h. After neutralization to
pH 5 with 1 M HOAc, the mixture was loaded onto LH-20 column. The compound was
eluted by CHCl3/MeOH/H2O (5:5:1). The fractions containing compounds were
combined and concentrated to dryness, which was redissolved in MeOH/H2O (1:1). The
pH of the solution was maintained around 9.5 by careful addition of 0.25 M NaOH
solution. After 6 h, the reaction was neutralized by 1 M HOAc and loaded to G-15
column. Fractions containing product was combined and concentrated to afford
1

compound 226 (1.5 mg, 80%). H-NMR (600 MHz, D2O): δ 1.98 (s, 9 H, NHCH3), 3.403.49 (m, 3 H), 3.61-3.68 (m, 2 H), 3.72-3.97 (m, 11 H), 4.02-4.07 (m, 1 H), 4.09-4.21 (m,
3 H), 4.22-4.42 (m, 4 H), 4.46-4.52 (m, 2 H), 4.59-4.61 (m, 1 H), 4.70-4.74 (m, 1 H),
4.77-4.81 (m, 20 H), 4.94-4.98 (m, 2 H), 5.22-5.26 (m, 2 H), 5.41-5.43 (m, 1 H). ESI-MS:

382

6-

C56H81N3O52S2
564.79, [M-4H]

4-

[M-2H]

2-

calcd: 847.66, obsd: 847.67, [M-3H]

3-

calcd: 564.77, obsd:

calcd: 423.33, obsd: 423.34.

N-(Acetyl)-O-(benzyl)-L-seryl-glycyl-O-[methyl 2-O-levulinoyl-3-O-benzyl-4O-p-methoxybenzyl-α-L-idopyranosyluronate-(1→4)-2-N-acetyl-3-O-benzyl-6-Olevulinoyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl 2-O-benzoyl-3-O-benzyl-α-Lidopyranosyluronate-(1→4)-2-N-acetyl-3,

6-di-O-benzyl-2-deoxy-α-D-

glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyl-(1→4)-2-Oacetyl-4,

6-O-benzylidene-β-D-galactopyranosyl-(1→3)-2-O-benzoyl-4,

6-O-

benzylidene-β-D-galactopyranosyl-(1→4)-2, 3-di-O-benzoyl-β-D-xylopyranosyl]-Lserine benzyl ester (227). Compoud 175 (33 mg, 0.0095 mmol) was dissolved in
THF/Ac2O/HOAc (3:2:1, 6 mL), followed by addition of Zn (480 mg), CuSO4 (saturated
solution, 30 μL). The resulting mixture was stirred under room temperature overnight.
After filtration, the mixture was diluted with DCM and washed with sat. NaHCO3. The
combined organic phase was dried over Na2SO4 and purified by silica gel column to
1

afford compound 227 (25 mg, 76%). H-NMR (600 MHz, CD3Cl): δ 1.28 (s, 3 H), 1.30 (s,
3 H), 1.34 (s, 3 H), 1.92 (s, 3 H, CH3COCH2CH2), 2.09 (s, 3 H, CH3COCH2CH2), 2.412.72 (m, 8 H, CH3COCH2CH2), 2.74 (s, 3 H), 2.82 (br, 1 H), 2.90 (br, 1 H), 3.12 (br, 1
H), 3.17-3.24 (m, 3 H), 3.26-3.93 (m, 66 H), 3.97-4.65 (m, 43 H), 4.72-4.78 (m, 2 H),
4.89-4.97 (m, 5 H), 5.02-5.07 (m, 5 H), 5.14-5.19 (m, 1 H), 5.24-5.30 (m, 5 H), 5.37-5.42
(m, 2 H), 5.49-5.52 (m, 1 H), 6.74-6.76 (m, 2 H), 6.97-7.51 (m, 66 H), 7.80-7.95 (m, 11

383

H).

13

C-NMR (125 MHz, CD3Cl): δ 19.9, 20.6, 22.2, 22.6, 27.8, 29.5, 29.6, 36.5, 37.6,

37.8, 38.4, 42.4, 51.6, 51.9, 52.4, 52.6, 53.3, 55.1, 62.0, 65.0, 66.5, 66.9, 67.2, 68.2,
68.5, 69.2, 69.6, 70.1, 70.7, 71.2, 71.5, 72.0, 72.8, 72.9, 73.1, 73.2, 73.2, 73.9, 74.5,
74.9, 75.5, 78.3, 81.6, 97.6, 97.8, 98.6, 100.0, 100.4, 102.1, 113.6, 125.9, 126.2, 126.9,
127.1, 127.5, 127.6, 127.7, 127.7, 127.7, 127.8, 127.9, 128.0, 128.0, 128.1, 128.2,
128.2, 128.3, 128.3, 128.4, 128.4, 128.8, 129.4, 129.6, 129.8, 133.1, 133.3, 134.9,
136.1, 136.9, 137.3, 137.5, 137.6, 137.7, 138.2, 159.2, 162.9, 164.6, 165.1, 165.8,
168.3, 168.7, 169.0, 169.1, 169.6, 170.4, 170.8, 171.0, 171.9, 172.3, 174.5, 206.7,
+

207.1. MALDI-MS: C189H199N5O60 [M+Na] calcd: 3523.60, obsd: 3523.87.
N-(Acetyl)-O-(benzyl)-L-seryl-glycyl-O-[methyl

3-O-benzyl-4-O-p-

methoxybenzyl-α-L-idopyranosyluronate-(1→4)-2-N-acetyl-3-O-benzyl-2-deoxy-αD-glucopyranosyl-(1→4)-methyl 2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate(1→4)-2-N-acetyl-3, 6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl3-O-benzyl-β-D-glucopyranosyl-(1→4)-2-O-acetyl-4,

6-O-benzylidene-β-D-

6-O-benzylidene-β-D-galactopyranosyl-

galactopyranosyl-(1→3)-2-O-benzoyl-4,

(1→4)-2, 3-di-O-benzoyl-β-D-xylopyranosyl]-L-serine benzyl ester (228). Compound
228 was synthesized from compound 227 in 84% yield following the general procedure
1

of Lev deprotection. H-NMR (500 MHz, CD3Cl): δ 1.22 (s, 3 H), 1.28 (s, 3 H), 1.37 (s, 3
H), 2.00 (s, 3 H), 2.39-2.48 (m, 2 H), 3.13-3.43 (m, 21 H), 3.50-3.61 (m, 10 H), 3.653.88 (m, 30 H), 3.97-4.12 (m, 12 H), 4.19-4.70 (m, 38 H), 4.76-4.86 (m, 3 H), 4.88-4.90
(m, 1 H), 4.92-5.01 (m, 5 H), 5.03-5.21 (m, 8 H), 5.27-5.33 (m, 5 H), 5.40-5.54 (m, 3 H),
6.58-6.60 (m, 1 H), 6.76-6.79 (m, 3 H), 6.99-7.54 (m, 61 H), 7.84-7.97 (m, 14 H).
384

13

C-

NMR (125 MHz, CD3Cl): δ 17.8, 19.9, 22.3, 22.7, 22.8, 24.8, 25.8, 42.5, 51.7, 51.8,
52.0, 52.4, 52.4, 52.6, 55.2, 61.0, 62.0, 66.5, 67.0, 67.2, 68.1, 68.2, 68.5, 69.1, 70.1,
70.7, 71.2, 71.4, 72.2, 72.5, 72.6, 72.8, 72.9, 73.1, 73.2, 73.5, 74.0, 74.4, 74.6, 74.8,
75.2, 75.4, 75.5, 75.7, 78.0, 78.6, 81.5, 96.1, 97.8, 98.8, 100.0, 100.4, 100.9, 102.1,
113.7, 125.9, 126.3, 126.6, 126.9, 127.0, 127.1, 127.5, 127.6, 127.7, 127.8, 127.8,
127.9, 128.0, 128.0, 128.1, 128.1, 128.2, 128.3, 128.3, 128.4, 128.4, 128.5, 128.7,
128.8, 128.9, 129.0, 129.3, 129.4, 129.5, 129.6, 129.7, 129.8, 133.2, 133.4, 133.7,
134.9, 136.1, 136.8, 137.3, 137.3, 137.4, 137.6, 137.8, 138.1, 138.2, 159.4, 164.5,
164.6, 165.1, 165.8, 168.2, 168.5, 169.0, 169.1, 169.6, 170.0, 170.2, 170.5, 170.7.
+

MALDI-MS: C179H187N5O56 [M+Na] calcd: 3327.40, obsd: 3327.95.
Glycopeptide 229. Compound 229 was synthesized from compound 228 in 90%
2-

yield following the general procedure of O-sulfation. ESI-MS: C179H185N5O62S2
2H]

2-

[M-

calcd: 1730.05, obsd: 1730.84.
Glycopeptide 230. Compound 230 was synthesized from compound 229 in 95%

yield

following

C101H120N5O61S2

the
3-

general
2-

[M-2H]

procedure

of

global

debenzylation.
3-

calcd: 1221.79, obsd: 1222.28, [M-3H]

ESI-MS:

calcd: 814.19,

obsd: 814.58.
Glycopeptide 24. Compound 230 (1 mg) was dissolved in THF/H2O (5:1, 300
o

μL) and cooled to 0 C. 17μL LiOH (1.25 M) solution was added to the reaction mixture
o

and the reaction was kept under 0 C for 1 h. After neutralization to pH 5 with 1 M
HOAc, the mixture was loaded onto LH-20 column. The compound was eluted by
385

CHCl3/MeOH/H2O (5:5:1). The fractions containing compounds were combined and
concentrated to dryness, which was redissolved in MeOH/H2O (1:1). The pH of the
solution was maintained around 9.5 by careful addition of 0.25 M NaOH solution. After 6
h, the reaction was neutralized by 1 M HOAc and loaded to G-15 column. Fractions
containing product was combined and concentrated to afford compound 24 (0.6 mg,
1

72%). H-NMR (600 MHz, D2O): δ 2.05 (s, 3 H, NHCH3), 2.07 (s, 3 H, NHCH3), 2.12 (s,
3 H, NHCH3), 3.35-3.46 (m, 2 H), 3.57-3.96 (m, 30 H), 4.01-4.13 (m, 10 H), 4.20-4.39
(m, 6 H), 4.43-4.73 (m, 3 H), 5.18-5.24 (m, 4 H), 5.36-5.41 (m, 2 H). ESI-MS:
6-

C61H89N5O55S2

2-

[M-6H+4Na]

calcd: 963.69, obsd: 963.60,
4-

634.66, obsd: 564.79, [M-6H+2Na]

[M-6H+3Na]

3-

calcd:

calcd: 470.35, obsd: 470.60.

N-Fluorenylmethyloxycarbonyl-O-[2, 3-di-O-levulinoyl-4, 6-O-benzylidene -

β-D-galactopyranosyl-(1→3)-2-O-benzoyl-4,
galactopyranosyl-(1→4)-2,

6-O-benzylidene-β-D-

3-di-O-benzoyl-β-D-xylopyranosyl]-L-serine

(239).

Compound 179 (110 mg, 0.071 mmol) was dissolved in DCM/MeOH (1:1, 4 mL),
followed by addition of Pd/C (6 mg) and NH4OAc (19 mg, 0.247 mmol). The resulting
mixture was stirred under H2 atomosphere. The reaction was carefully monitored by
TLC. After the complete disappearance of starting material, the reaction was diluted
with DCM and filtered. After concentration, the residue was purified by silica gel column
1

to afford compound 239 (82 mg, 78%). H-NMR (600 MHz, CD3Cl): δ 1.82 (s, 3 H,
CH3COCH2CH2), 1.86 (s, 3 H, CH3COCH2CH2), 1.92-2.56 (m, 8 H, CH3COCH2CH2),
386

3.04 (s, 1 H), 3.13-3.18 (m, 1 H), 3.30 (s, 1 H), 3.51-3.59 (m, 3 H), 3.69-3.72 (m, 1 H),
3.84-3.91 (m, 4 H), 3.98-4.07 (m, 5 H), 4.17 (d, 1 H, J = 2.5 Hz), 4.33 (d, 1 H, J = 6 Hz),
4.51-4.59 (m, 3 H), 4.93-4.96 (m, 1 H), 5.06-5.09 (m, 1 H), 5.25 (s, 1 H), 5.28-5.31 (m, 2
H), 5.41 (t, 1 H, J = 7.5 Hz), 5.61-5.63 (m, 1 H), 7.01-7.23 (m, 17 H), 7.27-7.34 (m, 7 H),
7.43-7.45 (m, 1 H), 7.59-7.62 (m, 2 H), 7.69-7.71 (m, 2 H), 7.76-7.78 (m, 2 H), 7.84-7.86
(m , 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 0.6, 27.0, 27.6, 29.0, 29.1, 37.3, 37.3, 46.8,

63.0, 66.1, 66.3, 66.7, 67.8, 68.1, 68.4, 69.7, 70.8, 71.3, 72.6, 72.9, 75.3, 75.9, 76.0,
100.4, 100.6, 100.7, 101.1, 101.6, 119.7, 124.6, 124.8, 125.9, 126.0, 126.7, 126.7,
127.4, 127.6, 127.8, 128.0, 128.3, 128.5, 128.7, 128.8, 129.1, 129.2, 129.3, 129.4,
132.8, 133.0, 133.3, 137.0, 137.4, 140.9, 143.4, 143.5, 164.7, 165.2, 165.6, 171.3,
-

172.0, 207.2, 207.5. ESI-MS: C80H77NO26 [M-H] calcd: 1466.47, obsd: 1467.10.
N-(Acetyl)-O-(benzyl)-L-seryl-glycyl-O-[methyl 2-O-levulinoyl-3-O-benzyl-4O-p-methoxybenzyl-α-L-idopyranosyluronate-(1→4)-2-azido-3-O-benzyl-6-Olevulinoyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl 2-O-benzoyl-3-O-benzyl-α-Lidopyranosyluronate-(1→4)-2-azido-3, 6-di-O-benzyl-2-deoxy-α-D-glucopyranosyl(1→4)-2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyl-(1→4)-2-O-acetyl-4,
benzylidene-β-D-galactopyranosyl-(1→3)-2-O-benzoyl-4,
galactopyranosyl-(1→4)-2,

6-O-

6-O-benzylidene-β-D-

3-di-O-benzoyl-β-D-xylopyranosyl]-L-serine

(243).

Compound 175 (32 mg, 0.009 mmol) was dissolved in DCM/MeOH (1:1, 2 mL), followed
by addition of Pd/C (3 mg) and NH4OAc (3 mg, 0.032 mmol). The resulting mixture was
stirred under H2 atomosphere. The reaction was carefully monitored by TLC. After the
complete disappearance of starting material, the reaction was diluted with DCM and
387

filtered. After concentration, the residue was purified by silica gel column to afford
1

compound 243 (25 mg, 82%). H-NMR (500 MHz, CD3Cl): δ 1.14 (s, 3 H), 1.18 (s, 3 H),
1.97 (s, 3 H, CH3COCH2CH2), 2.03 (s, 3 H, CH3COCH2CH2), 2.38-2.63 (m, 8 H,
CH3COCH2CH2), 3.12-3.18 (m, 5 H), 3.20 (s, 3 H), 3.24-3.27 (m, 6 H), 3.29 (s, 3 H),
3.35-3.42 (m, 9 H), 3.54-3.58 (m, 4 H), 3.63-3.72 (m, 8 H), 3.75-4.19 (m, 20 H), 4.244.69 (m, 22 H), 4.76-4.78 (m, 3 H), 4.91-4.96 (m, 2 H), 5.01-5.10 (m, 3 H), 5.20-5.28 (m,
4 H), 5.39-5.44 (m, 2 H), 6.71-6.73 (m, 2 H), 6.96-7.23 (m, 48 H), 7.25-7.49 (m, 14 H),
7.75-7.88 (m, 10 H).

13

C-NMR (125 MHz, CD3Cl): δ 27.6, 29.2, 29.4, 37.7, 51.6, 55.0,

55.1, 70.8, 72.1, 73.1, 73.6, 74.5, 75.3, 76.5, 97.6, 100.4, 113.5, 113.6, 125.7, 125.9,
126.1, 127.6, 127.7, 128.2, 128.9, 129.0, 129.2, 129.4, 129.5, 129.5, 133.3, 136.8,
137.1, 137.2, 137.4, 137.5, 137.6, 137.7, 159.2, 164.6, 164.9, 165.2, 165.3, 166.0,
168.7, 169.3, 169.8, 171.8, 172.3, 206.6, 207.2. MALDI-MS: C178H185N9O58 [M+Na]

+

calcd: 3401.40, obsd: 3401.65.
Peptide 244: Compound 244 was synthesized following the general procedure
+

for solid phase peptide synthesis. MALDI-MS: C63H78N10O16 [M+Na] calcd: 1254.35,
obsd: 1254.80.
Peptide 245: Compound 244 (20 mg, 0.0163 mmol) was dissolved in 1 mL dry
DMF, followed by addition of MeI (2.5 µL, 0.04 mmol) and K2CO3 (4.5 mg, 0.032 mmol).
The resulting mixture was stirred under room temperature overnight. The product was
precipitated from tert-butyl methyl ether and purified by silica gel column to afford

388

+

compound 245 (3 mg, 15%). MALDI-MS: C64H80N10O16 [M+Na] calcd: 1268.38, obsd:
1268.70.
Peptide 247: Compound 247 was a side product generated during the synthesis
+

of compound 245. MALDI-MS: C58H76N10O16 [M+Na] calcd: 1191.54, obsd: 1191.80.
Peptide 249: Compound 244 (6 mg, 0.00488 mmol) was dissolved in 1 mL dry
DMF, followed by addition of BnBr (1.2 µL, 0.00975 mmol) and DIPEA (1.7 µL, 0.00975
mmol). The resulting mixture was stirred under room temperature overnight. The
product was precipitated from tert-butyl methyl ether and purified by silica gel column to
+

afford compound 249 (4 mg, 60%). MALDI-MS: C70H84N10O16 [M+Na] calcd: 1344.47,
obsd: 1344.10.
Peptide 250: Compound 249 (4 mg, 0.003 mmol) was dissolved in 0.5 mL DMF,
followed by addition of 13 µL piperidine. The resulting mixture was stirred under room
temperature for 2 h and the product was precipitated from tert-butyl methyl ether and
used directly for next step without further purification. MALDI-MS: C55H74N10O14
+

[M+H] calcd: 1099.54, obsd: 1099.90.
Glycopeptide 251: Peptide 250 (13 mg, 0.011 mmol) and glycopeptide 243 (13
mg, 0.00385 mmol) were dissolved in 0.6 mL dry DMF, to which were added 2.2 mg
HATU. The resulting mixture was stirred under room temperature overnight and diluted
with DCM. The solution was washed with sat. NaHCO3 solution. The combined organic
phase was dried over Na2SO4 and concentrated, purified by silica gel column to afford
1

compound 251 (14 mg, 75%). H-NMR (500 MHz, CD3Cl): δ 0.75-0.94 (m, 15 H), 1.91
389

(br, 37 H), 2.04 (s, 3 H), 2.11 (s, 3 H), 2.46-2.74 (m, 8 H, CH3COCH2CH2), 3.19-3.34
(m, 8 H), 3.44-3.62 (m, 15 H), 3.68-3.98 (m, 23 H), 4.09-4.23 (m, 10 H), 4.35-4.78 (m,
22 H), 4.85-4.89 (m, 3 H), 5.02-5.21 (m, 6 H), 5.33-5.39 (m, 2 H), 5.54-5.57 (m, 1 H),
6.79-6.81 (m, 3 H), 7.01-7.48 (m, 80 H), 7.79-8.01 (m, 6 H). MALDI-MS:
+

C233H257N19O71 [M+Na] calcd: 4479.71, obsd: 4479.45.
O-[2, 3-Di-O-levulinoyl-4, 6-O-benzylidene -β-D-galactopyranosyl-(1→3)-2O-benzoyl-4, 6-O-benzylidene-α-D-galactopyranosyl-(1→4)-2, 3-di-O-benzoyl-β-Dxylopyranosyl]-L-serine benzyl ester (252). Compound 180 (181 mg, 0.116 mmol)
was dissolved in 4 mL DCM, followed by addition of 475 µL piperidine. The resulting
mixture was stirred under room temperature for 2 h and diluted with DCM. The solution
was washed with H2O. The combined organic phase was dried over Na2SO4 and
1

concentrated, purified by silica gel column to afford compound 252 (121 mg, 75%). HNMR (500 MHz, CD3Cl): δ 1.94 (s, 3 H, CH3COCH2CH2), 1.95 (s, 3 H,
CH3COCH2CH2), 2.08-2.69 (m, 8 H, CH3COCH2CH2), 3.44 (s, 1 H), 3.46-3.59 (m, 2 H),
3.73-3.79 (m, 2 H), 3.96-4.16 (m, 5 H), 4.22-4.33 (m, 4 H), 4.50 (d, 1 H, J = 3.0 Hz),
4.65-4.72 (m, 3 H), 5.04 (s, 2 H), 5.13-5.17 (m, 1 H), 5.23-5.27 (m, 1 H), 5.38-5.40 (m, 1
H), 5.45-5.48 (m, 2 H), 5.55-5.59 (m, 2 H), 7.13-7.20 (m, 5 H), 7.23-7.52 (m, 21 H),
7.62-7.65 (m, 2 H), 7.83-7.86 (m , 2 H).

13

C-NMR (125 MHz, CD3Cl): δ 27.1, 28.0, 29.5,

29.5, 37.5, 37.6, 54.6, 63.8, 64.0, 66.3, 66.8, 68.2, 68.8, 70.0, 71.1, 71.6, 72.1, 73.0,
73.2, 75.2, 76.1, 98.6, 100.4, 100.8, 101.0, 101.3, 126.1, 126.2, 128.0, 128.1, 128.1,
128.2, 128.3, 128.3, 128.5, 128.6, 128.7, 128.9, 129.1, 129.4, 129.4, 129.6, 133.0,
390

133.1, 133.1, 135.4, 137.4, 137.5, 164.9, 165.2, 165.3, 170.9, 172.0, 172.8, 206.3,
+

206.6. ESI-MS: C72H73NO24 calcd: [M+H] calcd: 1336.45, obsd: 1336.64.
N-(Acetyl)-O-(benzyl)-L-seryl-glycyl-O-[2,

3-di-O-levulinoyl-4,

benzylidene-β-D-galactopyranosyl-(1→3)-2-O-benzoyl-4,
galactopyranosyl-(1→4)-2,

6-O-

6-O-benzylidene-α-D-

3-di-O-benzoyl-β-D-xylopyranosyl]-L-serine

benzyl

ester (253). Peptide 120 (13 mg, 0.0415 mmol) and compound 252 (37 mg, 0.0277
mmol) were dissolved in 1 mL dry DMF, to which were added 15 mg HATU and 40 µL
DIPEA. The resulting mixture was stirred under room temperature overnight and diluted
with DCM. The solution was washed with sat. NaHCO3 solution. The combined organic
phase was dried over Na2SO4 and concentrated, purified by silica gel column to afford
compound 253 (35 mg, 78%).

1

H-NMR (500 MHz, CD3Cl): δ 1.94 (s, 3 H,

CH3COCH2CH2), 1.95 (s, 3 H, CH3COCH2CH2), 1.96 (s, 3 H, CH3CONH), 1.98-2.69
(m, 8 H, CH3COCH2CH2), 2.85 (br, 1 H), 2.92 (s, 1 H), 3.44-3.52 (m, 3 H), 3.60-3.65 (m,
1 H), 3.72-3.85 (m, 4 H), 3.88-3.95 (m, 1 H), 4.02-4.11 (m, 3 H), 4.20-4.33 (m, 5 H),
4.48-4.56 (m, 4 H), 4.59-4.62 (m, 1 H), 4.66-4.69 (m, 2 H), 4.71 (m, 1 H, J = 8 Hz), 5.055.14 (m, 3 H), 5.23-5.28 (m, 1 H), 5.36-5.38 (m, 1 H), 5.43-5.47 (m, 2 H), 5.54-5.60 (m,
2 H), 6.38-6.39 (m, 1 H), 6.66-6.68 (m, 1 H), 6.86-6.88 (m, 1 H), 7.13-7.23 (m, 4 H),
7.24-7.53 (m, 27 H), 7.64-7.66 (m, 2 H), 7.84-7.86 (m , 2 H).

13

C-NMR (125 MHz,

CD3Cl): δ 23.0, 27.1, 28.0, 29.5, 29.5, 37.5, 37.6, 42.6, 52.4, 52.7, 63.8, 63.9, 66.3,
67.4, 68.3, 68.8, 69.0, 70.0, 71.6, 71.9, 72.1, 72.6, 73.2, 73.3, 75.2, 76.2, 98.6, 100.4,
100.8, 100.9, 101.3, 126.1, 126.2, 127.7, 127.8, 127.9, 128.0, 128.1, 128.1, 128.1,
391

128.2, 128.4, 128.5, 128.5, 128.6, 128.6, 128.7, 128.9, 129.0, 129.4, 129.4, 129.6,
133.1, 133.6, 135.0, 137.3, 137.4, 137.5, 165.2, 165.3, 165.3, 168.2, 168.9, 170.1,
170.4, 170.9, 172.0, 206.3, 206.6.
N-(Acetyl)-O-(benzyl)-L-seryl-glycyl-O-[2,

3-di-O-levulinoyl-4,

benzylidene-β-D-galactopyranosyl-(1→3)-2-O-benzoyl-4,
galactopyranosyl-(1→4)-2,

6-O-

6-O-benzylidene-α-D-

3-di-O-benzoyl-β-D-xylopyranosyl]-L-serine

(254).

Compound 253 (35 mg, 0.022 mmol) was dissolved in DCM/MeOH (1:1, 2 mL), followed
by addition of Pd/C (2 mg) and NH4OAc (6 mg, 0.077 mmol). The resulting mixture was
stirred under H2 atomosphere. The reaction was carefully monitored by TLC. After the
complete disappearance of starting material, the reaction was diluted with DCM and
filtered. After concentration, the residue was purified by silica gel column to afford
compound 254 (30 mg, 90%).

1

H-NMR (500 MHz, CD3Cl): δ 1.78 (s, 3 H,

CH3COCH2CH2), 1.81 (s, 3 H, CH3COCH2CH2), 1.89 (s, 3 H, CH3CONH), 1.91-2.56
(m, 8 H, CH3COCH2CH2), 3.04-3.09 (m, 1 H), 3.22 (br, 1 H), 3.39-3.46 (m, 3 H), 3.493.52 (m, 1 H), 3.60-3.62 (m, 1 H), 3.67 (br, 1 H), 3.73-3.76 (m, 1 H), 3.83-3.87 (m, 1 H),
4.04-4.19 (m, 7 H), 4.26-4.41 (m, 5 H), 4.47-4.62 (m, 4 H), 4.85-4.89 (m, 1 H), 5.05-5.09
(m, 1 H), 5.23-5.28 (m, 2 H), 5.34 (s, 1 H), 5.41-5.46 (m, 2 H), 6.94-7.03 (m, 5 H), 7.117.23 (m, 13 H), 7.25-7.37 (m, 8 H), 7.44-7.46 (m , 2 H), 7.61-7.64 (m, 2 H).

13

C-NMR

(125 MHz, CD3Cl): δ 22.0, 22.3, 25.0, 26.7, 27.6, 29.0, 29.1, 29.3, 31.6, 37.1, 37.3, 42.1,
63.5, 64.2, 66.1, 68.1, 68.5, 69.7, 71.3, 72.3, 72.4, 73.0, 73.3, 75.1, 76.0, 98.4, 100.3,
100.6, 101.3, 125.8, 125.9, 127.5, 127.7, 127.8, 127.9, 128.0, 128.2, 128.3, 128.5,
392

128.8, 129.0, 129.2, 132.8, 133.0, 133.4, 136.9, 137.1, 137.3, 165.1, 165.2, 165.5,
-

171.1, 172.0, 206.8, 207.4. ESI-MS: C79H83N3O28 calcd: [M+Na] calcd: 1544.52, obsd:
1544.60.
Glycopeptide 255: Peptide 250 (2 mg, 0.002 mmol) and compound 254 (6 mg,
0.004 mmol) were dissolved in 0.5 mL dry DMF, to which were added 1.5 mg HATU and
3 µL DIPEA. The resulting mixture was stirred under room temperature overnight and
diluted with DCM. The solution was washed with sat. NaHCO3 solution. The combined
organic phase was dried over Na2SO4 and concentrated, purified by silica gel column to
afford compound 255 (2.6 mg, 52%). MALDI-MS: C134H155N13O41 [M+Na]

+

calcd:

2626.73, obsd: 2626.70.
Glycopeptide 256: Compound 256 was synthesized from compound 251
following the general procedure for Lev deprotection. MALDI-MS: C223H245N19O67
+

[M+Na] calcd: 4286.42, obsd: 4286.92.
Glycopeptide 257: Compound 257 was synthesized from compound 256
2-

following the general procedure for O-sulfation. ESI-MS: C223H243N19O73S2

[M-2H]

2-

calcd: 2210.76, obsd: 2211.58.
Glycopeptide 260: Compound 251 (7 mg, 0.0016 mmol) was dissolved in
THF/Ac2O/HOAc (3:2:1, 1.5 mL), followed by addition of Zn (100 mg), CuSO4
(saturated solution, 10 μL). The resulting mixture was stirred under room temperature
overnight. After filtration, the mixture was diluted with DCM and washed with sat.
NaHCO3. The combined organic phase was dried over Na2SO4 and purified by silica
393

gel column to afford compound 260 (5 mg, 62%). MALDI-MS: C237H265N15O73
+

[M+Na] calcd: 4514.70, obsd: 4514.91.
Glycopeptide 261: Compound 261 was synthesized from compound 260
following the general procedure for Lev deprotection. MALDI-MS: C227H253N15O69
+

[M+Na] calcd: 4318.50, obsd: 4318.75.
Glycopeptide 262: Compound 262 was synthesized from compound 261
2-

following the general procedure for O-sulfation. ESI-MS: C227H251N15O75S2
3H]

2-

[M+Li-

calcd: 2228.28, obsd: 2228.35.
Glycopeptide 263: Compound 263 was synthesized from compound 262

following

the

general
2-

C135H173N15O74S2

procedure
3-

[M-3H]

for

global

debenzylation.

calcd: 1084.32, obsd: 1084.93, [M-4H]

4-

ESI-MS:

calcd: 812.99,

obsd: 813.22.
Octasaccharide 265: Compound 205 (1 mg) was dissolved in freshly dried
MeOH (0.5 mL). 0.5 M NaOMe in MeOH was freshly prepared and added dropwisely to
the reaction to maintian pH around 9.5. The reaction was left under N2 atomosphere for
50 h and quenched by 1 M HOAc. The mixture was loaded onto LH-20 column.
Glycopeptide containing fractions were combined and concentrated to afford compound
3-

265. ESI-MS: C59H90N3O52S2

[M-2H]

2-

calcd: 868.69, obsd: 868.68.

Glycopeptide 266: Compound 230 (2 mg) was dissolved in freshly dried MeOH
(0.5 mL). 0.5 M NaOMe in MeOH was freshly prepared and added dropwisely to the

394

reaction to maintian pH around 9.5. The reaction was left under N2 atomosphere for 30
h and quenched by 1 M HOAc. The mixture was loaded onto LH-20 column.
Glycopeptide containing fractions were combined and concentrated to afford compound
3-

266. ESI-MS: C64H98N5O55S2

2-

[M+Na]

calcd: 951.72, obsd: 952.22, [M-3H]

3-

calcd:

626.81, obsd: 626.85.
Glycopeptide 264: Peptide 250 (4 mg, 0.004 mmol) and compound 266 (4 mg)
were dissolved in 0.5 mL dry DMF, to which were added 2 mg HATU and 2 µL 2, 4, 6collidine. The resulting mixture was stirred under room temperature overnight. The
solution was loaded onto LH-20 column. Glycopeptide containing fractions were
2-

combined and concentrated to afford compound 264. ESI-MS: C119H171N15O68S2
2-

[M-2H]

calcd: 1480.99, obsd: 1481.28.

Glycopeptide 275: Compound 275 was synthesized from compound 264
following the general procedure for global debenzylation. ESI-MS: C98H151N15O68S2
3-

[M-3H]

4-

calcd: 896.94, obsd: 897.30. [M-4H]

4-

calcd: 672.45, obsd: 672.99.

Glycopeptide 233: Compound 275 was dissolved in MeOH/H2O (1:1, 0.5 mL),
to which 0.5 M NaOH solution was added to maintain pH around 9.5. The resulting
mixture was left under room temperature for 3 h. The solution was loaded onto LH-20
column. Glycopeptide containing fractions were combined and concentrated to afford
1

compound 233. H-NMR (600 MHz, D2O): δ 0.88-0.94 (m, 10 H), 1.24-1.43 (m, 20 H),
1.51-1.79 (m, 13 H), 2.03 (s, 3 H), 2.06 (s, 3 H), 2.11 (s, 3 H), 2.12-2.38 (m, 14 H), 3.32-

395

7-

4.69 (m, 63 H), 5.12-5.42 (m, 11 H). ESI-MS: C95H142N15O68S2
calcd: 666.19, obsd: 665.45. [M-4H+NH4]

4-

calcd: 888.58, obsd: 888.19.

396

[M-6H+2NH4]

4-

Appendix B

397

NMR Data

1

Figure 3.22. H-NMR (CDCl3, 500 MHz) of 35

398

1

Figure 3.23. H-NMR (CDCl3, 500 MHz) of 36

399

1

Figure 3.24. H-NMR (CDCl3, 500 MHz) of 37

400

1

Figure 3.25. H-NMR (CDCl3, 500 MHz) of 38

401

Figure 3.26. H-NMR (CDCl3, 500 MHz) of 39

402

1

Figure 3.27. H-NMR (CD3Cl, 500 MHz) of 40

403

1

Figure 3.28. H-NMR (CDCl3, 500 MHz) of 41

404

1

Figure 3.29. H-NMR (CDCl3, 500 MHz) of 43

405

1

Figure 3.30. H-NMR (CDCl3, 500 MHz) of 44

406

1

Figure 3.31. H-NMR (CDCl3, 500 MHz) of 45

407

1

Figure 3.32. H-NMR (CDCl3, 500 MHz) of 46

408

1

Figure 3.33. H-NMR (CDCl3, 500 MHz) of 47

409

1

Figure 3.34. H-NMR (CDCl3, 500 MHz) of 48

410

1

Figure 3.35. H-NMR (CDCl3, 500 MHz) of 50

411

1

Figure 3.36. H-NMR (CDCl3, 500 MHz) of 51

412

1

Figure 3.37. H-NMR (CDCl3, 500 MHz) of 52

413

Figure 3.38.

13

C-NMR (CDCl3, 125 MHz) of 52

414

Figure 3.39. gCOSY (CDCl3, 500 MHz) of 52

415

Figure 3.40. gHMQC (CDCl3, 500 MHz) of 52

416

Figure 3.41. gHMBC (CDCl3, 500 MHz) of 52

417

Ph
O
O
O

O

STol

53 OH

1

Figure 3.42. H-NMR (CDCl3, 500 MHz) of 53

418

Ph
O
O
O

O

STol

53 OH

Figure 3.43.

13

C-NMR (CDCl3, 125 MHz) of 53

419

Ph
O
O
O

O

STol
54 OBz

1

Figure 3.44. H-NMR (CDCl3, 500 MHz) of 54

420

Ph
O
O
O

O

STol
OBz
54

Figure 3.45.

13

C-NMR (CDCl3, 125 MHz) of 54

421

Ph
O
O
O

O

STol
54 OBz

Figure 3.46. gCOSY (CDCl3, 500 MHz) of 54

422

1

Figure 3.47. H-NMR (CDCl3, 500 MHz) of 55

423

Figure 3.48.

13

C-NMR (CDCl3, 125 MHz) of 55

424

Figure 3.49. gCOSY (CDCl3, 500 MHz) of 55

425

1

Figure 3.50. H-NMR (CDCl3, 500 MHz) of 56

426

Figure 3.51.

13

C-NMR (CDCl3, 125 MHz) of 56

427

Figure 3.52. gCOSY (CDCl3, 500 MHz) of 56

428

1

Figure 3.53. H-NMR (CDCl3, 500 MHz) of 57

429

13

C-NMR (CDCl3, 125 MHz)

Figure 3.54.

13

C-NMR (CDCl3, 125 MHz) of 57

430

Figure 3.55. gCOSY (CDCl3, 500 MHz) of 57

431

Ph
O
O
HO

O

STol
OBz
58

1

Figure 3.56. H-NMR (CDCl3, 500 MHz) of 58

432

Ph
O
O
LevO

O

STol

59 OH

1

Figure 3.57. H-NMR (CDCl3, 500 MHz) of 59

433

Ph
O
O
LevO

O

STol
OBz
60

1

Figure 3.58. H-NMR (CDCl3, 500 MHz) of 60

434

Ph
O
O
HO

O

STol
OBz
61

1

Figure 3.59. H-NMR (CDCl3, 500 MHz) of 61

435

Ph
O
O
HO

O

STol
OBz
61

Figure 3.60.

13

C-NMR (CDCl3, 125 MHz) of 61

436

Ph
O
O
HO

O

STol
OBz
61

Figure 3.61. gCOSY (CDCl3, 500 MHz) of 61

437

1

Figure 3.62. H-NMR (CDCl3, 500 MHz) of 64

438

Figure 3.63.

13

C-NMR (CDCl3, 125 MHz) of 64

439

Figure 3.64. gCOSY (CDCl3, 500 MHz) of 64

440

1

Figure 3.65. H-NMR (CD3OD, 500 MHz) of 65

441

Figure 3.66.

13

C-NMR (CD3OD, 125 MHz) of 65

442

1

Figure 3.67. H-NMR (CDCl3, 500 MHz) of 66

443

Figure 3.68.

13

C-NMR (CDCl3, 125 MHz) of 66

444

Figure 3.69. gCOSY (CDCl3, 500 MHz) of 66

445

gHMQC (CDCl3, 500 MHz)

Figure 3.70. gHMQC (CDCl3, 500 MHz) of 66

446

Figure 3.71. gHMBC (CDCl3, 500 MHz) of 66

447

1

Figure 3.72. H-NMR (CDCl3, 500 MHz) of 67

448

1

Figure 3.73. H-NMR (CDCl3, 500 MHz) of 68

449

Figure 3.74.

13

C-NMR (CDCl3, 125 MHz) of 68

450

Figure 3.75. gCOSY (CDCl3, 500 MHz) of 68

451

Figure 3.76. gHMQC (CDCl3, 500 MHz) of 68

452

1

Figure 3.77. H-NMR (CDCl3, 500 MHz) of 69

453

Figure 3.78.

13

C-NMR (CDCl3, 125 MHz) of 69

454

Figure 3.79. gCOSY (CDCl3, 500 MHz) of 69

455

1

Figure 3.80. H-NMR (CDCl3, 500 MHz) of 70

456

Figure 3.81.

13

C-NMR (CDCl3, 125 MHz) of 70

457

Figure 3.82. gCOSY (CDCl3, 500 MHz) of 70

458

1

Figure 3.83. H-NMR (CDCl3, 500 MHz) of 71

459

Figure 3.84.

13

C-NMR (CDCl3, 125 MHz) of 71

460

Figure 3.85. gCOSY (CDCl3, 500 MHz) of 71

461

1

Figure 3.86. H-NMR (CDCl3, 500 MHz) of 72

462

Figure 3.87.

13

C-NMR (CDCl3, 125 MHz) of 72

463

Figure 3.88. gCOSY (CDCl3, 500 MHz) of 72

464

1

Figure 3.89. H-NMR (CDCl3, 500 MHz) of 73

465

Figure 3.90.

13

C-NMR (CDCl3, 125 MHz) of 73

466

Figure 3.91. gCOSY (CDCl3, 500 MHz) of 73

467

1

Figure 3.92. H-NMR (CDCl3, 500 MHz) of 74

468

Figure 3.93.

13

C-NMR (CDCl3, 125 MHz) of 74

469

Figure 3.94. gCOSY (CDCl3, 500 MHz) of 74

470

1

Figure 3.95. H-NMR (CDCl3, 500 MHz) of 75

471

1

Figure 3.96. H-NMR (CDCl3, 500 MHz) of 76

472

1

Figure 3.97. H-NMR (CDCl3, 500 MHz) of 77

473

1

Figure 3.98. H-NMR (CDCl3, 500 MHz) of 78

474

Figure 3.99.

13

C-NMR (CDCl3, 125 MHz) of 78

475

Figure 3.100. gCOSY (CDCl3, 500 MHz) of 78

476

Figure 3.101. gHMQC (CDCl3, 500 MHz) of 78

477

Figure 3.102. gHMBC (CDCl3, 500 MHz) of 78

478

1

Figure 3.103. H-NMR (CDCl3, 500 MHz) of 81

479

Figure 3.104.

13

C-NMR (CDCl3, 125 MHz) of 81

480

Figure 3.105. gCOSY (CDCl3, 500 MHz) of 81

481

1

Figure 3.106. H-NMR (CDCl3, 600 MHz) of 82

482

Figure 3.107.

13

C-NMR (CDCl3, 150 MHz) of 82

483

Figure 3.108. gCOSY (CDCl3, 600 MHz) of 82

484

1

Figure 3.109. H-NMR (CDCl3, 500 MHz) of 83

485

1

Figure 3.110. H-NMR (CDCl3, 500 MHz) of 84

486

1

Figure 3.111. H-NMR (CDCl3, 500 MHz) of 87

487

Figure 3.112.

13

C-NMR (CDCl3, 125 MHz) of 87

488

Figure 3.113. gCOSY (CDCl3, 500 MHz) of 87

489

Figure 3.114. gHMQC (CDCl3, 500 MHz) of 87

490

1

Figure 3.115. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 87

491

1

Figure 3.116. H-NMR (CDCl3, 500 MHz) of 89

492

Figure 3.117.

13

C-NMR (CDCl3, 125 MHz) of 89

493

Figure 3.118. gCOSY (CDCl3, 500 MHz) of 89

494

Figure 3.119. gHMQC (CDCl3, 500 MHz) of 89

495

1

Figure 3.120. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 89

496

1

Figure 3.121. H-NMR (CDCl3, 500 MHz) of 91

497

1

Figure 3.122. H-NMR (CDCl3, 500 MHz) of 92

498

Figure 3.123.

13

C-NMR (CDCl3, 150 MHz) of 92

499

Figure 3.124. gCOSY (CDCl3, 500 MHz) of 92

500

Figure 3.125. gHMQC (CDCl3, 600 MHz) of 92

501

1

Figure 3.126. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 92

502

Figure 3.127. gHMBC (CDCl3, 600 MHz) of 92

503

1

Figure 3.128. H-NMR (CDCl3, 500 MHz) of 93

504

Figure 3.129.

13

C-NMR (CDCl3, 125 MHz) of 93

505

Figure 3.130. gCOSY (CDCl3, 500 MHz) of 93

506

1

Figure 3.131. H-NMR (CDCl3, 500 MHz) of 94

507

1

Figure 3.132. H-NMR (CDCl3, 600 MHz) of 96

508

Figure 3.133.

13

C-NMR (CDCl3, 150 MHz) of 96

509

Figure 3.134. gCOSY (CDCl3, 600 MHz) of 96

510

Figure 3.135. gHMQC (CDCl3, 600 MHz) of 96

511

1

Figure 3.136. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 96

512

1

Figure 3.137. H-NMR (CDCl3, 500 MHz) of 97

513

Figure 3.138.

13

C-NMR (CDCl3, 125 MHz) of 97

514

Figure 3.139. gCOSY (CDCl3, 500 MHz) of 97

515

Ph
O
O
LevO

O
BzO
O
98 BzO

O

STol
OBz

1

Figure 3.140. H-NMR (CDCl3, 500 MHz) of 98

516

Ph
O
O
LevO

O
BzO
O
98 BzO

Figure 3.141.

O

STol
OBz

13

C-NMR (CDCl3, 125 MHz) of 98

517

1

Figure 3.142. H-NMR (CDCl3, 500 MHz) of 100

518

1

Figure 3.143. H-NMR (CDCl3, 600 MHz) of 101

519

Figure 3.144.

13

C-NMR (CDCl3, 150 MHz) of 101

520

Figure 3.145. gCOSY (CDCl3, 600 MHz) of 101

521

Figure 3.146. gHMQC (CDCl3, 600 MHz) of 101

522

1

Figure 3.147. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 101

523

Figure 3.148. gHMBC (CDCl3, 600 MHz) of 101

524

1

Figure 3.149. H-NMR (CDCl3, 500 MHz) of 102

525

1

Figure 3.150. H-NMR (CDCl3, 500 MHz) of 103

526

Figure 3.151.

13

C-NMR (CDCl3, 125 MHz) of 103

527

Figure 3.152. gCOSY (CDCl3, 500 MHz) of 103

528

Figure 3.153. gHMQC (CDCl3, 500 MHz) of 103

529

1

Figure 3.154. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 103

530

Figure 3.155. gHMBC (CDCl3, 500 MHz) of 103

531

1

Figure 3.156. H-NMR (CDCl3, 500 MHz) of 105

532

Figure 3.157.

13

C-NMR (CDCl3, 150 MHz) of 105

533

Figure 3.158. gCOSY (CDCl3, 600 MHz) of 105

534

Figure 3.159. gHMQC (CDCl3, 600 MHz) of 105

535

1

Figure 3.160. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 105

536

1

Figure 3.161. H-NMR (CDCl3, 600 MHz) of 106

537

Figure 3.162.

13

C-NMR (CDCl3, 150 MHz) of 106

538

Figure 3.163. gHMQC (CDCl3, 600 MHz) of 106

539

1

Figure 3.164. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 106

540

1

Figure 3.165. H-NMR (CDCl3, 600 MHz) of 107

541

Figure 3.166.

13

C-NMR (CDCl3, 150 MHz) of 107

542

Figure 3.167. gCOSY (CDCl3, 600 MHz) of 107

543

Figure 3.168. gHMQC (CDCl3, 600 MHz) of 107

544

1

Figure 3.169. H-NMR (CDCl3, 600 MHz) of 108

545

Figure 3.170.

13

C-NMR (CDCl3, 150 MHz) of 108

546

Figure 3.171. gCOSY (CDCl3, 600 MHz) of 108

547

Figure 3.172. gHMQC (CDCl3, 600 MHz) of 108

548

1

Figure 3.173. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 108

549

1

Figure 3.174. H-NMR (CDCl3, 600 MHz) of 109

550

Figure 3.175.

13

C-NMR (CDCl3, 150 MHz) of 109

551

Figure 3.176. gCOSY (CDCl3, 600 MHz) of 109

552

O
OBn
O
TBSO
OBz
111

1

Figure 3.177. H-NMR (CDCl3, 500 MHz) of 111

553

O
OBn
O
TBSO
OBz
111

Figure 3.178.

13

C-NMR (CDCl3, 125 MHz) of 111

554

O
OBn
O
TBSO
OBz
111

Figure 3.179. gCOSY (CDCl3, 500 MHz) of 111

555

HO

STol
OBn
O

TBSO
OBz
114

1

Figure 3.180. H-NMR (CDCl3, 500 MHz) of 114

556

HO

STol
OBn
O

TBSO
OBz
114

Figure 3.181.

13

C-NMR (CDCl3, 125 MHz) of 114

557

HO

STol
OBn
O

TBSO
OBz
114

Figure 3.182. gCOSY (CDCl3, 500 MHz) of 114

558

1

Figure 3.183. H-NMR (CDCl3, 600 MHz) of 115

559

Figure 3.184.

13

C-NMR (CDCl3, 125 MHz) of 115

560

Figure 3.185. gCOSY (CDCl3, 500 MHz) of 115

561

1

Figure 3.186. H-NMR (CDCl3, 600 MHz) of 116

562

Figure 3.187.

13

C-NMR (CDCl3, 125 MHz) of 116

563

Figure 3.188. gCOSY (CDCl3, 600 MHz) of 116

564

Figure 3.189. gHMQC (CDCl3, 500 MHz) of 116

565

1

Figure 3.190. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 116

566

1

Figure 3.191. H-NMR (CDCl3, 500 MHz) of 117

567

1

Figure 3.192. H-NMR (CDCl3, 600 MHz) of 124

568

Figure 3.193.

13

C-NMR (CDCl3, 150 MHz) of 124

569

Figure 3.194. gCOSY (CDCl3, 600 MHz) of 124

570

Figure 3.195. gHMQC (CDCl3, 600 MHz) of 124

571

1

Figure 3.196. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 124

572

Figure 3.197. gHMBC (CDCl3, 600 MHz) of 124

573

1

Figure 3.198. H-NMR (CDCl3, 500 MHz) of S1

574

Figure 3.199.

13

C-NMR (CDCl3, 125 MHz) of S1

575

Figure 3.200. gCOSY (CDCl3, 500 MHz) of S1

576

1

Figure 3.201. H-NMR (CDCl3, 500 MHz) of 126

577

Figure 3.202.

13

C-NMR (CDCl3, 125 MHz) of 126

578

Figure 3.203. gCOSY (CDCl3, 500 MHz) of 126

579

1

Figure 3.204. H-NMR (CDCl3, 600 MHz) of 127

580

Figure 3.205.

13

C-NMR (CDCl3, 150 MHz) of 127

581

Figure 3.206. gCOSY (CDCl3, 600 MHz) of 127

582

Figure 3.207. gHMQC (CDCl3, 600 MHz) of 127

583

1

Figure 3.208. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 127

584

Figure 3.209. gHMBC (CDCl3, 600 MHz) of 127

585

1

Figure 3.210. H-NMR (CDCl3, 600 MHz) of 128

586

Figure 3.211.

13

C-NMR (CDCl3, 150 MHz) of 128

587

Figure 3.212. gCOSY (CDCl3, 600 MHz) of 128

588

Figure 3.213. gHMQC (CDCl3, 600 MHz) of 128

589

1

Figure 3.214. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 128

590

Figure 3.215. gHMBC (CDCl3, 600 MHz) of 128

591

1

Figure 3.216. H-NMR (CDCl3, 600 MHz) of 130

592

Figure 3.217.

13

C-NMR (CDCl3, 150 MHz) of 130

593

Figure 3.218. gCOSY (CDCl3, 600 MHz) of 130

594

Figure 3.219. gHMQC (CDCl3, 600 MHz) of 130

595

1

Figure 3.220. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 130

596

Figure 3.221. gHMBC (CDCl3, 600 MHz) of 130

597

1

H-NMR (CDCl3, 500 MHz)

1

Figure 3.222. H-NMR (CDCl3, 500 MHz) of 131

598

1

Figure 3.223. H-NMR (CDCl3, 500 MHz) of 132

599

1

Figure 3.224. H-NMR (CDCl3, 600 MHz) of 133

600

Figure 3.225.

13

C-NMR (CDCl3, 150 MHz) of 133

601

Figure 3.226. gCOSY (CDCl3, 600 MHz) of 133

602

Figure 3.227. gHMQC (CDCl3, 600 MHz) of 133

603

1

Figure 3.228. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 133

604

Figure 3.229. gHMBC (CDCl3, 600 MHz) of 133

605

1

Figure 3.230. H-NMR (CDCl3, 600 MHz) of 134

606

Figure 3.231.

13

C-NMR (CDCl3, 150 MHz) of 134

607

Figure 3.232. gCOSY (CDCl3, 600 MHz) of 134

608

Figure 3.233. gHMQC (CDCl3, 600 MHz) of 134

609

1

Figure 3.234. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 134

610

Figure 3.235. gHMBC (CDCl3, 600 MHz) of 134

611

1

Figure 3.236. H-NMR (CDCl3, 500 MHz) of 135

612

Figure 3.237.

13

C-NMR (CDCl3, 125 MHz) of 135

613

Figure 3.238. gCOSY (CDCl3, 500 MHz) of 135

614

Figure 3.239. gHMQC (CDCl3, 500 MHz) of 135

615

1

Figure 3.240. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 135

616

Figure 3.241. gHMBC (CDCl3, 500 MHz) of 135

617

1

Figure 3.242. H-NMR (CDCl3, 500 MHz) of 136

618

1

Figure 3.243. H-NMR (CDCl3, 500 MHz) of 137

619

1

Figure 3.244. H-NMR (CDCl3, 500 MHz) of 140

620

Figure 3.245.

13

C-NMR (CDCl3, 125 MHz) of 140

621

Figure 3.246. gCOSY (CDCl3, 500 MHz) of 140

622

O
OBn
O
PMBO
OBz
142

1

Figure 3.247. H-NMR (CDCl3, 600 MHz) of 142

623

O
OBn
O
PMBO
OBz
142

Figure 3.248.

13

C-NMR (CDCl3, 150 MHz) of 142

624

O
OBn
O
PMBO
OBz
142

Figure 3.249. gCOSY (CDCl3, 600 MHz) of 142

625

1

Figure 3.250. H-NMR (CDCl3, 500 MHz) of 143

626

Figure 3.251.

13

C-NMR (CDCl3, 125 MHz) of 143

627

Figure 3.252. gCOSY (CDCl3, 500 MHz) of 143

628

1

Figure 3.253. H-NMR (CDCl3, 500 MHz) of 144

629

Figure 3.254.

13

C-NMR (CDCl3, 125 MHz) of 144

630

Figure 3.255. gCOSY (CDCl3, 500 MHz) of 144

631

Figure 3.256. gHMQC (CDCl3, 500 MHz) of 144

632

1

Figure 3.257. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 144

633

Figure 3.258. gHMBC (CDCl3, 500 MHz) of 144

634

1

Figure 3.259. H-NMR (CDCl3, 500 MHz) of 145

635

Figure 3.260.

13

C-NMR (CDCl3, 125 MHz) of 145

636

Figure 3.261. gCOSY (CDCl3, 500 MHz) of 145

637

Figure 3.262. gHMQC (CDCl3, 500 MHz) of 145

638

1

Figure 3.263. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 145

639

Figure 3.264. gHMBC (CDCl3, 500 MHz) of 145

640

1

Figure 3.265. H-NMR (CDCl3, 500 MHz) of 146

641

Figure 3.266.

13

C-NMR (CDCl3, 125 MHz) of 146

642

Figure 3.267. gCOSY (CDCl3, 500 MHz) of 146

643

1

Figure 3.268. H-NMR (CDCl3, 500 MHz) of 149

644

Figure 3.269.

13

C-NMR (CDCl3, 125 MHz) of 149

645

Figure 3.270. gCOSY (CDCl3, 500 MHz) of 149

646

1

Figure 3.271. H-NMR (CDCl3, 500 MHz) of 150

647

1

Figure 3.272. H-NMR ((CD3)2CO, 500 MHz) of 125

648

Figure 3.273.

13

C-NMR ((CD3)2CO, 150 MHz) of 125

649

Figure 3.274. gCOSY ((CD3)2CO, 600 MHz) of 125

650

Figure 3.275. gHMQC ((CD3)2CO, 600 MHz) of 125

651

1

Figure 3.276. gHMQC (without H decoupling) ((CD3)2CO, 600 MHz) of 125

652

Figure 3.277. gHMBC ((CD3)2CO, 600 MHz) of 125

653

1

Figure 3.278. H-NMR (CDCl3, 500 MHz) of 154

654

Figure 3.279.

13

C-NMR (CDCl3, 125 MHz) of 154

655

Figure 3.280. gCOSY (CDCl3, 500 MHz) of 154

656

LevO
TBSO

OBn O
BnO
O
BzO

OBn
O
N3
O
BnO

Ph

Ph

O
O
O
OLev O
O
O
O
STol
O
O
BzO
OH
BzO 154

Figure 3.281. gHMQC (CDCl3, 500 MHz) of 154

657

LevO
TBSO

OBn O
BnO
O
BzO

OBn
O

Ph

Ph

O
O
O
OLev O
O
O
O
STol
O
O
BzO
OH
BzO 154

N3
O
BnO

1

Figure 3.282. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 154

658

LevO
TBSO

OBn O
BnO
O
BzO

OBn
O
N3
O
BnO

Ph

Ph

O
O
O
OLev O
O
O
O
STol
O
O
BzO
OH
BzO 154

Figure 3.283. gHMBC (CDCl3, 500 MHz) of 154

659

1

Figure 3.284. H-NMR (CDCl3, 500 MHz) of 155

660

Figure 3.285.

13

C-NMR (CDCl3, 125 MHz) of 155

661

Figure 3.286. gCOSY (CDCl3, 500 MHz) of 155

662

1

Figure 3.287. H-NMR (CDCl3, 500 MHz) of 156

663

Figure 3.288.

13

C-NMR (CDCl3, 125 MHz) of 156

664

Figure 3.289. gCOSY (CDCl3, 500 MHz) of 156

665

1

Figure 3.290. H-NMR (CDCl3, 500 MHz) of 159

666

Figure 3.291.

13

C-NMR (CDCl3, 150 MHz) of 159

667

Figure 3.292. gCOSY (CDCl3, 600 MHz) of 159

668

1

Figure 3.293. H-NMR (CDCl3, 600 MHz) of 160

669

Figure 3.294.

13

C-NMR (CDCl3, 150 MHz) of 160

670

Figure 3.295. gCOSY (CDCl3, 600 MHz) of 160

671

1

Figure 3.296. H-NMR (CDCl3, 500 MHz) of 162

672

Figure 3.297.

13

C-NMR (CDCl3, 125 MHz) of 162

673

Figure 3.298. gCOSY (CDCl3, 500 MHz) of 162

674

1

Figure 3.299. H-NMR (CDCl3, 600 MHz) of 163

675

Figure 3.300.

13

C-NMR (CDCl3, 150 MHz) of 163

676

Figure 3.301. gCOSY (CDCl3, 600 MHz) of 163

677

1

Figure 3.302. H-NMR (CDCl3, 500 MHz) of 165

678

Figure 3.303.

13

C-NMR (CDCl3, 125 MHz) of 165

679

Figure 3.304. gCOSY (CDCl3, 500 MHz) of 165

680

1

Figure 3.305. H-NMR (CDCl3, 500 MHz) of 167

681

1

Figure 3.306. H-NMR (CDCl3, 600 MHz) of 168

682

1

Figure 3.307. H-NMR (CDCl3, 500 MHz) of 172

683

1

Figure 3.308. H-NMR (CDCl3, 500 MHz) of 174

684

1

Figure 3.309. H-NMR (CDCl3, 500 MHz) of 276

685

Figure 3.310. gCOSY (CDCl3, 500 MHz) of 276

686

1

Figure 3.311. H-NMR (CDCl3, 500 MHz) of 183

687

Figure 3.312.

13

C-NMR (CDCl3, 125 MHz) of 183

688

Figure 3.313. gCOSY (CDCl3, 500 MHz) of 183

689

1

Figure 3.314. H-NMR (CDCl3, 500 MHz) of 184

690

Figure 3.315.

13

C-NMR (CDCl3, 125 MHz) of 184

691

Figure 3.316. gCOSY (CDCl3, 500 MHz) of 184

692

1

Figure 3.317. H-NMR (CDCl3, 500 MHz) of 185

693

Figure 3.318.

13

C-NMR (CDCl3, 125 MHz) of 185

694

Figure 3.319. gCOSY (CDCl3, 500 MHz) of 185

695

1

Figure 3.320. H-NMR (CDCl3, 500 MHz) of 186

696

Figure 3.321.

13

C-NMR (CDCl3, 125 MHz) of 186

697

Figure 3.322. gCOSY (CDCl3, 500 MHz) of 186

698

1

Figure 3.323. H-NMR (CDCl3, 500 MHz) of 187

699

Figure 3.324.

13

C-NMR (CDCl3, 125 MHz) of 187

700

Figure 3.325. gCOSY (CDCl3, 500 MHz) of 187

701

1

Figure 3.326. H-NMR (CDCl3, 500 MHz) of 188

702

Figure 3.327.

13

C-NMR (CDCl3, 125 MHz) of 188

703

Figure 3.328. gCOSY (CDCl3, 500 MHz) of 188

704

1

Figure 3.329. H-NMR (CDCl3, 500 MHz) of 269

705

Figure 3.330.

13

C-NMR (CDCl3, 125 MHz) of 269

706

1

Figure 3.331. H-NMR (CDCl3, 500 MHz) of 120

707

Figure 3.332.

13

C-NMR (CDCl3, 125 MHz) of 120

708

1

Figure 3.333. H-NMR (CDCl3, 500 MHz) of 177

709

Figure 3.334.

13

C-NMR (CDCl3, 125 MHz) of 177

710

Figure 3.335. gCOSY (CDCl3, 500 MHz) of 177

711

Figure 3.336. gHMQC (CDCl3, 500 MHz) of 177

712

1

Figure 3.337. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 177

713

Figure 3.338. gHMBC (CDCl3, 500 MHz) of 177

714

1

Figure 3.339. H-NMR (CDCl3, 600 MHz) of 191

715

Figure 3.340.

13

C-NMR (CDCl3, 150 MHz) of 191

716

Figure 3.341. gCOSY (CDCl3, 600 MHz) of 191

717

1

Figure 3.342. H-NMR (CDCl3, 500 MHz) of 178

718

Figure 3.343.

13

C-NMR (CDCl3, 125 MHz) of 178

719

Figure 3.344. gCOSY (CDCl3, 500 MHz) of 178

720

1

Figure 3.345. H-NMR (CDCl3, 600 MHz) of 179

721

Figure 3.346.

13

C-NMR (CDCl3, 150 MHz) of 179

722

Figure 3.347. gCOSY (CDCl3, 600 MHz) of 179

723

Figure 3.348. gHMQC (CDCl3, 600 MHz) of 179

724

1

Figure 3.349. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 179

725

Figure 3.350. gHMBC (CDCl3, 600 MHz) of 179

726

1

Figure 3.351. H-NMR (CDCl3, 600 MHz) of 180

727

Figure 3.352.

13

C-NMR (CDCl3, 150 MHz) of 180

728

Figure 3.353. gCOSY (CDCl3, 600 MHz) of 180

729

Figure 3.354. gHMQC (CDCl3, 600 MHz) of 180

730

1

Figure 3.355. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 180

731

Figure 3.356. gHMBC (CDCl3, 600 MHz) of 180

732

1

Figure 3.357. H-NMR (CDCl3, 500 MHz) of 181

733

Figure 3.358.

13

C-NMR (CDCl3, 150 MHz) of 181

734

Figure 3.359. gCOSY (CDCl3, 500 MHz) of 181

735

1

Figure 3.360. H-NMR (CDCl3, 600 MHz) of 182

736

Figure 3.361.

13

C-NMR (CDCl3, 150 MHz) of 182

737

Figure 3.362. gCOSY (CDCl3, 600 MHz) of 182

738

Figure 3.363. gHMQC (CDCl3, 600 MHz) of 182

739

1

Figure 3.364. gHMQC (without H decoupling) (CDCl3, 600 MHz) of 182

740

Figure 3.365. gHMBC (CDCl3, 600 MHz) of 182

741

1

Figure 3.366. H-NMR (CDCl3, 600 MHz) of 176

742

Figure 3.367.

13

C-NMR (CDCl3, 150 MHz) of 176

743

Figure 3.368. gCOSY (CDCl3, 600 MHz) of 176

744

Figure 3.369. gHMQC (CDCl3, 500 MHz) of 176

745

1

Figure 3.370. gHMQC (without H decoupling) (CDCl3, 500 MHz) of 176

746

Figure 3.371. gHMBC (CDCl3, 500 MHz) of 176

747

1

Figure 3.372. H-NMR (CDCl3, 500 MHz) of 192

748

Figure 3.373.

13

C-NMR (CDCl3, 125 MHz) of 192

749

Figure 3.374. gCOSY (CDCl3, 500 MHz) of 192

750

1

Figure 3.375. H-NMR (CDCl3, 500 MHz) of 194

751

Figure 3.376.

13

C-NMR (CDCl3, 125 MHz) of 194

752

Figure 3.377. gCOSY (CDCl3, 500 MHz) of 194

753

1

Figure 3.378. H-NMR (CDCl3, 500 MHz) of 195

754

Figure 3.379.

13

C-NMR (CDCl3, 125 MHz) of 195

755

Figure 3.380. gCOSY (CDCl3, 500 MHz) of 195

756

1

Figure 3.381. H-NMR (CDCl3, 500 MHz) of 196

757

1

Figure 3.382. H-NMR (CDCl3, 500 MHz) of 175

758

Figure 3.383.

13

C-NMR (CDCl3, 125 MHz) of 175

759

1

Figure 3.384. H-NMR (CDCl3, 500 MHz) of 220

760

Figure 3.385.

13

C-NMR (CDCl3, 125 MHz) of 220

761

Figure 3.386. gCOSY (CDCl3, 500 MHz) of 220

762

1

Figure 3.387. H-NMR (CDCl3, 500 MHz) of 198

763

1

Figure 3.388. H-NMR (CDCl3, 500 MHz) of 205

764

1

Figure 3.389. H-NMR (CDCl3, 500 MHz) of 221

765

Figure 3.390.

13

C-NMR (CDCl3, 125 MHz) of 221

766

1

Figure 3.391. H-NMR (CDCl3, 500 MHz) of 222

767

1

Figure 3.392. H-NMR (CDCl3, 500 MHz) of 223

768

Figure 3.393.

13

C-NMR (CDCl3, 125 MHz) of 223

769

1

Figure 3.394. H-NMR (D2O, 600 MHz) of 226

770

1

Figure 3.395. H-NMR (CDCl3, 600 MHz) of 227

771

Figure 3.396.

13

C-NMR (CDCl3, 150 MHz) of 227

772

1

Figure 3.397. H-NMR (CDCl3, 500 MHz) of 228

773

Figure 3.398.

13

C-NMR (CDCl3, 125 MHz) of 228

774

1

Figure 3.399. H-NMR (D2O, 600 MHz) of 24

775

1

Figure 3.400. H-NMR (CDCl3, 600 MHz) of 239

776

Figure 3.401.

13

C-NMR (CDCl3, 125 MHz) of 239

777

Figure 3.402. gCOSY (CDCl3, 600 MHz) of 239

778

OLev
OBn
Ph
COOMe
O
O
O
O
OBn
O
MeOOC OBn BnO
O BnO
COOMe O
O
AcHN
AcHN
O
O
PMBO
243 BnO
O
O
BzO
OLev
OBz

Ph
O
O
O BzO
O
OBz

O

O
OAc

BnO

H
N

AcHN
O

1

Figure 3.403. H-NMR (CDCl3, 500 MHz) of 243

779

OBz
O
O

O
N
H

CO2H

OLev
OBn
Ph
COOMe
O
O
O
O
OBn
O
MeOOC OBn BnO
O BnO
COOMe O
O
AcHN
AcHN
O
O
PMBO
243 BnO
O
O
BzO
OLev
OBz

Ph
O
O
O BzO
O
OBz

O

O
OAc

BnO

H
N

AcHN
O

Figure 3.404.

13

C-NMR (CDCl3, 125 MHz) of 243

780

OBz
O
O

O
N
H

CO2H

1

Figure 3.405. H-NMR (CDCl3, 500 MHz) of 251

781

1

Figure 3.406. H-NMR (CDCl3, 500 MHz) of 252

782

Figure 3.407.

13

C-NMR (CDCl3, 125 MHz) of 252

783

Figure 3.408. gCOSY (CDCl3, 500 MHz) of 252

784

Ph

Ph

O
O

O
O

O

O
LevO

O
OLev
253

OBz
O
O

BzO BzO
O
BnO
H
N

AcHN
O

O
N
H

COOBn

1

Figure 3.409. H-NMR (CDCl3, 500 MHz) of 253

785

Ph

Ph

O
O

O
O

O

O
LevO

O
OLev
253

H
N

AcHN
O

Figure 3.410.

OBz
O
O

BzO BzO
O
BnO

O
N
H

COOBn

13

C-NMR (CDCl3, 125 MHz) of 253

786

Ph

Ph

O
O

O
O
O
LevO

O
OLev
253

O

OBz
O
O

BzO BzO
O
BnO
H
N

AcHN
O

O
N
H

COOBn

Figure 3.411. gCOSY (CDCl3, 500 MHz) of 253

787

Ph

Ph

O
O

O
O

O

O
LevO

O
OLev

OBz
O
O

BzO BzO
O
BnO
AcHN

H
N
O 254

O
N
H

COOH

1

Figure 3.412. H-NMR (CDCl3, 500 MHz) of 254

788

Ph

Ph

O
O

O
O

O

O
LevO

O
OLev

OBz
O
O

BzO BzO
O
BnO
AcHN

H
N
O 254

Figure 3.413.

O
N
H

COOH

13

C-NMR (CDCl3, 125 MHz) of 254

789

1

Figure 3.414. H-NMR (D2O, 600 MHz) of 233

790

HPLC chromatogram

Figure 3.415. HPLC curve for crude peptide 244 before purification

791

Figure 3.416. HPLC curve for crude glycopeptide 238 before purification

792

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793

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