DESIGN AND SYNTHESIS OF A HYALURONAN OLIGOSACCHARIDE AND
ANALOGUES AS POTENTIAL INHIBITORS OF CD44-HYALURONAN BINDING
By
Xiaowei Lu

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

ABSTRACT
DESIGN AND SYNTHESIS OF A HYALURONAN OLIGOSACCHARIDE AND
ANALOGUES AS POTENTIAL INHIBITORS OF CD44-HYALURONAN BINDING
By
Xiaowei Lu
Hyaluronan (HA) is a non-sulfated negatively charged linear polysaccharide, which
is composed of 2,000-25,000 repeating units of disaccharides: [-D-glucuronic acid-β13-N-acetyl-D-glucosamine-β-14-]. Tumor microenvironment contains different type
of cells and ECM. HA and HA binding proteins are the major components of
extraceullular matrix (ECM). CD44 is the primary receptor for HA. CD44, a single chain
transmembrane protein, is composed of four parts: the N-terminal hyaluronan binding
domain (HABD), the stem domain, the transmembrane domain and the C-terminal
cytoplasmic-tail domain. Multivalent HA-CD44 interactions induce signaling pathways
that promote tumor cell invasiveness, proliferation, survival and multidrug resistance
(MDR). Multivalent HA-CD44 interactions are required for formation of constitutive
signaling complexes. Replacement of the multivalent interactions with monovalent
interactions by the treatment with hyaluronan oligosaccharide (sHA) causes
disassembly of constitutive signaling complexes and attenuated signaling pathway.
These finally lead to the inhibition of tumor cell proliferation and MDR.
In this dissertation, the chemical synthesis of a hyaluronan decasaccharide (HA10)
using the pre-activation based chemoselective glycosylation strategy is described.
Assembly of large oligosaccharides is generally challenging due to the increased
difficulties in both glycosylation and deprotection. Indeed, the same building blocks

previously employed for hyaluronan hexasaccharide (HA6) synthesis failed to yield the
desired HA10. After extensive experimentation, the HA10 backbone was successfully
constructed with an overall yield of 37% from disaccharide building blocks. The
trichloroacetyl group was used as the nitrogen protective group for the glucosamine
units and the addition of trimethylsilyl triflate (TMSOTf) was found to be crucial to
suppress the formation of trichloromethyl oxazoline side-product and enable high
glycosylation yield. For deprotections, the combination of a mild basic condition and the
monitoring methodology using 1H-NMR allowed the removal of all base-labile protective
groups, which facilitated the generation of the fully deprotected HA10.
Based on the co-crystal structure of CD44 HABD and hyaluronan octasaccharides
(HA8), hyaluronan pentasaccharide (HA5) analogues were designed and synthesized.
Due to synthetic difficulties, the initial design of Library A was abolished. By rotating SC4 bond, new analogues, Library B and Library C, were generated. Eleven
compounds in Library B and Library C were synthesized by a cutting edge method,
and screened by inhibitory Enzyme-linked immunosorbent assay (ELISA). Finally, it was
found that the aromatic group in analogue 56 contributes to the binding of 56 to CD44.
And this interaction overcomes the loss of favorable enthalpy caused by the loss of Hbonds from COOH of glucronic acid 7 (GluUA7) and primary OH of NAcetylglucosamine 8 (GlcNAc8). Although the binding affinity of 56 is only comparable
to HA6 and less than HA8, this provides a new direction towards further design of novel
HA inhibitors.

DEDICATION

I dedicate this dissertation to my husband, Liping Liu, for his remarkable patience,
unwavering love and support over the course of my research. I dedicate this dissertation
to my parents, Yueyue Dong and Zhiwei Lu, for their sacrifices and support. Finally, I
dedicated this dissertation to my grandparents, Jixiang Lu and Longying Chen, who are
in heaven.

iv

ACKNOWLEDGEMENTS

I would like to thank all of those people who helped make this dissertation possible.
First, I wish to thank my advisor, Professor Xuefei Huang, for all his guidance,
encouragement, support, and patience. His sincere interests in science, aggressiveness,
personality and dedication have been a great influence on me. My advisor is a great
teacher who knows that real growth comes through pain. I appreciated my advisor for
challenging me and believing that I am able to go through it. By going through those
challenges, I have learned a lot and gained immense confidence. My parents brought
me to this world and provided me with good educations. Professor Huang taught me
how to be a professional scientist and how to thrive in the real world.
Also, I would like to thank my committee members Professor Gregory Baker,
Professor Gary Blanchard and Professor John McCracken for their input, valuable
discussions and accessibility.
I am very thankful to the Department of Chemistry at Michigan State University,
especially Professor Gary Blanchard, associate Chair for education, for been a strong
support to me throughout my graduate school career.
I give many thanks to Dr. Daniel Holmes and Mr. Kermit Johnson for the help on
NMR, Dr. Daniel Jones and Ms. Lijun Chen for the help on mass spectrometer. I would
like to thank all the staffs in department of chemistry for their hard work and dedication.
Finally, I would like to thank all my lab mates for all the help over the years.

v

TABLE OF CONTENTS
LIST OF TABLES…………………………….....…………………………………………..viii
LIST OF FIGURES ……………………….....………………………………………….......ix
LIST OF ABBREVIATIONS …………………….....……………………………………….xix
LIST OF SCHEMES ………………….......………………………………………….…....xxviii
CHAPTER 1 Functions of CD44-Hyaluronan Interactions in Inflammation and
Malignancy..........................................................................................................……….1
1.1. CD 44, a major receptor for HA ..........................................................1
1.1.1. HA .............................................................................................1
1.1.2. CD 44.........................................................................................2
1.1.3. CD44-HA interactions.................................................................3
1.2. Functions of CD44 and HA interactions in the immune system .........…… 4
1.2.1. Pro-inflammatory role ……...………………………………...….. 4
1.2.2. Anti-inflammatory role …………………..……………………..…6
1.2.2.1. HA uptake..................................................................................6
1.2.2.2. AICD..................................................................................... .....6
1.3. Functions of CD44 and HA interactions in cancer …...........………....7
1.3.1. Tumor microenvironment ...........................…………………..…7
1.3.2. Tumor initiation and progression ..................………………...…7
1.3.2.1. HA in invasion............................................................................7
1.3.2.2. CD44 in extravagation of tumor cells during metastasis............8
1.3.2.3. CD44 reduces Fas-mediated killing of tumor cells.....................8
1.3.2.4. EMT............................................................................................8
1.3.2.5. Cancer stem cell.........................................................................8
1.3.3. MDR ...........................………………………………………….....9
1.3.4. Signaling pathway .............…………………………………….......10
1.3.5. Perturbation of HA-CD44 with sHA.............................................11
References …………………………………………………………...……...13
CHAPTER 2 Syntheses of HA Oligosaccharides ….............................................…22
2.1. Enzymatic synthesis …………………………………….…………………..22
2.2. Chemical synthesis ....................….…………………………………………25
2.2.1. The first method with glucose building blocks...............………...26
2.2.1.1. Synthesis by Vliegenthart’s group...............................................26
2.2.1.2. Synthesis by Petillo’s group........................................................29
2.2.1.3. Interactive one-pot synthesis by Huang’s group.........................31
2.2.2. The second method with glucuronic acid building blocks...........37
2.2.2.1. Synthesis by Jacquinet’s group..................................................37
2.2.2.2. Synthesis by van der Marel’s group............................................39
2.2.2.3. Synthesis by Huang’s group......................................................44
References
……………………………………………………………........45
vi

CHAPTER 3
3.1.
3.2.
3.3.
3.4.

Chemical Synthesis of HA10...............................................................49
Introduction
………………………………………………………….......... 48
Results and Discussion ………………………………................................49
Conclusion ..........................................................……………………......59
Experimental Section ............................………………………...………...61
Appendix A (NMR Spectra)..........................…………………………….…85
References ………………………………………………………………....139

CHAPTER 4 Design and Synthesis of HA5 Analogues as Potential Inhibitors of
CD44-HA binding .........................................................................................143
4.1. Introduction
………………………………………………………….........143
4.2. Results and Discussion ………………………………...............................148
4.2.1. HA2 Library.................................................................................148

4.3.
4.4.

4.2.1.1. The Design and Synthesis of HA2 Library................................148
4.2.1.2. The ELISA..................................................................................150
4.2.1.3. Results and Discussion..............................................................153
4.2.2. HA5 Library...............................................................................155
4.2.2.1. The Design.................................................................................155
4.2.2.2. The Syntheses...........................................................................156
4.2.2.3. Results and Discussion..............................................................170
Conclusion ..........................................................……………………... 173
Experimental Section ............................………………………...………. 175
Appendix B (NMR Spectra) ……................……………………………....204
References ………………………………………………………………....271

vii

LIST OF TABLES
Table 2.1.

One-pot synthesis ...............................…………………………………….34

Table 3.1.

The effects of additives on glycosylation product distribution ..…………56

viii

LIST OF FIGURES
Figure 1.1.

CD44 pre-mRNA ...................................…………………………….……….2

Figure 1.2.

CD44s and CD44v1-10 ............……………………………………….………3

Figure 1.3. Co-crystal of CD44 HABD and HA 8 …….......…………………….…..….4
Figure 1.4.

H-Bonds between CD44 HABD and HA8…..............……………….……...5

Figure 1.5.

Leukocyte recruitment ……........................................…………………….…..6

Figure 1.6.

CD44-HA signaling pathway

Figure 1.7.

Perturbation of HA-CD44 interaction........................................................11

.................................................................10

Figure 2.1. Scheme of catalyst generation and dual-enzyme reactor. A, mutagenesis
was used to transform the dual-action HAS into two single-action catalysts, GlcNActransferase (GN-T) and GlcUA-transferase (GA-T). B, a starting acceptor was combined
with the UDP-GlcNAc precursor and circulated through the GlcNAc-transferase reactor
(white circle, GlcNAc; black circle, GlcUA). After coupling, UDP-GlcUA precursor was
added to the mixture and circulated through the GlcUA-transferase reactor. This
stepwise synthesis can be repeated as desired (dashed line) until the target
oligosaccharide size is reached......................................................................................24
Figure 2.2.

Compounds 1, 2, 3 and 4…................................……...................……….28

Figure 2.3.

Compounds 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21……..................….29

Figure 2.4.

Compounds 22, 23 and 24……............................................................….30

Figure 2.5.

Compounds 32, 33, 34, 35 and 36….............................…...................….33

Figure 2.6.

Compounds 41, 42, 43 and 44…...................................…...................….35

Figure 2.7.

Compounds 45, 46, 47 and 48…...................................…...................….35

Figure 2.8.

Compounds 49, 50, 51 and 52…...................................…...................….37

Figure 2.9.

Compounds 53, 54 and 55….........................................…...................….39

Figure 2.10. Compounds 63, 64 and 65….............................…….................……….40
Figure 2.11. Compounds 71, 72 and 73.................................……...................……….41
Figure 2.12. Compounds 81, 82 and 83…..............................……...................……….44

ix

Figure 3.1.

Compound 4..................….................................……...................……….51

Figure 3.2.

Compounds 25, 26, 27 and 28….........................…...................……….57

Figure 3.3.
Figure 3.4.
Figure 3.5.
Figure 3.6.
Figure 3.7.
Figure 3.8.

1
1

H-NMR of compound 9 (500 MHz, D2O).............................................. 86

H-NMR of compound 10 (500 MHz, CDCl3) ........................................ 87

13
1

C-NMR of compound 10 (125 MHz, CDCl3).........................................88
1

H– H gCOSY of compound 10 (500 MHz, CDCl3)................................ 89

1

13

H– C gHMQC of compound 10 (500 MHz, CDCl3) ........................... 90

1

H–

13

C gHMQC (without 1H decoupling) of compound 10 (500 MHz,

CDCl3)........................................................................................................................... 91
Figure 3.9.

1

13

H– C gHMBC of compound 10 (500 MHz, CDCl3) ..............................92

1

Figure 3.10.

H-NMR of compound 19 (500 MHz, CDCl3) .........................................93

13

Figure 3.11.

C-NMR of compound 19 (125 MHz, CDCl3) ......................................94

Figure 3.12. 1H–1H gCOSY of compound 19 (500 MHz, CDCl3)..................................95
1

13

Figure 3.13. H– C gHMQC of compound 19 (500 MHz, CDCl3)...............................96
1

Figure 3.14. H–

13

C gHMQC (without 1H decoupling) of compound 19 (500 MHz,

CDCl3)............................................................................................................................97
Figure 3.15.
Figure 3.16.
Figure 3.17.

1

13

H– C gHMBC of compound 19 (500 MHz, CDCl3) ...........................98

1

H-NMR of compound 11 (500 MHz, CDCl3) .........................................99

13

C-NMR of compound 11 (125 MHz CDCl3) .......................................100

x

Figure 3.18.
Figure 3.19.

1

1

H– H gCOSY of compound 11 (500 MHz, CDCl3) ..............................101

1

13

H– C gHMQC of compound 11 (500 MHz, CDCl3) ...........................102
1

Figure 3.20.

13

H– C gHMQC (without 1H decoupling) of compound 11 (500 MHz,

CDCl3)..........................................................................................................................103
Figure 3.21.
Figure 3.22.
Figure 3.23.
Figure 3.24.
Figure 3.25.

1

13

H– C gHMBC of compound 11 (500 MHz, CDCl3) ............................104

1

H-NMR of compound 12 (500 MHz, CDCl3) ........................................105

13
1

C-NMR of compound 12 (125 MHz, CDCl3) ......................................106
1

H– H gCOSY of compound 12 (500 MHz, CDCl3) ..............................107

1

13

H– C gHMQC of compound 12 (500 MHz, CDCl3) ..........................108
1

Figure 3.26.

13

H– C gHMQC (without 1H decoupling) of compound 12 (500 MHz,

CDCl3)..........................................................................................................................109
Figure 3.27.
Figure 3.28.
Figure 3.29.
Figure 3.30.
Figure 3.31.

1

13

H– C gHMBC of compound 12 (500 MHz, CDCl3) ...........................110

1

H-NMR of compound 13 (500 MHz, CDCl3) ........................................111

13
1
1

C-NMR of compound 13 (125 MHz, CDCl3) ......................................112
1

H– H gCOSY of compound 13 (500 MHz, CDCl3) .............................113
13

H– C gHMQC of compound 13 (500 MHz, CDCl3)............................114
1

Figure 3.32.

13

H– C gHMQC (without 1H decoupling) of compound 13 (500 MHz,

CDCl3)..........................................................................................................................115
Figure 3.33.

1

13

H– C gHMBC of compound 13 (500 MHz, CDCl3) ............................116

xi

Figure 3.34.
Figure 3.35.
Figure 3.36.
Figure 3.37.

1

H-NMR of compound 25 (500 MHz, CDCl3) ........................................117

13
1

C-NMR of compound 25 (125 MHz, CDCl3).......................................118
1

H– H gCOSY of compound 25 (500 MHz, CDCl3) ..............................119

1

13

H– C gHMQC of compound 25 (500 MHz, CDCl3) ...........................120
1

Figure 3.38.

13

H– C gHMQC (without 1H decoupling) of compound 25 (500 MHz,

CDCl3)..........................................................................................................................121
Figure 3.39.
Figure 3.40.
Figure 3.41.
Figure 3.42.
Figure 3.43.

1

13

H– C gHMBC of compound 25 (500 MHz, CDCl3).............................122

1

H-NMR of compound 26 (500 MHz, CDCl3).........................................123

13
1

C-NMR of compound 26 (125 MHz, CDCl3).......................................124
1

H– H gCOSY of compound 26 (500 MHz, CDCl3) ..............................125

1

13

H– C gHMQC of compound 26 (500 MHz, CDCl3) ...........................126
1

Figure 3.44.

13

H– C gHMQC (without 1H decoupling) of compound 26 (500 MHz,

CDCl3)..........................................................................................................................127
Figure 3.45.
Figure 3.46.
Figure 3.47.
Figure 3.48.
Figure 3.49.
Figure 3.50.

1

13

H– C gHMBC of compound 26 (500 MHz, CDCl3) ............................128

1

H-NMR of compound 27 (500 MHz, CDCl3) ........................................129

13
1

C-NMR of compound 27 (125 MHz, CDCl3).......................................130
1

H– H gCOSY of compound 27 (500 MHz, CDCl3) .............................131

1
1

13

H– C gHMQC of compound 27 (500 MHz, CDCl3) ..........................132
H-NMR of compound 28 (500 MHz, CDCl3) ........................................133
xii

Figure 3.51.
Figure 3.52.
Figure 3.53.
Figure 3.54.

13
1

C-NMR of compound 28 (125 MHz, CDCl3) .....................................134
1

H– H gCOSY of compound 28 (500 MHz, CDCl3) ..............................135

1

13

H– C gHMQC of compound 28 (500 MHz, CDCl3) ...........................136
1

13

H– C gHMQC (without 1H decoupling) of compound 28 (500 MHz,

CDCl3)..........................................................................................................................137
Figure 3.55.

1

13

H– C gHMBC of compound 28 (500 MHz, CDCl3) ...........................138

Figure 4.1.

Inhibitors reported by Takahashi's group...............................................144

Figure 4.2.

H-bonds in CD44-HA8 co-crystal structure............................................145

Figure 4.3.

Ligands of Galectin-3............................................................................147

Figure 4.4.

Co-crystal structure of galectin-3 and analogue 2.................................147

Figure 4.5.

MAG Ligands.........................................................................................148

Figure 4.6.

The hydrophobic pocket in CD44-HA8 co-crystal structure...................148

Figure 4.7.

Docking results of HA2 library ...............................................................149

Figure 4.8.

HA8 inhibition curve ..............................................................................152

Figure 4.9.

Inhibition ELISA of compounds 10 ~ 16.................................................154

Figure 4.10.

Alternative Inhibition ELISA of compound 10, Linker 1 and 2...............155

Figure 4.11. Compound 10, Linker 1 and 2............................................. .................155
Figure 4.12.

HA5 backbone.......................................................................................156

Figure 4.13. Visualization of the amide linked hydrophobic groups in CD44 HABD..156
Figure 4.14. HA5 analogues a) Library A; b) Library B; c) Library C .........................164

xiii

Figure 4.15.

Library B ..............................................................................................165

Figure 4.16.

Library C ..............................................................................................165

Figure 4.17.

Visualizations of compounds 49 and 50 in CD44 HABD......................166

Figure 4.18.

Visualizations of compounds 51 - 56 in CD44 HABD...........................166

Figure 4.19.

Visualizations of compounds 56 - 59 in CD44 HABD...........................167

Figure 4.20. Inhibition ELISA of sHA and analogues ................................................171
Figure 4.21.

sHA and analogues...............................................................................172

Figure 4.22

Inhibition curve of compound 56, HA6 and HA8....................................172

Figure 4.23.
Figure 4.24.
Figure 4.25.
Figure 4.26.
Figure 4.27.
Figure 4.28.
Figure 4.29.
Figure 4.30.
Figure 4.31.
Figure 4.32.
Figure 4.33.

1
1

H-NMR of compound 10 (500 MHz, CDCl3).........................................205
H-NMR of compound 11 (500 MHz, CDCl3).........................................206

1
1
1
1
1
1

H-NMR of compound 12 (500 MHz, CDCl3)........................................207
H-NMR of compound 13 (500 MHz, CDCl3)........................................208
H-NMR of compound 14 (500 MHz, CDCl3)........................................209
H-NMR of compound 15 (500 MHz, CDCl3)........................................210
H-NMR of compound 16 (500 MHz, CDCl3)........................................211

H-NMR of NMR of compound 38 (500 MHz, CDCl3) ..........................212

13
1

C-NMR of compound 38 (125 MHz CDCl3) ......................................213
1

H– H gCOSY of compound 38 (500 MHz, CDCl3) .............................214

1

13

H– C gHMQC of compound 38 (500 MHz, CDCl3)...........................215

xiv

Figure 4.34.

1

H–

13

C gHMQC (without 1H decoupling) of compound 38 (500 MHz,

CDCl3)..........................................................................................................................216
Figure 4.35.
Figure 4.36.
Figure 4.37.
Figure 4.38.

1

13

H– C gHMBC of compound 38 (500 MHz, CDCl3)............................217

1
1
1

H-NMR of compound 40 (500 MHz, CDCl3)........................................218
1

H– H gCOSY of compound 40 (500 MHz, CDCl3)..............................219

H–

13

C gHMQC (without 1H decoupling) of compound 40 (500 MHz,

CDCl3)..........................................................................................................................220
Figure 4.39.
Figure 4.40.
Figure 4.41.
Figure 4.42.
Figure 4.43.

1

13

H– C gHMBC of compound 40 (500 MHz, CDCl3) ...........................221

1

H-NMR of compound 72 (500 MHz, CDCl3)........................................222

13
1
1

C-NMR of compound 72 (125 MHz, CDCl3) .....................................223
1

H– H gCOSY of compound 72 (500 MHz, CDCl3) .............................224

H–

13

C gHMQC (without 1H decoupling) of compound 72 (500 MHz,

CDCl3)..........................................................................................................................225
Figure 4.44.
Figure 4.45.
Figure 4.46.
Figure 4.47.

1

H-NMR of compound 73 (500 MHz, CDCl3) ......................................226

13

C-NMR of compound 73 (125 MHz, CDCl3)......................................227

1

H–1H gCOSY of compound 73 (500 MHz, CDCl3) ..............................228

1

13

H– C gHMQC (without 1H decoupling) of compound 73 (500 MHz,

CDCl3)..........................................................................................................................229
Figure 4.48.

1

H-NMR of compound 61 (500 MHz, CDCl3) .......................................230

xv

Figure 4.49.
Figure 4.50.
Figure 4.51.
Figure 4.52.

13
1

C-NMR of compound 61 (125 MHz, CDCl3) .....................................231
1

H– H gCOSY of compound 61 (500 MHz, CDCl3)..............................232

1

13

H– C gHMQC of compound 61 (500 MHz, CDCl3) ..........................233

1

13

H– C gHMQC (without 1H decoupling) of compound 61 (500 MHz,

CDCl3)..........................................................................................................................234
Figure 4.53.
Figure 4.54.
Figure 4.55.
Figure 4.56.
Figure 4.57.

1

13

H– C gHMBC of compound 61 (500 MHz, CDCl3) ...........................235

1

H-NMR of compound 62 (500 MHz, CDCl3) ......................................236

13
1

C-NMR of compound 62 (125 MHz, CDCl3) .....................................237
1

H– H gCOSY of compound 62 (500 MHz, CDCl3)..............................238

1

13

H– C gHMQC (without 1H decoupling) of compound 62 (500 MHz,

CDCl3)..........................................................................................................................239
Figure 4.58.
Figure 4.59.
Figure 4.60.
Figure 4.61.
Figure 4.62.

1
1

13

H– C gHMBC of compound 62 (500 MHz, CDCl3) ..........................240

H-NMR of compound 63 (500 MHz, CDCl3) .......................................241

13
1

C-NMR of compound 63 (125 MHz, CDCl3) .....................................242
1

H– H gCOSY of compound 63 (500 MHz, CDCl3) ............................243

1

13

H– C gHMQC of compound 63 (500 MHz, CDCl3) ..........................244
1

Figure 4.63.

13

H– C gHMQC (without 1H decoupling) of compound 63 (500 MHz,

CDCl3)..........................................................................................................................245
Figure 4.64.

1

H-NMR of compound 64 (500 MHz, CDCl3) .......................................246

xvi

Figure 4.65.
Figure 4.66.
Figure 4.67.

13
1
1

C-NMR of compound 64 (125 MHz, CDCl3)......................................247
1

H– H gCOSY of compound 64 (500 MHz, CDCl3).............................248
13

H– C gHMQC (without 1H decoupling) of compound 64 (500 MHz,

CDCl3)..........................................................................................................................249
Figure 4.68.
Figure 4.69.
Figure 4.70.
Figure 4.71.
Figure 4.72.
Figure 4.73.
Figure 4.74.
Figure 4.75.
Figure 4.76.
Figure 4.77.
Figure 4.78.
Figure 4.79.
Figure 4.80.
Figure 4.81.

1
1

1

H– H gCOSY of compound 49 (600 MHz, D2O) ................................251

1
1

1

1

1

1

H-NMR of compound 54 (600 MHz, D2O) ..........................................260
1

H– H gCOSY of compound 54 (600 MHz, D2O) ................................261

1
1

H-NMR of compound 53 (600 MHz, D2O)...........................................258

H– H gCOSY of compound 53 (600 MHz, D2O) ................................259

1
1

H-NMR of compound 52 (600 MHz, D2O) ..........................................256

H– H gCOSY of compound 52 (600 MHz, D2O) ................................257

1
1

H-NMR of compound 51 (600 MHz, D2O) ..........................................254

H– H gCOSY of compound 51 (600 MHz, D2O).................................255

1
1

H-NMR of compound 50 (600 MHz, D2O) ..........................................252

H– H gCOSY of compound 50 (600 MHz, D2O).................................253

1
1

H-NMR of compound 49 (600 MHz, D2O) ........................................250

H-NMR of compound 55 (600 MHz, D2O) ..........................................262
1

H– H gCOSY of compound 55 (600 MHz, D2O).................................263

xvii

Figure 4.82.
Figure 4.83.
Figure 4.84.
Figure 4.85.
Figure 4.86.
Figure 4.87.
Figure 4.88.

1
1

1

H– H gCOSY of compound 56 (600 MHz, D2O) ................................265

1
1

H-NMR of compound 56 (600 MHz, D2O) ..........................................264

H-NMR of compound 57 (600 MHz, D2O) ..........................................266
1

H– H gCOSY of compound 57 (600 MHz, D2O).................................267

1
1
1

H-NMR of compound 58 (600 MHz, D2O) ..........................................268
H-NMR of compound 59 (600 MHz, D2O) ..........................................269
H-NMR of compound 67 (600 MHz, D2O)...........................................270

xviii

LIST OF ABBREVIATIONS
Acetic acid (AcOH)
Acetic anhydride (Ac2O)
Acetonitrile (MeCN)
Acetyl (Ac)
Activation-induced cell death (AICD)
Adipic dihydrazide (ADH)
Alanine (Ala)
Allyl (All)
Allyloxycarbonyl (Alloc)
Ammonium acetate (NH4OAc)
Arginine (Arg, R)
Avidin conjugated horseradish peroxidase (Avidin-HRP)
Azobisisobutyronitrile azobisisobutylonitrile (AIBN)
Benzoyl (Bz)
Benzoyl chloride (BzCl)
Benzyl (Bn)
[Bis(acetoxy)iodo]benzene (BAIB)
Boron trifluoride etherate (BF3.Et2O)
Bovine serum albumin (BSA)
Breast cancer resistance protein (BCRP)
Calcium carbonate (CaSO4)
Camphorsulfonic acid (CSA)
xix

Catalytic amount (Cat.)
Ceric ammonium nitrate (CAN)
Chloroform (CHCl3)
Concentrated (conc.)
Copper sulfate (CuSO4)
Correlation spectroscopy (COSY)
Cysteine (Cys)
Cytotoxic T lymphocytes (CTL)
Degree celsius (°C)
Dichloromethane (DCM)
Deuterated chloroform (CDCl3)
Deuterated methanol (CD3OD)
Deuterated water (D2O)
1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU)
2,3-Dichloro-5,6-dicyano-1, 4-benzoquinone (DDQ)
Diethyl ether (Et2O)
4-Dimethylaminopyridine (DMAP)
Dimethylformamide (DMF)
Dimethyl(methy1-thio)sulfonium triflate (DMTST)
Dimethyl sulfoxide (DMSO)
Di-tert-butylsilylidene (DTBS)
Doublet (d)
xx

Electrospray ionization mass spectrometry (ESI-MS)
Epidermal growth factor receptor (EGFR)
Epithelial-mesenchymal transition (EMT)
Equivalent (eq.)
Erizin-radixin-moiesin (ERM)
Ethanol (EtOH)
Ethyl acetate (EA)
1-Ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride (EDCI)
Extraceullular matrix (ECM)
Enzyme-linked immunosorbent assay (ELISA)
Galactose (Gal)
Glucronic acid (GlcUA)
Glycosaminoglycan (GAG)
Gram (g)
Grb2-associated binder (Gab)
Growth factor receptor-bound protein (Grb)
Guanine nucleotide exchange factor (Vav)
Glucose (Glc)
Glucuronic acid (GlcUA)
Hepatocyte growth factor (HGF)
Heteronuclear multiple bond coherence (HMBC)
Heteronuclear multiple quantum coherence (HMQC)
High performance liquid chromatography (HPLC)
Hour (hr)
xxi

Human epidermal growth factor receptor 2 (ErbB2)
Hyaluronan (HA)
Hyaluronan binding domain (HABD)
Hyaluronan decasaccharide (HA10)
Hyaluronan disaccharide (HA2)
Hyaluronan heptasaccharide (HA7)
Hyaluronan hexasaccharide (HA6)
Hyaluronan-mediated motility receptor (RHAMM)
Hyaluronan octasaccharides (HA8)
Hyaluronan oligosaccharide (sHA)
Hyaluronan pentasaccharide (HA5)
Hyaluronic acid receptor for endocytosis (HARE)
Hyaluronan synthases (HAS)
Hyaluronan tetrasaccharide (HA4)
Hyaluronan trisaccharide (HA3)
.

Hydrazine hydrate (NH2NH2 H2O)
.

Hydrazine acetate (NH2NH2 HOAc)
Hydrochloric acid (HCl)
Hydrogen bond (H-Bond)
Hydrogen fluoride (HF)
Hydrogen peroxide (H2O2)
xxii

Hydroxyl (OH)
Isoleucine (Ile)
Levulinoyl (Lev)
Lymphatic vessel endothelial hyaluronan receptor (LYVE)
Molecular sieve (MS)
Megahertz (MHz)
Methanol (MeOH)
Methyl (Me)
Methyl amine (MeNH2)
Methoxyphenyl (MP)
Microliter (μl)
Milligram (mg)
Millimolar (mmol)
Mitogen-activated protein kinases (MAPK)
Multiplet (m)
Multidrug resistance (MDR)
Multidrug resistance-associated protein (MRP)
Myelin associated glycoprotein (MAG)
N-Acetylglucosamine (GlcNAc)
N-hydroxysuccinimide (NHS)
N-iodosuccinimide (NIS)
N, N’-Dicyclohexylcarbodiimide (DCC)
Nuclear magnetic resonance (NMR)
xxiii

Palladium hydroxide (Pd(OH)2)
Palladium on activated carbon (Pd/C)
P-glycoprotein (P-gp)
p-Methoxybenzyl (PMB)
p-toluoyl (Tol)
Phenyl (Ph)
Phenyl diazomethane (PhCHN2)
Phthalimido (Phth)
Phosphate buffered saline (PBS)
PBS solution with Tween 20 (PBST)
Phosphatidylinositol 3-kinase (PI3K)
Potassium bicarbonate (KHCO3)
Potassium carbonate (K2CO3)
Potassium iodide (KI)
Protein kinase B (AKT)
Proteoglycan (PG)
Proto-oncogene tyrosine-protein kinase (Src)
p-Toluenesulfenyl chloride (p-TolSCl)
p-Toluenesulfenyl triflate (p-TolSOTf)
p-Toluenesulfonic acid (p-TsOH)
Pyridine (Py)
Pyridinum chlorochromate (PCC)
Pyridinium dichromate (PDC)
xxiv

Ras homolog gene family, member A (RhoA)
Ras-related C3 botulinum toxin substrate 1 (Rac1)
Receptor tyrosine kinase (RTK)
RNA triphosphatase (RTPase)
Room temperature (r.t.)
Quantitatively convert (quant.)
Serine (Ser)
Serum-derived HA-binding protein (SHAP)
Sialic acid (Sia)
Silver triflate (AgOTf)
Silver carbonate (Ag2CO3)
Singlet (s)
Size-exclusion column (G-15)
Sodium acetate (NaOAc)
Sodium bicarbonate (NaHCO3)
Sodium carbonate (Na2CO3)
Sodium chlorite (NaClO2)
Sodium hydride (NaH)
Sodium hydroxide (NaOH)
Sodium hypochlorite (NaClO)
Sodium methoxide (NaOMe)
Sodium sulfate (Na2SO4)
xxv

Sodium thiosulfate (Na2S2O3)
Structure–activity relationship (SAR)
n-Butanol (n-BuOH)
t-Butanol (t-BuOH)
Tetrabutylammonium hydroxide (TBAOH)
Tetrabutylammonium thioacetate (TBASAc)
tert-butyl diphenyl silyl (TBDPS)
tert-butyl diphenylchlorosilane (TBDPSCl)
2, 2, 6, 6-Tetramethylpiperidin-1-oxyl (TEMPO)
Tetrahydrofuran (THF)
3,3′,5,5′-Tetramethylbenzidine (TMB)
Thin layer chromatography (TLC)
Thioacetic acid (HSAc)
Threonine (Thr)
TNF-α-stimulated protein 6 (TSG-6)
TNF receptor superfamily, member 6 (Fas)
Toll-like receptors (TLR)
Tosyl (Ts)
tributyltin hydride (Bu3SnH)
Trichloroethoxycarbonyl (Troc)
Triethylamine (TEA)
.

Triethylamine tris-hydrofluoride (TEA 3HF)
Triflic acid (TfOH)
xxvi

Trifluoromethanesulfonic anhydride (Tf2O)
Trimethylsilyl triflate (TMSOTf)
Triphenylphosphine oxide (Ph2SO)
tri-tert-butyl pyrimidine (TTBP)
Triplet (t)
Trichloroacetyl (TCA)
Triethyl phosphite (TEP)
Triethylsilane (Et3SiH)
Trifluoroacetic acid (TFA)
tri-tert-butyl silyl (TBS)
Tyrosine (Tyr, Y)
Uridine diphosphate (UDP)
Uridine triphosphate (UTP)
Very late antigen (VLA)
Zinc (Zn)

xxvii

LIST OF SCHEMES
Scheme 2.1.

Enzymatic synthesis of HA with regeneration of sugar nucleotides..….23

Scheme 2.2.

Transglycosylation reaction by testicular hyaluronidase...................….23

Scheme 2.3.

HA synthesis with oxazoline building block............................................25

Scheme 2.4.

Two general strategies for sHA assembly...............…………….……….25

Scheme 2.5.

Synthesis by Vliegenthart’s group...........................…………….……….27

Scheme 2.6.

Deprotections of compound 12 and 13...................…………….……….28

Scheme 2.7.

Synthesis by Petillo’s group....................................…………….……….31

Scheme 2.8.

Interactive one-pot synthesis ................................................................32

Scheme 2.9.

Synthesis of HA2 building blocks............................…………….……….33

Scheme 2.10. Deprotection of compound 44..............……………...................……….37
Scheme 2.11. Synthesis by Jacquinet’s group..............................…………….……….38
Scheme 2.12. Synthesis of compound 70.....................................…………….……….40
Scheme 2.13. Synthesis of compound 80…….…................................................…….41
Scheme 2.14. Synthesis of compound 86 and 87.........................…………….……….43
Scheme 3.1.

Previous synthesis on HA6 3.................................................................51

Scheme 3.2.

Previous synthesis on HA10...................................…………….……….52

Scheme 3.3.

Retrosynthetic scheme towards HA10 9...............…………….……….53

Scheme 3.4.

Synthesis of monosaccharide building blocks 14 and 15...............……53

Scheme 3.5.

Assembly of HA2 building blocks............................…………….……….54

Scheme 3.6.

Oxazoline formation................................................…………….……….55

Scheme 3.7.

Deprotection............................................................…………….……….59
xxviii

Scheme 4.1.

Synthesis of Biotin-HA...............................................…………….……150

Scheme 4.2.

Retrosynthetic scheme of compound 1.....................…………….……151

Scheme 4.3.

Retrosynthetic scheme of compound 17...................…………….……157

Scheme 4.4.

Synthesis of compound 23........................................…………….……158

Scheme 4.5.

Synthesis towards HA5 with STol donor...................…………….……159

Scheme 4.6.

Synthesis towards HA5 with Br donor.....................…………….……160

Scheme 4.7.

Synthesis towards HA5 with trichloroacetimidate donor.......... …..…161

Scheme 4.8.

Synthesis route towards HA5 thioglycoside.....................…........ ……162

Scheme 4.9.

Synthesis toward HA2 thioglycoside .....................…...………….……163

Scheme 4.10.

Synthesis of library B.....................……..........................……….……168

Scheme 4.11.

Synthesis of library C.....................……..........................……….……169

Scheme 4.12.

Deprotection of compound 57.....................………….............….……170

Scheme 4.13. Deprotection Preparation of monosaccharide 35, 36, 37, 38, 15 and
23................................................................................................................................184

xxix

CHAPTER 1
Functions of CD44-HA Interactions in Inflammation and Malignancy
1.1. CD 44, a major receptor for HA
1.1.1. HA
HA is a non-sulfated negatively charged linear polysaccharide, which is
composed

of

2,000-25,000

repeating

units

of

disaccharides:

[-D-glucuronic
6

7

acid-β-13-N-acetyl-D-glucosamine-β-14-]. The molecular weight is around 10 - 10
1

Da and the polymer length is 2-25 μm . Unlike other glucosaminoglycan, HA is not linked
to core proteins. HA is synthesized by three hyaluronan synthases (HAS) (HAS1, HAS2
and HAS3) on the plasma membrane and the chains are extruded into the extracellular
space

2-5

.

HA is a major component of ECM. HA is tethered to cell surface by either
cell-surface receptors such as CD44 or HAS. This HA ‘coat’ can incorporate
proteoglycan (PG) and other HA binding proteins, such as TNF-α-stimulated protein 6
(TSG-6) and link protein

6-7

.

There are multiple HA binding proteins, also called hyaladherins: lymphatic vessel
endothelial hyaluronan receptor 1 (LYVE1) is mainly restricted to lymphatic vessel and
8

lymph node ; hyaluronic acid receptor for endocytosis (HARE) clears HA from
9

circulation ; toll-like receptors (TLRs) and TSG-6 are related to the immune system
CD44

is

related

to

the

immune

system

1

atherosclerosis

and

cancer

8, 10

;

11-12

;

hyaluronan-mediated motility receptor (RHAMM) is important to wound healing and
13

cancer .
1.1.2. CD 44
Figure 1.1. CD44 pre-mRNA. Adapted with permission from Ref 14. Copyright 2003
Nature Publishing Group. For interpretation of the references to color in this and all
other figures, the reader is referred to the electronic version of this dissertation.

CD44 is the primary receptor for HA. CD44 is a single chain transmembrane
protein. The CD44 pre-mRNA is encoded by 20 exons (Figure 1.1)

14

. The first five

non-variable exons encode the N-terminal HABD. In this region, 90 amino acids (32-123),
15

which are conserved in many hyaladherins, are named ‘link module’

14

(Figure 1.2 ).

Between HABD and the transmembrane domain is the stem domain. In the standard
isoform CD44s, this region is composed of 46 amino acids. By alternative splicing, this
domain can be enlarged by different combination of variant exons (V1-V10) to generate

2

different isoforms of CD44

16-17

. The transmembrane domain is composed of 13

hydrophobic amino acids and a cysteine. This domain participates in CD44
condensation

18

and CD44-lipid rafts interaction

19-20

. The C-terminal cytoplasmic-tail

domain plays a crucial role in signaling.
Figure 1.2. CD44s and CD44v1-10. Adapted with permission from Ref 14. Copyright
2003 Nature Publishing Group.

1.1.3. CD44-HA interactions
In 2007, Jackson’s group reported the co-crystal structure of murine CD44 HABD
21

and HA8 (Figure 1.3) . HA8 binds to a shallow groove and the interactions are
dominated by H-Bonds and hydrophobic interactions. The hydrophobic interactions are
3

from sugar rings from GlcNAc4 to GlcUA7 and the methyl group in GlcNAc6. The
H-Bonds are from GlcUA5 to GlcNAc8 as summarized in Figure 1.4. In addition, two
conformers are identified. These two forms differed in the orientation of arginine 45
(Arg45) which move closer to HA8.

Figure 1.3. Co-crystal of CD44 HABD and HA8. Adapted with permission from Ref 21.
Copyright 2008 Nature Publishing Group.

1.2. Functions of CD44 and HA interactions in the immune system
1.2.1. Pro-inflammatory role
22

Leukocytes are recruited to inflammatory site in four steps (Figure 1.5) . First,
4

leukocytes in the blood stream are tethered to endothelial cells through binding with cell
adhesion molecules. Second, tethering and rolling reduce the velocity and allow further
interactions with endothelial cells, which leads to adhesion. Third, adhesion is followed
by the arrest step. Finally leukocytes move outward through intact vessel walls into
surrounding body tissue, which is also called diapedesis (Figure 1.5). CD44-HA
interactions and selectins-ligands interactions contribute to the initial tethering and rolling
23

steps leading to integrin-mediated adhesion .
Figure 1.4. H-Bonds between CD44 HABD and HA8

Siegelman’s group reported that T cell rolling is CD44 and HA dependent

23-25

. In

inflammation sites, endothelial cell surface CD44 binds HA, and present HA to T cell
5

surface CD44. This CD44-HA-CD44 ‘sandwich’ interaction leads to rolling

26-27

. CD44

also participates in T cell adhesion. This process is mediated by integrin very late antigen
4 (VLA-4) and CD44 cytoplasmic domain

28-29

.

Neutrophil recruitment is reduced in CD44 null mice
macrophages is observed in CD44 null macrophages

30-31

. Reduced recruitment of

32-33

. However, how CD44 and HA

involved in neutrophil and macrophage recruitments is not very clear

23, 34

.

Figure 1.5. Leukocyte recruitment. Adapted with permission from Ref 22. Copyright
2002 Elsevier.

1.2.2. Anti-inflammatory role
1.2.2.1. HA uptake
Successful repair after inflammation requires removal of the ECM break down
products. During inflammation, accumulated HA fragments have proinflammatory
10

functions . CD44 positive macrophages play important roles in HA fragment clearance
35-37

and maintain HA homeostasis

. Since HA fragments up-regulate TLR-2 and 4

responses, HA uptake can down-regulate TLR-2 and 4 signaling and reduce
6

38-39

inflammatory response

.

1.2.2.2. AICD
Stimulation from T cell receptors to already expanded T cell without

appropriate
40

co-stimulation could lead to T cell death, named activation-induced cell death (AICD) .
AICD removes activated T cells to maintain immune homeostasis. High MW HA-CD44
binding enhances AICD in T cells

23, 41-42

. This CD44-mediated AICD is TNF receptor

43

superfamily member 6 (Fas)-independent .
1.3. Functions of CD44 and HA interactions in cancer
1.3.1. Tumor microenvironment
The tumor microenvironment contains different type of cells, including vascular
44

cells, fibroblasts, immune cells and ECM . HA and HA binding proteins are the major
components of ECM

45-46

. HA provides a favorable environment for cell proliferation and

migration. HA binding proteins regulate HA functions

47-48

. serum-derived HA-binding

protein (SHAP), TSG-6 and versican contribute to the formation of HA meshwork. CD44,
LYVE-1 and RHAMM are the cell surface receptor for anchoring

49-51

. In addition, tumor

cell and other types of cells communicate with each other to adjust the components in
tumor microenvironment for tumor
metastasis

52-53

cell proliferation, migration, invasion, and

.

7

1.3.2. Tumor initiation and progression
1.3.2.1. HA in invasion
HA promotes tumor cell

migration

and

invasion from

three

aspects.

Cancer-associated fibroblasts amplify the synthesis of HA and proteoglycans, and the
expanded ECM leads to increased viscoelasticity and malleability which support the
54

cancer cell shape change and tissue penetration . HA is involved in the protease
production and presentation. Finally, HA participates in the induction of cytoskeleton
1, 6

rearrangements

.

1.3.2.2. CD44 in tumor cells extravagation during metastasis
Metastatic process is similar to leukocyte recruitment. Tumor cells must tether and
adhere to endothelial cells of the blood vessel wall and then transmigrate to the tissue.
HA-CD44 interaction leads to the amplification of integrin-mediated adhesion to the
vessel wall

55-57

.

1.3.2.3. CD44 reduces Fas-mediated killing of tumor cells
Although cytotoxic T lymphocytes (CTL)-mediated cytotoxicity plays a major role
in tumor rejection, tumors often protect themselves against CTL recognition and attack.
CD44-HA interactions reduce both Fas expression and Fas-mediated apoptosis, leading
55, 58

to less susceptibility to CTL-mediated cytotoxicity

8

.

1.3.2.4. Epithelial-mesenchymal transition (EMT)
EMT is the transition of cells which involves decreased intercellular junctions,
escaped apoptosis and enhanced invasiveness which CD44-HA interaction is important
for EMT. Disruption of HA-CD44 interaction can block the EMT induction from HGF
1, 53

treatment and overexpresion of beta-catenine

.

1.3.2.5. Cancer stem cell
Cancer stem cells are stem cells within tumors. Very small number of cancer
stem cells can rapidly regenerate a fully grown tumor when implanted in an animal
host

59-61

. CD44 is the common marker for cancer stem cell isolation. It was reported

that CD44 has important functions in leukemia stem cells, brain tumor stem cells,
pancreatic cancer stem cells and breast cancer stem cells

59, 62-65

.

However, nothing

66

is known about the role of HA-CD44 in cancer stem cells .
1.3.3. MDR
Four ways could generate MDR: the reduced uptake of drugs from the tissue
barriers; enhanced repair systems; increased anti-apoptosis; and finally, enhanced drug
67-69

efflux

.

HA and CD44 promote MDR in many cancer cell types

66, 70-72

. The possible

mechanisms are: decreased tissue barrier and enhanced drug penetration

9

73-75

;

enhanced cell survival signaling pathways

76

; and finally, enhanced drug efflux by

regulating the expression and membrane stabilization of dug transporters, including
P-glycoprotein, multidrug resistance-associated protein 2 (MRP2) and BCRP

70, 77

.

P-gp is a well characterized ATP-binding cassette transporter (ABC-transporter) in
78

MDR subfamily. P-gp is in close vicinity to CD44 in lipid rafts . Perturbation of HA-CD44
interaction induces internalization of P-gp and CD44 into the cell. In addition, expression
of CD44 and P-gp are co-regulated

79-80

.

1.3.4. Signaling pathway
Multivalent HA-CD44 interactions induce direct and indirect interactions with RTK
receptor tyrosine kinases (RTKs) including human epidermal growth factor receptor 2
(ErbB2) and epidermal growth factor receptor (EGFR), and non-receptor kinases of
proto-oncogene tyrosine-protein kinase (Src) family or Ras family GTPases (Figure 1.6)
1, 13, 81

. Adaptor proteins such as guanine nucleotide exchange factor 2 (Vav2), growth

factor receptor-bound protein 2 (Grb2), and Grb2-associated binder 1 (Gab-1) mediate
the formation of above complex and mediate the interaction of CD44 with upstream
effectors such as Ras homolog gene family member A (RhoA), Ras-related C3 botulinum
toxin substrate 1 (Rac1) and Ras, which eventually influence down stream
mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K)/
82

protein kinase B (AKT) signaling pathways . The above signaling pathways promote

10

tumor cell invasiveness, proliferation, survival and MDR

13, 83

.

In addition, CD44 can influence cellular events including tumor cell proliferation
13-14, 84

and motility by cross-linking to actin cytoskeleton through ankyrin or ERM family

.

Figure 1.6. CD44-HA signaling pathway

1.3.5. Perturbation of HA-CD44 with HA oligomers (sHA)
Multivalent HA-CD44 interaction is required for formation of constitutive signaling
complexes containing CD44, RTKs, Src, adaptor proteins, erizin-radixin-moiesin (ERM)
76, 85-86

and other signaling components (Figure 1.7a)

. Replacement of the multivalent

interactions with monovalent interaction by the treatment with sHA causes disassembly

11

83

of constitutive signaling complexes and attenuated signaling pathway (Figure 1.7b) .
87

These finally lead to the inhibition of cell proliferation and MDR .

Figure 1.7. Perturbation of HA-CD44 interaction by. Adapted with permission from Ref 1.
Copyright 2004 Nature Publishing Group.

12

References

13

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Steven,

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21

CHAPTER 2
Syntheses of sHA
2.1. Enzymatic syntheses
sHA can be assembled via either chemoenzymatic or chemical synthesis. Earlier
chemoenzymatic approaches utilized the biosynthetic pathways through HAS and
1

associated accessory enzymes (Scheme 2.1)

or a transglycosylation reaction

2

catalyzed by hyaluronidases (Scheme 2.2) . Recently, oxazolidine containing building
blocks, which are transition state analogues for hyaluronidases, have been polymerized
3-5

to yield HA (Scheme 2.3)

. In order to control the length of HA from enzymatic

reactions, a HA synthase was converted by mutagenesis into two single-action
6

glycosyltransferases (GlcUA transferase and GlcNAc transferase) (Figure 2.1) . The
alternating stepwise usage of these two novel enzymes led to the construction of a
series of monodisperse synthetic sHA. Despite these successes, the inherent substrate
specificities of enzymes limit the structural diversity of sHA analogues that can be
generated. Chemical syntheses can complement the chemoenzymatic approaches to
create greater varities of sHA structures, facilitating the structure–activity relationship
(SAR) studies.

22

Scheme 2.1. Enzymatic synthesis of HA with regeneration of sugar nucleotides

Scheme 2.2. Transglycosylation reaction by testicular hyaluronidase

23

Figure 2.1. Scheme of catalyst generation and dual-enzyme reactor. A, mutagenesis
was used to transform the dual-action HAS into two single-action catalysts,
GlcNAc-transferase (GN-T) and GlcUA-transferase (GA-T). B, a starting acceptor was
combined with the UDP-GlcNAc precursor and circulated through the
GlcNAc-transferase reactor (white circle, GlcNAc; black circle, GlcUA). After coupling,
UDP-GlcUA precursor was added to the mixture and circulated through the
GlcUA-transferase reactor. This stepwise synthesis can be repeated as desired (dashed
line) until the target oligosaccharide size is reached. Adapted with permission from Ref 6.
Copyright 2003 American Society for Biochemistry and Molecular Biology

24

Scheme 2.3. HA synthesis with oxazoline building block

2.2. Chemical synthesis
Scheme 2.4. Two general strategies for sHA assembly

There
are three central factors need to be taken into consideration in designing a synthetic

25

route for sHA: 1) the stereocontrolled construction of the

oligosaccharide backbone; 2)

introduction of GlcUA; and 3) installation of acetamido groups. Two general strategies for
sHA assembly are typically adapted depending upon the order of transformations
7

(Scheme 2.4) .

In the first method, more reactive glucose is used as building blocks
8-14

with post glycosylation conversion to GlcUA via oxidation (Scheme 2.4a)

, while the
15

second method utilizes GlcUA directly as the glycosyl donor (Scheme 2.4b) .
Currently, both approaches have been successfully applied in preparation of sHA.

2.2.1. The first method with glucose building blocks
2.2.1.1. Synthesis by Vliegenthart’s group
For the first approach, a selectively removable protective group must be installed
on the 6- hydroxyl (OH) group of glucose to allow for later oxidation state adjustment.
As an example, Vliegenthart and coworkers have reported the chemical synthesis of
11

tetrasaccharide 1 with GlcUA at reducing end (Scheme 2.5) . The desired 1,2-trans
linkages were controlled by the presence of participating neighboring groups, i.e.
N-phthalimido (Phth) on glucosamines 2 and 3 and Tol on glucoside 4. The Lev moiety
was installed on 6-O of glucoside 4 as a selectively removable protective group.
Following glycosylation of 4 by donor 2, adjustment of the aglycon group at the reducing
end by removal of the anomeric methoxyphenyl (MP) group and imidation of the resulting
hemiacetal yielded disaccharide donor 6 (Scheme 2.5a). Reaction of 4 with 3 and
subsequent deprotection gave disaccharide acceptor 8, which was followed by
26

glycosylation by donor 6 and isopropylidene removal producing tetrasaccharide core 9
(Scheme 2.5b). Acetylation and selective cleavage of the two levulinoyl (Lev) groups
by hydrazine acetate (NH2NH2.HOAc) yielded diol 10, which was oxidized to the
corresponding di-carboxylic acid 11 by tandem Swern and sodium chlorite (NaClO2)
oxidations. Treatment of 11 by methyl amine (MeNH2) and subsequent N-acetylation
produced the target tetrasaccharide 1.
Scheme 2.5. Synthesis by Vliegenthart’s group

27

Figure 2.2. Compounds 1, 2, 3 and 4

Scheme 2.6. Deprotections of compounds 12 and 13

This strategy has been expanded to synthesize pentasaccharide 12 and
10

hexasaccharide 17 with glucosamine at the reducing end . However, after
delevulinoylation of 12 with NH2NH2.HOAc, oxidation of the diol 13 by the tandem Swern
and NaClO2 oxidations did not give satisfactory results. Instead, the usage of pyridinum
chlorochromate (PCC) and acetic anhydride (Ac2O) was necessary producing the
desired diacid 14 in good yield (Scheme 2.6a). Following the same oxidation method
with PCC, the conversion of triol 18 to triacid 19 was achieved (Scheme 2.6b).
28

Dephthaloylation/ deacylation of triacid 19 using MeNH2 in ethanol gave a complex
mixture. Ultimately, the removal of N-phthaloyl moieties was carried out with treatment of
ethylenediamine in n-butanol (n-BuOH) followed by N-acetylation to afford the fully
deprotected hexasaccharide 21. Besides oxidation, the free OH groups in 13 and 18 can
be sulfated generating analogues 15 and 20, which will be difficult to obtain through the
chemoenzymatic approach, thus highlighting the advantage of chemical synthesis
approach.
Figure 2.3. Compounds 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21

2.2.1.2. Synthesis by Petillo’s group
29

Petillo and coworkers have reported syntheses of hyaluronan trisaccharide (HA3)
with

the

combination

of

the
9

methodologies (Scheme 2.7) .

sulfoxide

and

trichloroacetimidate

glycosylation

Glycosylation of acceptor 22 by glycosyl sulfoxide 23

produced disaccharide 25. The protective groups on 25 were adjusted leading to
acceptor 26 with a PMB group masking the 6-OH of the glucose unit. Troc was employed
as the 1,2-trans directing N-protective group for glucosamine trichloroacetimidate donor
24, which glycosylated alcohol 26 yielding trisaccharide core 27. Conversion of the
N-trichloroethoxycarbonyl (Troc) and azido into acetamido moieties gave trisaccharide
28. The p-methoxybenzyl (PMB) group was selectively removed by ceric ammonium
nitrate (CAN) exposing the 6-OH group of the glucose, which was directly converted to
carboxylic acid by a 2, 2, 6, 6-Tetramethylpiperidin-1-oxyl (TEMPO) mediated sodium
hypochlorite (NaClO) oxidation in a modest 57% yield. Subsequent deacetylation and
hydrogenolysis produced trisaccharide 31.

Figure 2.4. Compounds 22, 23 and 24

30

Scheme 2.7. Synthesis by Petillo’s group

2.2.1.3. Interative one-pot synthesis by Huang’s group
As described above, traditional oligosaccharide synthesis required multiple
modifications

of

aglycons,

adjustments

of

protective

groups

on

advanced

oligosaccharide intermediates as well as purification of these intermediates. Our group
16

developed a novel iterative one-pot oligosaccharide assembly strategy , in which a
thioglycosyl donor is pre-activated by a thiophilic promoter in the absence of an acceptor,

31

generating a reactive intermediate.

Upon addition of a thioglycosyl acceptor to the

reaction, the free OH group of the acceptor will attack on the reactive intermediate
leading to a disaccharide. With its anomeric thioacetal moiety, the disaccharide can be
subjected to another round of pre-activation and nucleophilic substitution extending the
oligosaccharide chain. Multiple glycosylations can be sequentially carried out in the
same flask without intermediate separations, thus greatly facilitating glyco-assembly
(Scheme 2.8). Furthermore, because donor activation and glycosylation are carried out
in two distinct steps, anomeric reactivities of thioglycosyl donor and acceptor do not need
17

to be differentiated , granting greater flexibilities in selecting of protective groups to
achieve high yields in glycosylation.
Scheme 2.8. Interactive one-pot synthesis

The one-pot method was successfully applied to the HA6 synthesis, which was
described below. Donor 32 was pre-activated by the promoter p-toluenesulfenyl triflate
32

(p-TolSOTf), formed in situ through reaction of silver triflate (AgOTf) with p-TolSCl
(Scheme 2.9a). Subsequent addition of acceptor 35 and a sterically hindered
non-nucleophilic base tri-tert-butyl pyrimidine (TTBP) to the reaction formed disaccharide
37, which was deprotected to give disaccharide 38. Disaccharide acceptor 40 with a
methoxy group at the reducing end was prepared by reacting 32 with acceptor 36,
followed by removal of tri-tert-butyl silyl (TBS) (Scheme 2.9b).
Scheme 2.9. Synthesis of disaccharide building blocks

Figure 2.5. Compounds 32, 33, 34, 35 and 36

With all necessary building blocks in hand, one pot syntheses were performed.
Pre-activation of donor 32 by p-TolSOTf was followed by addition of acceptor 35 and
TTBP. Upon completion of the reaction, addition of acceptor 40, TTBP and promoter
33

p-TolSOTf to the same reaction flask produced tetrasaccharide core 41 in excellent overall
yield in just 3 hrs (Table 2.1, entry 1), which was the only compound needed purification
in this three component one-pot synthesis. sHA core sequences containing odd number
of monosaccharide units can also be synthesized. Two pentasaccharide 42 and 43 with
different sequences were constructed through three and four component one-pot
reactions in excellent yields (Table 2.1, entries 2, 3). A hexasaccharide 44 was easily
accessed as well in high yield following the pre-activation protocol (Table 2.1, entry 4).
Table 2.1.

One-pot synthesis

34

Figure 2.6. Compounds 41, 42, 43 and 44

Figure 2.7.

Compounds 45, 46, 47 and 48

After the establishment of sHA core, the next stages were deprotections and
oxidation state adjustments. Initial efforts of removing the three PMB groups from
hexasaccharides 45 and 48 failed using either CAN or 2, 3-Dichloro-5, 6-dicyano-1,
4-benzoquinone (DDQ), although similar reactions have been reported such as CAN
9

oxidation of trisaccharide 28 (Scheme 2.10) . After repeated trials, it was found that the
presence of TBS moiety was beneficial for deprotection of PMB, as CAN oxidation of
hexasaccharide 44 yielding triol 46 in good yield (Scheme 2.10). Conversion of 47 into
35

tricarboxylic acids turned out to be challenging. Several methods such as
TEMPO/NaClO,

NaClO/NaClO2

catalyzed

by

TEMPO,

TEMPO/[bis(acetoxy)iodo]benzene (BAIB), and Dess-Martin oxidation followed by
NaClO2 did not give the desired product presumably due to the need to oxidize multiple
OH groups in the same molecule. Instead, multiple partially oxidized products were often
obtained from these reactions. Finally, it was discovered that a convenient 2 step
one-pot protocol using TEMPO/NaClO followed by treatment of NaClO2 afforded the
desired tri-carboxylic acid, which was isolated as benzyl ester 47 by subsequent
treatment with phenyl diazomethane (PhCHN2) in high yield and good purity (Scheme
18

2.10) . This protocol was also found to be compatible with a variety of sensitive
functional groups, such as allyl, thioacetal, PMB and isopropylidene. Final deprotections
of sHA 47 was carried out smoothly by the reaction sequence of desilylation,
hydrogenation, saponification and N-acetylation to produce the sHA hexasaccharide 49.
This procedure for deprotection and oxidation state adjustment has also been extended
to generate tetrasaccharide 50 and pentasaccharide 51 and 52.
Scheme 2.10. Deprotection of compound 44

36

Figure 2.8. Compounds 49, 50, 51 and 52

2.2.2. The second method with GlcUA building blocks
As an alternative to glucosyl donors, GlcUA building blocks can be directly utilized
for sHA assembly. Due to the presence of electron withdrawing carboxylic acid on the
pyranose ring, GlcUA building blocks are known to be less reactive than glucoside both
as glycosyl donors and acceptors.

2.2.2.1. Synthesis by Jacquinet’s group
Jacquinet and coworkers developed a nice strategy using the reactive GlcUA
15

trichloroacetimidate donors . Trichloroacetyl (TCA) was employed as the N-protective
group of glucosamine, which can be readily converted into the corresponding
acetamides under the neutral tributyltin hydride (Bu3SnH) reduction condition without
going through amine intermediates.

37

Scheme 2.11. Synthesis by Jacquinet’s group

The two key disaccharides 56, 58 in Jacquinet synthesis were prepared from the
coupling of glycosyl donor 53 with acceptors 54 and 55 respectively (Scheme 2.11a, b).
Disaccharide 56 was transformed into trichloroacetimidate donor 57 over 2 steps, while
disaccharide 58 was subjected to dechloroacetylation to afford acceptor 59. Coupling of
59 with 57 followed by dechloroacetylation gave tetrasaccharide acceptor 60 (Scheme
2.11). Repetition of glycosylation by 57, deprotection of the chloroacetyl group and
glycosylation afforded octasaccharide 61. Subsequent deprotections were performed in
38

the following order: removal of the chloroacetyl group by thiourea, transformation of
NH-TCA into NHAc via Bu3SnH reduction, debenzylidene and saponification to provide
sHA octasaccharide 62 (Scheme 2.11c).
Figure 2.9. Compounds 53, 54 and 55

2.2.2.2. Synthesis by van der Marel’s group
Van

der

Marel

and

co-workers

synthesized

HA

pentasaccharide

by

chemoselective strategies based on their previous finding that 1-hydroxysugar donors
can be condensed with 1-thioglycosides acceptor using Gin’s activator system for
dehydative

glycosylations

19-23

.

Triphenylphosphine

oxide

(Ph2SO)/

trifluoromethanesulfonic anhydride (Tf2O)/TTBP mediated condensation of donor 63 and
acceptor 65 yield thiodisaccharide 66, which was condensed with acceptor glucuronide
64 under the same condition provide trisaccharide 67 (Scheme 2.12). Donor 66 was
condensed with acceptor 68 yielding HA pentasaccharide 69. The yield for glycosidic
bond formation was moderate, because the acid instability of benzylidene acetal
required careful tuning of the amount of TTBP. Too little TTBP gave cleavage of
benzylidene groups, whereas orthoester/oxazoline formation would be observed with
excess of TTBP. After acid cleavage of the benzylidene group, saponification or the ester
39

and NHTCA groups and N-acetylation, 70 was obtained in good 48% yield.
Figure 2.10.

Compounds 63, 64 and 65

Scheme 2.12. Synthesis of compound 70

40

Figure 2.11.

Compounds 71, 72 and 73

Scheme 2.13. Synthesis of compound 80

This strategy has been expanded to synthesize heptasaccharide 80 through
41

chemoselective

strategies

with

glucuronate

ester

thioglycoside

and

trifluoro-N-phenylimidate glucosamine building blocks (Scheme 2.13). More acid stable
24

di-tert-butylsilylidene (DTBS) groups were used to replace benzylidene groups .
Imidate donor 71 was condensed with acceptor 72 using a catalytic amount of TfOH to
yield disaccharide 74 in 90% yield. Under N-iodosuccinimide (NIS)/TfOH condition,
disaccharide 74 and acceptor 73 were condensed to give trisaccharide 75 in 75% yield.
In the same condition, disaccharide 74 and trisaccharide 76 were condensed to give
pentasaccharide 77 in 98% yield. Disaccharide 74 and pentasaccharide 78 were
condensed to give heptasaccharide 79 in 61% yield. The deprotection was similar to
their previous pentasaccharide synthesis, except triethylamine tris-hydrofluoride (TEA.
3HF) was used for DTBS removal. Fully deprotected heptasaccharide 80 was produced
in a very good 46% yield. Compared to their previous pentasaccharide synthesis, the
yields for glycosidic bond formation was significantly improved.
The HA heptasaccharide synthesis was adapted to the syntheses of HA
tetrasaccharide analogues with 4-methylumbelliferyl group at the reducing end, with
25

4-OH at the non-reducing glucuronate either removed or methylated . Imidate donor 82
or 83 condensed with acceptor 81 using a catalytic amount of TfOH to yield disaccharide
84 or 85 in good yield (Scheme 2.14.). Global deprotection was more difficult than
previous heptasaccharide synthesis due to the co-existence of 4-methylumbelliferyl
group and two NHTCA groups. To remove TCA groups, pervious strong basic condition

42

had to be replaced with reductive dehalogenation with zinc (Zn)/ acetic acid (AcOH).
Since monochloroacetamide were obtained as the major product, KI was used to transfer
monochloroacetamide to monoiodideacetamide, which was transferred to acetamide
group by dehalogenation. The overall yields for deprotections were good.

Scheme 2.14. Synthesis of compounds 86 and 87

43

Figure 2.12. Compounds 81, 82 and 83

2.2.2.3.. Synthesis by Huang’s group

We reported the first chemical synthesis of a fully deprotected HA 10, which is
26

elaborated in chapter 3 .

44

References

45

References
1. De Luca, C.; Lansing, M.; Martini, I.; Crescenzi, F.; Shen, G.-J.; O'Regan, M.; Wong,
C.-H., Enzymic synthesis of hyaluronic acid with regeneration of sugar nucleotides. J Am
Chem Soc 1995, 117 (21), 5869-5870.
2. Saitoh, H.; Takagaki, K.; Majima, M.; Nakamura, T.; Matsuki, A.; Kasai, M.; Narita, H.;
Endo, M., Enzymic reconstruction of glycosaminoglycan oligosaccharide chains using
the transglycosylation reaction of bovine testicular hyaluronidase. J Biol Chem 1995, 270
(8), 3741-3747.
3. Kobayashi, S.; Ohmae, M.; Ochiai, H.; Fujikawa, S., A hyaluronidase supercatalyst
for the enzymatic polymerization to synthesize glycosaminoglycans. Chem-Eur J 2006,
12 (23), 5962-5971.
4. Ochiai, H.; Ohmae, M.; Mori, T.; Kobayashi, S., Bottom-up synthesis of hyaluronan
and its derivatives via enzymatic polymerization: Direct incorporation of an amido
functional group. Biomacromolecules 2005, 6 (2), 1068-1084.
5. Kobayashi, S.; Morii, H.; Itoh, R.; Kimura, S.; Ohmae, M., Enzymatic polymerization
to artificial hyaluronan: A novel method to synthesize a glycosaminoglycan using a
transition state analogue monomer. J Am Chem Soc 2001, 123 (47), 11825-11826.
6. DeAngelis, P. L.; Oatman, L. C.; Gay, D. F., Rapid chemoenzymatic synthesis of
monodisperse hyaluronan oligosaccharides with immobilized enzyme reactors. J Biol
Chem 2003, 278 (37), 35199-35203.
7. Karst, N. A.; Linhardt, R. J., Recent chemical and enzymatic approaches to the
synthesis of glycosaminoglycan oligosaccharides. Curr Med Chem 2003, 10 (19),
1993-2031.
8. Codee, J. D. C.; van den Bos, L. J.; Litjens, R. E. J. N.; Overkleeft, H. S.; Van Boom,
J. H.; van der Marel, G. A., Sequential one-pot glycosylations using 1-hydroxyl and
1-thiodonors. Org Lett 2003, 5 (11), 1947-1950.
9. Yeung, B. K. S.; Hill, D. C.; Janicka, M.; Petillo, P. A., Synthesis of two hyaluronan
trisaccharides. Org Lett 2000, 2 (9), 1279-1282.
10. Halkes, K. M.; Slaghek, T. M.; Hypponen, T. K.; Kruiskamp, P. H.; Ogawa, T.;
Kamerling, J. P.; Vliegenthart, J. F. G., Synthesis of hyaluronic-acid-related
oligosaccharides and analogs, as their 4-methoxyphenyl glycosides, having
46

N-acetyl-β-D-glucosamine at the reducing end. Carbohydrate Res 1998, 309 (2),
161-174.
11. Slaghek, T. M.; Hypponen, T. K.; Ogawa, T.; Kamerling, J. P.; Vliegenthart, J. F. G.,
Synthesis of hyaluronic acid related di- and tetrasaccharides having a glucuronic acid at
the reducing end. Tetrahedron Asymmetry 1994, 5 (11), 2291-2301.
12. Slaghek, T. M.; Nakahara, Y.; Ogawa, T.; Kamerling, J. P.; Vliegenthart, J. F. G.,
Synthesis of hyaluronic acid-related di-, tri-, and tetrasaccharides having an
N-acetylglucosamine residue at the reducing end. Carbohydrate Res 1994, 255, 61-85.
13. Slaghek, T. M.; Hyppoenen, T. K.; Ogawa, T.; Kamerling, J. P.; Vliegenthart, F. G.,
Synthesis of a tetrasaccharide fragment of hyaluronic acid having a glucuronic acid at
the reducing end. Tetrahedron Lett 1993, 34 (49), 7939-7942.
14. Slaghek, T.; Nakahara, Y.; Ogawa, T., Stereocontrolled synthesis of hyaluronan
tetrasaccharide. Tetrahedron Lett 1992, 33 (34), 4971-4974.
15. Blatter, G.; Jacquinet, J.-C., 2-Deoxy-2-trichloroacetamido-Dglucopyranose derivatives in syntheses of hyaluronic acid-related tetra-, hexa-, and
octa-saccharides having a methyl β-D-glucopyranosiduronic acid at the reducing end.
Carbohydrate Res 1996, 288, 109-125.
16. Huang, X.; Huang, L.; Wang, H.; Ye, X.-S., Iterative one-pot synthesis of
oligosaccharides. Angew Chem, Int Ed 2004, 43 (39), 5221-5224.
17. Zhang, Z.; Ollmann, I. R.; Ye, X.-S.; Wischnat, R.; Baasov, T.; Wong, C.-H.,
Programmable one-pot oligosaccharide synthesis. J Am Chem Soc 1999, 121 (4),
734-753.
18. Huang, L.; Teumelsan, N.; Huang, X., A facile method for oxidation of primary
alcohols to carboxylic acids and its application in glycosaminoglycan syntheses.
Chem-Eur J 2006, 12 (20), 5246-5252.
19. Dinkelaar, J.; Codee, J. D. C.; Van den Bos, L. J.; Overkleeft, H. S.; Van der Marel, G.
A., Synthesis of hyaluronic acid oligomers using Ph2SO/Tf2Omediated glycosylations. J Org Chem 2007, 72 (15), 5737-5742.
20. Codee, J. D. C.; Stubba, B.; Schiattarella, M.; Overkleeft, H. S.; Van Boeckel, C. A. A.;
Van Boom, J. H.; Van der Marel, G. A., A modular strategy toward the synthesis of
47

heparin-like oligosaccharides using monomeric building blocks in a sequential
glycosylation strategy. J Am Chem Soc 2005, 127 (11), 3767-3773.
21. Garcia, B. A.; Poole, J. L.; Gin, D. Y., Direct glycosylations with 1-hydroxy glycosyl
donors using trifluoromethanesulfonic anhydride and diphenyl sulfoxide. J Am Chem Soc
1997, 119 (32), 7597-7598.
22. Codee, J. D. C.; Litjens, R. E. J. N.; den Heeten, R.; Overkleeft, H. S.; van Boom, J.
H.; van der Marel, G. A., Ph2O/Tf2O: A powerful promotor system in chemoselective
glycosylations using thio glycosides. Org Lett 2003, 5 (9), 1519-1522.
23. Crich, D.; Smith, M., 1-Benzenesulfinyl piperidine/trifluoromethanesulfonic anhydride:
A potent combination of shelf-stable reagents for the low-temperature conversion of
thioglycosides to glycosyl triflates and for the formation of diverse glycosidic linkages. J
Am Chem Soc 2001, 123 (37), 9015-9020.
24. Dinkelaar, J.; Gold, H.; Overkleeft, H. S.; Codee, J. D. C.; van der Marel, G. A.,
Synthesis of hyaluronic acid oligomers using chemoselective and one-pot strategies. J
Org Chem 2009, 74 (11), 4208-4216.
25. Gold, H.; Munneke, S.; Dinkelaar, J.; Overkleeft, H. S.; Aerts, J. M. F. G.; Codee, J. D.
C.; van der Marel, G. A., A practical synthesis of capped 4-methylumbelliferyl hyaluronan
disaccharides and tetrasaccharides as potential hyaluronidase substrates. Carbohydrate
Res 2011, 346 (12), 1467-1478.
26. Lu, X.; Kamat, M. N.; Huang, L.; Huang, X., Chemical synthesis of a hyaluronic acid
decasaccharide. J Org Chem 2009, 74 (20), 7608-7617.

48

CHAPTER 3
Chemical Synthesis of a HA10
3.1. Introduction:
HA is the major ligand of CD44. Evidence is accumulating suggesting that the size
of HA fragments recognized by CD44 provides a physiologically important switch
1

between its adhesion and signaling functions . Binding of HA polymers to CD44 usually
leads to cell adhesion

2-3

rather than activation. In contrast, recognition of HA fragments

instead of the polymer leads to CD44 signaling, which is involved in diseases such as
cancer and inflammation

4-7

. CD44 requires at least a HA6 sequence for binding. and
8

only a HA10 or higher can effectively compete with polymeric HA . Therefore, access to
synthetic HA with variable length and sequence can greatly facilitate the establishment of
9

its SAR .
HA can be synthesized via either enzymatic or chemical methods. Although
impressive enzymatic synthesis has been achieved, the inherent substrate specificities
of enzymes limit the structural diversity of HA analogs that can be generated. Chemical
synthesis can thus complement the enzymatic approaches to create greater varieties of
HA structures.
10

The longest sHA chemically assembled to date are an HA8 . Previously, our
group has developed a synthetic strategy to acquire sHA ranging from di- to
11

hexa-saccharides . With the fascinating biological activities of longer sHA, we became
interested in pursuing their chemical synthesis, which is not a trivial extension of shorter
49

oligomers as elongation of the sugar sequences but can create significant new
challenges in both glycosylation and deprotection. Herein, we report our results on
overcoming the obstacles for chemical synthesis of an HA10.
3.2. Results and Discussion:
The design of a successful route to HA oligosaccharides must take into account
three factors: 1) stereochemical control in glycosylation; 2) introduction of the GlcUA;
and 3) protective groups for the glucosamine nitrogen moieties. In our previous HA6
synthesis, we used two key building blocks 1 and 2, which led to high yield of
11

hexasaccharide 3 in one pot (Scheme 3.1) . The Bz and Phth groups were crucial to
facilitate the formation of 1,2-trans glycosidic linkages.
12

reactivities ,

Due to its high inherent
11

glucoside building block 1 was utilized as a GlcUA surrogate . The

PMB groups in 3 masking the glucoside 6-O-position were selectively removed by DDQ
oxidation and the resulting triol was successfully oxidized and deprotected producing
HA6. Following the success of HA6 synthesis, our initial attempt towards HA10 hinged
upon the usage of monosaccharides 1 and 2, from which decasaccharide 4 was
assembled. However, removal of the five PMB groups in 4 turned out to be very
problematic (Scheme 3.2a). The previously employed DDQ oxidation
13

the oligosaccharide. CAN oxidation
14

acid (TfOH)

11

decomposed

or the combination of p-tolyl sulfonamide and triflic

also failed to lead to the desired penta-ol. To circumvent this problem, the

6-OH group liberation and oxidation state adjustment were performed on disaccharide 6
(Scheme 3.2b). DDQ mediated PMB removal followed by primary OH oxidation and
50

benzyl ester formation produced disaccharide 7 containing the GlcUA moiety.
Condensations of the resulting disaccharides afforded decasaccharide 8 with the GlcUA
already installed, which set the stage for deprotection. Although it has been shown
previously that Phth groups could be deprotected in molecules containing carboxylic
11, 15-16

esters

,

despite repeated trials and screening of a variety of reagents and

reaction conditions, the conditions required to remove all five Phth groups in
disaccharide 7 turned out to be incompatible with the five carboxylic ester moieties
present and vice versa. This prompted us to abandon the Phth group and utilize the TCA
group

10, 17

as an alternative protective group for nitrogen.

Based

on

these

considerations,

a

new

retrosynthetic

route

towards

decasaccharide 9 was designed (Scheme 3.3). The fully protected decasaccharide 10
would be assembled from disaccharides 11, 12 and 13, which in turn would be derived
from monosaccharide building blocks 1, 14 and 15.
Scheme 3.1. Previous synthesis on HA6 3

51

Figure 3.1. Compound 4

Scheme 3.2. Previous synthesis on HA10

Glucoside donor 1 was prepared according to a literature procedure

11

and the

syntheses of glucosamine building blocks 14 and 15 were outlined in Scheme 3.4.
Peracetylated N-TCA-glucosamine 16

10

was transformed into the corresponding

1-β-p-tolyl thioglycoside 17 and the 1-β-methoxy derivative 18. Compound 17 was then
52

deacetylated and protected with a benzylidene group to afford 14, whereas compound
18 was protected by benzylidene to give 15.
Scheme 3.3. Retrosynthetic scheme towards HA10 9

Scheme 3.4. Synthesis of monosaccharide building blocks 14 and 15

53

Scheme 3.5. Assembly of HA2 building blocks

With the monosaccharides in hand, disaccharide synthesis was performed.
Pre-activation of glucoside donor 1 by the reagent combination of AgOTf and
11, 18

p-Toluenesulfenyl chloride (p-TolSCl)

at – 78 C was performed followed by the

addition of acceptor 14 and a bulky base TTBP

19

leading to disaccharide 19 in 80% yield.

The primary OH group in 19 was liberated by treatment with DDQ and oxidized by PDC
in DMF to afford its carboxylic acid derivative, which was protected as a benzyl ester
(disaccharide 11) in 65% yield for the 3 steps. Subsequent removal of the TBS group led
to the disaccharide acceptor 12 bearing a free secondary OH group (Scheme 3.5a).
Disaccharide 13 was constructed through a similar sequence from 1 and 15 in an overall
54

yield of 48% for the 5 steps (Scheme 3.5b).
Following the same glycosylation protocol as used in the preparation of
disaccharide 19, coupling of disaccharide donor 11 with acceptor 12 was attempted
(Scheme 3.7a). However, no desired tetrasaccharide was obtained with full recovery
of acceptor 12.

3

20-21

Oxazoline derivative 20 ( JH1-H2 = 8.0 Hz

) was isolated from the

reaction in quantitative yield with the possible mechanism for its formation depicted in
Scheme 3.6b.

Neighboring group participation by the TCA group through attachment

of its carbonyl oxygen to the oxacarbenium ion forms the oxazolinium ion intermediate
22, which can react with an acceptor to afford the expected 1,2-trans-glycoside 23
(pathway a, Scheme 3.6b). Alternatively, deprotonation of the oxazolinium ion produces
the stable trichloro-oxazoline 24 (pathway b).
Scheme 3.6. Oxazoline formation

55

Table 3.1. The effects of additives on glycosylation product distribution
Entries
1

Reaction conditions

Donor 11 + Donor 25 + Donor 25 +
acceptor 12
acceptor 13
acceptor 28
20 (100%)
-

st

1 Generation
TTBP was added after
donor was activated

2

2

nd

25 (55%) +
20 (40%)

26 (40%) +
27 (50%)

10 (10%) +
27 (85%)

25 (55%)

26 (60%)

10 (40%)

25 (82%)

Generation

26 (71%)

10 (77%)

No TTBP was added after
donor was activated
3

3

rd

Generation

TfOH was added after
donor was activated
4

4

th

Generation

TMSOTf was added after
donor was activated
Following this mechanism, the failure of glycosylation could be attributed to the
17

addition of TTBP , which is used to scavenge the TfOH accumulated upon productive
glycosylation. Therefore, a 2

nd

omitting TTBP from the reaction.

generation glycosylation protocol was adopted by
Following this protocol, 11 (1 eq.) was condensed

with 12 (1 eq.) to produce tetrasaccharide 25 in 55% yield with 40% oxazoline by-product
20 (Table 3.1, entry 2). The next condensation of tetrasaccharide donor 25 (1 eq.) with
disaccharide acceptor 13 gave hexasaccharide 26 in 40% yield and oxazoline 27 (50%
yield). Subsequent cleavage of TBS group in 26 gave the hexasaccharide acceptor 28 in
90% yield. Disappointingly, the final coupling of 25 (1.25 eq.) and 28 (1 eq.) only gave
decasaccharide 10 in 10% yield, with the majority of 25 converted to oxazoline 27 (85%

56

yield, Table 3.1, entry 2). The substantial increase of the amounts of oxazoline side
products is presumably because with the increasing sizes of the glycosyl donor and
acceptor, the compounds become less reactive towards glycosylation (Scheme 3.6b,
pathway a), which in turn favors the competing reaction of oxazoline formation even in
the absence of base (Scheme 3.6b, pathway b).
Figure 3.2. Compound 25, 26, 27 and 28

In order to suppress the amount of oxazoline, exogenous TfOH (~ 0.4 eq.) was
introduced to the reaction mixture after the donor activation to shift the equilibrium from
oxazoline 24 to the oxazolinium ion 22 (Scheme 3.6b). Following this 3

rd

generation

procedure, although the reaction of 11 and 12 was not affected, the yields for 25 with 13
and 28 were enhanced to 60% and 40% respectively (Table 3.1, entry 3). The acid
sensitive benzylidene moieties were found to be stable under this condition. To further
improve on the glycosylation, Lewis acid TMSOTf was examined as an alternative to
57

TfOH. Based on this protocol, significant enhancements were observed for all three
reactions (Table 3.1, entry 4), with the yield of decasaccharide 10 reaching 77%, which
enabled us to acquire over 200 mg of this decasaccharide. These results suggest that
the addition of TMSOTf successfully suppressed the formation of trichloro-oxazoline
by-products.
Deprotection of large complex oligosaccharides could be very challenging due to
the presence of multiple protective groups, as observed in our attempts on
decasaccharides 4 and 8. To deprotect decasaccharide 10, its TBS group was removed
first by HF in Py to give 29 in 79% yield. This was followed by a mild basic condition (20
eq. of KOH added in 10 portions to a solution of 29 over 2 days

22

) in order to cleave all

ten carboxylic esters and five trichloroacetamides (Scheme 3.7). Harsh basic conditions
should be avoided to prevent epimerization of the GlcUA. The progress of the hydrolysis
turned out to be difficult to monitor by TLC for two reasons. First, the liberated amino
groups gave no ninhydrin test presumably due to steric hindrance. Secondly, the
compounds with free carboxylic acid and amino groups became zwitterionic, which
streaked on TLC. MS analysis of the crude reaction mixture did not yield much
information resulting from signal suppression by the presence of salts. NMR analysis of
the reaction mixture turned out to be most informative. The cluster of peaks around 8.0
ppm in

1

H-NMR assigned to the ortho-hydrogens of Bz groups, merged into a doublet

like pattern over time signifying the cleavage of benzoyl esters to benzoate salt.
Another characteristic of the NMR is that the set of four doublets around 7.0 ppm
58

representing the amide protons of trichloroacetamide groups in 29, became smaller and
eventually disappeared as the reaction proceeded. The rate of trichloroacetamide
cleavage was much slower compared with that of the benzoates. When the crude NMR
spectra did not show much change after week 5, the reaction was stopped. The reaction
mixture was N-acetylated followed by hydrogenation and size exclusion chromatography
to produce the fully deprotected HA decasaccharide 9 in 35% yield from 29. NMR
analysis indicated all the glycosyl linkages were beta, confirming no epimerizations
occurred despite the prolonged reaction time.
Scheme 3.7. Deprotection

59

3.3. Conclusions
We reported the first chemical synthesis of a fully deprotected HA10. Our
pre-activation based thioglycoside strategy with p-TolSCl/AgOTf as the promoter was
successfully applied for efficient construction of the decasaccharide backbones.
However, considerable difficulties were encountered in deprotection, with the selection
of nitrogen protective group turned out to be crucial. While removal of all five Phth was
found to be incompatible with the carboxylic esters present in the decasaccharide, TCA
could be removed together with the esters under a mild basic condition. The TCA
associated oxazoline formation side reaction during glycosylation was suppressed by
addition of TMSOTf to the reaction mixture. Our experience highlights the challenges
associated with assembly of large oligosaccharides, as the conditions employed for
synthesis of shorter counterparts may not be directly translatable and each synthesis
needs individual optimization and development. We believe our strategy on the HA
decasaccharide assembly can open up possibilities to chemical synthesis of longer sHA
and facilitate the structure-activity relationship studies of this important class of
molecules.

60

3.4. Experimental Section:
General procedure for deprotection of PMB group: PMB-protected compound
(1 mmol) and DDQ (1.3 eq.) were dissolved in a solvent mixture of dichloromethane
(DCM) and a saturated aqueous solution of sodium bicarbonate (NaHCO3) (v/v 19:1),
which was kept in dark. The reaction mixture is stirred for 2 hrs from 0 °C to room
temperature (r.t.). Then a 2
mixture, and a 3
the 2

nd

rd

nd

portion of DDQ (0.5 eq.) was added to the reaction

portion of DDQ (0.5 eq.) could be added 15 min after the addition of

portion to further push the reaction to completion. When most starting material

disappeared, the reaction mixture was diluted with DCM and washed repeatedly with a
saturated aqueous solution of NaHCO3 until the organic layer become colorless. The
organic layer was concentrated, and the resultant residue was purified by silica gel
chromatography to give the desired compound.
General procedure for oxidation of alcohols to carboxylic acids: After a
mixture of alcohol (1 mmol) and MS-4Ǻ (5 g) in anhydrous dimethylformamide (DMF)
was stirred at r.t. for 2 hrs, a solution of pyridinium dichromate (PDC) (6 mmol) in DMF
was added dropwise to the reaction. The reaction mixture was stirred at r.t. until thin
layer chromatography (TLC) indicated completion of the reaction, which typically
required overnight. The reaction was diluted with ethyl acetate (EA) and filtrated to
remove the insoluble PDC.

The filtrate was washed with brine to remove DMF. The

crude product was then purified by silica gel chromatography with EA/DCM/methanol
(MeOH) solvent systems.
61

General procedure for benzyl ester formation: The carboxylic acid containing
compound was dissolved in DCM (5 ml) and treated with PhCHN2 solution in diethyl
ether (Et2O) (~2 eq.) for 2–3 hrs until the disappearance of all the starting material. The
crude product was purified by silica gel chromatography.
General procedure for removal of TBS groups: The TBS-protected compound
(0.5 mmol) was dissolved in pyridine (Py) (4 ml) in a plastic flask followed by the addition
of 65–70% hydrogen fluoride (HF)/Py solution (2 ml) at 0 °C. The solution was stirred for
24 hrs until TLC indicated completion of the reaction. The reaction mixture was diluted
with EA (50 ml) and washed with 10% aqueous copper sulfate (CuSO4) solution (20 ml).
The aqueous phase was extracted with EA (30 ml) twice and the combined organic
layers were washed with a saturated aqueous solution of NaHCO 3 to remove HF. The
crude product was purified by silica gel chromatography.
p-Tolyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-Dglucopyranoside (14): After D-glucosamine hydrochloride (10.8 g, 50.0 mmol) and
sodium methoxide (NaOMe) (50.0 ml, 1M NaOMe/MeOH, 50.0 mmol) in MeOH were
stirred at r.t. under N2 protection for 45 min, trichloroacetyl chloride (5.61 ml, 50.0 mmol)
and triethylamine (TEA) (6.95 ml, 50.0 mmol) were added. After overnight when
electrospray ionization mass spectrometry (ESI-MS) indicated that D-glucosamine
hydrochloride completely disappeared, MeOH was removed by a rotary evaporator and
the flask was placed under high vacuum for 2 hrs. Ac2O and Py (150 ml, v/v 1:2) were
then added and the reaction mixture was stirred for 2 days. The mixture was
62

concentrated, and the resulting residue was diluted with EA and washed with a saturated
aqueous solution of NaHCO3, 10% HCl, H2O, and brine. The organic phase was dried
over

Na2SO4,

filtered,

and

concentrated.

After

1,3,4,6-tetra-O-acetyl-2-deoxy-2-trichloroacetamido-D-glucopyranoside

recrystallization,
(16)

was

obtained as a white solid (16.3 g, 33.0 mmol, 66% for 2 steps). Boron trifluoride etherate
(BF3.Et2O) (7.75 ml, 89 mmol) was added to a solution of 16 (4.93 g, 10 mmol) and
p-toluenethiol (1.99 g, 16.0 mmol) in dry DCM (200 ml). The reaction mixture was stirred
at r.t. under N2 protection overnight, diluted with DCM (300 ml), washed with saturated
NaHCO3, dried over Na2SO4, filtered, and concentrated. Recrystallization afforded
p-tolyl 3,4,6-tri-O-acetyl-2-deoxy-2-trichloroacetamido-1-thio-β-D-glucopyranoside (17)
as a white solid (5.01 g, 9.00 mmol, 90%). 17 (5.01 g, 9.00 mmol) was dissolved in mixed
solvent of DCM/MeOH (v/v 1:1, 200 ml).

NaOMe (2.70 ml, 1M in MeOH, 2.70 mmol)

was added, and the mixture was stirred at r.t. under N2 protection for 2 hrs, neutralized
with Amberlite IR-120, concentrated and dried under vacuum. Benzaldehyde dimethyl
acetal (1.52 ml, 10.8 mmol) was added to a solution of resulting residue and
camphorsulfonic acid (CSA) (0.750 g, 4.05 mmol) in anhydrous toluene (200 ml). The
reaction mixture was stirred under N2 protection at 80 °C for 1 hr. When TLC showed
around 70% conversion of starting material to product, the reaction flask was placed on a
high vacuum rotary evaporator to further push the reaction to completion. When toluene
was completely removed by the high vacuum rotary evaporator, the reaction mixture was
neutralized with TEA in DCM.

The mixture was diluted with DCM (300 ml) and washed
63

with saturated NaHCO3, dried over Na2SO4, filtered, and concentrated. Recrystallization
afforded 14 as a white solid (3.27 g, 6.30 mmol, 70% for 2 steps).
Methyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-Dglucopyranoside (15): Compound 16 (4.93 g, 10 mmol) was dissolved in HBr in AcOH
(30.0 ml, 33% wt., 174 mmol).

After 6 hrs, the mixture was diluted with DCM (240 ml)

and poured onto crushed ice in saturated NaHCO3 (600 ml). The organic phase was
separated and washed again with saturated NaHCO3 until the pH reached 7, which was
then dried over sodium sulfate (Na2SO4), filtered, and concentrated. The resulting crude
residue was mixed with silver carbonate (Ag2CO3) (2.76 g, 10.0 mmol), calcium
carbonate (CaSO4) (2.72 g, 20.0 mmol) and anhydrous MeOH (100 ml). The mixture was
stirred at r.t. under N2 protection for 1 day until TLC showed one major product, on which
all three acetyl groups has already been cleaved because of the basic reaction condition.
The mixture was concentrated and purified by silica gel chromatography with
DCM/MeOH system. Methyl 2-deoxy-2-trichloroacetamido-β-D-glucopyranoside (18)
was afforded (2.70 g, 8.00 mmol, 80% for 2 steps). Compound 18 (2.70 g, 8.00 mmol)
was mixed with CSA (0.84 g, 3.60 mmol), benzaldehyde dimethyl acetal (1.35 ml, 9.60
mmol) in anhydrous toluene (200 ml). The reaction mixture was stirred under N2
protection at 80 °C for 1 hr. When TLC showed around 70% conversion of starting
material to product, the reaction flask was placed on a high vacuum rotary evaporator to
further push the reaction to completion. When toluene was completely removed by the
high vacuum rotary evaporator, the reaction mixture was neutralized with TEA in DCM.
64

The mixture was diluted with DCM (300 ml) and washed with saturated NaHCO3, dried
over Na2SO4, filtered, and concentrated. Recrystallization afforded 15 as a white solid
20

(2.39 g, 5.6 mmol, 70%). [α]D

-29.3

o

1

(c 0.5, DCM); H NMR (500 MHz, CDCl3): δ =

3.46-3.59 (m, 6 H, -OCH3, H-2, H-4, H-5), 3.80-3.84 (m, 1 H, H-6), 4.38-4.42 (m, 2 H,
H-3, H-6), 4.87 (d, 1 H, J = 8.0 Hz, H-1), 5.57 (s, 1 H, CHPh), 6.93 (d, 1 H, J = 7.0 Hz,
NH), 7.38-7.51 (m, 5 H, CHarom);

13

C NMR (125 MHz, CDCl3): δ = 57.8 (-OCH3), 59.8

(C-2), 66,4 (C-5), 68,8 (C-6), 69.8 (C-3), 81.8 (C-4), 92.5 (CCl3), 101,2 (C-1), 102,2
(CHPh), 126.5 -129.6 (Carom), 137.12 (Cq Carom), 162.6 (C=O TCA); HRMS [M + Na]+:
m/z: calcd for C16H18Cl3NNaO6 448.0097, found 448.0088.
p-Tolyl O-(2-O-benzoyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-6-O-pmethoxybenzyl-β- D-glucopyranosyl)-(13)-4,6-O-benzylidene-2-deoxy-1-thio-2trichloroacetamido-β-D- glucopyranoside (19): The mixture of donor 1 (715 mg, 1
mmol) and freshly activated molecular sieve (MS)-4Ǻ (800 mg) in Et2O (20.0 ml) was
stirred in a dry ice–isopropanol bath for 1 hr, followed by the addition of AgOTf (771 mg,
3.00 mmol) in Et2O (10.0 ml). After 5 min, orange colored p-TolSCl (157 μl, 1.00 mmol)
was added via a micro-syringe. 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. The temperature at which pre-activation was performed was not crucial as
long as it did not exceed -50 °C. After the donor was completely consumed according to
65

TLC analysis (about 5 min at −78 °C), a solution of acceptor 14 (467 mg, 0.900 mmol)
and TTBP (248 mg, 1.00 mmol) in a mixture solvent of DCM and MeCN (v/v 19:1, 3.00
ml) was then added dropwise within 10 min to the reaction mixture (MeCN was
necessary as acceptors 14 were not freely soluble in DCM). The reaction mixture was
warmed to −10 °C under stirring in 2 hrs. TEA (0.500 ml) was then added and the
mixture was diluted with DCM (100 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
NaHCO3 (100 ml) and twice with H2O (100 ml). The organic layer was concentrated and
the crude product was purified by silica gel chromatography. Compound 19 (800 mg,
0.720 mmol, 80%) was afforded as white solid. [α]D

20

+22.2

o

1

(c 0.5, DCM); H NMR

(500 MHz, CDCl3): δ = -0.08 (s, 3 H, SiCH3), -0.05 (s, 3 H, SiCH3), 0.79 (s, 9 H,
SiC(CH3)3), 2.35 (s, 3 H, PhCH3), 3.35-3.43 (m, 3 H, H-5’, H-6’, H-2), 3.49 (ddd, 1 H, J =
4.5 Hz, 5.0 Hz, 9.0 Hz, H-5), 3.56-3.68 (m, 4 H, H-3’, H-4’, H-6, H-6’), 3.72 (t, 1 H, J = 9.0
Hz, H-4), 3.80 (s, 3 H, CH3 PMB), 4.27 (dd, 1 H, J = 5.0 Hz, 10.5 Hz, H-6), 4.34 (d, 1 H, J
= 12.0 Hz, CH2PMP), 4.54 (d, 1 H, J = 11.0 Hz, CH2Ph), 4.58 (d, 1 H, J = 11.0 Hz,
CH2Ph), 4.63 (d, 1 H, J = 12.0 Hz, CH2PMP), 4.66 (t, 1 H, J = 9.0 Hz, H-3 ) ,4.93 (d, 1 H,
J = 8.0 Hz, H-1’), 5.20 (t, 1 H, J = 8.0 Hz, H-2’), 5.32 (s, 1 H, CHPh), 5.44 (d, 1 H, J =
10.5 Hz, H-1), 6.89 (d, 1 H, J = 8.5 Hz, NH), 7.04-7.11 (m, 7 H, CHarom), 7.20-7.42 (m,
14 H, CHarom), 7.82-7.84 (m, 2 H, CHarom Bz);

66

13

C NMR (125 MHz, CDCl3): δ = -4.5

(SiCH3), -3.6(SiCH3), 18.1 (SiC(CH3)3), 21.4 (PhCH3), 26.0 (x3, SiC(CH3)3), 55.5
(PhOCH3), 57.6 (C-5’), 68.7 (C-6), 69.1 (C-6’), 70.9 (C-5), 71.7 (C-4’), 73.3, (CH2PMP),
74.4 (C-2’), 75.0 (CH2Ph), 76.0 (C-3), 77.4 (C-2), 78.8 (C-4), 83.5 (C-3’), 85.4 (C-1), 92.5
(CCl3), 98.0 (C-1’), 101.3 (CHPh), 114.0 (Carom-2 PhOCH3), 126.4-130.2 (CHarom),
133.3, 133.8 (x2), 137.3, 137.9, 139.0 (Cq SPhCH3, Cq CHPh, Cq Bn, Cq Bz, Carom-4
PhOCH3), 159.6 (Carom-1 PhOCH3), 161.7, 165.3 (C=O TCA, C=O Bz); HRMS [M +
Na]+: m/z: calcd for C56H64Cl3NNaO12SSi 1130.2882, found 1130.2845.
p-Tolyl

O-(benzyl

2-O-benzoyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-β-D-

glucopyranosyluronate)-(13)-4,6-O-benzylidene-2-deoxy-1-thio-2-trichloroaceta
mido-β-D-glucopyranoside (11): 19 was converted to 11 in 3 steps (65% for 3 steps)
following the general procedures for deprotection of PMB, oxidation of alcohol to
20

carboxylic acid and benzyl ester formation. [α]D

+10.2

o

1

(c 0.5, DCM); H NMR (500

MHz, CDCl3): δ = -0.16 (s, 3 H, SiCH3), -0.13 (s, 3 H, SiCH3), 0.74 (s, 9 H, SiC(CH3)3),
2.36 (s, 3 H, PhCH3), 3.30 (ddd, 1 H, J = 7.5 Hz, 9.0 Hz, 10.5 Hz, H-2), 3.49-3.55 (m, 2
H, H-3’, H-5), 3.65-3.70 (m, 2 H, H-6, H-4), 3.83 (d, J = 6.5 Hz, H-5’), 4.13 (t, J = 6.5 Hz,
H-4’), 4.32 (dd, 1 H, J = 5.0 Hz, 10.0 Hz, H-6), 4.54 (t, 1 H, J = 9.0 Hz, H-3) ,4.59 (d, 1 H,
J = 11.5 Hz, -CH2Ph ), 4.65 (d, 1 H, J = 11.5 Hz, -CH2Ph), 5.03 (d, 1 H, J = 7.0 Hz, H-1’),
5.13 (d, 1 H, J = 10.5 Hz, -COOCH2Ph ), 5.17 (d, 1 H, J = 10.5 Hz, -COOCH2Ph ), 5.18
(dd, 1 H, J = 6.0 Hz, 7.0 Hz, H-2’), 5.29 (d, 1 H, J = 10.5 Hz, H-1), 5.32 (s, 1 H, CHPh),
6.96 (d, 1 H, J = 7.5 Hz, NH), 7.10-7.18 (m, 7 H, CHarom), 7.26-7.38 (m, 14 H, CHarom),

67

7.51-7.54 (m, 1 H, CHarom), 7.88-7.89 (m, 2 H, CHarom Bz);

13

C NMR (125 MHz,

CDCl3): δ = -5.0 (SiCH3), -4.3 (SiCH3), 18.0 (SiC(CH3)3), 21.4 (PhCH3), 25.9 (x3,
SiC(CH3)3), 57.5 (C-2), 67.4 (COOCH2Ph), 68.8 (C-6), 70.8 (C-5), 71.4 (C-4’), 73.7
(OCH2Ph), 74.4 (C-2’), 76.5 (C-3), 77.6 (C-5’), 80.2 (C-4), 81.3 (C-3’), 84.6 (C-1), 92.2
(CCl3), 99.0 (C-1’), 101.8 (CHPh), 126.5-130.2 (CHarom), 133.5, 134.2, 135.3, 137.3,
137.9, 139.2 (Cq STol, Cq CHPh, Cq OBn, Cq Bz, Cq COOBn), 161.8, 165.4, 168.6 (C=O
TCA, C=O COOBn, C=O Bz); HRMS [M + Na]+ calcd for C55H60Cl3NNaO12SSi
1114.2563, found 1114.2560.
p-Tolyl O-(benzyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyluronate)(13)- 4,6-O-benzylidene-2-deoxy-1-thio-2-trichloroacetamido-β-Dglucopyranoside (12): 11 was converted to 12 following the general procedure for TBS
removal (90%). [α]D

20

+5.5 o (c 1.0, DCM); 1H NMR (500 MHz, CDCl3): δ = 2.36 (s, 3 H,

PhCH3), 3.02 (s, 1 H, OH), 3.30 (ddd, 1 H, J = 7.0 Hz, 9.0 Hz, 10.5 Hz,H-2), 3.52 (ddd, 1
H, J = 4.0 Hz, 4.5 Hz, 9.0 Hz, H-5), 3.55-3.60 (m, 3 H, H-3’, H-5’, H-6), 3.70 (t, 1 H, J =
9.0 Hz, H-4), 4.03 (t, 1 H, J = 9.0 Hz, H-4’), 4.27 (dd, 1 H, J = 4.0 Hz, 10.0 Hz, H-6), 4.59
(t, 1 H, J = 9.0 Hz, H-3 ) ,4.62 (d, 1H, J = 11.5 Hz, -CH2Ph ), 4.72 (d, 1 H, J = 11.5 Hz,
-CH2Ph ), 4.86 (d, 1 H, J = 8.0 Hz, H-1’), 5.15 (d, 1 H, J = 11.5 Hz, -COOCH2Ph ), 5.17
(t, 1 H, J = 8.0 Hz, H-2’), 5.26 (d, 1 H, J = 11.5 Hz, -COOCH2Ph ), 5.32 (s, 1 H, CHPh),
5.37 (d, 1 H, J = 10.5 Hz, H-1), 7.05 (d, 1 H, J = 7.0 Hz, NH), 7.08-7.18 (m, 7 H, CHarom),
7.30-7.45 (m, 14 H, CHarom), 7.54-7.57 (m, 1 H, CHarom), 7.92-7.94 (m, 2 H, CHarom

68

Bz);

13

C NMR (125 MHz, CDCl3): δ = 21.4 (PhCH3), 57.4 (C-2), 68.1 (COOCH2Ph), 68.7

(C-6), 70.7 (C-5), 72.2 (C-4’), 73.2 (C-2’), 74.0 (OCH2Ph), 74.7 (C-3), 76.9 (C-5’), 80.1
(C-4), 81.0 (C-3’), 84.4 (C-1), 92.1 (CCl3), 99.6 (C-1’), 101.4 (CHPh), 126.3-130.2
(CHarom), 133.6, 134.1, 134.8, 137.3, 137.8, 139.2 (Cq STol, Cq CHPh, Cq OBn, Cq Bz,
Cq COOBn), 161.8, 165.3, 169.3 (C=O TCA, C=O COOBn, C=O Bz); HRMS [M + Na]+:
m/z: calcd for C49H46Cl3NNaO12S 1000.1699, found 1000.1706.
Methyl O-(benzyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyluronate)
-(13)-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-glucopyranoside (13):
The mixture of donor 1 (715 mg, 1.00 mmol), acceptor 15 (427 mg, 1.00 mmol) and
freshly activated MS-4Ǻ (800 mg) in a mixture solvent of DCM and MeCN (v/v 40:1, 20.0
ml) was stirred for 1 hr at -78 °C, followed by the addition of AgOTf (771 mg, 3.00 mmol)
in Et2O (10 ml). After 5 min, p-TolSCl (157 μl, 1.00 mmol) was added via a syringe to
activate the donor. The yellow color of the reaction disappears quickly and TLC analysis
showed the donor was completely consumed. A solution of TTBP (248 mg, 1.00 mmol)
in DCM (1.00 ml) was then added dropwise to the reaction mixture. The reaction mixture
was warmed to −10 °C under stirring in 2 hrs, followed by the same workup and
purification procedures described above for the synthesis of compound 19. The resulting
disaccharide was subjected to deprotection of PMB, oxidation of alcohol to carboxylic
acid, benzyl ester formation and TBS removal reactions following the general procedures
20

to afford compound 13 (426 mg, 0.480 mmol, 48% for 5 steps) as a white solid. [α]D

69

+8.2 o (c 0.5, DCM); 1H NMR (500 MHz, CDCl3): δ = 3.00 (d, 1 H, J = 2.5 Hz, OH), 3.30
(ddd, 1 H, J = 7.0 Hz, 8.0 Hz, 9.5 Hz, H-2), 3.48-3.50 (m, 4 H, -OCH3, H-5), 3.57-3.60 (m,
3 H, H-3’, H-5’, H-6), 3.76 (t, 1 H, J = 9.0 Hz, H-4), 4.03 (ddd, 1 H, J = 2.5 Hz, 9.0 Hz, 9.0
Hz, H-4’), 4.26 (dd, 1 H, J = 5.0 Hz, 10.5 Hz, H-6), 4.59 (dd, 1 H, J = 9.0 Hz, 9.5 Hz, H-3 )
,4.63 (d, 1 H, J = 11.5 Hz, -CH2Ph ), 4.73 (d, 1 H, J = 11.5 Hz, -CH2Ph ), 4.88 (d, 1 H, J =
7.5 Hz, H-1’), 4.99 (d, 1 H, J = 8.0 Hz, H-1), 5.14 (d, 1 H, J = 12.0 Hz, -COOCH2Ph ),
5.20 (dd, 1 H, J = 7.5 Hz, 9.5 Hz, H-2’), 5.26 (d, 1 H, J = 12.0 Hz, -COOCH2Ph ), 5.36 (s,
1 H, CHPh), 7.05 (d, 1 H, J = 7.0 Hz, NH), 7.08-7.09 (m, 3 H, CHarom), 7.27-7.40 (m, 14
H, CHarom), 7.54-7.57 (m, 1 H, CHarom), 7.92-7.94 (m, 2 H, CHarom Bz);

13

C NMR (125

MHz, CDCl3): δ = 57.8 (-OCH3) 59.3 (C-2), 66.3 (C-5), 68.0 (COOCH2Ph), 68.8 (C-6),
72.3 (C-4’), 73.2 (C-2’), 74.0 (C-5’), 74.7 (OCH2Ph), 76.2 (C-3), 80.3 (C-4), 81.1 (C-3’),
92.1 (CCl3), 99.7 (C-1), 100.0 (C-1’), 101.4 (CHPh), 126.2-130.2 (CHarom), 133.5, 134.9,
137.4, 138.0 (Cq CHPh, Cq OBn, Cq Bz, Cq COOBn), 162.3, 165.3, 169.3 (C=O TCA,
C=O COOBn, C=O Bz); HRMS [M + Na]+: m/z: calcd for C43H42Cl3NNaO13 908.1619,
found 908.1616.
2-Trichloromethyl O-(benzyl 2-O-benzoyl-3-O-benzyl-4-O-tertbutyldimethylsilyl-β-D- glucopyranosyluronate)-(13)-4,6-O-benzylidene-1,220

dideoxy-α-D-glucopyrano-[2,1-d]-2-oxazoline (20): [α]D

+54.1

o

(c 1.0, DCM); 1H

NMR (500 MHz, CDCl3): δ = -0.06 (s, 3 H, SiCH3), -0.05 (s, 3 H, SiCH3), 0.81 (s, 9 H,
SiC(CH3)3), 3.53 (dd, 1 H, J = 5.0 Hz, 10.0 Hz, H-6), 3.61 (t, 1 H, J = 10.0 Hz, H-5), 3.68
(t, 1 H, J = 7.5 Hz, H-3’), 3.91 (dd, 1 H, J = 7.5 Hz, 10.0 Hz, H-4), 4.01 (d, 1 H, J = 8.0 Hz,
70

H-5’) ,4.15-4.18 (m, 2 H, H-3, H-4’), 4.22 (dd, 1 H, J = 5.0 Hz, 8.0 Hz, H-2), 4.34 (dd, 1 H,
J = 5.0 Hz, 10.0 Hz, H-6), 4.66 (d, 1 H, J = 12.5 Hz, CH2Ph ), 4.69 (d, 1 H, J = 12.5 Hz,
CH2Ph ), 5.02 (d, 1 H, J =7.5 Hz, H-1’), 5.11 (d, 1 H, J = 12.0 Hz, COOCH2Ph), 5.15 (d,
1 H, J = 12.0 Hz, COOCH2Ph), 5.38 (t, 1 H, J = 7.5 Hz, H-2’), 5.39 (s, 1 H, CHPh), 6.08
(d, 1 H, J = 8.0 Hz, H-1), 7.13-7.17 (m, 4 H, CHarom), 7.31-7.43 (m, 13 H, CHarom),
7.53-7.70 (m, 1 H, CHarom), 8.00-8.10 (m, 2 H, CHarom Bz);

13

C NMR (125 MHz,

CDCl3): δ = -4.9 (SiCH3), -4.0 (SiCH3), 18.1 (SiC(CH3)3), 26.1 (x3, SiC(CH3)3), 63.4
(C-5), 67.5 (COOCH2Ph), 68.7 (C-6), 69.0 (C-2), 72.1 (C-4’), 74.1 (C-2’), 74.6
(OCH2Ph), 77.4 (C-5’), 78.7 (C-4), 80.3 (C-3), 82.3 (C-3’), 101.1 (C-1’), 101.5 (CHPh),
105.3 (C-1), 110.0 (CCl3), 125.5-128.7 (CHarom), 133.5, 135.2, 137.1, 137.8 (Cq CHPh,
Cq OBn, Cq Bz, Cq COOBn), 162.5, 165.5, 168.2 (C=N, C=O COOBn, C=O Bz); HRMS
[M + Na]+: m/z: calcd for C48H52Cl3NNaO12Si 990.2222, found 990.2231.
p-Tolyl

O-(benzyl

2-O-benzoyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-β-D-

glucopyranosyluronate)-(13)-O-(4,6-O-benzylidene-2-deoxy-2-trichloroacetamid
o-β-D-glucopyranosyl)-(14)-O-(benzyl 2-O-benzoyl-3-O-benzyl-β-Dglucopyranosyluronate) -(13)-4,6-O-benzylidene-2-deoxy-1-thio-2trichloroacetamido-β-D-glucopyranoside (25): The mixture of donor 11 (37.0 mg,
33.9 μmol) and freshly activated MS-4Ǻ (300 mg) in Et2O (2.00 ml) was stirred for 1 hr at
-78 °C, followed by the addition of AgOTf (26.0 mg, 102 μmol) in Et2O (1.00 ml). After 5
min, p-TolSCl (5.30 μl, 33.9 μmol) was added via a micro-syringe to activate the donor.
The characteristic yellow color of p-TolSCl in the reaction solution dissipated rapidly
71

within a few seconds indicating depletion of p-TolSCl and TLC analysis showed the
donor was completely consumed.

A solution of acceptor 12 (30.0 mg, 30.5 μmol) in a

mixture solvent of DCM and acetonitrile (MeCN) (v/v 19:1, 0.700 ml) was then added
dropwise to the reaction mixture. After 5 min, TMSOTf (2 μl, 11.0 μmol) was added via
micro-syringe. The reaction mixture was warmed to −10 °C under stirring in 2 hrs
followed by the same workup and purification procedures described above for the
synthesis of compound 19. Compound 25 (54.0 mg, 27.8 μmol, 82%) was afforded as
20

white solid. [α]D

+2.9

o

(c 0.5, DCM); 1H NMR (500 MHz, CDCl3): δ = -0.14 (s, 3 H,

SiCH3), -0.11 (s, 3 H, SiCH3), 0.78 (s, 9 H, SiC(CH3)3), 2.31 (s, 3 H, PhCH3), 3.09 (ddd,
1 H, J = 5.0 Hz, 5.0 Hz, 10.0 Hz, H-5’’), 3.17-3.22 (m, 1 H, H-6’’), 3.44-3.50 (m, 2 H, H-2,
H-5), 3.53 (dd, 1 H, J = 9.5 Hz, 10.0 Hz, H-3), 3.55 (t, 1 H, J = 7.0 Hz, H-3’’’), 3.58-3.61
(m, 2 H, H-4’’, H-6), 3.64 (t, 1 H, J = 8.0 Hz, H-3’), 3.72 (t, 1 H, J = 9.0 Hz, H-2’’), 3.81
(dd, 1 H, J = 9.0 Hz, 9.5 Hz H-4), 3.86 (d, 1 H, J = 7.0 Hz, H-5’’’), 3.87 (d, 1 H, J = 8.0
Hz, H-5’), 4.02 (dd, 1 H, J = 5.0 Hz, 10.5 Hz, H-6’’), 4.17 (t, 1 H, J = 7.0 Hz, H-4’’’), 4.20
(t, 1 H, J = 8.0 Hz, H-4’ ), 4.29 (dd, 1 H, J = 5.0 Hz, 10.5 Hz, H-6 ), 4.41 (t, 1 H, J = 9.0
Hz, H-3’’ ), 4.53 (d, 1 H, J = 11.5 Hz, -CH2Ph ), 4.61 (d, 1 H, J = 9.0 Hz, H-1’’), 4.61 (d, 1
H, J = 11.5 Hz, -CH2Ph ), 4.68 (d, 1 H, J = 11.5 Hz, -CH2Ph ), 4.74 (d, 1 H, J = 11.5 Hz,
-CH2Ph ), 4.97 (d, 1 H, J = 10.0 Hz, -COOCH2Ph ), 5.00 (s, 1 H, CHPh ), 5.00 (d, 1 H, J =
7.0 Hz, H-1’’’), 5.06 (t, 1 H, J = 8.0 Hz, H-2’), 5.12 (d, 1 H, J = 7.5 Hz, H-1), 5.12 (d, 1 H, J
= 10.0 Hz, -COOCH2Ph), 5.16 (d, 1 H, J = 10.0 Hz, -COOCH2Ph), 5.17 (d, 1 H, J = 8.0

72

Hz, H-1’ ), 5.19 (d, 1 H, J = 11.5 Hz, -COOCH2Ph ), 5.20 (t, 1 H, J = 7.0 Hz, H-2’’’), 5.32
(s, 1 H, CHPh), 6.49 (d, 1 H, J = 9.0 Hz, NH), 7.08 (d, 1 H, J = 7.5 Hz, NH’’), 7.10-7.20
(m, 13 H, CHarom), 7.26-7.42 (m, 25 H, CHarom), 7.53-7.58 (m, 2 H, CHarom), 7.89-7.91
(m, 2 H, CHarom Bz), 7.95-7.97 (m, 2 H, CHarom Bz);

13

C NMR (125 MHz, CDCl3): δ =

-5.0 (SiCH3), -4.2 (SiCH3), 18.1 (SiC(CH3)3), 21.4 (PhCH3), 26.0 (x3, SiC(CH3)3), 56.8
(C-2), 57.6 (C-2’’), 66.4 (C-5’’), 67.4 (COOCH2Ph ), 68.4 (COOCH2Ph ),68.0 (C-6),
68.0 (C-6’’), 70.8 (C-5), 71.4 (C-4’’’), 73.7 (OCH2Ph), 74.6 (OCH2Ph), 74.6 (C-2’’’), 75.0
(C-2’), 75.0 (C-5’), 75.8 (C-4), 77.0 (C-3’’), 77.3 (C-4’), 77.8 (C-5’’’), 78.5 (C-3’), 79.8
(C-4’’), 80.2 (C-3), 81.4 (C-3’’’), 85.6 (C-1), 92.4 (CCl3), 92.7 (CCl3), 99.3 (CHPh), 100.0,
100.0 (C-1’’, C-1’’’), 101.0 (CHPh), 101.7 (C-1’), 126.2-130.1 (CHarom), 133.5, 133.5,
134.1, 135.4, 135.4, 137.4, 137.4, 138.0, 138.4, 138.4 (Cq STol, Cq CHPh x2, Cq OBn
x2, Cq Bz x2, Cq COOBn x2), 161.7, 165.0, 165.3, 165.7, 168.5, 169.1 (C=O TCA x2,
C=O COOBn x2, C=O Bz x2); HRMS [M + H]+: m/z: calcd for C97H99Cl6N2O24SSi
1949.4191, found 1949.4142.
Methyl

O-(benzyl

2-O-benzoyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-β-D-

glucopyranosyluronate)-(13)-O-(4,6-O-benzylidene-2-deoxy-2-trichloroacetamid
o-β-D-glucopyranosyl)-(14)-O-(benzyl 2-O-benzoyl-3-O-benzyl-β-Dglucopyranosyluronate)-(13)-O-(4,6-O-benzylidene-2-deoxy-2-trichloroacetamid
o-β-D-glucopyranosyl)-(14)-O-(benzyl 2-O-benzoyl-3-O-benzyl-β-Dglucopyranosyluronate)-(13)-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-βD-glucopyranoside (26): After a mixture of donor 25 (33.4 mg, 17.1 μmol), acceptor 13

73

(15.2 mg, 17.1 μmol) and MS-AW-300 (200 mg) in a mixture solvent of DCM and MeCN
(v/v 19:1, 2.00 ml) was stirred at -78 °C for 1 hr, AgOTf (13.0 mg, 51.4 μmol) in Et2O (0.7
00 ml) was added. When the temperature of the reaction mixture reduced to -78 °C,
p-TolSCl (17.1 μmol, 2.46 μl) was added via a micro-syringe. After 5 min when the
yellowish color disappears, TMSOTf (0.750 μl, 4.13 μmol) was added via micro-syringe.
The reaction mixture was warmed to −10 °C under stirring in 2 hrs, followed by the same
workup and purification procedures described above for the synthesis of compound 19.
20

Compound 26 (33.0 mg, 12.0 μmol, 71%) was afforded as white solid. [α]D

-1.7

o

(c

1

0.5, DCM); H NMR (500 MHz, CDCl3): δ = -0.14 (s, 3 H, SiCH3), -0.10 (s, 3 H, SiCH3),
0.78 (s, 9 H, SiC(CH3)3), 3.00 (dd, 1 H, J = 4.5 Hz, 9.5 Hz, H-6
4.5 Hz, 5.0 Hz, 9.5 Hz, H-5
(m, 1 H, H-5

a/c/e

a/c/e

a/c/e

a/c/e

3.72-3.78 (m, 2 H, H-2

, H-3

b/d/f

a/c/e

, H-6

a/c/e

H-5

b/d/f

), 3.85 (d, 1 H, J = 6.5 Hz, H-5
b/d/f

Hz, H-6
H-1

c/e

a/c/e

b/d/f

a/c/e

), 3.18-3.23

), 3.50 (s, 3 H, OCH3),

a/c/e

4.04 (dd, 1 H, J = 4.5 Hz, 10.5 Hz, H-6
b/d/f

a/c/e

, H-3

x2), 3.81 (dd, 1 H, J = 9.0 Hz, 9.5 Hz, H-4

), 3.90 (d, 1 H, J = 8.5 Hz, H-5

H, J = 8.5 Hz, H-4

, H-2

x2), 3.61-3.70 (m, 3 H, H-3

a/c

= 9.0 Hz, 9.5 Hz, H-4

), 3.13 (ddd, 1 H, J =

), 3.15 (dd, 1 H, J = 4.5 Hz, 9.5 Hz, H-6

), 3.42-3.48 (m, 3 H, H-5

3.52-3.61 (m, 3 H, H-3

a/c/e

a/c

b/d/f

, H-4

), 3.86 (d, 1 H, J = 8.5 Hz,

), 4.17 (t, 1 H, J = 6.5 Hz, H-4

), 4.25 (t, 1 H, J = 8.5 Hz, H-4

b/d/f

), 4.47 (dd, 1 H, J = 9.0 Hz, 9.5 Hz, H-3

),

), 3.81 (dd, 1 H, J

), 4.02 (dd, 1 H, J = 4.5 Hz, 10.0 Hz, H-6

a/c/e

a/c/e

b/d/f

a/c/e

),

), 4.19 (t, 1

), 4.29 (dd, 1 H, J = 5.5 Hz, 10.5

a/c/e

), 4.52 (d, 1 H, J = 8.0 Hz,

), 4.56 (d, 1 H, J = 11.5 Hz, -CH2Ph ), 4.57 (d, 1 H, J = 12.0 Hz, -CH2Ph ), 4.62 (d,

74

1 H, J = 11.5 Hz, -CH2Ph ), 4.68 (d, 1 H, J = 12.0 Hz, -CH2Ph ), 4.72 (d, 1 H, J = 8.0 Hz,
H-1

c/e

), 4.75 (d, 1 H, J = 11.5 Hz, -CH2Ph ), 4.78 (d, 1 H, J = 11.5 Hz, -CH2Ph ), 4.84 (d,
a

1 H, J = 8.5 Hz, H-1 ),4.94 (d, 1 H, J = 6.0 Hz, H-1
H-1

b/d/f

), 4.99 (d, 1 H, J = 7.0 Hz, H-1

b/d/f

b/d/f

), 4.97 (d, 1 H, J = 6.5 Hz,

), 5.01 (d, 1 H, J = 12.0 Hz, -COOCH2Ph), 5.04

(d, 1 H, J = 12.0 Hz, -COOCH2Ph), 5.07 (dd, 1 H, J = 6.0 Hz, 8.0 Hz, H-2
H, J = 12.0 Hz, -COOCH2Ph ), 5.12 (dd, 1 H, J = 6.5 Hz, 8.5 Hz, H-2

b/d/f

b/d/f

), 5.11 (d, 1

), 5.14 (s, 1 H,

CHPh), 5.18 (d, 1 H, J = 12.0 Hz, -COOCH2Ph), 5.19 (s, 1 H, CHPh), 5.20 (d, 1 H, J =
12.0 Hz, -COOCH2Ph), 5.21 (d, 1 H, J = 12.0 Hz, -COOCH2Ph), 5.21 (dd, 1 H, J = 6.0
Hz, 7.0 Hz, H-2

b/d/f

), 5.32 (s, 1 H, CHPh), 6.48 (d, 1 H, J = 8.5 Hz, NH), 6.60 (d, 1 H, J =

8.5 Hz, NH), 6.99 (d, 1 H, J = 8.0 Hz, NH), 7.13-7.60 (m, 54 H, CHarom), 7.89-7.98 (m, 6
H, CHarom Bz);

13

C NMR (125 MHz, CDCl3): δ = -5.0 (SiCH3), -4.2 (SiCH3), 18.1 (SiC(C

3)3), 26.0 (x3, SiC(CH3)3), 57.2 (C-2

66.3, 66.4, 66.4 (C-5

a/c/e

, C-6

a/c/e

a/c/e

, C-5

), 57.5 (C-2

a/c/e

a/c/e

(C-2

b/d/f

), 75.1 (C-2

77.6 (C-4
(C-4
(C-1

b/d

b/d/f

b/d/f

b/d/f

a/c/e

b/d/f

b/d/f

c/e

), 81.5 (C-3

), 100.0 (C-1

, C-6

a/c/e

a/c/e

, C-5

a/c/e

), 78.8 (C-3

), 76.8 (C-4

b/d/f

c/e

),

f

a/c/e

), 79.0 (C-3

a/c/e

), 77.0 (C-4

b/d/f

b/d/f

), 100.3 (C-1

b/d/f

x2), 75.0

), 77.5 (C-4

), 80.0 (x2, C-3

a/c/e

), 92.4 (CCl3), 92.6 (CCl3), 92.7 (CCl3), 99.3 (C-1

), 100.1 (C-1

a/c/e

), 71.4 (C-4 ), 73.7

), 74.7 (OCH2Ph), 74.7 (OCH2Ph), 74.7 (x2, C-5

), 76.0 (C-3

), 77.8 (C-5

), 57.7 (OCH3), 58.8 (C-2

), 67.4 (COOCH2Ph ), 68.0 (COOCH2Ph ),

68.1 (COOCH2Ph ), 68.3, 68.6, 68.7 (C-6
(OCH2Ph), 74.3 (C-2

a/c/e

b/d

),

x2), 82.2

b/d/f

), 99.9

a

), 100.8 (C-1 ), 100.9 (CHPh),

101.1 (CHPh), 101.7 (CHPh), 126.3-130.4 (CHarom), 133.4, 133.5, 133.6, 135.0 (x2),

75

135.4, 137.4, 137.5, 137.5, 137.9, 138.3, 138.5 (Cq CHPh x3, Cq OBn x3, Cq Bz x3, Cq
COOBn x3), 161.6, 162.0, 162.3, 165.3, 165.6, 165.6, 168.6, 169.0, 169.1 (C=O TCA x3,
C=O COOBn x3, C=O Bz x3); HRMS [M + Na]+: m/z: calcd for C133H132Cl9N3NaO37Si
2734.5383, found 2734.5469.
2-Trichloromethyl O-(benzyl 2-O-benzoyl-3-O-benzyl-4-O-tertbutyldimethylsilyl- β-D-glucopyranosyluronate)-(13)-O-(4,6-O-benzylidene2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(14)-O-(benzyl 2-O-benzoyl
-3-O-benzyl-β-D-glucopyranosyl- uronate)-(13)-4,6-O-benzylidene-1,2-dideoxy
-α-D-glucopyrano-[2,1-d]-2-oxazoline (27): [α]D

20

+19.3 o (c 0.5, DCM); 1H NMR (500

MHz, CDCl3): δ = -0.15 (s, 3 H, SiCH3), -0.12 (s, 3 H, SiCH3), 0.77 (s, 9 H, SiC(CH3)3),
3.02 (ddd, 1 H, J = 4.5 Hz, 5.0 Hz, 9.5 Hz, H-5’’),

3.26-3.31 (m, 1 H, H-6’’), 3.44-3.48

(m, 2 H, H-6, H-5), 3.55 (t, 1 H, J = 7.0 Hz, H-3’’’), 3.63 (t, 1 H, J = 9.5 Hz, H-4’’),
3.73-3.68 (m, 2 H, H-3’, H-2’’), 3.82-3.91 (m, 4 H, H-3, H-4, H-5’, H-5’’’), 4.04 (dd, 1 H, J
= 5.0 Hz, 11.0 Hz, H-6’’), 4.13-4.23 (m, 4 H, H-2, H-3’’, H-4’, H-4’’’), 4.28-4.29 (m, 1 H,
H-6), 4.55 (d, 1 H, J = 8.0 Hz, H-1’’), 4.57 (d, 1 H, J = 11.5 Hz, -CH2Ph), 4.60 (d, 1 H, J =
11.5 Hz, -CH2Ph ), 4.67 (d, 1 H, J = 11.5 Hz, -CH2Ph ), 4.77 (d, 1 H, J = 11.5 Hz,
-CH2Ph ), 4.89 (d, 1 H, J = 7.5 Hz, H-1’’’), 4.95 (d, 1 H, J = 12.0 Hz, -COOCH2Ph), 5.02
(d, 1 H, J = 7.0 Hz, H-1’), 5.15 (d, 1 H, J = 12.0 Hz, -COOCH2Ph ), 5.17 (s, 1 H, CHPh),
5.19 (d, 1 H, J = 12.0 Hz, -COOCH2Ph), 5.20 (d, 1 H, J = 12.0 Hz, -COOCH2Ph ), 5.20 (t,
1 H, J = 7.0 Hz, H-2’ ), 5.24 (dd, 1 H, J = 7.5 Hz, 8.5 Hz, H-2’’’), 5.34 (s, 1 H, CHPh), 6.09
(d, 1 H, J = 7.5 Hz, H-1), 6.58 (d, 1 H, J = 8.5 Hz, NH’’), 7.10-7.18 (m, 10 H, CHarom),
76

7.29-7.45 (m, 22 H, CHarom), 7.59-7.52 (m, 2 H, CHarom), 7.95-8.00 (m, 4 H, CHarom Bz);
13

C NMR (125 MHz, CDCl3): δ = -5.0 (SiCH3), -4.2 (SiCH3), 18.1 (SiC(CH3)3), 26.0 (x3,

SiC(CH3)3), 57.6 (C-2’’), 63.4 (C-5), 66.4 (C-5’’), 67.4 (COOCH2Ph ), 67.8 (COOCH2Ph
), 68.6 (C-6), 68.6 (C-6’’), 68.9 (C-2), 71.4 (C-4’’’), 73.1 (C-2’’’), 73.7 (OCH2Ph), 74.7
(C-2’), 74.8 (OCH2Ph), 75.2 (C-5), 76.7 (C-4’’), 77.1 (C-3’’), 77.8 (C-5’’’), 78.5 (C-4), 79.9
(C-3’), 80.1 (C-4’), 80.2 (C-3), 81.4 (C-3’’’), 92.6 (CCl3, TCA), 99.2 (C-1’), 99.4 (C-1’’),
101.0 (C-1’’’), 101.3 (CHPh), 101.7 (CHPh), 110.0 (N=C-CCl3), 105.2 (C-1), 126.2-130.1
(CHarom), 133.4x2, 135.1, 135.4, 137.1, 137.4, 138.0, 138.2 (Cq CHPh x2, Cq OBn x2,
Cq Bz x2, Cq COOBn x2), 161.8, 162.5, 165.3, 165.4, 168.2, 168.5 (C=N-CCl3, C=O
TCA, C=O COOBn x2, C=O Bz x2); HRMS [M + Na]+: m/z: calcd for
C90H90Cl6N2NaO24Si 1843.3682, found 1843.3724.
Methyl O-(benzyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyluronate)-(1
3)-O- (4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(1
4)-O-(benzyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyluronate)-(13)-O-(4,6
-O-benzylidene-2- deoxy-2-trichloroacetamido-β-D-glucopyranosyl)-(14)-O(benzyl 2-O-benzoyl-3-O- benzyl-β-D-glucopyranosyluronate)-(13)-4,6-Obenzylidene-2-deoxy-2-trichloroacetamido-β-D-glucopyranoside

(28):

26

was

converted to 28 following the general procedure for TBS removal as described before
(90%). [α]D

20

-3.0 o (c 0.5, DCM); 1H NMR (500 MHz, CDCl3): δ = 3.00 (ddd, 1 H, J = 5.0

Hz, 5.0 Hz, 10.0 Hz, H-5

a/c/e

), 3.07 (s, 1 H, OH), 3.12-3.20 (m, 3 H, H-6

77

a/c/e

x3),

3.43-3.48 (m, 3 H, H-5
H-3

a/c/e

a/c/e

x2, H-2

a/c/e

x2), 3.63-3.70 (m, 3 H, H-3

b/d/f

3.82 (dd, 1 H, J = 8.5 Hz, 9.0 Hz, H-4
3.87 (d, 1 H, J = 8.5 Hz, H-5
Hz, 9.5 Hz, H-6
H-3
H-5
H-3

b/d/f
b/d/f

, H-4

a/c/e

b/d/f

), 3.50 (s, 3 H, OCH3), 3.61 (t, 2 H, J = 9.0 Hz,
x2, H-4

a/c/e

a/c/e

), 3.72-3.79 (m, 2 H, H-2

), 3.82 (dd, 1 H, J = 8.5 Hz, 9.5 Hz, H-4

b/d

), 3.90 (d, 1 H, J = 8.0 Hz, H-5

b/d

), 4.05 (dd, 1 H, J = 4.5 Hz, 11.0 Hz, H-6

x2), 4.19 (dd, 1 H, J = 8.0 Hz, 8.5 Hz, H-4

), 4.29 (dd, 1 H, J = 5.5 Hz, 10.5 Hz, H-6

a/c/e

), 4.52 (d, 1 H, J = 8.0 Hz, H-1

c/e

a/c/e

a/c/e

b/d/f

x2),

a/c/e

),

), 4.02 (dd, 1 H, J = 4.5

a/c/e

), 4.08-4.18 (m, 3H,

), 4.25 (d, 1 H, J = 8.5 Hz,

), 4.48 (dd, 1 H, J = 9.0 Hz, 9.5 Hz,

), 4.56 (d, 1 H, J = 12.0 Hz, -CH2Ph ), 4.57 (d, 1

H, J = 12.0 Hz, -CH2Ph ), 4.66 (d, 1 H, J = 11.5 Hz, -CH2Ph ), 4.75 (d, 1 H, J = 7.5 Hz,
H-1

c/e

), 4.75 (d, 1 H, J = 11.5 Hz, -CH2Ph ), 4.76 (d, 1 H, J = 12.0 Hz, -CH2Ph ), 4.79
a

(d, 1 H, J = 12.0 Hz, -CH2Ph ), 4.85 (d, 1 H, J = 8.0 Hz, H-1 ), 4.85 (d, 1 H, J = 7.5 Hz,
H-1

b/d/f

), 4.94-4.98 (m, 2 H, H-1

b/d/f

x2), 5.02 (d, 1 H, J

= 11.5 Hz, -COOCH2Ph), 5.04

(d, 1 H, J = 12.0 Hz, -COOCH2Ph), 5.06 (dd, 1 H, J = 8.0 Hz, 8.5 Hz, H-2

b/d/f

), 5.10 (d, 1

H, J = 12.0 Hz, -COOCH2Ph ), 5.13 (s, 1 H, CHPh), 5.16 (d, 1 H, J = 12.0 Hz,
-COOCH2Ph), 5.16 (d, 1 H, J = 12.0 Hz, -COOCH2Ph), 5.17 (dd, 1 H, J = 7.5 Hz, 8.5 Hz,
H-2

b/d/f

), 5.19 (dd, 1 H, J = 6.5 Hz, 8.0 Hz, H-2

b/d/f

), 5.21 (s, 1 H, CHPh ), 5.25 (d, 1 H, J

= 11.5 Hz, -COOCH2Ph), 5.32 (s, 1 H, CHPh), 6.45 (d, 1H, J = 8.5 Hz, NH), 6.63 (d, 1 H,
J = 9.0 Hz, NH), 7.02 (d, 1 H, J = 7.5 Hz, NH), 7.13-7.61 (m, 54 H, CHarom), 7.90-8.04
(m, 6 H, CHarom Bz);

13

C NMR (125 MHz, CDCl3): δ = 57.1 (C-2

57.7 (OCH3), 58.8 (C-2

a/c/e

), 66.3, 66.4 (C-5

78

a/c/e,

C-6

a/c/e

a/c/e

), 57.5 (C-2

a/c/e

),

), 66.4 (COOCH2Ph ), 68.0

(COOCH2Ph ), 68.1 (COOCH2Ph ), 68.1, 68.3, 68.5, 68.7 (C-6
f

(C-4 ), 73.6 (C-2

b/d/f

), 74.2 (C-2

b/d/f

), 74.3 (C-5

b/d

a/c/e

x2, C-5

a/c/e

x2), 72.0

), 74.6 (OCH2Ph), 74.8 (OCH2Ph),

74.7 (OCH2Ph), 74.9 (C-5b/d), 75.2 (C-2b/d/f), 76.0 (C-3f), 76.9 (C-4a/c/e), 77.2 (C-4b/d),
77.4 (C-4b/d), 77.6 (C-5f), 78.8 (C-3a/c/e), 79.0 (C-3a/c/e), 80.1, 80.1, 80.1 x2 (C-3
C-4

a/c/e

(C-1

b/d/f

x2), 81.1 (C-3
), 100.0 (C-1

a/c/e

c/e

), 92.4 (CCl3), 92.6 (CCl3), 92.7 (CCl3), 99.8 (C-1

), 100.0 (C-1

c/e

), 100.4 (C-1

b/d/f)

b/d/f

b/d/f

x2,

), 99.9

1a

, 100.8 (C- ), 100.9 (CHPh),

101.1 (CHPh), 101.4 (CHPh), 126.1-130.3 (CHarom), 133.5, 133.6, 133.6, 134.9, 135.0,
135.1, 137.4, 137.5 x2,

138.0, 138.3, 138.5 (Cq CHPh x3, Cq OBn x3, Cq Bz x3, Cq

COOBn x3), 161.7, 162.0, 162.4, 165.1, 165.6, 165.7, 169.0, 169.3, 169.3 (C=O TCA x3,
C=O COOBn x3, C=O Bz x3); HRMS [M + Na]+: m/z: calcd for C127H118Cl9N3NaO37
2620.4513, found 2620.4595.
Methyl

O-(benzyl

2-O-benzoyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-β-D-

glucopyranosyluronate)-(13)-O-(4,6-O-benzylidene-2-deoxy-2-trichloroacetamid
o-β-D -glucopyranosyl)-(14)-O-(benzyl 2-O-benzoyl-3-O-benzyl-β-Dglucopyranosyluronate) -(13)-O-(4,6-O-benzylidene-2-deoxy-2trichloroacetamido-β-D-glucopyranosyl)-(14)-O-(benzyl 2-O-benzoyl-3-O-benzylβ-D-glucopyranosyluronate)-(13)-O-(4,6-O- benzylidene-2-deoxy-2trichloroacetamido-β-D-glucopyranosyl)-(14)-O-(benzyl 2-O- benzoyl-3-O-benzyl
-β-D-glucopyranosyluronate)-(13)-O-(4,6-O-benzylidene-2-deoxy-2-trichloroacet
amido-β-D-glucopyranosyl)-(14)-O-(benzyl 2-O-benzoyl-3-O-benzyl -β-D-

79

glucopyranosyluronate)-(13)-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-βD-glucopyranoside (10): After a mixture of donor 25 (33.4 mg, 17.1 μmol), acceptor 28

(25.0 mg, 9.62 μmol) and MS-AW-300 (200 mg) in a mixture solvent of DCM and MeCN
(v/v 19:1, 2.00 ml) was stirred at -78 °C for 1 hr, AgOTf (51.4 mg, 20.0 μmol) in Et2O (0.7
00 ml) was added. When the temperature of the reaction mixture reduces to -78 °C,
p-TolSCl (17.1 μmol, 2.50 μl) was added via a micro-syringe. After 5 min, when the
yellowish color disappeared, TMSOTf (1.25 μl, 6.90 μmol) was added via micro-syringe.
The reaction mixture was warmed to −10 °C under stirring in 2 hrs, followed by the same
workup and purification procedures described above for the synthesis of compound 19.
Compound 10 (32.6 mg, 7.37 μmol, 76.6%) was afforded as white solid. [α]D

20

-2.7 o (c

0.5, DCM); 1H NMR (500 MHz, CDCl3): δ = 1H NMR (500 MHz, CDCl3): δ = -0.14 (s, 3 H,
SiCH3), -0.10 (s, 3 H, SiCH3), 0.79 (s, 9 H, SiC(CH3)3), 3.01 (dd, 1 H, J = 5.0 Hz, 9.0 Hz,
H-5

a/c/e/g/i

Hz, H-5
a/c/e/g/i

), 3.03 (dd, 1 H, J = 5.0 Hz, 9.0 Hz, H-5

a/c/e/g/i

, H-5

), 3.12-3.22 (m, 5 H, H-5

a/c/e/g/i

, H-6

a/c/e/g/i

a/c/e/g/i

a/c/e/g/i

, H-6

), 3.05 (dd, 1 H, J = 5.0 Hz, 9.0

a/c/e/g/i

x4), 3.42-3.48 (m, 3 H, H-2

), 3.50 (s, 3 H, OCH3), 3.53 (dd, 1 H, J = 7.0 Hz, 7.5 Hz),

3.55 (dd, 1 H, J = 6.0 Hz, 7.0 Hz), 3.56 (dd, 1 H, J = 5.5 Hz, 6.0 Hz), 3.60 (dd, 2 H, J =
8.5 Hz, 10.0 Hz), 3.66 (dd, 1 H, J = 6.5 Hz, 8.5 Hz), 3.67 (t, 2 H, J = 8.0 Hz), 3.68 (dd, 2
H, J = 8.5 Hz, 9.0 Hz) (H-4
a/c/e/g/i

a/c/e/g/i

, H-3

a/c/e/g/i

x4, H-3

x4), 3.80 (dd, 2 H, J = 9.0 Hz, 9.5 Hz, H-4

9.5 Hz, H-4

b/d/f/h/j

b/d/f/h/j

x2), 3.85 (d, 1 H, J = 6.5 Hz, H-5

80

b/d/f/h/j

x5), 3.72-3.78 (m, 4 H, H-2

x2), 3.81 (dd, 2 H, J = 9.0 Hz,

b/d/f/h/j

), 3.86 (d, 1 H, J = 7.0 Hz,

H-5

b/d/f/h/j

), 3.88 (d, 2 H, J = 9.0 Hz, H-5

4.01-4.06 (m, 4 H, H-6
7.0 Hz, H-4
H-4

b/d/f/h/j

Hz, H-6
H-1

b/d/f/h/j

a/c/e/g/i

b/d/f/h/j

x2), 3.90 (d, 1 H, J = 8.5 Hz, H-5

x4), 4.14 (t, 1 H, J = 7.0 Hz, H-4

), 4.18 (t, 1 H, J = 8.5 Hz, H-4

b/d/f/h/j

), 4.25 (dd, 1 H, J = 8.5 Hz, 9.0 Hz, H-4

a/c/e/g/i

a/c/e/g/i

b/d/f/h/j

b/d/f/h/j

),

), 4.16 (t, 1 H, J =

), 4.24 (dd, 1 H, J = 8.5 Hz, 9.0 Hz,

b/d/f/h/j

), 4.48 (dd, 1 H, J = 9.0 Hz, 9.5 Hz, H-3

), 4.28 (dd, 1 H, J = 5.0 Hz, 10.5

a/c/e/g/i

), 4.53 (d, 1 H, J = 8.0 Hz,

), 4.56 (d, 1 H, J = 11.5 Hz, -CH2Ph ), 4.56 (d, 1 H, J = 11.5 Hz, -CH2Ph ),

4.56 (d, 1 H, J = 8.5 Hz, H-1

a/c/e/g/i

), 4.57 (d, 1 H, J = 11.5 Hz, -CH2Ph ), 4.62 (d, 1 H, J

= 11.5 Hz, -CH2Ph ), 4.63 (d, 1 H, J = 8.0 Hz, H-1

a/c/e/g/i

), 4.63 (d, 1 H, J = 11.5 Hz,

-CH2Ph ), 4.69 (d, 1 H, J = 11.5 Hz, -CH2Ph), 4.75 (d, 1 H, J = 8.0 Hz, H-1

a/c/e/g/i

), 4.76

(d, 1 H, J = 11.5 Hz, -CH2Ph ), 4.79 (d, 1 H, J = 11.5 Hz, -CH2Ph x3 ), 4.83 (d, 1 H, J =
8.0 Hz, H-1
H-1

b/d/f/h/j

a/c/e/g/i

), 4.91 (t, 2 H, J = 6.0 Hz, H-2

), 4.96 (d, 1 H, J = 6.0 Hz, H-1

b/d/f/h/j

b/d/f/h/j

x2), 4.95 (d, 1 H, J = 6.0 Hz,

), 5.00 (d, 1 H, J = 7.0 Hz, H-1

b/d/f/h/j

),

4.98 (d, 1 H, J = 11.0 Hz, -COOCH2Ph), 5.00 (d, 1 H, J = 12.0 Hz, -COOCH2Ph), 5.02
b/d/f/h/j

(d, 1 H, J = 11.5 Hz, -COOCH2Ph x2), 5.06 (d, 1 H, J = 8.0 Hz, H-1
J = 8.0 Hz, H-1

b/d/f/h/j

), 5.06 (d, 1 H,

), 5.06 (d, 1 H, J = 12.0 Hz, -COOCH2Ph), 5.09 (dd, 1 H, J = 8.0

Hz, 9.0 Hz, H-2b/d/f/h/j), 5.12 (d, 1 H, J = 11.5 Hz, -COOCH2Ph ), 5.13 (s, 3 H, CHPh x3),
5.15 (d, 1 H, J = 11.5 Hz, -COOCH2Ph), 5.15 (dd, 1 H, J = 8.0 Hz, 9.0 Hz, H-2b/d/f/h/j),
5.19 (d, 1 H, J = 12.0 Hz, -COOCH2Ph ), 5.19 (s, 1 H, CHPh ), 5.20 (d, 1 H, J = 11.0 Hz,
-COOCH2Ph ), 5.21 (d, 1 H, J = 12.0 Hz, -COOCH2Ph ), 5.21 (dd, 1 H, J = 7.0 Hz, 8.5
Hz, H-2

b/d/f/h/j

), 5.31 (s, 1H, CHPh), 6.50 (d, 1 H, J = 9.0 Hz, NH), 6.55 (d, 2 H, J = 8.0

Hz, NH x2), 6.64 (d, 1 H, J = 9.0 Hz, NH), 7.02 (d, 1 H, J = 7.5 Hz, NH), 7.10-7.58 (m, 90
81

H, CHarom), 7.90-7.95 (m, 10 H, CHarom Bz);

13

C NMR (125 MHz, CDCl3): δ = -5.0

(SiCH3), -4.2 (SiCH3), 18.1 (SiC(C 3)3), 26.0 (x3, SiC(CH3)3), 57.1, 57.1 (x2) (C-2
x3), 57.8 (C-2
(C-5
C-6

a/c/e/g/i

a/c/e/g/i

(C-2

b/d/f/h/j

), 57.8 (OCH3), 58.7 (C-2

x2), 68.6 (C-6
), 74.6 (C-2
b/d/f/h/j

77.4 (x2) (C-4

b/d/f/h

(x2), 78.8 (C-3
x5), 99.3 (C-1
a/c/e/g/i

a/c/e/g/i

), 66.4, 66.3 (x2), 66.3 (x2)

x5), 67.4 (COOCH2Ph ), 68.1 (x5, COOCH2Ph x4, C-6

x5), 75.0 (C-2

(C-1

a/c/e/g/i

a/c/e/g/i

b/d/f/h/j

), 68.7 (C-6

b/d/f/h/j

), 75.2 (C-2

x4), 77.1 (x3), 76.9 (C-4

b/d/f/h/j

x5, C-3

a/c/e/g/i

), 100.0 (C-1

x2), 100.3 (C-1

a/c/e/g/i

), 68.4 (x2,

j

), 71.4 (C-4 ), `73.7 (OCH2Ph), 74.3

), 74.7 (OCH2Ph), 74.8 (x7), 74.9 (OCH2Ph x3, C-5

), 75.0 (C-2

b/d/f/h/j

a/c/e/g/i

a/c/e/g/i

b/d/f/h/j

a/c/e/g/i

x4, C-4

a/c/e/g/i

b/d/f/h/j

a/c/e/g/i

), 77.8, 77.6,

x4), 81.4, 83.3, 80.1 (x4), 79.0, 79.0

a/c/e/g/i

), 92.4, 92.6, 92.7, 92.8 (x2) (CCl3

), 100.0 (C-1

x3), 100.8 (C-1

), 76.0 (C-3

b/d/f/h/j

a/c/e/g/i

a/c/e/g/i

), 100.2 (C-1

b/d/f/h/j

), 100.3

), 100.9 (CHPh), 100.9 (CHPh

x2), 101.1 (CHPh), 101.7 (CHPh), 126.2-130.2 (CHarom), 133.5, 133.5 (x2), 133.5,
133.6, 135.0 (x2), 135.0 (x2), 135.4, 137.4, 137.5 (x2), 137.5, 137.5, 138.0, 138.3, 138.5
(x2), 138.5 (Cq CHPh x5, Cq OBn x5, Cq Bz x5, Cq COOBn x5), 161.6, 161.9 (x2), 162.0,
162.3, 165.3, 165.6, 165.6, 165.7, 165.6, 168.6, 169.1 (x2), 169.3, 169.4 (C=O TCA x5,
C=O COOBn x5, C=O Bz x5); MALDI [M + Na]+: m/z: calcd for C217H208Cl15N5NaO61Si
4442.85, found 4443.15.
Methyl O-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2-deoxy-β-Dglucopyranosyl)-(14)-O-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2deoxy-β-D-glucopyranosyl)-(14)-O-(β-D-glucopyranosyluronic acid)-(13)-O(2-N- acetyl-2-deoxy-β-D-glucopyranosyl)-(14)-O-(β-D-glucopyranosyluronic
82

acid)-(13)-O-(2-N-acetyl-2-deoxy-β-D-glucopyranosyl)-(14)-O-(β-Dglucopyransyluronic acid)-(13)-2-N-acetyl-2-deoxy-β-D-glucopyranoside (9): 10
was converted to 29 following the general procedure for TBS removal as described
before (79%). Compound 29 (44.0 mg, 10.0 μmol) was dissolved in THF (2 ml). 0.2 M
KOH aqueous solution (1.00 ml, 200 μmol) was added in 10 portions during 2 days. The
reaction mixture was stirred at r.t. during which the ratio of H2O to THF was gradually
increased for a better solubility of the reaction mixture. Reaction was checked by
comparisons of the crude 1H NMR spectra which was taken by the end of each week.
Reaction was stopped by the neutralization with 0.2 M AcOH in H2O by the end of the
fifth week when peaks around 7.0 ppm in 1H-NMR of the crude NMR indicating NHTCA
groups disappeared and peaks around 8.0 peaks for ortho-hydrogens on benzoyl groups
changed from several equally height peaks into two main peaks. The solvent was
completely removed by high vacuum rotary evaporator, and the crude residue was
dissolved in anhydrous MeOH (3.00 ml) and cooled to 0°C. TEA (7.0 μl, 50 μmol) and
acetic anhydride (Ac2O) (118 μl, 1.25 mmol) were added, and the reaction mixture was
stirred for two days under N2 protection at r.t.. The reaction was stopped when TLC
(AcOH/ DCM/ MeOH solvent systems) show one main product forms. The reaction
mixture was concentrated and purified by silica gel chromatography (AcOH/ DCM/
MeOH solvent systems) to yield a mixture mainly composed of 31. Pd(OH)2 (50.0 mg)
was added to a solution of the 31 in THF (0.5 ml), MeOH (0.7 ml) and AcOH (0.5 ml).
The reaction flask was evacuated using a water aspirator and filled with hydrogen. This
83

process was repeated three times and the reaction mixture was stirred under hydrogen
atmosphere for 3 days. The solution was filtered and concentrated, and the crude
product was purified by Sephadex G-15 size exclusion chromatography to give the
20

desired product 9 (6.60 mg, 3.50 μmol, 35% from 29 for 3 steps). [α]D

-27

o

(c 0.05,

1

H2O); H NMR (500 MHz, D2O): δ = 1.90-1.92 (m, 15 H, NHCOCH3 x5), 3.20-3.26 (m, 5
H), 3.35-3.43 (m, 15 H), 3.45-3.48 (m, 5 H), 3.56-3.66 (m, 20 H), 3.70-3.82 (m, 10 H),
4.34 (d, 2 H, J = 8.5 Hz, anomeric H x2), 4.36 (d, 4 H, J = 7.0 Hz, anomeric H x4), 4.44
(d, 2 H, J = 8.5 Hz, anomeric H x2), 4.45 (d, 2 H, J = 7.5 Hz, anomeric H x2); HRMS
3

[M-3H] -: m/z: calcd for C71H106N5O56 641.5200, found 641.5198.

84

Appendix A

85

NMR Data
1

Figure 3.3. H-NMR of compound 9 (500 MHz, D2O)

86

1

Figure 3.4. H-NMR of compound 10 (500 MHz, CDCl3)

87

Figure 3.5.

13

C-NMR of compound 10 (125 MHz, CDCl3)

88

1

1

Figure 3.6. H– H gCOSY of compound 10 (500 MHz, CDCl3)

89

1

13

Figure 3.7. H– C gHMQC of compound 10 (500 MHz, CDCl3)

90

1

13

1

Figure 3.8. H– C gHMQC (without H decoupling) of compound 10 (500 MHz, CDCl3)

91

1

13

Figure 3.9. H– C gHMBC of compound 10 (500 MHz, CDCl3)

92

1

Figure 3.10. H-NMR of compound 19 (500 MHz, CDCl3)

93

Figure 3.11.

13

C-NMR of compound 19 (125 MHz, CDCl3)

94

1

1

Figure 3.12. H– H gCOSY of compound 19 (500 MHz, CDCl3)

95

1

13

Figure 3.13. H– C gHMQC of compound 19 (500 MHz, CDCl3)

96

1

Figure 3.14. H–

13
1
C gHMQC (without H decoupling) of compound 19 (500 MHz,CDCl3)

97

1

13

Figure 3.15. H– C gHMBC of compound 19 (500 MHz, CDCl3)

98

1

Figure 3.16. H-NMR of compound 11 (500 MHz, CDCl3)

99

Figure 3.17.

13

C-NMR of compound 11 (125 MHz CDCl3)

100

1

1

Figure 3.18. H– H gCOSY of compound 11 (500 MHz, CDCl3)

101

Figure 3.19.

1

13

H– C gHMQC of compound 11 (500 MHz, CDCl3)

102

1

13

1

Figure 3.20. H– C gHMQC (without H decoupling) of compound 11 (500 MHz,CDCl3)

103

1

13

Figure 3.21. H– C gHMBC of compound 11 (500 MHz, CDCl3)

104

1

Figure 3.22. H-NMR of compound 12 (500 MHz, CDCl3)

105

Figure 3.23.

13

C-NMR of compound 12 (125 MHz, CDCl3)

106

1

1

Figure 3.24. H– H gCOSY of compound 12 (500 MHz, CDCl3)

107

1

13

Figure 3.25. H– C gHMQC of compound 12 (500 MHz, CDCl3)

108

1

13

1

Figure 3.26. H– C gHMQC (without H decoupling) of compound 12 (500 MHz,CDCl3)

109

Figure 3.27.

1

13

H– C gHMBC of compound 12 (500 MHz, CDCl3)

110

1

Figure 3.28. H-NMR of compound 13 (500 MHz, CDCl3)

111

Figure 3.29.

13

C-NMR of compound 13 (125 MHz, CDCl3)

112

1

1

Figure 3.30. H– H gCOSY of compound 13 (500 MHz, CDCl3)

113

1

13

Figure 3.31. H– C gHMQC of compound 13 (500 MHz, CDCl3)

114

1

13

1

Figure 3.32. H– C gHMQC (without H decoupling) of compound 13 (500 MHz,CDCl3)

115

1

13

Figure 3.33. H– C gHMBC of compound 13 (500 MHz, CDCl3)

116

1

Figure 3.34. H-NMR of compound 25 (500 MHz, CDCl3)

117

Figure 3.35.

13

C-NMR of compound 25 (125 MHz, CDCl3)

118

1

1

Figure 3.36. H– H gCOSY of compound 25 (500 MHz, CDCl3)

119

1

13

Figure 3.37. H– C gHMQC of compound 25 (500 MHz, CDCl3)

120

1

13

1

Figure 3.38. H– C gHMQC (without H decoupling) of compound 25 (500 MHz,CDCl3)

121

1

13

Figure 3.39. H– C gHMBC of compound 25 (500 MHz, CDCl3)

122

1

Figure 3.40. H-NMR of compound 26 (500 MHz, CDCl3)

123

Figure 3.41.

13

C-NMR of compound 26 (125 MHz, CDCl3)

124

1

1

Figure 3.42. H– H gCOSY of compound 26 (500 MHz, CDCl3)

125

1

13

Figure 3.43. H– C gHMQC of compound 26 (500 MHz, CDCl3)

126

1

13

Figure 3.44. H– C gHMQC (without 1H decoupling) of compound 26 (500 MHz,CDCl3)

127

Figure 3.45. 1H–13C gHMBC of compound 26 (500 MHz, CDCl3)

128

1

Figure 3.46. H-NMR of compound 27 (500 MHz, CDCl3)

129

Figure 3.47.

13

C-NMR of compound 27 (125 MHz, CDCl3)

130

1

1

Figure 3.48. H– H gCOSY of compound 27 (500 MHz, CDCl3)

131

1

13

Figure 3.49. H– C gHMQC of compound 27 (500 MHz, CDCl3)

132

1

Figure 3.50. H-NMR of compound 28 (500 MHz, CDCl3)

133

Figure 3.51.

13

C-NMR of compound 28 (125 MHz, CDCl3)

134

1

1

Figure 3.52. H– H gCOSY of compound 28 (500 MHz, CDCl3)

135

1

13

Figure 3.53. H– C gHMQC of compound 28 (500 MHz, CDCl3)

136

1
1 13
Figure 3.54. H– C gHMQC (without H decoupling) of compound 28 (500 MHz,CDCl3)

137

1

13

Figure 3.55. H– C gHMBC of compound 28 (500 MHz, CDCl3)

..

138

References

139

References
1. Aruffo, A., CD44: One ligand, two functions. J Clin Invest 1996, 98, 2191-2192.
2. DeGrendele, H. C.; Estess, P.; Picker, L. J.; Siegelman, M. H., CD44 and its ligand
hyaluronate mediate rolling under physiologic flow: A novel lymphocyte-endothelial cell
primary adhesion pathway. J Exp Med 1996, 183, 1119-1130.
3. Jalkanen, S.; Bargatze, R. F.; de los Toyos, J.; Butcher, E. C., CD44 and hyaluronan
dependent rolling interactions of lymphocytes on tonsillar stroma. J Cell Biol 1987, 105,
983–990.
4. Jiang, D.; Liang, J.; Fan, J.; Yu, S.; Chen, S.; Luo, Y.; Prestwich, G. D.;
Mascarenhas, M. M.; Garg, H. G.; Quinn, D. A.; Homer, R. J.; Goldstein, D. R.; Bucala,
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6. Ghatak, S.; Misra, S.; Toole, B. P., Hyaluronan oligosaccharides inhibit
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3-kinase/Akt cell survival pathway. J Biol Chem 2002, 277 (41), 38013-38020.
7. McKee, C. M.; Penno, M. B.; Cowman, M.; Bao, C.; Noble, P. W., Hyaluronan (HA)
gragments induce chemokine gene expression in alveolar macrophages. J Clin Invest
1996, 98, 2403–2413.
8. Tammi, R.; MacCallum, D.; Hascall, V. C.; Pienimäki, J. P.; Hyttinen, M.; Tammi, M.,
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decasaccharides and not hexasaccharides. J Biol Chem 1998, 273, 28878-28888.
9. Asari, A., Novel functions of hyaluronan oligosaccharides. Glycoforum Hyaluronan
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10. Blatter, G.; Jacquinet, J.-C., The use of 2-deoxy-2-trichloroacetamidoD-glucopyranose derivatives in dyntheses of hyaluronic acid-related tetra-, hexa-, and
octa-saccharides having a methyl -D-glucopyranosiduronic acid at the reducing end.
Carbohydrate Res 1996, 288, 109-125.

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11. Huang, L.; Huang, X., Highly efficient syntheses of hyaluronic acid oligosaccharides.
Chem Eur J 2007, 13, 529-540.
12. Zeng, Y.; Wang, Z.; Whitfield, D.; Huang, X., Installation of electron donating
protective groups, a strategy for glycosylating unreactive thioglycosyl acceptors using
the pre-activation based glycosylation method. J Org Chem 2008, 73, 7952-7962.
13. Wuts, P. G. M.; Greene, T. W., Protective groups in organic synthesis. 4th ed.;
Wiley-Interscience: New York, 2006.
14. Hinklin, R. J.; Kiessling, L. L., p-Methoxybenzyl ether cleavage by polymersupported sulfonamides. Org Lett 2002, 4, 1131-1133.
15. Sun, B.; Srinivasan, B.; Huang, X., Pre-activation based one-pot synthesis of an
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core-fucosylated
complex
type
bi-antennary
N-glycan
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16. Halkes, K. M.; Slaghek, T.; Hypponen, T. K.; Kruiskamp, P. H.; Ogawa, T.;
Kamerling, J. P.; Vliegenthart, J. F. G., Synthesis of hyaluronic-acid-related
oligosaccharides and analogues, as their 4-methoxyphenyl glycosides, having
N-acetyl--D-glucosamine at the reducing end. Carbohydrate Res 1998, 309, 161-174.
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Synthesis of hyaluronic acid oligomers using chemoselective and one-pot strategies. J
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19. Crich, D.; Smith, M.; Yao, Q.; Picione, J., 2,4,6-Tri-tert-butylpyrimidine (TTBP): A
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22. Dinkelaar, J.; Codee, J. D. C.; van den Bos, L. J.; Overkleeft, H. S.; van Boom, J. H.;
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142

CHAPTER 4
Design and Synthesis of HA5 Analogues as Potential Inhibitors of CD44-HA
Binding
4.1. Introduction
HA is a non-sulfated negatively charged linear polysaccharide, which is
composed

of

2,000-25,000

repeating

units

acid-β-13-N-acetyl-D-glucosamine-β-14-].

of

Tumor

disaccharide:

[-D-glucuronic

microenvironments

contain

different type of cells and ECM. HA and HA binding proteins are the major components of
1

ECM . CD44 is the primary receptor for HA. Multivalent HA-CD44 interactions induce
direct and indirect interactions with RTKs including ErbB2 and EGFR, and non-receptor
kinases of Src family or Ras family GTPases. Adaptor proteins such as Vav2, Grb2, and
Gab-1 mediate the formation of the signaling complex and mediate the interaction of
CD44 with upstream effectors such as RhoA, Rac1 and Ras, which eventually influence
downstream MAPK and PI3K/Akt signaling pathways

2-4

. The above signaling pathways
5-6

promote tumor cell invasiveness, proliferation, survival and MDR

.

Multivalent HA-CD44 interactions are required for formation of constitutive
signaling complexes. Replacement of the multivalent interactions with monovalent
interactions by the treatment with sHA causes disassembly of constitutive signaling
2, 7-10

complexes and attenuated signaling pathway

. These finally led to the inhibition of

tumor cell proliferation and MDR.
Other than sHAs, the first reported CD44-HA inhibitors are F-16438s isolated from
143

fungus strain SANK 30502 by Takahashi’s group in 2006. The IC50 values were reported
in the μM range

7-8

.

A year later, the Takahashi’s group reported another inhibitor,
9

F-19848A from fungus strain SANK 20204, with IC50 values in the μM range .
However none of those IC50 values was compared with sHAs in the same inhibitory
assay, and how those inhibitors bind CD44 was not characterized.
Figure 4.1. Inhibitors reported by Takahashi's group

In 2007, Jackson’s group reported the co-crystal structure of the CD44
10

hyaluronan binding domain and HA8 . In the co-crystal structure, HA8 binds to a
shallow groove and the interactions are dominated by hydrogen bonds and hydrophobic
interactions. The hydrophobic interactions are from sugar rings from GlcNAc4 to GlcUA7
144

and the methyl group in GlcNAc6. The H-bonds are from GlcUA5 to GlcNAc8 as
10

10

summarized in Figure 4.2 . That HA10 (Kd = 61 μM ) binds slightly stronger than HA8
10

(Kd = 125 μM ) can not be explained by the co-crystal structure, as the terminal GlcUA1,
GlcNAc2 and GlcUA3 do not show much interaction with the protein.
Figure 4.2. H-bonds in CD44-HA8 co-crystal structure

The other factor that may play a role is HA dynamics. The HA polymer has been
characterized as stiffened worm-like coil in solution. The presence of intramolecular
11

H-bonds in aqueous solution leads to stiffening of the HA polymer . In the case of
oligomers, conformations become more rigid as the chain length is increased due to the
terminal rings being less constrained by having fewer intramolecular H-bonds. The more
145

dynamical nature generate a greater entropic penalty which might explain the affinity
11-16

difference between HA10 and HA8

.

Nilsson's group accomplished beautiful work on the discovery of Galectin-3
ligands 2

(Kd = 0.88 μM), which bound 76 times stronger than the native ligand 1 (Kd =
17-18

0.88 μM) (Figure 4.3)

. The co-crystal structure of Galectin-3 and analogue 2

showed that the affinity enhancement came from the arginine-arene interaction between
18

Arg 144 and the aromatic group in analogue 2 (Figure 4.4) . Based on this discovery,
Nilsson and co-workers addressed the second arginine-arene interaction between
Arg186 and another aromatic group (Figure 4.3), and found ligand 3 (Kd = 46 nM),
19

which bound 1457 times stronger than ligand 1 . Paulson and co-workers reported the
20

a MAG ligand 5 (Figure 4.5), which bound 83 times stronger than ligand 4 . Docking
results suggested that the increased affinity is due to a stacking interaction of the
para-fluorobenzyl moiety with Arg118. Inspired by their work, our goal was is to add extra
functionality for approaching extra hydrophobic interaction with the hydrophobic pocket
formed by tyrosine 46 (Tyr46), Tyr83, isoleucine 111 (Ile111), serine 114 (Ser114),
threonine 116 (Thr116) and Tyr119 (shown orange in Figure 4.6).

146

Figure 4.3. Ligands of Galectin-3

Figure 4.4. Co-crystal structure of galectin-3 and analogue 2. Adapted with permission
from Ref 18. Copyright 2005 American Chemical Society.

147

Figure 4.5. MAG Ligands

Figure 4.6. The hydrophobic pocket in CD44-HA8 co-crystal structure

4.2. Results and Discussion
4.2.1. HA2 Library
4.2.1.1. The Design and Synthesis of HA2 Library
We started with a HA2 library which is synthetically more available than sHA
analogues with more sugars. The docking result (Figure 4.7) showed that the linker
might be too long for directing the aromatic groups into the hydrophobic pocket (colored

148

in red). Syntheses of the HA2 library proceeded smoothly (Scheme 4.1). Coupling
monosaccharide 6 and 7 yielded disaccharide 8 in 70% yield. PMB groups in compound
8 were removed by DDQ, and then the primary OH was oxidized to COOH. The benzoyl
(Bz) and Phth groups were cleaved by MeNH2, and the exposed OH groups were
acetylated. Finally the COOH group was protected as a methyl ester to produce
disaccharide 9. Various hydrophobic functionalities were then conjugated to compound 9
by azide alkyne cycloaddition. After hydrolysis, the hyaluronan disaccharide (HA2) library
(Compounds 10 - 16) was generated.
Figure 4.7. Docking results of HA2 library

149

Scheme 4.1. The syntheses of HA2 library

4.2.1.2. The ELISA
150

Scheme 4.2. Synthesis of Biotin-HA

The design of a screening assay for lectin is challenging by their tendency to bind
weakly to carbohydrate ligands. An inhibitory ELISA was established for studying the
binding affinity of the analogues. First anti-IgG antibody coated 96-well plate was used to
catch CD44/IgGFc chimera. Compared to direct coating of the plate with CD44, this
xIgG–IgGFc/CD44 layout helps to fully expose the CD44 HA binding domain and reduce
the amount of expensive CD44 used. The mixture of biotin-HA and sHA analogues were
then added to the plate. Biotin-HA polymer with different percentages of biotin was

151

21

synthesized based on Prestwich’s work . HA with 10% biotin turned out to be the best
for ELISA (Scheme 4.2). Too much biotin decreases the binding affinity between HA and
CD44, while too little biotin reduces the binding affinity between biotin-HA and
avidin-HRP. Finally, avidin conjugated horseradish peroxidase (avidin-HRP) was added
to quantify the amount of biotin-HA attached to the CD44.
To test the efficiency of this ELISA method, the inhibitory potency of HA8 was
measured (Figure 4.8). Since HA8 inhibits the binding between biotin-HA and CD44,
when the HA8 concentration increased, less biotin-HA binds to the CD44 coated plate,
leading to decreased signal. The IC50 of HA8 by this inhibitory ELISA is 136.8 µM.
Figure 4.8. HA8 inhibition curve

HA8 Inhibition Curve

A 450nm

2
1.5
1
0.5
0
1

10

100

1000

HA8 Concentration (µM)

4.2.1.3. Results and Discussion
The results of the inhibition ELISA for compounds 10 - 16 are shown in Figure 4.9.
Inhibitor NEG was the group without HA inhibitor, which was used as minimum inhibition.
152

HA 16 kDa was the group with HA polymer 16 kDa 2.5 µg per well, which was used as
maximum inhibition. HA8 (25 µg) was the group with 25 µg HA8 per well. HA4 (200 µg)
was the group with 200 µg HA4 per well. All other groups were measured in the presence
of 200 µg compounds 10 - 16 per well. Based on these results, 25 µg per well HA8 and
200 µg per well HA4 could inhibit HA-CD44 binding. However, 200 µg per well
compounds 10 - 16 did not show any inhibition to HA-CD44 interaction.
An unexpected result is that compound 10 could obviously increase the signal. To
confirm this result, an alternative ELISA method was used. First the plate was coated
with HA (from rooster comb) 5μg/well in pH 9.3 buffer at 220C overnight. Then the plate
was blocked with 1% bovine serum albumin (BSA) in PBS for 90 min at 370C. Next, the
mixture of CD44 and inhibitor was added and the plate was incubated at 370C for 45 min.
Then MEM-85, a CD44 monoclonal antibody, 0.1 μg/well was added and the plate was
incubated at 220C for 1 hr. Next, anti-mouse IgG Fc-HRP (1x 2000 dilution), which bound
MEM-85, was added and the plate was incubated at 220C for 1 hr. Finally TMB solution
was added and the plate was read at 450 nm. In this inhibitory ELISA, the wells with
inhibitors, which reduce the binding between the HA coated plate and free CD44 protein,
would show decreased signals. The inhibitory or enhancive ability of compound 10,
linker 1 and 2 (Figure 4.11) were evaluated by this ELISA method. The results are
shown in Figure 4.10. Inhibitor NEG was the group without HA inhibitor, which was
used as minimum inhibition. HA 16 kDa (50 µg) was the group with HA polymer 16 kDa
50 µg per well, which was used as maximum inhibition. Various amount (1.56 - 200 µg
153

per well ) of compound 10 were tested, but none of them show any inhibitory or
enhancive effects. Various amount (1.5625 - 200 µg per well ) of linker 1 or linker 2,
which are parts of compound 10, were tested as well. Linker 1 showed moderate
inhibitory effect at 100 µg per well, and linker 2 showed moderate inhibitory effect at 200
µg per well.
In summary, the HA2 library failed to show any inhibitory or enhancive effect on
CD44-HA interaction. This result might be attributed to two reasons. Most interactions
between sHA and CD44 were missing in HA2 analogues, or our previous docking results
indicated that the linker in the HA2 library might be too long for directing the hydrophobic
groups into CD44 hydrophobic pocket.
Figure 4.9. Inhibition ELISA of compounds 10 - 16

154

Figure 4.10. Alternative Inhibition ELISA of compound 10, Linker 1 and 2

Figure 4.11. Compound 10, Linker 1 and 2

4.2.2. HA5 Library
4.2.2.1. The Design
Based on the results of HA2 library. The design philosophy for the HA5 library is to
keep most functionality required to address CD44-HA8 interactions, and add extra
functionality to approach extra hydrophobic interaction with the hydrophobic pocket
formed by Tyr46, Tyr83, Ile111, Ser114, Thr116 and Tyr119 (shown orange in Figure 4.6).
HA5 backbone GlcUA3 to GlcUA7 (Figure 4.12) was kept and the hydrophobic group
was connected to an artificial 3-axial-amino group on GlcUA7. The analogues were
155

carefully evaluated by manually written into the co-crystal PDB file (2JCQ) by Pymol and
Chemdraw 3D with the assumption that the conformation and interactions of HA5
backbone remain exactly the same as it is in the CD44-HA8 co-crystal structure.
Visualization of the amide linked hydrophobic groups contacting protein hydrophobic
pocket is shown in Figure 4.13.
Figure 4.12. HA5 backbone

Figure 4.13. Visualization of the amide linked hydrophobic groups in CD44 HABD

4.2.2.2. The Syntheses
The most challenging part in this project is synthesis. With traditional synthetic
methods which build up oligosaccharide from monosaccharide building blocks, it takes
years to get one oligosaccharide synthesized. To simplify the synthesis of HA5 library, a
cutting edge route was chose. HA5 17 would be directly built up from HA4 building blocks
156

18. Compound 18 would be acquired several steps from compound 20. Compound 20
would be generated by enzymatic digestion of HA polymer 21 (Scheme 4.3).
Scheme 4.3. Retrosynthetic scheme of compound 17

Based on the DeAngelis’ and Asari’s work

22-23

, the enzymatic reaction from 21 to

20 was scaled up to 80 g (Scheme 4.4). Since only HA4 not the longer oligomers is the
target molecule, the reaction time was elongated up to 8 weeks to push the reaction to
completion. Compound 20 was precipitated out of the reaction mixture by mixed solvent
of H2O/EtOH (v/v 1:9), while the acetate salt in the digestion buffers stay in the mother
157

liquor. Crude compound 20 was converted to its TBA form 22 with Dowex strong acidic
resin TBA form for improving the solubility in DMF. Subsequential methylation and
acetylation yielded compound 23. Compound 23 was purified by silica gel column
chromatography with DCM/EA/MeOH system, and the overall yield from crude 20 was
35%.
Scheme 4.4. Synthesis of compound 23

The synthesis toward pentasaccharide 27 with donor 25 was unsuccessful
(Scheme 4.5). The anomeric OAc in tetrasaccharide 23 was converted to α-bromide,
and then converted to β-STol in 47% yield for 2 steps (Scheme 4.5a). The NHAc groups
in compound 24 were converted to NAc2 in 65% yield. However, donor 25 was not be
able to coupled to acceptor 26 with both AgOTf/p-TolSCl and AgOTf/DMTST promoting
systems (Scheme 4.5b).

158

Scheme 4.5. Synthesis towards HA5 with STol donor

The syntheses on the glycosylation with Br donor failed as well. The coupling
reaction between model donor 28 and acceptor 26 failed to produce disaccharide 29
(Scheme 4.6a). Donor 28 decomposed in the glycosylation condition. Model donor 30
quantitatively converted to oxzoline 31 in the glycosylation condition (Scheme 4.6b).
Anomeric Cl donor 32 might be an alternative to Br donor. However, the transformation
from 23 to 32 failed. Glycosidic bonds in compound 23 got cleaved in the strong acidic
conditions used for the above transformation (Scheme 4.6c).

159

Scheme 4.6. Synthesis towards HA5 with Br donor

Trichloroacetimidate donor might be a good alternative to Br donor and STol donor.
The NHAc groups in tetrasaccharide 23 was converted to NAC2 groups to produce
compound 33 (Scheme 4.7a). To get the trichloroacetimidate donor, the anomeric Ac
group in compound 33 had to be removed to get 34. However, NH4OAc 5 eq. was not
able to cleave the anomeric Ac group in compound 33. And there was no selectivity
.

between the anomeric Ac and NAc2 group in presence of 1.5 eq. NH2NH2 HOAc
(Scheme 4.7b).
So far, all the attempts toward the construction of an O-linkage between a HA4
and a glucose failed. The failure of coupling with STol donor 25 and Br donor 30 could be
attributed to the highly disarmed nature of the donor. Not only the acetyl groups but also
the NAc2 groups next to the anomeric position contribute to its disarmed nature.
160

Alternatively an S-linkage could be used to mimic the O-linkage. Thioglycoside 35 could
be generated from compound 36 and compound 37 (Scheme 4.8a).
Scheme 4.7. Synthesis towards HA5 with trichloroacetimidate donor

Compound 36 was generated in 3 steps from compound 23 in 30% overall yield
(Scheme 4.8b). First, the anomeric OAc groups was converted the α-bromide in strong
acidic condition, in which part of the glycosidic linkages got cleaved to generate
trisaccharide and disaccharide side products. This was followed by conversion of
α-bromide to β-SAc by a combination of tetrabutylammonium thioacetate (TBASAc) and
thioacetic acid (HSAc). HSAc was used to adjust the acidity of the reaction mixture for
minimizing the oxazoline formation. In the third step, in low temperature, 0.5 eq. NaOMe
was used to selectively remove anomeric acetic group in quantitative yield to give
compound 36.
161

Scheme 4.8. Synthesis route towards HA5 thioglycoside

Glucosamine 39 was used as a model molecule of compound 36 (Scheme 4.9a).
Coupling reaction between glucosamine 39 and galactose 37 failed to yield thioglycoside
44. The azido group in galactose 37 was not stable in the presence of compound 39 and
thioaziridine 40 was formed as the major product. As 3-axial-NH2 was synthetically
challenging, 3-axial-OH could be an alternative functionality to direct the hydrophobic
groups into the hydrophobic pocket. However, coupling between compound 39 and
galactose 41 did not work. The main side products were the disulfide 42 and elimination
product 43 (Scheme 4.9b). Fortunately coupling between Glucosamine 39 and
galactose 46 worked. After delevulinoylation, compound 47 was produced in 56% yield
162

for 2 steps. However, the conversion from compound 47 to compound 48 failed possibly
due to the steric hindrance. Therefore, the attempt to acquire 3-axial-OH from compound
47 through 3-triflate compound 48 failed. (Scheme 4.9c)
Scheme 4.9. Synthesis toward HA2 thioglycoside

Due to difficulties in the syntheses of analogues with 3-axial-NH2 or 3-axial-OH groups
(Figure 4.14a library A), alternative functional groups, COOH or 3-equatorial-OH in
163

GlcUA7, could be used to direct the hydrophobic group into the pocket by simply rotating
S-C4 bond (Figure 4.14b library B, Figure 4.14c library C). However, in those alternative
libraries, the hydrogen bonds (H-bonds) from COOH in GlcUA7 would be missing, and
the conformation after S-C4 rotation might not be favored.
Figure 4.14. HA5 analogues

Five compounds 49 - 53 in library B (Figure 4.15) and six compounds 54 - 59 in
library C (Figure 4.16) were selected to be synthesized by manually drawing the R
groups into the co-crystal structure with Pymol and Chemdraw 3D. Results are shown in
Figure 4.17 - 4.19. The compounds 49, 53, 57 and 58 may have hydrophobic interaction
164

with the pocket. Compound 50 may have π-π stacking with the aromatic ring of Tyr46.
Compounds 51 and 54 may have π-π stacking with the aromatic ring of Tyr83.
Compounds 52, 55 and 59 may have π-π stacking with the aromatic ring of Tyr83 and
H-bond with side chain OH in S134. One benzene ring in compound 56 may have
hydrophobic contact with the pocket and the other benzene ring may possess T-shaped
π-π stacking with Tyr46 (blue). Compound 53 may have both hydrophobic interaction
and H-bond with OH group in Thr116.
Figure 4.15. Library B

Figure 4.16. Library C

165

Figure 4.17. Visualizations of compounds 49 and 50 in CD44 HABD
Compound 49
Compound 50

Figure 4.18. Visualizations of compounds 51 - 55 in CD44 HABD
Compound 51
Compound 52

Compound 53

Compound 54

Compound 55

166

Figure 4.19. Visualizations of compounds 56 - 59 in CD44 HABD
Compound 56
Compound 57

Compound 58

Compound 59

The synthesis of the compounds 49 - 59 proceeded smoothly as shown in
Scheme 4.10 and Scheme 4.11. Tetrasaccharide 36 was coupled with galactose 60 to
generate pentasaccharide 61. The TBDPS group in pentasaccharide 61 was removed to
yield pentasaccharide 62. The primary OH in pentasaccharide 62 was oxidized to COOH
and coupled to the hydrophobic groups by amide linkage. Compounds 49 - 52 were
yielded by one step deprotection (Scheme 4.10). Similarly, compound 36 was coupled
with galactose 46 to yield pentasaccharide 63. Compound 63 was converted to
compound 64 in 3 steps. The 3-OH in compound 64 was conjugated to the hydrophobic
groups though carbamate linkages. Compound 54 - 59 were yielded by one step
deprotection (Scheme 4.11). The result of deprotection of compound 65 was different
167

Scheme 4.10. Synthesis of library B

from other compounds in library C (Scheme 4.12). In exactly the same condition, part of
3-carbamate group migrated to the 2 position to generate compound 66. The mixture of
compounds 59 and 66, which could not be separated by high performance liquid
chromatography (HPLC), was used for ELISA screening.
168

Scheme 4.11. Synthesis of library C

Compared to traditional synthesis in chapter 3, this synthetic strategy is more
efficient in generating oligosaccharide libraries due to two main advantages. First,
pentasaccharides were constructed directly from tetrasaccharide building blocks.
Second, the very simple one step deprotection is much more convenient compared to
169

the complicated multiple step deprotections in chapter3.
Scheme 4.12. Deprotection of compound 65

4.2.2.3. Results and discussion
The inhibitory ELISA was used to study the binding between compounds 49 - 59
and CD44 (Figure 4.20). CD44 NEG was the groups without CD44, which was used as
negative control. Inhibitor NEG was the group without HA inhibitor, which was used as
minimum inhibition. HA 16 kDa was the group with HA polymer 16 kDa 2.5 µg per well,
which was used as maximum inhibition. All other groups were measured in the presence
of 235 µM of HA5 analogues as potential inhibitors. Pentasaccharide 67 without
hydrophobic functionality was used to compare with compounds 49 - 59.
First, there was clear trend that HA8 bound stronger than HA6 and HA6 bound
stronger than HA4. Second, the binding affinity of 67 was similar to HA4. Third, the
binding affinities of compounds 49, 50, 51, 52, 53, 54, 55, 57, 58 and 59 were about the
same as 67. Fourth, the binding affinity of compound 56 was comparable to HA6.

170

Inhibition curve of compound 56, HA6 and HA8 was shown in Figure 4.22.
Figure 4.20. Inhibition ELISA of sHA and analogues

Figure 4.21. sHA and analogues

The difference in binding affinity between compound 67 and compound 56
indicated that aromatic group in compound 56 clearly contributes to the binding of
171

compound 56 to CD44. H-bonds in HA6 from COOH of GlcUA7 and the primary OH of
GlcNA8 are missing in compound 56, which may contribute to the loss of favored
enthalpy in binding. Computer screening results (Figure 4.17 - 4.19) shown that all
hydrophobic groups in compounds 49 - 59 have interactions with CD44, but only the
binding affinity of compound 56 was comparable to HA6. The explanation might be that
interactions of the hydrophobic group in compounds 49, 50, 51, 52, 53, 54, 55, 57, 58
and 59 to CD44 was not strong enough to overcome the loss of favored enthalpy caused
by the loss of H-bond mentioned before. The special orientation of the two benzene rings
in compound 56, which might contribute to the T-shaped π-π stacking with Tyr46, might
make compound 56 a better inhibitor than other HA5 analogues. Following this result,
beyond this thesis, analogues similar to compound 56 with subtle difference on the
aromatic functionality could be synthesized to further enhance binding.
Figure 4.22. Inhibition curve of compound 56, HA6 and HA8
1.4
1.2

O. D.

1
0.8

56
HA6

0.6

HA8

0.4
0.2
0
1

10

100
uM

172

1000

4.3. Conclusion
The purpose for this project is to design and synthesize analogues based on
co-crystal structure of CD44 and HA8. Due to synthetic difficulties, the initial design of
library A was abolished. By rotating S-C4 bond, new analogues, library B and library C,
were generated. 11 compounds in library B and library C were synthesized by a cutting
edge method, and screened by inhibitory ELISA. Finally, it was found that the aromatic
group in compound 56 contributes to the binding of compound 56 to CD44. And this
interaction overcomes the loss of favored enthalpy caused by the loss of H-bonds from
COOH of GluUA7 and primary OH of GlcNAc8. Although the binding affinity of 56 is only
comparable to HA6 and less than HA8, this provides a new direction towards further
design of novel HA inhibitors.

173

4.4. Experimental Section:
1-[3-(O-(2-N-acetyl-2-deoxy-β-D-glucopyranosyl)-(14)-α-D-glucopyranosyl
uronic acid-1-yl)-propyl]-1H-[1,2,3]-triazole-4-carboxylic acid octylamide (10): The
mixture of donor 6 (595 mg, 1.10 mmol), acceptor 7 (325 mg, 0.550 mmol, 0.5 eq.) and
freshly activated MS-4Ǻ (4 g) in a mixture solvent of DCM and MeCN (v/v 40:1, 20 ml)
was stirred for 1 hr at -78 °C, followed by the addition of AgOTf (848 mg, 3.30 mmol, 3
eq.) in Et2O (10 ml). After 5 min, p-TolSCl (173 μl, 1.10 mmol, 1 eq.) was added via a
syringe to activate the donor. The yellow color of the reaction disappears quickly and
TLC analysis showed the donor was completely consumed. The reaction mixture was
warmed to −10 °C under stirring in 2 hrs. TEA (0.5 ml) was then added and the mixture
was diluted with DCM (100 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
NaHCO3 (100 ml) and twice with H2O (100 ml). The organic layer was concentrated and
the crude product was purified by silica gel chromatography to produce disaccharide 8 in
70% yield. Disaccharide 8 was subjected to deprotection of PMB and oxidation of alcohol
to carboxylic acid following the general procedures on page 61. The yielding
disaccharide (230 mg, 0.255 mmol) was dissolved in 2M MeNH2 (14 ml). The reaction
was stirred at r.t. for 2 days. The solvent was removed and the crude mixture was
co-evaporated with toluene, then DMAP (188 mg, 1.53 mmol, 6 eq. ), Ac2O (0.200 ml,
23.0 mmol, 90 eq.) and DCM (16 ml) were added. The reaction mixture was stirred at r.t.
174

overnight, concentrated and co-evaporated with toluene. EDCI (158 mg, 0.826 mmol, 3
eq.) and anhydrous MeOH (3 ml) were added. The reaction mixture was stirred at r.t.
overnight, concentrated, and purified by silica gel chromatography to yield compound 9
in 15% yield for 5 steps. Compound 9 (6 mg, 0.008 mmol), alkyne (7 mg, 0.04 mmol, 5
eq.), CuSO4 (2 mg, 0.008 mmol, 1 eq.) and sodium ascorbate (3.2 mg, 0.016 mmol, 2
eq.) were dissolved in a mixture solvent of tBuOH and H2O (2 ml, v/v=1:1). The reaction
mixture was stirred at r.t. overnight, concentrated, and purified by silica gel
chromatography. The resulting disaccharide was dissolved in a mixture solvent of THF
and H2O (2 ml, v/v=1:1) and KOH (41 μl 0.1M KOH in H2O, 41 μmol, 1.5 eq.) was added.
The reaction mixture was stirred at r.t. overnight, concentrated and purified by Sephadex
G-15 size exclusion chromatography to produce compound 10 in quant. yield for 2 steps.
1

H NMR (500 MHz, D2O): δ = 0.74 (t, 3H, J = 7.0 Hz), 1.14-1.27 (m, 10H), 1.52 (t, 2H, J

= 7.5 Hz), 1.92 (s, 3H, NHCOCH3), 2.16-2.20 (m, 2H), 3.28-3.81 (m, 13H), 3.92 (d, 1H, J
= 10.0 Hz 1H, H-5), 4.43 (d, 1 H, J = 9.0 Hz, H-1'), 4.52 (t, 2 H, J = 7.0 Hz), 4.78 (d, 1 H, J
= 3.5 Hz, H-1), 8.32 (s, 1H, CHarom); HRMS [M]: m/z: calcd for C28H47N5O13 661.3170,
found 661.3145.
1-[3-(O-(2-N-acetyl-2-deoxy-β-D-glucopyranosyl)-(14)-α-D-glucopyranosyl
uronic acid-1-yl)-propyl]-1H-[1,2,3]-triazole-4-carboxylic acid 3-phenylpropylamide
(11). Compound 11 was produced following the same procedure for the synthesis of
1

compound 10. H NMR (500 MHz, D2O): δ = 1.92 (t, 2H, J = 7.0 Hz,), 1.98 (s, 3H,

175

NHCOCH3), 2.14-2.22 (m, 2H), 2.67 (t, 2H, J = 8.0 Hz), 3.31-3.77 (m, 15H), 3.82 (d, 1H,
J = 12.5 Hz 1H, H-5), 4.43 (d, 1 H, J = 8.5 Hz, H-1'), 4.50-4.56 (m, 2 H), 4.76 (d, 1 H, J =
3.5 Hz, H-1), 7.10-7.25 (m, 5H, CHarom), 8.27 (s, 1H, CHarom); HRMS [M]: m/z: calcd for
C29H41N5O13 667.2701, found 667.2718.
1-[3-(O-(2-N-acetyl-2-deoxy-β-D-glucopyranosyl)-(14)-α-D-glucopyranosyl
uronic acid-1-yl)-propyl]-1H-[1,2,3]-triazole-4-carboxylic acid 4-phenylbutanyl
amide (12). Compound 12 was produced following the same procedure for the synthesis
1

of compound 10. H NMR (500 MHz, D2O): 1.10 (t, 2H, J = 7.5 Hz), 1.52-1.56 (m, 4H),
1.93 (s, 3H, NHCOCH3), 2.18-2.25 (m, 2H), 2.54 (t, 2H, J = 7.5 Hz), 3.28-3.72 (m, 15H),
3.78 (d, 1H, J = 11.5 Hz 1H, H-5), 4.38 (d, 1 H, J = 8.5 Hz, H-1'), 4.46-4.49 (m, 2 H), 4.70
(m, 1 H, H-1), 7.09-7.22 (m, 5H, CHarom), 8.26 (s, 1H, CHarom); HRMS [M]: m/z: calcd
for C30H43N5O13 681.2857, found 681.2869.
1-[3-(O-(2-N-acetyl-2-deoxy-β-D-glucopyranosyl)-(14)-α-D-glucopyranosyl
uronic acid-1-yl)-propyl]-1H-[1,2,3]-triazole-4-carboxylic acid 2-(2-chlorophenyl)
ethanylamide (13). Compound 13 was produced following the same procedure for the
1

synthesis of compound 10. H NMR (500 MHz, D2O): δ = 1.89 (s, 3H, NHCOCH3),
2.08-2.14 (m, 2H), 2.93 (t, 2H, J = 6.5 Hz), 3.26-3.70 (m, 15H), 3.76 (d, 1H, J = 11.5 Hz
1H, H-5), 4.35 (d, 1 H, J = 8.5 Hz, H-1'), 4.41-4.47 (m, 2H, NH), 4.58 (d, 1 H, J = 4.0 Hz,
H-1), 7.07-7.26 (m, 4H, CHarom), 8.17 (s, 1H, CHarom); HRMS [M]: m/z: calcd for
C28H38ClN5O13 687.2155, found 687.2124.
1-[3-(O-(2-N-acetyl-2-deoxy-β-D-glucopyranosyl)-(14)-α-D-glucopyranosyl
176

uronic acid-1-yl)-propyl]-1H-[1,2,3]-triazole-4-carboxylic acid 2-(4-chlorophenyl)
ethanylamide (14). Compound 14 was produced following the same procedure for the
1

synthesis of compound 10. H NMR (500 MHz, D2O): δ = 1.89 (s, 3H, NHCOCH3),
2.08-2.14 (m, 2H), 2.77 (t, 2H, J = 7.0 Hz), 3.27-3.69 (m, 15H), 3.75 (d, 1H, J = 11.0 Hz
1H, H-5), 4.35 (d, 1 H, J = 8.5 Hz, H-1'), 4.43-4.47 (m, 2H, NH), 4.81 (s, 1 H, H-1),
7.10-7.18 (m, 4H, CHarom), 8.18 (s, 1H, CHarom); HRMS [M]: m/z: calcd for
C28H38ClN5O13 687.2155, found 687.2201.
1-[3-(O-(2-N-acetyl-2-deoxy-β-D-glucopyranosyl)-(14)-α-D-glucopyranosyl
uronic acid-1-yl)-propyl]-1H-[1,2,3]-triazole-4-carboxylic acid 2-(naphthalen-2-yl)
ethanylamide (15). Compound 15 was produced following the same procedure for the
synthesis of compound 10.

1

H NMR (500 MHz, D2O): 1.93 (s, 3H, NHCOCH3),

2.08-2.14 (m, 2H), 3.00 (t, 2H, J = 6.0 Hz), 3.27-3.74 (m, 15H), 3.79 (d, 1H, J = 12.5 Hz
1H, H-5), 4.40 (d, 1 H, J = 8.0 Hz, H-1'), 4.43-4.45 (m, 2H, NH), 4.61 (s, 1 H, H-1),
7.39-7.77 (m, 7H, CHarom), 8.13 (s, 1H, CHarom); HRMS [M]: m/z: calcd for
C32H41N5O13 703.2701, found 703.2674.
1-[3-(O-(2-N-acetyl-2-deoxy-β-D-glucopyranosyl)-(14)-α-D-glucopyranosyl
uronic acid-1-yl)-propyl]-1H-[1,2,3]-triazole-4-carboxylic acid 2-(4-hydroxyphenyl)
ethanylamide (16). Compound 16 was produced following the same procedure for the
1

synthesis of compound 10. H NMR (500 MHz, D2O): δ = 1.90 (s, 3H, NHCOCH3),
2.08-2.14 (m, 2H), 2.72 (t, 2H, J = 6.5 Hz), 3.28-3.78 (m, 16H), 4.36 (d, 1 H, J = 8.5 Hz,

177

H-1'), 4.48-4.45 (m, 2H), 6.67-7.05 (m, 7H, CHarom), 8.20 (s, 1H, CHarom);HRMS [M]:
m/z: calcd for C28H39N5O14 669.2494, found 669.2481.
Preparation of HA-ADH-biotin:
1) HA-ADH: HA (16 kDa, 100 mg, 0.260 mmol HA2) was dissolved in H2O (25
ml). To this mixture was added adipic dihydrazide (ADH) (905 mg, 20 eq., 5.20 mmol),
and 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride (EDCI) (8.0 mg, 0.2
eq. 0.052 mmol). The pH of the reaction mixture was maintained at 4.75 by addition of
0.1 N HCl until no further rise in pH was observed. The pH of the reaction mixture was
then raised to 7.0 by addition of 1 N NaOH. After dialysis and lyophilization, HA-ADH
1

was yielded as a white solid with 16% ADH per HA2 unit. In H-NMR spectra, the
integrations of CH2 groups (from ADH) and NHAc groups (from HA) were used to
21

characterize the degree of substitution .
2) NHS-biotin: Biotin (100 mg, 0.4 mmol), N-hydroxysuccinimide (NHS) (47 mg, 1
eq., 0.4 mmol) and N, N’-Dicyclohexylcarbodiimide (DCC) (110 mg, 1.3 eq. 0.520 mmol)
were dissolved in DMF (3 ml). The reaction mixture was sonicated for 10 hrs. The crude
product was concentrated and precipitated out with Et2O (15 ml) for 3 times to yield a
24

white solid 150 mg. This crude mixture was used for the synthesis of biotin-ADH-HA .
3) HA-ADH-biotin: HA-ADH (50 mg) and NHS-biotin (150 mg) were dissolved in
H2O (10 ml). The reaction mixture was sonicated overnight. After dialysis and
lyophilization, HA-ADH-biotin was yielded as a white solid with 16% ADH and 10% biotin

178

1

per HA2 unit. In H-NMR spectra, the integrations of CH2 groups (from ADH and biotin)
and NHAc groups (from HA) were used to characterize the degree of substitution.
Procedure for inhibitory ELISA
Goat anti-human IgG (Fc) (3 μg in 100 μl phosphate buffered saline (PBS) per
well) was added to the 96-well microtiter plate, and the plate was incubated in 4 °C
overnight. The plate was washed with 0.5% PBS-Tween 20 (PBST) (200 μl per well) for
3 times. Then 5% BSA in PBS (200 μl per well) was added and the plate was incubated
at 37 °C for 90 min. The plate was washed with 0.5% PBST (200 μl per well) for 3 times
and PBS (200 μl per well) once. Then recombinant human CD44/Fc chimera (0.2 μg in
100 μl PBS per well) was added, and the plate was incubated at 37 °C for 45 min. The
plate was washed with 0.05% PBST (200 μl per well) for 3 times and PBS (200 μl per
well) once. Then HA-ADH-biotin (0.5 μg per well) was pre-mixed with sHA or analogues
in 100 μl PBS and the mixtures were added. The plate was incubated at 22 °C for 2 hrs,
then washed with 0.05% PBST (200 μl per well) for 3 times and PBS (200 μl per well)
once. Avidin-HRP 1 x 2000 (100 μl per well) in 0.2% BSA-PBS was added, and the plate
was incubated at 22 °C for 1 hr. The plate was washed with 0.05% PBST (200 μl per
well) for 3 times. Fresh prepared 3,3′,5,5′-tetramethylbenzidine (TMB) solution (200 μl
per well) was added. And the plate was kept in dark at 22 °C for 15 min. 0.5 M H2SO4
(50 μl per well) was added, and the plate was read at 450nm.
Digestion of HA polymer:

179

80 g HA (sodium salt, pure powder, food grade) was partially dissolved in 1L
digestion buffer (0.15M NaCl, 0.1M NaOAc, pH was adjusted to 5.2 by AcOH). Bovine
testicular hyaluronidase (200 mg, 400-1000 u/mg) was added. The reaction was stirred
at 37 °C in an oil bath for 8 weeks. Hyaluronidase 75 mg was added every 3 days. The
reaction was monitored by TLC with t-butanol (tBuOH)/H2O/AcOH (1.5:1:1) and stained
by 1, 3-dihydroxylnaphalene (0.2 g in 50 ml 5% H2SO4/ethanol (EtOH)). When the major
product was HA4, the reaction mixture was heated up to 100 °C. 900 ml H2O was
removed by high vacuum rotary evaporator, and crude HA4 50 g was precipitated out by
EtOH (900 ml), sodium acetate (NaOAc) was left in mother liquor, and the crude HA4
was directly used for next step

21-22

.

Methyl S-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside)
-(13)- 2-O-benzoyl-3,4-dideoxy-3,4-epimino-6-O-p-methoxybenzyl-β-Dallopyranoside (40): 39 (0.025 mmol, 9.0 mg) and 37 (0.0375 mmol, 1.5 eq., 21.0 mg )
were co-evaporated with toluene, and dissolved in anhydrous DMF (3 ml). TEA (1 mmol,
4 eq., 14 μl) was added at 0 °C and the reaction mixture was stirred at r.t. overnight.
Then the crude mixture was concentrated and purified by silica gel chromatography with
1

toluene/acetone system to yield 40 (32%). H NMR (500 MHz, CDCl3): δ = 1.83 (s, 3H),
1.99 (s, 3H), 2.00 (s, 3H), 2.01 (s, 3H) (OCOCH3 x3, NHCOCH3), 2.54 (dd, 1H, J = 1.5
Hz, 7.5 Hz, H-4), 2.83 (dd, 1H, J = 4.0 Hz, 7.5 Hz, H-3), 3.31-3.35 (m, 1H, H-5), 3.45 (s,
3H, C6H4OCH3), 3.58 (dd, 1H, J = 6.5 Hz, 10.0 Hz, H-6), 3.67 (dd, 1H, J = 5.0 Hz, 10.0

180

Hz, H-6), 3.80 (s, 3H, OCH3 anomeric), 3.97-4.03 (m, 2H, H-6’ x2), 4.07-4.13 (m, 2H,
H-2’, H-5), 4.52 (d, 1H, J = 7.0 Hz, CH2PMP), 4.53 (d, 1H, J = 7.0 Hz, CH2PMP), 4.53 (d,
1H, J = 9.5 Hz, H-1), 4.66 (d, 1H, J = 11.0 Hz, H-1’), 4.88 (t, 1H, J = 9.5 Hz, H-3’),
4.95-5.00 (m, 2H, H-2, H-4’), 5.55 (d, 1H, J = 9.5 Hz, NH’), 6.87-6.88 (m, 2H, CHarom),
7.48-7.51 (m, 2H, CHarom), 7.59-7.61 (m, 1H, CHarom), 7.24-7.26 (m, 2H, CHarom),
8.12-8.14 (m, 2H, CHarom); 13C NMR (gHMQC decoupling, 500 MHz, CDCl3): δ = 17.2 x
3, 19.2 (OCOCH3 x3, NCOCH3), 37.8 (C-3), 39.8 (C-4), 46.1 (C6H4OCH3), 47.4 (OCH3
anomeric), 51.8 (C-6’), 56.7 (C-2), 58.4 (C-6), 58.9 (C-4’), 60.8 (C-5), 61.0 (C-2’), 61.4
(C-3’), 63.2 (C-5’), 75.1 (C-1’), 82.8 (C-1), 113.9 x 2, 127.9 x 2, 128.7, 129.3 x 2, 129.8 x
2, (Carom x9); HRMS [M ]: m/z: calcd for C36H44N2O14S 760.2513, found 760.2519.
Acetyl O-(methyl 2,3,4-tri-O-acetyl- β-D-glucopyranosyluronate)-(13)-O-(2acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(14)-O-(methyl 2,3-di
-O-acetyl-β-D-glucopyranosyluronate)-(13)-(2-acetamido-3,6-di-O-acetyl-2-deoxy
+

- 1-thio- β-D-glucopyranoside (38): Ion exchange resin (Dowex 50WX4 Na form, 39
g) was washed with H2O (500 ml) for 3 times and stirred in 5% HCl (500 ml) at r.t. for 1
hr. The filtered resin was washed with H2O (500 ml) for 3 times. Tetrabutylammonium
hydroxide (TBAOH) (40% wt. H2O, 45 ml) was added to adjust the pH to 12. Then the
filtered resin was washed with H2O (500 ml) for 3 times. TBA resin was stirred with crude
20 (15 g) in H2O (300 ml) for one day. The resin was filtered out and could be reused.
H2O was removed and the HA4-TBA (22) (17 g) was yielded as a white solid with 80%

181

1

TBA per HA2. The degree of substitution was characterized by H-NMR. Compound 22
(17 g) was co-evaporated with toluene, and dissolved in anhydrous DMF (150 ml). MeI
(30 ml) was added and the reaction mixture was stirred at 35 °C for 3 days. DMF was
removed by high vacuum rotary evaporator, and the solid was dissolved in MeOH (300
ml). After filtration, the filtrate was collected and the crude HA methyl ester (15 g) was
obtained as light yellow solid. Crude HA methyl ester (12 g) was co-evaporated with
toluene, and dissolved in anhydrous DMF (25 ml), Py (25 ml) and Ac2O (40 ml). The
reaction mixture was stirred at 40 °C for 2 days. The solvents were removed, and the
crude product was dissolved in DCM (70 ml). The solution was poured into Et2O (250 ml)
to precipitate the product. After filtration and purification by silica gel chromatography
(DCM/MeOH), 23 (8.2 g) was yielded as α/β mixture (35% from crude 20). Compound 23
(0.48 mmol, 590 mg) was co-evaporated with toluene and dissolved in DCM (3 ml). Ac2O
(0.2 ml) was added and the reaction mixture was stirred at r.t. for 30 min. HBr (33% wt. in
AcOH, 3 ml) was added at 0 °C dropwise and the reaction was stirred at r.t. for 6 hrs. The
reaction was diluted with DCM (200 ml) and poured onto crushed ice in saturated NaCl
(200 ml). The organic phase was separated and washed with saturated NaHCO 3 until
the pH reached 7, which was then dried over Na2SO4, filtered, and concentrated. The
resulting crude residue was co-evaporated with toluene, and dissolved in anhydrous
MeCN (3 ml). The mixture of TBASAc (2.60 mmol, 5 eq., 777 mg) and HSAc (7.34 mmol,
15 eq., 525 μl) in anhydrous MeCN (1 ml) was added to the reaction mixture dropwise at
0 °C. The reaction was stirred at r.t. for 4.5 hrs. The crude mixture was concentrated and
182

1

purified with silica gel chromatography (DCM/MeOH) to yield 38 (370 mg, 30%). H NMR
(500 MHz, CDCl3): δ = 1.95 (s, 3H) x2, 1.96 (s, 3H) x2, 1.72 (s, 3H), 2.00 (s, 3H), 2.02,
(s, 3H), 2.03 (s, 3H) x2, 2.33 (s, 3H, SCOCH3), 3.05-3.10 (m, 1H, H-2’’), 3.60-3.62 (m,
1H, H-5’’), 3.67 (s, 3H, COO CH3), 3.73-3.83 (m, 4H, H-5, COOCH3), 3.93-4.00 (m, 4H,
H-5’’’, H-5’, H-3, H-6’’), 4.02-4.09 (m, 3H, H-4’, H-6, H-2), 4.13 (dd, 1H, J = 5.0 Hz, 12.5
Hz, H-6), 4.28 (dd, 1H, J = 4.5 Hz, 12.5 Hz, H-6’’), 4.43 (t, 1H, J = 9.5 Hz, H-3’), 4.59 (d,
1H, J = 8.0 Hz, H-1’’’), 4.75-4.80 (m, 3H, H-1’’, H-1’, H-2’), 4.4 (t, 1H, J = 9.5 Hz, H-2’’’),
4.86 (t, 1H, J = 9.5 Hz, H-4’’), 4.88 (t, 1H, J = 9.5 Hz, H-4), 5.03 (t, 1H, J = 8.5 Hz, H-3’),
5.08 (t, 1H, J = 9.5 Hz, H-4’’’), 5.16 (d, 1H, J = 9.0 Hz, H-1), 5.17 (t, 1H, J = 9.5Hz, H-3’’’),
6.22 (d, 1H, J = 6.5 Hz, NH’’), 6.35 (d, 1H, J = 8.5 Hz, NH); 13C NMR (125 MHz, CDCl3):
δ = 20.3 x2, 20.4, 20.5 x3, 20.7 x2 (OCOCH3 x9), 23.2, 23.5 (NCOCH3 x2), 30.8
(SOCH3), 52.6 (COOCH3), 53.0 (COOCH3), 57.5 (C-2’’), 61.9 (C-6’’), 62.2 (C-6), 68.1
(C-4’’), 68.3 (C-4), 69.3 (C-4’’’), 71.2 (C-2’), 71.5 (C-2’’’), 71.6 (C-5’’), 72.1 x2 (C-3’,
C-3’’’), 72.1 x2 (C-5’, C-5’’’), 74.7 (C-4’), 75.8 (C-2), 76.2 (C-5), 76.8 (C-3’’), 79.7 (C-3),
81.2 (C-1), 98.9 (C-1’), 100.6 (C-1’’), 100.1 (C-1’’’), 177.9, 167.8, 169.2, 169.3, 169.4,
169.7 x2, 170.0 x2, 170.1, 170.4 (OCOCH3 x9, NHCOCH3 x2, SCOCH3, COOCH3 x2);
HRMS [M + Na]+: m/z: calcd for C50H68N2O32SNa 1263.3374, found 1263.3434.

183

Preparation of monosaccharide 35, 36, 37, 38, 15 and 23:
Scheme 4.13. Preparation of monosaccharide 35, 36, 37, 38, 15 and 23

Methyl-β-D-galactoside 68 (7.7 mmol, 1.5 g) and CSA (3.2 mmol, 0.42 eq., 0.75 g)
were co-evaporated with toluene and dissolved in anhydrous DMF (80 ml). Anisaldehyde
dimethyl acetal (8.8 mmol, 1.15 eq, 1.5 ml) was added, and the reaction was stirred at 80
°C for 2 hrs. TEA (3.2 mmol, 0.42 eq., 0.45 ml) was added to adjust the pH to 7. The
crude mixture was concentrated and purified by silica gel chromatography
(toluene/acetone) to yield compound 69 (1.95 g, 81%). Compound 69 (17.4 mmol, 5.4 g)
184

and DCC (26.0 mmol, 1.5 eq., 5.57 g) were co-evaporated with toluene and dissolved in
anhydrous DCM (260 ml). Levulinic acid (20.8 mmol, 1.2 eq., 2.10 ml) and DMAP (8.7
0mmol, 0.5 eq., 1.08 g) were added. The reaction mixture was stirred at r.t. overnight,
and dilute with DCM (600 ml). The solid was removed by filtration. The crude product
was concentrated and purified by silica gel chromatography (toluene/acetone) to yield
the a mixture of 3-O-Lev and 2-O-Lev (1:1, 11.8 mmol. 4.85 g), which was co-evaporated
with toluene and dissolved in anhydrous DCM (80 ml). Benzoyl chloride (BzCl) (14.2
mmol, 1.2 eq, 1.65 ml) and 4-dimethylaminopyridine (DMAP) (17.8 mmol, 1.5 eq., 2.17
g) were added to the reaction mixture. After 4 hrs, the reaction mixture was concentrated
to yield a mixture of compound 70 and 71. Compound 70 (5.65 mmol. 2.90 g) and 71
(5.65 mmol. 2.90 g) were separated by the combination of gel chromatography
purification (toluene/acetone v/v 10:1) and crystallization (EA/Hexane, DCM/Et2O). 70
(1.17 mmol. 0.600 g) was dissolved in MeOH (3ml), and p-toluenesulfonic acid (TsOH)
(0.117 mmol, 0.1 eq., 22.0 mg) was added. The reaction mixture was stirred at r.t. for 2
hrs, and quenched with TEA (0.117 mmol, 0.1 eq., 11.4 μl). The crude mixture was
concentrated and purified by silica gel chromatography (toluene/acetone) yielding a diol
(330 mg, 0.84 mmol). The diol (330 mg, 0.84 mmol) was co-evaporated with toluene and
dissolved in anhydrous DCM (5 ml). Imidazole (1.68 mmol, 2 eq.,113 mg) and tert-butyl
diphenylchlorosilane (TBDPSCl) (0.870 mmol, 1.05 eq., 0.23 ml) were added, and the
reaction mixture was stirred at r.t. overnight. The crude mixture was directly purified by
silica gel chromatography (Hexane/EA/DCM) to yield 72 (0.390 g, 0.615 mmol).
185

Compound 73 was synthesized in 53% yield from 71 following the same procedures as
72. Compound 72 (51 mg, 0.080 mmol) was dissolved in DCM (1.5 ml). Py (0.26 mmol, 7
eq, 15 μl) and Tf2O (0.12 mmol, 1.5 eq, 20 μl) were added sequentially at 0 °C. The
reaction was stirred at r.t. for 1 hrs. The crude mixture was directly purified by silica gel
chromatography (Hexane/EA/DCM) to produce compound 46 (45 mg, 0.07 mmol).
Compound 66 was formed in 87% yield from compound 73 following the same procedure
as compound 46.
Methyl 2-O-benzoyl-3-O-levulinyl-4, 6-O-p-methoxybenzylidene -β-D1

glucopyranoside (70). H NMR (500 MHz, CDCl3): δ = 1.88 (s, 3H CH2CH2COCH3),
2.42-2.63 (m, 4H, CH2CH2COCH3), 3.50 (s, 3H, C6H4OCH3), 3.55 (t, 1H, J = 1.0 Hz,
H-5), 3.79 (s, 3H, OCH3 anomeric), 4.06 (dd, 1H, J = 1.0 Hz, 12.5 Hz, H-6’), 4.33 (dd,
1H, J = 1.0 Hz, 12.5 Hz, H-6), 4.36 (dd, 1H, J = 1.0 Hz, 3.5 Hz, H-4), 4.56 (d, 1H, J = 8.0
Hz, H-1), 5.15 (dd, 1H, J = 3.5 Hz, 10.5 Hz, H-3), 5.47 (s, 1H, CHPh), 5.62 (dd, 1H, J =
8.0 Hz, 10.5 Hz, H-2), 6.87-6.90 (m, 2H, CHarom), 7.41-7.47 (m, 4H, CHarom), 7.53-7.57
(m, 1H, CHarom), 7.99-8.01 (m, 2H, CHarom);

13

C NMR (125 MHz, CDCl3): δ = 28.2

(CH2CH2COCH3), 29.4 (CH2CH2COCH3), 37.8 (CH2CH2COCH3), 55.3 (OCH3
anomeric), 56.5 (C6H4COCH3), 66.4 (C-5), 68.8 (C-6), 68.9 (C-2), 72.0 (C-3), 73.4 (C-4),
101.0 (CHPh), 101.9 (C-1), 113.5 x2, 127.8 x2, 128.4 x2, 129.2 x2, 129.7, 130.1, 133.1,
160.1

(Carom

x12),

165.2,

172.1

(COPh,

OCOCH2CH2COCH3),

206.1

(OCOCH2CH2COCH3); HRMS [M + Na]+: m/z: calcd for C27H30O10Na 537.1737, found
537.1763.
186

Methyl 2-O-benzoyl -6-O-tert-butyldiphenylsilyl-3-O-levulinyl -β-D1

glucopyranoside (72). H NMR (500 MHz, CDCl3): δ = 1.04 (s, 9H, SiC(CH3)3), 2.05 (s,
3H, CH2CH2COCH3), 2.41-2.56 (m, 2H, CH2CH2COCH3), 2.63-2.56 (m, 2H,
CH2CH2COCH3), 3.01 (d, 1H, J = 4.0 Hz, OH), 3.46 (s, 3H, OCH3 anomeric), 3.65 (dt,
1H, J = 1.0 Hz, 5.5 Hz, H-5), 3.93 (dd, 1H, J = 5.5 Hz, 10.5 Hz, H-6), 3.99 (dd, 1H, J = 5.5
Hz, 10.5 Hz, H-6’), 4.29 (dd, 1H, J = 1.0 Hz, 4.0 Hz, H-4), 4.49 (d, 1H, J = 8.5 Hz, H-1),
5.06 (dd, 1H, J = 3.0 Hz, 10.5 Hz, H-3), 5.56 (dd, 1H, J = 8.5 Hz, 10.5 Hz, H-2), 7.36-7.45
(m, 8H, CHarom), 7.53-7.57 (m, 1H, CHarom), 7.70-7.71 (m, 4H, CHarom), 8.00-8.01 (m,
2H CHarom);

13

C NMR (125 MHz, CDCl3): δ = 19.2 (SiC(CH3)3), 26.7 (SiC(CH3)3), 28.3

(CH2CH2COCH3), 29.6 (CH2CH2COCH3), 38.1 (CH2CH2COCH3), 56.5 (OCH3), 63.2
(C-6), 67.4 (C-4), 67.7 (C-2), 74.2 x2 (C-3, C-5), 102.1 (C-1), 127.8 x2, 127.8 x2, 128.4
x2, 129.8, 129.3 x3, 132.8, 133.0, 133.1 x2, 135.5 x3, 135.7 x3(Carom x18), 165.3,
172.0 (COPh, OCO CH2CH2COCH3), 207.2 (CH2CH2COCH3); HRMS [M + Na]+ calcd
for C35H42O9SiNa 657.2496, found 657.2514.
Methyl 3-O-benzoyl-2-O-levulinyl-4, 6-O-p-methoxybenzylidene -β-D1

glucopyranoside (71). H NMR (500 MHz, CDCl3): δ = 2.02 (s, 3H CH2CH2COCH3),
2.49-2.65 (m, 4H, CH2CH2COCH3), 3.54 (s, 3H, C6H4OCH3), 3.58 (d, 1H, J = 1.0 Hz,
H-5), 3.77 (s, 3H, OCH3 anomeric), 4.07 (dd, 1H, J = 2.1 Hz, 12.5 Hz, H-6’), 4.34 (dd,
1H, J = 1.5 Hz, 12.5 Hz, H-6), 4.50-4.52 (m, 1H, H-4), 4.51 (d, 1H, J = 8.0 Hz,H-1), 5.15
(dd, 1H, J = 3.5 Hz, 10.5 Hz, H-3), 5.45 (s, 1H, CHPh), 5.57 (dd, 1H, J = 8.0 Hz, 10.5 Hz,

187

H-2), 6.83-6.84 (m, 2H, CHarom), 7.38-7.55 (m, 5H, CHarom), 7.99-8.01 (m, 2H,
CHarom);

13

C

NMR

(125

MHz,

CDCl3):

δ

=

28.0

(CH2CH2COCH3),

29.7

(CH2CH2COCH3), 37.9 (CH2CH2COCH3), 55.3 (C6H4COCH3), 56.6 (OCH3 anomeric),
66.4 (C-5), 68.9 (C-6), 72.8 (C-3), 73.5 (C-4), 100.8 (CHPh), 101.7 (C-1), 113.4 x2,
127.6 x2, 127.8, 128.4 x2, 129.9, 130.0 x2, 133.4, 160.0 (Carom x12), 166.2, 171.6
(COPh, OCOCH2CH2COCH3), 205.9 (CH2CH2COCH3); HRMS [M + Na]+ :m/z: calcd for
C27H30O10 Na 537.1737, found 537.1739
Methyl 3-O-benzoyl -6-O-tert-butyldiphenylsilyl-2-O-levulinyl -β-D1

glucopyranoside (73). H NMR (500 MHz, CDCl3): δ = 1.03 (s, 9H, SiC(CH3)3), 2.01 (s,
3H, CH2CH2COCH3), 2.45-2.64 (m, 4H, CH2CH2COCH3), 2.80 (d, 1H, J = 4.5 Hz, OH),
3.50 (s, 3H, OCH3 anomeric), 3.64-3.66 (m, 1H, H-5), 3.92 (dd, 1H, J = 4.5 Hz, 10.5 Hz,
H-6), 3.98 (dd, 1H, J = 6.0 Hz, 10.5 Hz, H-6’), 4.39 (t, 1H, J = 3.0 Hz, H-4), 4.43 (d, 1H, J
= 8.0 Hz, H-1), 5.11 (dd, 1H, J = 3.0 Hz, 10.0 Hz, H-3), 5.49 (dd, 1H, J = 8.0 Hz, 10.0 Hz,
H-2), 7.34-7.45 (m, 8H, CHarom), 7.54-7.58 (m, 1H, CHarom), 7.66-7.70 (m, 4H CHarom),
8.02-8.04 (m, 4H CHarom) ;

13

C NMR (125 MHz, CDCl3): δ = 19.1 (SiC(CH3)3), 26.7

(SiC(CH3)3) 28.0 (CH2CH2COCH3), 29.6 (CH2CH2COCH3), 37.9 (CH2CH2COCH3),
56.6 (OCH3), 63.4 (C-5), 68.0 (C-2), 69.3 (C-6), 73.8 (C-3), 74.4 (C-4), 102.0 (C-1),
127.8 x2, 127.8 x2, 128.5 x2, 129.4, 129.9 x2, 130.0 x2, 132.6, 132.8, 133.4, 135.5 x2,
135.6

x2

(Carom

x18),

165.9,

171.6

(COPh,

OCOCH2CH2COCH3),

205.9

(CH2CH2COCH3); HRMS [M + Na]+ calcd for C35H42O9SiNa 657.2496, found
657.2474.
188

Methyl S-(methyl 2,3,4-tri-O-acetyl- β-D- glucopyranosyluronate)-(13)-O(2-acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(14)-O-(methyl
2,3-di-O-acetyl-β-D-glucopyranosyluronate)-(13)-(2-acetamido-3,6-di-O-acetyl2-deoxy- β-D-glucopyranosyl)-(14)-3-O-benzoyl-6-O-tert-butyldiphenylsilyl-2-Olevulinyl-β-D-glucopyranoside (61): 38 (0.04 mmol, 50 mg) was dissolved in MeOH (1
ml) and DCM (1 ml). NaOMe (1M in MeOH, 0.02 mmol, 0.5 eq.) was added in 5 portions
at -40 °C. After the starting material was consumed, acidic resin (IR 120) was added to
adjust the pH to 4.5. The crude 36 was concentrated, and co-evaporated with toluene.
Crude 36 (0.04 mmol) and freshly prepared compound 60 (0.08 mmol, 2 eq.) were
dissolved in anhydrous DMF (2 ml). TEA (0.12 mmol, 3 eq, 15 μl) was added at 0 °C, and
the reaction mixture was stirred at r.t. for 2 days. The reaction mixture was concentrated
and purified by silica gel chromatography (toluene/ acetone) to yield compound 61 in
34% yield. 1H NMR (500 MHz, CDCl3): δ = 1.01 (s, 9H, SiC(CH3)3), 1.51 (s, 3H,
NHCOCH3), 1.81 (s, 3H, OCOCH3), 1.92 (s, 3H), 1.95 (s, 3H), 1.96 (s, 3H), 1.97 (s, 3H),
1.98 (s, 6H), 1.99 (s, 3H), 2.02 (s, 3H), 2.03 (s, 3H), 2.04 (s, 3H), 2.44-2.64 (m, 4H,
CH2CH2COCH3), 3.04-3.10 (m, 1H, H-2’’’), 3.31 (t, 1H, J = 11.0 Hz, H-4), 3.47 (s, 3H,
OCH3 anomeric), 3.58-3.69 (m, 2H, H-5’, H-5’’’), 3.69 (s, 3H, COOCH3), 3.69-3.75 (m,
1H, H-5), 3.78-3.84 (m, 1H, H-3’), 3.84 (s, 3H, COOCH3), 3.92-4.09 (m, 8H, H-6 x2, H-2’,
H-6’, H-4’’, H-5’’, H-6’’’, H-5’’’’), 4.28 (dt, 2H, J = 4.5 Hz, 13.0 Hz, H-6’, H-6’’’), 4.36 (d, 1H,
J = 8.5 Hz, H-1), 4.44 (t, 1H, J = 9.5 Hz, H-3’’’), 4.61 (d, 1H, J = 8.0 Hz, H-1’’’’), 4.63 (d,
1H, J = 8.0 Hz, H-1’’), 4.73-4.78 (m, 3H, H-4’, H-2’’’’, H-2’’), 4.80 (d, 1H, J = 8.0 Hz,
189

H-1’’’), 4.82 (d, 1H, J = 10.5 Hz, H-1’), 4.87 (t, 1H, J = 9.5 Hz, H-4’’’), 5.06 (t, 1H, J = 9.0
Hz, H-3’’’’), 5.10 (t, 1H, J = 9.0 Hz, H-4’’’’), 5.13 (t, 1H, J = 8.5 Hz, H-2), 5.18 (t, 1H, J =
9.0 Hz, H-3’’), 5.41 (dd, 1H, J = 9.0 Hz, 11.0 Hz, H-3), 5.50 (d, 1H, J = 10.0 Hz, NH’’’),
5.67 (d, 1H, J = 8.0 Hz, NH’), 7.33-7.48 (m, 8H, CHarom), 7.58-7.61 (m, 1H, CHarom),
7.69-7.73 (m, 4H, CHarom), 7.97-8.05 (m, 2H, CHarom);

13

C NMR (125 MHz, CDCl3): δ

= 19.3 (Cq, SiC(CH3)3), 20.4 x3, 20.5 x3, 20.6 x2, 20.7 (OCOCH3 x9), 22.9, 23.5
(NHCOCH3 x2), 26.8 (SiC(CH3)3), 27.9 (CH2C H2COCH3), 29.4 (CH2C H2COCH3),
37.9 (CH2CH2COCH3), 45.2 (C-4), 52.7 (COOC H3), 52.9 (C-5), 53.0 (COOCH3), 56.3
(OCH3 anomeric), 58.0 (C-2’’’), 61.9, 62.6 (C-6’, C-6’’’), 62.9 (C-6), 68.0 (C-4’’’), 68.8,
69.4, 70.9, 71.0, 71.8, 71.9, 72.0, 72.1 x 2, 72.3 x 2, 72.7, 74.5, 75.7, 75.7 (C-2, C-3,
C-2’, C-4’, C-5’, C-2’’, C-3’’, C-4’’, C-5’’ C-5’’’, C-2’’’’, C-3’’’’, C-4’’’’, C-5’’’’), 76.2 (C-3’’’),
80.3 (C-3’), 81.5 (C-1’), 98.5 (C-1’’’), 99.9 (C-1’’’’), 101.1 (C-1’’), 101.2 (C-1), 127.5,
127.6, 127.7, 127.8, 128.6, 128.9, 129.6, 129.7, 130.1, 133.1, 133.4, 134.0, 135.5,135.6
x2, 135.7, 135.8 x2 (Carom x18),166.9, 167.5, 167.8,169.0, 169.2, 169.3 x2, 169.7,
169.9, 170.2 x2, 170.6, 170.6, 170.9, 171.4 (OCOCH3 x9, NHCOCH3 x2, COOCH3 x2,
OCOCH2CH2COCH3, COPh), 205.9 (OCOCH2CH2COCH3); HRMS [M + NH4]+: m/z:
calcd for C83H110N3O39SSi 1832.6206, found 1832.6115.
Methyl S-(methyl 2,3,4-tri-O-acetyl- β-D- glucopyranosyluronate)-(13)-O-(2acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(14)-O-(methyl 2,3-di
-O-acetyl-β-D-glucopyranosyluronate)-(13)-(2-acetamido-3,6-di-O-acetyl-2-deoxy
- β-D-glucopyranosyl)-(14)-2-O-benzoyl-6-O-tert-butyldiphenylsilyl-3-O-levulinyl
190

-β-D-glucopyranoside (63): Crude 36 (0.04 mmol) and freshly prepared 46 (0.04 mmol,
1.25 eq.) were dissolved in anhydrous DMF (2 ml). TEA (0.12 mmol, 2.5 eq, 12.5 μl) was
added at 0 °C, and the reaction mixture was stirred at r.t. for 2 days. The reaction mixture
was concentrated and purified by silica gel chromatography (toluene/ acetone) to yield
compound 63 in 27% yield. 1H NMR (500 MHz, CDCl3): δ = 1.02 (s, 9H, SiC(CH3)3),
1.95 (s, 3H), 1.96 (s, 9H), 1.98 (s, 9H), 2.00 (s, 3H), 2.03 (s, 3H), 2.04 (s, 3H), 2.06 (s,
3H), 2.20 (s, 3H), 2.29-2.34 (m, 1H, CH2CH2COCH3), 2.51-2.56 (m, 1H,
CH2CH2COCH3), 2.66-2.86 (m, 2H, CH2CH2COCH3), 2.96-3.01 (m, 1H, H-2’’’),
3.33-3.46 (m, 4H, H-4, OCH3 anomeric), 3.55 (t, 1H, J = 9.5 Hz, H-3’), 3.60-3.65 (m, 2H,
H-5’, H-5’’’), 3.69 (s, 3H, COOCH3), 3.72-3.87 (m, 7H, H-5, COOCH3, H-6 x 2, H-5’’),
3.91-4.00 (m, 3H, H-5’’’’, H-6’, H-6’’’), 4.01 (t, 1H, J = 9.5 Hz, H-4’’) 4.14-4.25 (m, 2H,
H-2’, H-6’), 4.33 (dd, 1H, J = 5.0 Hz, 12.5 Hz, H-6’’’), 4.45 (d, 1H, J = 8.0 Hz, H-1), 4.46
(d, 1H, J = 10.0 Hz, H-1’), 4.49 (t, 1H, J = 9.5 Hz, H-3’’’), 4.59 (d, 1H, J = 8.0 Hz, H-1’’’’),
4.68 (d, 1H, J = 8.5 Hz, H-1’’), 4.76 (dd, 1H, J = 8.0 Hz, 9.5 Hz, H-2’’’’), 4.80 (d, 1H, J =
8.5 Hz, H-1’’’), 4.81 (dd, 1H, J = 8.5 Hz, 9.5 Hz, H-2’’), 4.82 (t, 1H, J = 9.5 Hz, H-4’), 4.86
(t, 1H, J = 9.5 Hz, H-4’’’), 5.06 (t, 1H, J = 9.5 Hz, H-3’’), 5.10 (t, 1H, J = 9.5 Hz, H-4’’’’),
5.17 (dd, 1H, J = 8.0 Hz, 11.5 Hz, H-2), 5.18 (t, 1H, J = 9.5 Hz, H-3’’’’), 5.33 (dd, 1H, J =
10.0 Hz, 11.5 Hz, H-3), 5.62 (d, 1H, J = 7.0 Hz, NH’’’), 6.89 (d, 1H, J = 10.0 Hz, NH’),
7.31-7.46 (m, 8H, CHarom), 7.57-7.60 (m, 1H, CHarom), 7.69-7.74 (m, 4H, CHarom)
7.98-8.00 (m, 2H, CHarom);

13

C NMR (125 MHz, CDCl3): δ = 19.3 (Cq, SiC(CH3)3), 20.4

191

x2, 20.5 x2, 20.6 x2, 21.0 x3 (OCOCH3 x9), 23.2, 23.5 (NHCOCH3 x2), 26.8
(SiC(CH3)3), 27.7 (CH2C H2COCH3), 29.7 (CH2C H2COCH3), 37.7 (CH2CH2COCH3),
43.7 (C-4), 52.7 (COOC H3), 53.0 (C-2’), 53.3 (COOCH3), 56.3 (OCH3 anomeric), 58.2
(C-2’’’), 61.9 (C-6’’’), 62.9 (C-6), 63.3 (C-6’), 67.9 (C-4’’’), 68.5 (C-2’’), 69.4 (C-4’’’’), 70.1
(C-3), 70.7 (C-4’), 72.0 (C-2’’’’), 72.1 (C-5’/ C-5’’’), 72.3 (C-3’’), 72.3 (C-5’’’’), 74.0 (C-2),
74.0 (C-3’’’’), 74.6 (C-5’’), 75.8 (C-4’’), 75.8 (C-5’/ C-5’’’), 75.9 (C-3’’’), 76.5 (C-5), 80.2
(C-3’), 80.6 (C-1’), 98.4 (C-1’’’), 99.9 (C-1’’’’), 101.2 (C-1), 101.4 (C-1’’), 127.5 x2, 127.7
x3, 128.5, 129.5, 129.6, 129.7, 129.8, 133.2, 133.3, 133.4, 135.6 x2, 135.8 x3 (Carom
x18),164.5, 165.3, 166.9,167.5, 168.9, 169.2, 169.3, 169.7, 169.8, 169.9, 170.2, 170.3,
170.3,

170.6,

170.9,

172.7

(OCOCH3

x9,

NHCOCH3

x2,

COOCH3

x2,

OCOCH2CH2COCH3, COPh), 209.6 (OCOCH2CH2COCH3); HRMS [M + Na]+: m/z:
calcd for C83H106N2O39SSNai 1837.5760, found 1837.5684.
Methyl S-(methyl 2,3,4-tri-O-acetyl- β-D- glucopyranosyluronate)-(13)-O-(2acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(14)-O-(methyl 2,3-diO-acetyl-β-D-glucopyranosyluronate)-(13)-(2-acetamido-3,6-di-O-acetyl-2-deoxyβ-D-glucopyranosyl)-(14)-3-O-benzoyl-2-O-levulinyl-β-D-glucopyranoside

(62):

61 (60 mg, 0.033 mmol) was dissolved in Py (1.5 ml) in a plastic flask followed by the
addition of 65–70% HF/Py solution (0.3 ml) at 0 °C. The solution was stirred for 4 hrs,
diluted with DCM (15 ml) and washed with 10% aqueous CuSO4 solution (15 ml). The
aqueous phase was extracted with DCM (30 ml) twice and the combined organic layers

192

were washed with a saturated aqueous solution of NaHCO 3 to remove HF. The crude
product was purified by silica gel chromatography to yield compound 62 in 85% yield. 1H
NMR (500 MHz, CDCl3): δ = 1.55 (s, 3H), 1.91 (s, 3H), 1.96 (s, 3H), 1.97 (s, 6H), 1.98 (s,
6H), 2.00 (s, 3H), 2.02 (s, 3H), 2.04 (s, 3H), 2.06 (s, 3H), 2.43-2.60 (m, 4H,
CH2CH2COCH3), 3.05-3.35 (m, 2H, H-4, H-2’), 3.50 (s, 3H, OCH3 anomeric), 3.60-3.63
(m, 2H, H-5, H-5’’’), 3.70 (s, 3H, COOCH3), 3.70-3.74 (m, 1H, H-5), 3.84 (s, 3H,
COOCH3), 3.79-3.93 (m, 1H, H-3’’’), 3.94-4.09 (m, 8H, H-6 x2, H-2’’’, H-5’’’’, H-4’’, H-5’’,
H-6’, H-6’’’ ), 4.18-4.31 (m, 2H, H-6’, H-6’’’) 4.42 (d, 1H, J = 8.0 Hz, H-1),

4.44 (dd, 1H,

J = 7.0 Hz, 10.5 Hz, H-3’), 4.61 (d, 1H, J = 7.5 Hz, H-1’’’’), 4.66 (d, 1H, J = 7.5 Hz, H-1’’),
4.76 (dd, 1H, J = 7.5 Hz, 9.0 Hz, H-2’’’’), 4.78 (dd, 1H, J = 7.5 Hz, 9.0 Hz, H-2’’), 4.79 (d,
1H, J = 8.0 Hz, H-1’’’), 4.81 (t, 1H, J = 9.0 Hz, H-4’’’), 4.87 (t, 1H, J = 10.0 Hz, H-4’), 4.89
(d, 1H, J = 10.5 Hz, H-1’), 5.06 (t, 1H, J = 9.0 Hz, H-3’’’), 5.11 (t, 1H, J = 9.0 Hz, H-4’’’’),
5.11 (t, 1H, J = 10.0 Hz, H-2), 5.19 (t, 1H, J = 9.5 Hz, H-3’’), 5.40 (dd, 1H, J = 9.0 Hz,
11.0 Hz, H-3), 5.70 (d, 1H, J = 8.5 Hz, NH’’’), 5.76 (d, 1H, J = 7.5 Hz, NH’), 7.43-7.46 (m,
2H, CHarom), 7.57-7.60 (m, 1H, CHarom), 8.00-8.01 (m, 2H, CHarom);

13

C NMR (125

MHz, CDCl3): δ = 20.4 x2, 20.5 x2, 20.6 x2, 20.7 x3 (OCOCH3 x9), 22.9, 23.5
(NHCOCH3

x2),

27.8

(CH2C

H2COCH3),

29.7

(CH2C

H2COCH3),

37.9

(CH2CH2COCH3), 45.7 (C-4), 52.7 (COOC H3), 53.0 (COOCH3), 57.0 (OCH3
anomeric), 57.9 (C-2’), 61.9, 62.1 (C-6’, C-6’’’), 62.7 (C-6), 68.0 (C-4’), 68.7 (C-4’’’), 69.4
(C-2), 71.0 x2 (C-3, C-2’’’’), 71.9 x2, 72.0 (C-2’’, C-5’, C-5’’’), 72.1 (C-3’’’’), 72.3 x2, 72.6,
(C-2’’’, C-3’’, C-4’’’’), 74.5 (C-5’’’’), 75.7, 75.9 (C-4’’, C-5’’), 76.2 (C-5), 77.0 (C-3’), 79.9
193

(C-3’’’), 81.6 (C-1’), 98.5 (C-1’’’), 99.9 (C-1’’’’), 101.1 (C-1’’), 101.6 (C-1), 128.2, 128.6,
128.8, 129.0, 130.0, 134.0 (Carom x6), 167.0, 167.2, 167.9, 169.0 169.3 x2, 169.6,
169.7, 170.1 x2, 170.2, 170.6 x2, 171.1, 171.4 (OCOCH3 x9, NHCOCH3 x2, COOCH3
x2, OCOCH2CH2COCH3, COPh), 205.9 (OCOCH2CH2COCH3); HRMS [M + Na]+: m/z:
calcd for C67H88N2O39SNa 1599.4583, found 1599.4655.
Methyl S-(methyl 2,3,4-tri-O-acetyl- β-D- glucopyranosyluronate)-(13)-O-(2acetamido-3,6-di-O-acetyl-2-deoxy-β-D-glucopyranosyl)-(14)-O-(methyl 2,3-di
-O-acetyl-β-D-glucopyranosyluronate)-(13)-(2-acetamido-3,6-di-O-acetyl-2-deoxy
-

β-D-glucopyranosyl)-(14)-6-O-acetyl-2-O-benzoyl-β-D-glucopyranoside

(64):

The TBDPS group in 63 (0.017 mmol, 30 mg) was removed following the same
procedure as the synthesis of 62. Without column purification, the crude mixture was
co-evaporated with toluene and dissolved in Py (0.7 ml) and Ac2O (0.7 ml). After 3 hrs at
r.t., the solvent was removed. The reaction mixture was co-evaporated with toluene to
remove trace amount of Ac2O, and dissolved in AcOH (0.4 ml) and Py (1.6 ml).
Hydrazine monohydrate (0.085 mmol, 5 eq., 4.3 μl) was added and the reaction mixture
was stirred at r.t. overnight. Acetone (0.1 ml) was added, and reaction mixture was
concentrated and purified by silica gel chromatography (DCM/ MeOH) to yield 64 (70%
for 3 steps). 1H NMR (500 MHz, CDCl3): δ = 1.91 (s, 3H), 1.94 (s, 3H), 1.98 (s, 3H) x2,
1.99 (s, 3H), 2,.00 (s, 3H), 2.01 (s, 3H) , 2.02 (s, 3H), 2.03 (s, 3H), 2.04 (s, 6H), 2.13 (s,
6H), 2.77 (t, 1H, J = 10.5 Hz, H-4), 2.92-2.18 (m, 1H, H-2’), 3.48 (s, 3H, OCH3 anomeric),

194

3.55-3.62 (m, 3H, H-2’’’, H-5, H-5’’’), 3.69 (s, 3H, COOCH3), 3.68-3.74 (m, 1H, H-5’’’),
3.80 (s, 3H, COOCH3), 3.76-3.81 (m, 1H, H-3), 3.93-4.31 (m, 8H, H-5’’’’, H-5’’, H-4’’, H-6’
x2, H-6’’’ x2, H-3’’’), 4.46 (d, 1H, J = 8.0 Hz, H-1’), 4.46 (d, 1H, J = 8.0 Hz, H-1),
4.50-4.54 (m, 3H, H-6 x2, H-3’) 4.59 (d, 1H, J = 8.0 Hz, H-1’’’’), 4.65 (d, 1H, J = 8.0 Hz,
H-1’’), 4.77 (d, 1H, J = 8.0 Hz, H-1’’’), 4.78 (dd, 1H, J = 8.0 Hz, 9.5 Hz, H-2’’’’), 4.78 (dd,
1H, J = 8.0 Hz, 9.5 Hz, H-2’’), 4.78 (t, 1H, J = 9.0 Hz, H-4’’’), 4.84 (t, 1H, J = 9.5 Hz,
H-4’’’), 4.86 (t, 1H, J = 9.5 Hz, H-4’), 5.03 (t, 1H, J = 9.0 Hz, H-3’), 5.05 (t, 1H, J = 9.5 Hz,
H-2), 5.01 (t, 1H, J = 9.5 Hz, H-4’’’’), 5.18 (t, 1H, J = 9.5 Hz, H-3’’’’), 5.87 (d, 1H, J = 8.5
Hz, NH’), 6.30-6.33 (m, 1H, NH’’’), 7.40-7.43 (m, 2H, CHarom), 7.53-7.56 (m, 1H,
CHarom), 8.01-8.03 (m, 2H, CHarom);

13

C NMR (125 MHz, CDCl3): δ = 20.4 x 3, 20.5,

20.6 x3, 20.7 x2, 21.0 (OCOCH3 x10), 23.5 x2 (NHCOCH3 x2), 50.5 (C-4), 52.7 (COOC
H3), 53.0 (COOCH3), 56.9 (OCH3 anomeric), 58.2 (C-2’), 61.9, 62.6 (C-6’, C-6’’’), 63.7
(C-6), 68.0 (C-4’), 68.1 (C-4’’’), 68.6 (C-2’’/ C-2’’’’), 69.4 (C-4’’’’), 71.4 (C-2’’/ C-2’’’’), 71.8,
71.9 ( C-5’, C-5), 72.1 (C-3’’’’), 72.3 x2 (C-2, C-5’’’’), 73.0 (C-2’’’), 74.5 x2 (C-3, C-4’’/
C-5’’), 74.8 (C-3), 75.4 (C-3’’’), 75.7 (C-4’’/ C-5’’), 75.9 (C-5’’’), 76.6 (C-3’, C-3’’’), 77.0
(C-1’), 98.4 (C-1’’’), 100.0 (C-1’’’’), 100.8 (C-1’’), 101.4 (C-1), 128.4 x2, 129.7, 129.9 x2,
133.3 (Carom x6), 166.1, 167.0, 167.9, 169.0 169.3 x3, 169.7, 170.2 x2, 170.6, 170.9,
171.2, 171.3, 171.4 (OCOCH3 x9, NHCOCH3 x2, COOCH3 x2, OCOCH2CH2COCH3,
COPh), 205.9 (OCOCH2CH2COCH3); HRMS [M + Na]+: m/z: calcd for C64H84N2O38S
Na 1543.4320, found 1543.4391.

195

Methyl S-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2-deoxy-β-Dglucopyranosyl)-(14)-O-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2deoxy-β-D-glucopyranosyl)-(14)-N-cyclohexyl-β-D-glucopyranuronamide (49): 62
(0.0036 mmol, 5.7 mg) was dissolved in DCM (0.5 ml), tBuOH (0.5 ml) and H2O (0.25
ml). TEMPO (0.0036 mmol, 1 eq., 0.60 mg) and BAIB (0.0036 mmol, 1 eq., 3.0 mg) were
added and reaction mixture was stirred at r.t. overnight. The crude mixture was
concentrated and dissolved in DCM (1 ml). Cyclohexylamine (0.0043 mmol, 1.2 eq, 0.43
mg), EDCI (0.0054 mmol, 1.5 eq. 1.0 mg) and DMAP (0.001 mmol, 0.3 eq, 0.2 mg) were
added. The reaction mixture was stirred at r.t. overnight and purified by silica gel
chromatography. The product was dissolved in MeOH (0.3 ml) and H2O (0.3 ml). The pH
was adjusted to 9.5 by 1 M NaOMe in MeOH, and reaction mixture was stirred at r.t. for 2
days. 1 M HCl was added to adjust the pH to 7 and the reaction mixture was
concentrated and purified by Sephadex G-15 size exclusion chromatography to give the
1

desired product 49 in 40% yield from 62 for three steps). H NMR (600 MHz, D2O): δ =
1.20-1.38 (m, 6 H, CH2 x3), 1.63-1.65 (m, 1 H, CH), 1.74-1.79 (m, 2 H, CH2), 1.88-1.91
(m, 2 H, CH2), 2.02 (s, 3H, NHCOCH3), 2.05 (s, 3H, NHCOCH3), 3.03 (t, 1H, J = 10.8
Hz, H-2’), 3.33-3.39 (m, 3H, H-2, H-2’’, H-2’’’’), 3.51-3.69 (m, 12 H), 3.73-3.78 (m, 7 H),
3.86-3.89 (m, 1H, H-2’’’), 3.92 (d, 1 H, J = 14.4 Hz, H-5, H-5’’ or H-5’’’’), 3.94 (d, 1 H, J =
12.0 Hz, H-5, H-5’’ or H-5’’’’), 3.97 (d, 1 H, J = 12.6 Hz, H-5, H-5’’ or H-5’’’’), 4.08 (d, 1 H,
J = 10.8 Hz, H-1’), 4.41 (d, 1 H, J = 7.8 Hz, H-1, H-1’’ or H-1’’’’), 4.49 (d, 1 H, J = 8.4 Hz,
H-1, H-1’’ or H-1’’’’), 4.53 (d, 1 H, J = 8.4 Hz, H-1, H-1’’ or H-1’’’’), 4.59 (d, 1 H, J = 8.4 Hz,
196

H-1’’’), 4.71 (d, 1 H, J = 10.8 Hz, NH’’’); HRMS [M]: m/z: calcd for C41H65N3O27S
1063.3526, found 1063.3520.
Methyl S-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2-deoxy-β-Dglucopyranosyl)-(14)-O-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2deoxy-β-D-glucopyranosyl)-(14)-N-2-(1H-indol-3-yl)ethyl-β-D-glucopyranuronami
de (50): Compound 50 was synthesized in 45% from compound 62 following the
1

procedure for the synthesis of compound 49. H NMR (600 MHz, D2O): δ = 1.84 (s, 3H,
NHCOCH3), 1.89 (s, 3H, NHCOCH3), 2.76 (t, 1H, J = 10.8 Hz, H-2’), 2.85-2.89 (m, 1 H,
CH2), 2.91-2.93 (m, 1 H, CH2), 3.13-3.24 (m, 3H, H-2, H-2’’, H-2’’’’), 3.34-3.65 (m, 19 H),
3.67-3.71 (m, 1H, H-2’’’), 3.71 (d, 1 H, J = 10.2 Hz, H-5, H-5’’ or H-5’’’’), 3.78 (d, 1 H, J =
12.6 Hz, H-5, H-5’’ or H-5’’’’), 3.78 (d, 1 H, J = 12.6 Hz, H-5, H-5’’ or H-5’’’’), 3.87 (d, 1 H,
J = 10.8 Hz, H-1’), 4.18 (d, 1 H, J = 7.8 Hz, H-1, H-1’’ or H-1’’’’), 4.30 (d, 1 H, J = 8.4 Hz,
H-1, H-1’’ or H-1’’’’), 4.32 (d, 1 H, J = 7.8 Hz, H-1, H-1’’ or H-1’’’’),4.38 (d, 1 H, J = 10.2
Hz, NH’’’), 4.43 (d, 1 H, J = 8.4 Hz, H-1’’’), 7.02-7.05 (m, 1H, CHarom), 7.10-7.12 (m, 1H,
CHarom), 7.14 (s, 1H, CHalkene), 7.36-7.38 (m, 1H, CHarom), 7.57-7.59 (m, 1H, CHarom);
HRMS [M]: m/z: calcd for C45H64N4O27S 1124.3479, found 1124.3564.
Methyl S-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2-deoxy-β-Dglucopyranosyl)-(14)-O-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2deoxy-β-D-glucopyranosyl)-(14)-N-phenyl-β-D-glucopyranuronamide

(51):

compound 51 was synthesized in 40% from compound 62 following the procedure for the

197

1

synthesis of compound 49. H NMR (600 MHz, D2O): δ = 1.76 (s, 3H, NHCOCH3),
2.05 (s, 3H, NHCOCH3), 3.14 (t, 1H, J = 10.8 Hz, H-2’), 3.32-3.35 (m, 2H, H-2, H-2’’ or
H-2’’’’), 3.42-3.45 (m, 1H, H-2, H-2’’ or H-2’’’’), 3.48-3.80 (m, 19 H), 3.80-3.86 (m, 1H,
H-2’’’), 3.88 (d, 1 H, J = 10.2 Hz, H-5, H-5’’ or H-5’’’’), 3.93 (d, 1 H, J = 12.6 Hz, H-5, H-5’’
or H-5’’’’), 3.93 (d, 1 H, J = 12.6 Hz, H-5, H-5’’ or H-5’’’’), 4.26 (d, 1 H, J = 9.0 Hz, H-1’),
4.45 (d, 1 H, J = 7.0 Hz, H-1, H-1’’ or H-1’’’’), 4.48 (d, 1 H, J = 7.8 Hz, H-1, H-1’’ or H-1’’’’
), 4.51 (d, 1 H, J = 7.8 Hz, H-1, H-1’’ or H-1’’’’), 4.57 (d, 1 H, J = 9.0 Hz, H-1’’’), 4.67 (d, 1
H, J = 9.6 Hz, NH’’’), 7.32-7.34 (m, 1H, CHarom), 7.49-7.51 (m, 1H, CHarom), 7.58-7.59
(m, 2H, CHarom); HRMS [M]: m/z: calcd for C41H59N3O27S 1057.3057, found
1057.3108.
Methyl S-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2-deoxy-β-Dglucopyranosyl)-(14)-O-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2deoxy-β-D-glucopyranosyl)-(14)-N-4-hydroxyphenyl-β-D-glucopyranuronamide
(52): Compound 52 was synthesized in 35% from compound 62 following the procedure
for the synthesis of compound 49.

1

H NMR (600 MHz, D2O): δ = 1.93 (s, 3H,

NHCOCH3), 2.05 (s, 3H, NHCOCH3), 3.11 (t, 1H, J = 10.8 Hz, H-2’), 3.34-3.48 (m, 3H,
H-2, H-2’’, H-2’’’’), 3.51-3.80 (m, 19 H), 3.84-3.88 (m, 1H, H-2’’’), 3.88 (d, 1 H, J = 12.6
Hz, H-5, H-5’’ or H-5’’’’), 3.93 (d, 1 H, J = 10.2 Hz, H-5, H-5’’ or H-5’’’’), 3.94 (d, 1 H, J =
10.8 Hz, H-5, H-5’’ or H-5’’’’), 4.19 (d, 1 H, J = 10.5 Hz, H-1’), 4.46 (d, 1 H, J = 7.8 Hz,
H-1, H-1’’ or H-1’’’’), 4.49 (d, 1 H, J = 8.4 Hz, H-1, H-1’’ or H-1’’’’), 4.49 (d, 1 H, J = 8.4 Hz,
H-1, H-1’’ or H-1’’’’), 4.58 (d, 1 H, J = 8.4 Hz, H-1’’’), 4.67 (d, 1 H, J = 10.8 Hz, NH’’’),
198

6.64-6.65 (m, 2H, CHarom), 7.23-7.24 (m, 2H, CHarom); HRMS [M]: m/z: calcd for
C42H61N3O28S 1073.3006, found 1073.3060.
Methyl S-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2-deoxy-β-Dglucopyranosyl)-(14)-O-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2deoxy-β-D-glucopyranosyl)-(14)-N-3-methoxyphenyl-β-D-glucopyranuronamide
(53): Compound 53 was synthesized in 35% from compound 62 following the procedure
1

for the synthesis of compound 49. H NMR (600 MHz, D2O): δ = 2.05 (s, 6H, NHCOCH3
x2), 3.03 (t, 1H, J = 10.8 Hz), 3.32-3.36 (m, 2H), 3.50-3.61 (m, 11H), 3.70-3.97 (m, 16 H),
4.24 (d, 1 H, J = 10.8 Hz, H-1’), 4.44 (d, 1 H, J = 8.4 Hz, H-1, H-1’’ or H-1’’’’), 4.48 (d, 1 H,
J = 7.2 Hz, H-1, H-1’’ or H-1’’’’), 4.51 (d, 1 H, J = 7.8 Hz, H-1, H-1’’ or H-1’’’’), 4.57 (d, 1 H,
J = 8.4 Hz, H-1’’’), 4.66 (d, 1 H, J = 10.8 Hz, NH’’’), 6.92-6.94 (m, 1H, CHarom), 7.18-7.20
(m, 1H, CHarom), 7.28-7.29 (m, 1H, CHarom), 7.41-7.44 (m, 1H, CHarom); HRMS [M]:
m/z: calcd for C42H61N3O28S 1087.3162, found 1087.3182.
Methyl S-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2-deoxy-β-Dglucopyranosyl)-(14)-O-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2deoxy-β-D-glucopyranosyl)-(14)-3-naphthalen-1-ylcarbamate-β-D-glucopyranosi
de (54): Compound 64 (0.0035 mmol, 5.2 mg) was co-evaporated with toluene and
dissolved in DCM (1 ml). Naphthalen-1-yl isocyanate (0.17 mmol, 50 eq., 25 μl) was
added and the reaction mixture was stirred for 2 days. The crude mixture was purified by
silica gel chromatography and dissolved in MeOH (0.3 ml) and H2O (0.3 ml). The pH was
adjusted to 9.5 by 1 M NaOMe in MeOH and the reaction mixture was stirred at r.t. for 4
199

days. 1 M HCl was added to adjust the pH to 7 and the reaction mixture was
concentrated and purified by Sephadex G-15 size exclusion chromatography to give the
1

desired product compound 54 in 50% yield from 64 for 2 steps. H NMR (600 MHz,
D2O): δ = 2.05 (s, 6H, NHCOCH3 x2), 3.32-3.37 (m, 3H, H-2, H-2’’, H-2’’’’), 3.47-3.80 (m,
19 H), 3.84-3.94 (m, 7H, H-2’’’, H-2’, H-5’’, H-5’’’’, C6H4OCH3), 4.20 (d, 1 H, J = 12.0 Hz,
H-1’), 4.48 (d, 3 H, J = 7.8 Hz, H-1, H-1’’, H-1’’’’), 4.57 (d, 1 H, J = 8.4 Hz, H-1’’’),
7.60-7.70 (m, 4H, CHarom), 7.93-7.41 (m, 1H, CHarom), 8.03-8.04 (m, 1H, CHarom),
8.16-8.17 (m, 1H, CHarom); HRMS [M]: m/z: calcd for C46H63N3O28S 1137.3319, found
1137.3282.
Methyl S-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2-deoxy-β-Dglucopyranosyl)-(14)-O-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2deoxy-β-D-glucopyranosyl)-(14)-3-(3-carboxyphenyl)carbamate-β-D-glucopyran
oside (55): Compound 55 was synthesized in 40% from 64 following the procedure for
1

the synthesis of compound 54. H NMR (600 MHz, D2O): δ = 1.64 (s, 3H, NHCOCH3 ),
2.06 (s, 3H, NHCOCH3), 3.32-3.37 (m, 3H, H-2, H-2’’, H-2’’’’), 3.47-3.80 (m, 19 H),
3.84-3.94 (m, 7H, H-2’’’, H-2’, H-5’’, H-5’’’’, C6H4OCH3), 4.22 (d, 1 H, J = 10.8 Hz, H-1’),
4.48 (d, 1 H, J = 12.6 Hz, H-1, H-1’’ or H-1’’’’), 4.49 (d, 1 H, J = 7.8 Hz, H-1, H-1’’ or
H-1’’’’), 4.52 (d, 1 H, J = 8.4 Hz, H-1, H-1’’ or H-1’’’’), 4.58 (d, 1 H, J = 8.4 Hz, H-1’’’),
4.85-4.86 (m, 1H), 5.02-5.08 (m, 1H), (NH’ or NH’’’), 7.46-8.11 (m, 4H, CHarom); HRMS
[M]: m/z: calcd for C43H61N3O30S 1131.3061, found 1131.3084.

200

Methyl

S-(β-D-glucopyranosyluronic

acid)-(13)-O-(2-N-acetyl-2-deoxy-β-D-

glucopyranosyl)-(14)-O-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2deoxy-β-D-glucopyranosyl)-(14)-3-(3-benzylphenyl)carbamate-β-D-glucopyranos
ide (56): Compound 56 was synthesized in 45% yield from compound 64 following
1

procedure for the synthesis of compound 54. H NMR (600 MHz, D2O): δ = 1.54 (s, 3H,
NHCOCH3), 2.05 (s, 3H, NHCOCH3), 3.29-3.30 (m, 1H), 3.33-3.35 (m, 2H) 3.49-3.60
(m, 13 H), 3.74-4.02 (m, 15H), 4.20-4.22 (m, 1H, H-1’), 4.41-4.43 (m, 1H, H-1, H-1’’ or
H-1’’’’), 4.49 (d, 2H, J = 7.2 Hz, H-1, H-1’’ or H-1’’’’), 4.59 (d, 1 H, J = 8.4 Hz, H-1’’’),
5.00-5.01 (m, 1H, NH’/NH’’’), 7.14-7.41 (m, 9H, CHarom); HRMS [M]: m/z: calcd for
C49H67N3O28S 1177.3632, found 1177.3612.
Methyl S-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2-deoxy-β-Dglucopyranosyl)-(14)-O-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2deoxy-β-D-glucopyranosyl)-(14)-3-2,5-dimethoxyphenethylcarbamate-β-D-gluco
pyranoside (57): Compound 57 was synthesized in 35% from compound 64 following
1

procedure for the synthesis of compound 54. H NMR (600 MHz, D2O): δ = 1.97 (s, 3H,
NHCOCH3), 2.05 (s, 3H, NHCOCH3), 2.66-2.95 (m, 4H, CH2CH2), 3.34-3.61 (m, 16H),
3.72-3.89 (m, 19H), 3.94 (d, 1H, J = 11.4 Hz, H-1’), 4.15 (d, 1H, J = 11.4 Hz NH’/NH’’’),
4.20 (d, 1H, J = 10.8 Hz, NH’/NH’’’), 4.49 (d, 1H, J = 7.8 Hz, H-1, H-1’’ or H-1’’’’), 4.50 (d,
2 H, J = 7.2 Hz, H-1, H-1’’ or H-1’’’’), 4.59 (d, 1 H, J = 7.8 Hz, H-1’’’), 6.86-6.92 (m, 2H,
CHarom), 7.01-7.05 (m, 1H, CHarom); HRMS [M]: m/z: calcd for C46H69N3O30S
1175.3687, found 1175.3688.
201

Methyl S-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2-deoxy-β-Dglucopyranosyl)-(14)-O-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2deoxy-β-D-glucopyranosyl)-(14)-3-2,3,3-trimethylbutan-2-ylcarbamate-β-Dglucopyranoside (58): Compound 58 was synthesized in 20% from compound 64
1

following the procedure for the synthesis of compound 54. H NMR (600 MHz, D2O): δ =
0.56 (s, 3H, CH3), 0.86 (s, 6H, CH3), 1.20 (s, 6H, CH3), 1.87 (s, 6H, NHCOCH3),
3.18-3.79 (m, 33H), 4.20-4.27 (m, 2H, NH), 4.31-4.43 (m, 5H, H-1, H-1, H-1’’, H-1’’’ and
H-1’’’’); HRMS [M]: m/z: calcd for C44H73N3O28S 1123.4101, found 1123.4058.
Methyl S-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2-deoxy-β-Dglucopyranosyl)-(14)-O-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2deoxy-β-D-glucopyranosyl)-(14)-3- (4-methoxyphenyl)methanylcarbamate-β-Dglucopyranoside (59): Compound 59 was synthesized in 50% (as a mixture of
compound 59 and 61) from compound 64 following the procedure for the synthesis of
compound 54. For the deprotection step, side product 61 was generated by 3-carbamate
group migration. The mixture of compound 59 and 61 could not be separated by HPLC.
HRMS [M]: m/z: calcd for C65H73N3O29S 1131.3424, found 1131.3374.
Methyl S-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2-deoxy-β-Dglucopyranosyl)-(14)-O-(β-D-glucopyranosyluronic acid)-(13)-O-(2-N-acetyl-2deoxy-β-D-glucopyranosyl)-(14)-β-D-glucopyranoside (67): Compound 64 was
dissolved in MeOH (0.3 ml) and H2O (0.3 ml), and the pH was adjusted to 9.5 by 1 M
NaOMe in MeOH. The reaction mixture was stirred at r.t. for 2 days. 1 M HCl was added
202

to adjust the pH to 7 and the reaction mixture was concentrated and purified by
Sephadex G-15 size exclusion chromatography to give the desired product 67 in 70%
1

yield from compound 64. H NMR (600 MHz, D2O): δ = 2.05 (s, 3H, NHCOCH3), 2.06 (s,
3H, NHCOCH3), 2.85 (t, 1H, J = 11.4 Hz), 3.29-3.40 (m, 3H), 3.51-3.95 (m, 27H), 4.10
(d, 1H, J = 12.0 Hz, H-1’), 4.37 (d, 1H, J = 7.8 Hz 1H, H-1, H-1’’ or H-1’’’’), 4.49 (d, 1 H, J
= 7.8 Hz, H-1, H-1’’ or H-1’’’’) 4.52 (d, 1 H, J = 7.8 Hz, H-1, H-1’’ or H-1’’’’), 4.59 (d, 1 H, J
= 7.8 Hz, H-1’’’); HRMS [M]: m/z: calcd for C35H56N2O27S 968.2791, found 928.2816.

203

Appendix B

204

NMR Spectra
1

Figure 4.23. H-NMR of compound 10 (500 MHz, CDCl3)

205

1

Figure 4.24. H-NMR of compound 11 (500 MHz, CDCl3)

206

1

Figure 4.25. H-NMR of compound 12 (500 MHz, CDCl3)

207

1

Figure 4.26. H-NMR of compound 13 (500 MHz, CDCl3)

208

1

Figure 4.27. H-NMR of compound 14 (500 MHz, CDCl3)

209

1

Figure 4.28. H-NMR of compound 15 (500 MHz, CDCl3)

210

1

Figure 4.29. H-NMR of compound 16 (500 MHz, CDCl3)

211

1

Figure 4.30. H-NMR of compound 38 (500 MHz, CDCl3)

212

Figure 4.31.

13

C-NMR of compound 38 (125 MHz CDCl3)

213

1

1

Figure 4.32. H– H gCOSY of compound 38 (500 MHz, CDCl3)

214

1

13

Figure 4.33. H– C gHMQC of compound 38 (500 MHz, CDCl3)

215

1

13

Figure 4.34. H– C gHMQC (without 1H decoupling) of compound 38 (500 MHz,CDCl3)

216

1

13

Figure 4.35. H– C gHMBC of compound 38 (500 MHz, CDCl3)

\

217

1

Figure 4.36. H-NMR of compound 40 (500 MHz, CDCl3

218

1

1

Figure 4.37. H– H gCOSY of compound 40(500 MHz, CDCl3)

219

1

13

Figure 4.38. H– C gHMQC (without 1H decoupling) of compound 40 (500 MHz,CDCl3)

220

1

13

Figure 4.39. H– C gHMBC of compound 40 (500 MHz, CDCl3)

221

1

Figure 4.40. H-NMR of compound 72 (500 MHz, CDCl3)

222

Figure 4.41.

13

C-NMR of compound 72 (125 MHz, CDCl3)

223

1

1

Figure 4.42. H– H gCOSY of compound 72 (500 MHz, CDCl3)

224

1

13

Figure 4.43. H– C gHMQC (without 1H decoupling) of compound 72 (500 MHz,CDCl3)

225

1

Figure 4.44. H-NMR of compound 73 (500 MHz, CDCl3)

226

Figure 4.45.

13

C-NMR of compound 73 (125 MHz, CDCl3)

227

Figure 4.46. 1H–1H gCOSY of compound 73 (500 MHz, CDCl3)

228

1

13

Figure 4.47. H– C gHMQC (without 1H decoupling) of compound 73 (500 MHz,CDCl3)

229

1

Figure 4.48. H-NMR of compound 61 (500 MHz, CDCl3)

230

Figure 4.49.

13

C-NMR of compound 61 (125 MHz, CDCl3)

231

1

1

Figure 4.50. H– H gCOSY of compound 61 (500 MHz, CDCl3)

232

1

13

Figure 4.51. H– C gHMQC of compound 61 (500 MHz, CDCl3)

233

1

13

Figure 4.52. H– C gHMQC (without 1H decoupling) of compound 61 (500 MHz,CDCl3)

234

1

13

Figure 4.53. H– C gHMBC of compound 61 (500 MHz, CDCl3)

235

1

Figure 4.54. H-NMR of compound 62 (500 MHz, CDCl3)

236

Figure 4.55.

13

C-NMR of compound 62 (125 MHz, CDCl3)

237

1

1

Figure 4.56. H– H gCOSY of compound 62 (500 MHz, CDCl3)

238

1

13

Figure 4.57. H– C gHMQC (without 1H decoupling) of compound 62 (500 MHz,CDCl3)

239

1

13

Figure 4.58. H– C gHMBC of compound 62 (500 MHz, CDCl3)

240

1

Figure 4.59. H-NMR of compound 63 (500 MHz, CDCl3)

241

Figure 4.60.

13

C-NMR of compound 63 (125 MHz, CDCl3)

242

1

1

Figure 4.61. H– H gCOSY of compound 63 (500 MHz, CDCl3)

243

1

13

Figure 4.62. H– C gHMQC of compound 63 (500 MHz, CDCl3)

244

1

13

Figure 4.63. H– C gHMQC (without 1H decoupling) of compound 63 (500 MHz,CDCl3)

245

1

Figure 4.64. H-NMR of compound 64 (500 MHz, CDCl3)

246

Figure 4.65.

13

C-NMR of compound 64 (125 MHz, CDCl3)

247

1

1

Figure 4.66. H– H gCOSY of compound 64 (500 MHz, CDCl3)

248

1

13

Figure 4.67. H– C gHMQC (without 1H decoupling) of compound 64 (500 MHz,CDCl3)

249

1

Figure 4.68. H-NMR of compound 49 (600 MHz, D2O)

250

1

1

Figure 4.69. H– H gCOSY of compound 49 (600 MHz, D2O)

251

1

Figure 4.70. H-NMR of compound 50 (600 MHz, D2O)

252

1

1

Figure 4.71. H– H gCOSY of compound 50 (600 MHz, D2O)

253

1

Figure 4.72. H-NMR of compound 51(600 MHz, D2O)

254

1

1

Figure 4.73. H– H gCOSY of compound 51 (600 MHz, D2O)

255

1

Figure 4.74. H-NMR of compound 52 (600 MHz, D2O)

256

1

1

Figure 4.75. H– H gCOSY of compound 52 (600 MHz, D2O)

257

1

Figure 4.76. H-NMR of compound 53 (600 MHz, D2O)

258

1

1

Figure 4.77. H– H gCOSY of compound 53 (600 MHz, D2O)

259

1

Figure 4.78. H-NMR of compound 54 (600 MHz, D2O)

260

1

1

Figure 4.79. H– H gCOSY of compound 54 (600 MHz, D2O)

261

1

Figure 4.80. H-NMR of compound 55 (600 MHz, D2O)

262

1

1

Figure 4.81. H– H gCOSY of compound 55 (600 MHz, D2O)

263

1

Figure 4.82. H-NMR of compound 56 (600 MHz, D2O)

264

1

1

Figure 4.83. H– H gCOSY of compound 56 (600 MHz, D2O)

265

1

Figure 4.84. H-NMR of compound 57 (600 MHz, D2O)

266

1

1

Figure 4.85 H– H gCOSY of compound 57 (600 MHz, D2O)

267

1

Figure 4.86. H-NMR of compound 58 (600 MHz, D2O)

268

1

Figure 4.87. H-NMR of compound 58 (600 MHz, D2O)

269

1

Figure 4.88. H-NMR of compound 67 (600 MHz, D2O)

270

References

271

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