STEREOSELECTIVE GLYCOSYLATIONS AND SYNTHESIS OF
HYALURONAN BIOSYNTHESIS INHIBITORS
By
Gilbert Ochieng Wasonga

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
CHEMISTRY
2010

1

ABSTRACT
STEREOSELECTIVE GLYCOSYLATIONS AND SYNTHESIS OF HYALURONAN
SYNTHESIS INHIBITORS
By
Gilbert Ochieng Wasonga
Stereochemical control is an important issue in carbohydrate synthesis. Glycosyl donors
with participating acyl protective groups on 2-O have been shown to give 1,2-trans
glycosides reliably under the pre-activation based reaction condition. In this work, the
effects of additives and reaction solvent on stereoselectivity was examined using donors
without participating protective groups on 2-O.

We have established that the

stereoselectivity could be directed by reaction solvent. The trend of stereochemical
dependence on reaction solvent was applicable to a variety of reactions including the
selective formation of β-mannosides. In the second part, 3-MeO-GlcNAc is efficiently
prepared using a furanose oxazoline intermediate which is well suited for large scale
synthesis without the need for extensive column chromatography. In addition, we have
developed a robust and rapid procedure for the synthesis of 3-F-GlcNAc derivative
required for inhibition studies of hyaluronan biosynthesis. In the course of our synthesis,
we have shown the expanded utility of Lattrell-Dax method for carbohydrate
epimerization reactions.

II

ACKNOWLEDGMENTS
I would like to thank my advisor Professor Xuefei Huang for his guidance and
support that have been instrumental in my graduate studies. Along the way, I have had
the opportunity to be mentored by Doctor Youlin Zeng who was a very helpful mentor at
the beginning of my chemistry research experience and I will always be grateful to him.
Dr. Daniel Holmes was very helpful with 2D-NMR spectroscopy training which have
been vital for my research. In addition, Dr. Daniel Jones and Lijun Chen have been very
helpful in training and running mass spectroscopy.
My experience and friendships developed with my past and present lab mates is
something to cherish for a lifetime.

From our interactions, I now have a better

appreciation of people from different cultures which in many ways are similar to my
own.
I also want to thank my guidance committee members Dr. Babak Borhan, Dr.
Gary Blanchard and Dr. William Wulff for their patience and serving on my committee.
Finally, I would like to thank my family for I will not be this far without your love and
support.

iii
1

TABLE OF CONTENTS
LIST OF TABLES .................................................................................................. v
LIST OF FIGURES ................................................................................................ vi
LIST OF SCHEMES............................................................................................... xi
LIST OF ABBREVIATIONS AND SYMBOLS ................................................... xii
CHAPTER 1
PRE-ACTIVATION BASED STEREOSELECTIVE GLYCOSYLATION
1.1 Introduction .................................................................................................. 1
1.2 Result and Discussions ................................................................................. 14
1.3 Conclusion .................................................................................................... 24
1.4 Experimental Section ................................................................................... 25
Appendix 1: Spectral data .................................................................................. 46
References .......................................................................................................... 96
CHAPTER 2
STEREOSELECTIVE GLYCOSYLATION OF AZIDO GLUCOSIDE
2.1 Introduction .................................................................................................. 107
2.2 Result and Discussion .................................................................................. 109
2.3 Experimental Section ................................................................................... 115
Appendix 2: Spectral data .................................................................................. 125
References .......................................................................................................... 137
CHAPTER 3
SYNTHESIS OF HYALURONAN SYNTHESIS INHIBITORS
3.1 Introduction .................................................................................................. 140
3.2 Result and Discussion .................................................................................. 144
3.3 Conclusion .................................................................................................... 151
3.4 Experimental Section ................................................................................... 152
Appendix 3: Spectral data .................................................................................. 165
References .......................................................................................................... 184

iv

List of Tables
Table 1.1 Effects of triflate salt additives on stereoselectivity ............... 20
Table1.2 Solvent effects on stereoselectivity .......................................... 21
Table 2.1 Glycosylations of donor 6 ....................................................... 111
Table 2.2 Glycosylations of donor 14 ..................................................... 113

v

List of Figures
1

Figure 1.1 H-NMR compound 3 .......................................................................... 48
1

Figure 1.2 H-NMR compound 4 .......................................................................... 49
1

Figure 1.3 H-NMR compound 5 .......................................................................... 50
1

Figure 1.4 H-NMR compound 6 .......................................................................... 51
1

Figure 1.5 H-NMR compound ............................................................................. 52
1

Figure 1.6 H-NMR compound 8 .......................................................................... 53
1

Figure 1.7 H-NMR compound 9 .......................................................................... 54
1

Figure 1.8 H-NMR compound 10 ........................................................................ 55
1

Figure 1.9 H-NMR compound 13 ........................................................................ 56
1

Figure 1.10 H-NMR compound 15 ...................................................................... 57

Figure 1.11

13

C-NMR compound 15 ..................................................................... 58

1

Figure 1.12 H-NMR compound 15 ...................................................................... 59

Figure 1.13

13

C-NMR compound 16 ..................................................................... 60

Figure 1.14 gCOSY compound 16 ......................................................................... 61

vi

Figure 1.15 gHMQC-coupled compound 16 ......................................................... 62
Figure 1.16 gHMQC-coupled (anomeric region expansion) compound 16 .......... 63
Figure 1.17 gHMQC-decoupled compound 16...................................................... 64
Figure 1.18 gHMBC compound 16........................................................................ 65
1

Figure 1.19 H-NMR compound 17 ...................................................................... 66

Figure 1.20

13

C-NMR compound 17 ..................................................................... 67

Figure 1.21 gCOSY compound 17 ......................................................................... 68
Figure 1.22 gHMQC-coupled compound 17 ......................................................... 69
Figure 1.23 gHMQC-coupled (anomeric region expansion) compound 17 .......... 70
Figure 1.24 gHMQC-decoupled compound 17...................................................... 71
Figure 1.25 gHMBC compound 17........................................................................ 72
1

Figure 1.26 H-NMR compound 18 ...................................................................... 73

Figure 1.27

13

C-NMR compound 18 ..................................................................... 74

Figure 1.28 gCOSY compound 18 ......................................................................... 75
Figure 1.29 gHMQC-coupled compound 18 ......................................................... 76
Figure 1.30 gHMQC-coupled (anomeric region expansion) compound 18 .......... 77
Figure 1.31 gHMQC-decoupled compound 18...................................................... 78
vii

Figure 1.32 gHMBC compound 18........................................................................ 79
1

Figure 1.33 H-NMR compound 20 ...................................................................... 80
1

Figure 1.34 H-NMR compound 21 ...................................................................... 81

Figure 1.35

13

C-NMR compound 21 ..................................................................... 82

Figure 1.36 gHMQC-coupled compound 21 ......................................................... 83
Figure 1.37 gHMQC-coupled (anomeric region expansion) compound 21 .......... 84
Figure 1.38 gHMQC-decoupled compound 21...................................................... 85
Figure 1.39 gHMBC compound 21........................................................................ 86
1

Figure 1.40 H-NMR compound 22 ...................................................................... 87

Figure 1.41

13

C-NMR compound 22 ..................................................................... 88

Figure 1.42 gCOSY compound 22 ......................................................................... 89
Figure 1.43 gHMQC-coupled compound 22 ......................................................... 90
Figure 1.44 gHMQC-coupled (anomeric region expansion) compound 22 .......... 91
Figure 1.45 gHMQC-decoupled compound 22...................................................... 92
Figure 1.46 gHMBC compound 22........................................................................ 93
1

Figure 1.47 H-NMR compound 23 ...................................................................... 94

viii

1

Figure 1.48 H-NMR compound 24 ...................................................................... 95
1

Figure 2.1 H-NMR compound 2 .......................................................................... 127
1

Figure 2.2 H-NMR compound 3 .......................................................................... 128
1

Figure 2.3 H-NMR compound 4 .......................................................................... 129
1

Figure 2.4 H-NMR compound 6 .......................................................................... 130
1

Figure 2.5 H-NMR compound 9 .......................................................................... 131
1

Figure 2.6 H-NMR compound 10 ........................................................................ 132
1

Figure 2.7 H-NMR compound 12 ........................................................................ 133
1

Figure 2.8 H-NMR compound 13 ........................................................................ 134
1

Figure 2.9 H-NMR compound 14 ........................................................................ 135
1

Figure 2.10 H-NMR compound 15 ...................................................................... 136
1

Figure 3.1 H-NMR compound 2 .......................................................................... 167
1

Figure 3.2 H-NMR compound 3 .......................................................................... 168
1

Figure 3.3 H-NMR compound 4 .......................................................................... 169

Figure 3.4

13

C-NMR compound 4 ......................................................................... 170
ix

1

Figure 3.5 H-NMR compound 6 .......................................................................... 171
1

Figure 3.6 H-NMR compound 7 .......................................................................... 172
1

Figure 3.7 H-NMR compound 8 .......................................................................... 173

Figure 3.8

13

C-NMR compound 8 ......................................................................... 174

1

Figure 3.9 H-NMR compound 11 ........................................................................ 175
1

Figure 3.10 H-NMR compound 12 ...................................................................... 176
1

Figure 3.11 H-NMR compound 13 ...................................................................... 177
1

Figure 3.12 H-NMR compound 14 ...................................................................... 178
1

Figure 3.13 H-NMR compound 16 ...................................................................... 179
1

Figure 3.14 H-NMR compound 17 ...................................................................... 180
1

Figure 3.15 H-NMR compound 18 ...................................................................... 181
1

Figure 3.16 H-NMR compound 19 ...................................................................... 182

Figure 3.17

13

C-NMR compound 19 ..................................................................... 183

x

List of Schemes
Scheme 1.1 Classical neighboring group participation by a 2-O ester....... 2
Scheme 1.2 Halide ion catalyzed glycosylation ......................................... 3
Scheme 1.3 Neighboring group participation chiral auxiliary at O-2 ........ 5
Scheme 1.4 Benzylidene effect .................................................................. 8
Scheme 1.5 Intramolecular aglycon delivery ............................................. 10
Scheme 1.6 Preactivation-based glycosylation strategy............................. 12
Scheme 1.7 Synthesis of building blocks 4, 7 and 9 .................................. 15
Scheme 1.8 Building blocks and glycosylation products ........................... 22
Scheme 1.9 Proposed mechanism of the effects of solvent and AgOTf .... 23
Scheme 2.1 Sulfonium ion promoted glycosylation................................... 108
Scheme 2.2 Synthesis of donor 6 ............................................................... 110
Scheme 2.3 Synthesis of donor 14 ............................................................. 113
Scheme 3.1 Synthesis of compound 4 ........................................................ 145
Scheme 3.2 Synthesis of compound 8 ........................................................ 146
Scheme 3.3 Proposed mechanism for synthesis of compound 9 ................ 146
Scheme 3.4 Synthesis of compound 19 ...................................................... 150

xi

List of Abbreviations and Symbols
Å

Angstrom

α

alpha

Ac

Acetyl

AcCl

Acetyl chloride

Ac2O

Acetic anhydride

AgOTf

Silver trifluoromethanesulfonate

β

beta

BF3.OEt2

Boron trifluoride etherate

Bn

Benzyl

BnBr

Benzyl bromide

BnOH

Benzyl alcohol

Bz

Benzoyl

BzCl

Benzoyl chloride

C

Carbon

C

Concentration

CSA

Camphorsulfonic acid

CCl4

Carbon tetrachloride

CH2Cl2

Dichloromethane

CD3OD

Deuterated methanol

CDCl3

Deuterated chloroform

CCl3CN

Trichloroacetonitrile
xii

Ce

Cerium

COCl2

Oxalyl chloride

conc.

Concentration

CuSO4

Copper (II) sulfate

°

C

Degree celsius

δ

Delta

d

doublet

DBU

1,8-Diazabicyclo[5.4.0]undec-7-ene

DCM

Dichloromethane

D2O

Deuterated water

DMAP

4-Dimethylaminopyridine

DMF

Dimethylformamide

DMSO

Dimethyl sulfoxide

DTBMP

2, 6-di- tert-butyl-4-methylpyridine

ESI

Electron spray ionization

EtOAc

Ethyl acetate

Et3N

Triethylamine

Et4NBr

Tetraethylammonium bromide

Et2O

Diethyl ether

EtOH

Ethanol

EtSH

Ethanethiol

xiii

eq

Equivalence

g

grams

gCOSY

Gradient correlation spectroscopy

gHMBC

Gradient heteronuclear bond correlation

gHMQC

Gradient heteronuclear multiple quantum correlation

H

Hydrogen

Hz

Hertz

HCl

Hydrochloric acid

HF

Hydrogen fluoride

HfOTf

Hafnium trifluoromethanesulfonate

hr

Hour

HRMS

High resolution mass spectroscopy

H2SO4

Sulfuric acid

L

Litre

m

Multi

M

Mega

Me

Methyl

Me4Si

Tetramethylsilane

MeOH

Methanol

mg

Milligram

min

Minute

mL

Millilitre

xiv

mmol

Millimolar

MS

Molecular sieves

Mo

Molybdenum

µ

Micro

Na

Sodium

N2

Nitrogen

N3

Azide

NBS

N-bromosuccinimide

NaHCO3

Sodium bicarbonate

NaOMe

Sodium methoxide

NH4OAc

Ammonium acetate

NMR

Nuclear magnetic resonance

NaN3

Sodium azide

Ph

Phenyl

PhSEt

Ethyl phenyl thioether

PPh3

Triphenyl phosphine

ppm

Part per million

PhSOTf

Benzenesulfenyl trifluoromethanesulfonate

PhSeOTf

Benzeneselenenyl trifluoromethanesulfonate

p-TolSCl

para-Toluenesulfenyl chloride

rt

room temperature

xv

s

Singlet

t

Triplet

Temp

Temperature

TFA

Trifluoroacetic acid

Tf2O

Trifluoromethanesulfonic anhydride

TLC

Thin layer chromatography

TMSOTf

Trimethylsilyl trifluoromethanesulfonate

xvi

Chapter 1: Pre-activation Based Stereoselective Glycosylation
1.1 Introduction
The inherent biological importance of carbohydrates in processes such as
fertilization, embryogenesis, neuronal development and tumor progression has generated
significant interest in understanding oligosaccharide function in living systems.

1

While

there is potential in developing oligosaccharides as new therapeutic agents and as
biomarkers for understanding pathology, growth in glycobiology is severely hampered by
the lack of pure and structurally well defined carbohydrates and glycoconjugates. The
difficulty in recovering these compounds as homogeneous extracts from their natural
sources has prompted an alternative approach which entails obtaining well defined
oligosaccharides by chemical synthesis .

2

Advances have been made in establishing methods, such as automated solid phase
synthesis,

3, 4

glycosylation

orthogonal glycosylation
9, 10,11

5, 6

, chemoselective glycosylation

and one-pot multistep glycosylations

8, 12-22

7, 8

,iterative

which have been applied

in complex oligosaccharides assemblies.
A major challenge still remains in the development of a general method for
stereoselective glycosylation.

23

The synthesis of oligosaccharides containing 1,2-trans

glycosidic linkages is relatively straightforward since neighboring group participation by
an acryl protecting group allows the formation of 1,2-trans linkages with a high degree of
stereocontrol.

1

The glycosyl donor with an anomeric leaving group is activated by a promoter
(A+B-) resulting in the departure of the anomeric leaving group (X) and formation of an
oxacarbenuim ion intermediate. Subsequent intramolecular stabilization by a 2-O-acyl
protecting group gives a more stable acetoxonium ion. A sugar alcohol (ROH) attacks
the dioxolenium ion predominantly from one face to provide a 1,2-trans glycoside.
While this is an effective strategy for accessing β−glycosides for D-glucose, D-galactose
and α−glycoside for mannose, a general method for synthesis of 1, 2-cis glycoside has
yet to emerge. This problem is also inextricably linked to the formation of 1,2-trans
glycoside in the absence of neighboring group participation (Scheme 1.1).

Scheme 1.1 Classical neighboring group participation by a 2-O ester
In an attempt to overcome this problem, the last two decades has seen the
emergence of various synthetic strategies for stereoselective glycosylation. Lemieux and
coworkers established the halide ion catalyzed glycosylation for the synthesis of
α−linked disaccharides.

24

A typical characteristic of this reaction involves the use of a

tetra-O-benzyl-α-glycopyranosyl

bromide

as

the

glycosyl

donor

and

N-

tetraethylammonium bromide as the catalyst. The α-glycosylation involves an in situ
anomerization of the donor in the presence of the catalyst as the key step to give
the β−glycosyl bromide in equilibrium which is more reactive than its α counterpart.

2

This equilibrium is shifted strongly towards the α-halide since this compound is
stabilized by an endo-anomeric effect. However, the energy barrier for nucleophilic
attack by an alcohol is lower for the β-halide leading to the formation of α glycoside.
Although in situ anomerization has proved useful in enhancing α-selectivity of Sialyl
Lewis

x 25

and N-glycan dodecasaccharide building blocks,

26

stereocontrol using this

approach is not absolute, often resulting in an anomeric mixture.

An important

requirement of this reaction is that the rate of equilibration is much faster than that of
glycosylation which entails careful manipulation of reactivity of both the donor and
acceptor for optimization of stereochemical outcomes (Scheme 1.2).

24

Scheme 1.2 Halide ion catalyzed glycosylation
Another significant advancement in stereoselective glycosylation was achieved by
Boons who developed a novel approach using a chiral auxiliary participating neighboring
group at O-2 of the glycosyl donor.

27

Using (1S)-phenyl-2-(phenylsulfanyl) ethyl moiety

to trap the oxacarbenium ion intermediate upon activation of glycosyl donor resulted in a
sulfonium ion intermediate formed either as a trans- or cis-decalin system. However, the
trans-decalin system predominates due to favorable steric interactions. Displacement of
equatorial anomeric sulfonium ion by sugar alcohol leads to the formation of 1, 2-cisglycoside. Using the S-auxiliary they were able to exclusively form α−linked glycosides
in very good yields. However, the R-auxiliary expected to give β-linked glycosides due
to favorable the formation of cis-decalin system, only resulted in a 1:1 mixture of
3

anomers indicating the importance of the stereogenic center in controlling
stereoselectivity (Scheme 1.3).

27

4

Scheme 1.3 Neighboring group participation by chiral auxilliary at O-2

5

Protecting groups have evolved from their simple role of masking hydroxyl
functional groups to playing a prominent role in stereoselective glycosylations. It has
emerged that using protecting groups which can conformationally lock the pyranose ring
system into a chair conformation, stereoselective coupling of oligosaccharides can be
accessed. This was first reported by Fraser-Reid who proposed that 4, 6-O-benzylidene
protected pyranosyl systems resist the formation of oxacarbenium ion.

28, 29

Taking

advantage of this benzylidene effect, Crich made a significant breakthrough in βmannoside synthesis by developing a direct approach for the formation of βmannopyranosides.

He employed mannopyranosyl sulfoxides or thioglycosides as

glycosyl donors leading to high selectivity (Scheme 1.4 a-b).

30-33

This was envisaged to

occur due to rigidification of the pyranoside ring by the 4, 6-O-benzylidene thus favoring
the triflate intermediate over the oxacarbenium ion resulting in an SN2 displacement of
the mannosyl α−triflates intermediate.

34

The effect of 4, 6-O-acetal group in the

construction of β-mannosyl linkages has been extended to other mannosyl donors by Kim
and coworkers in developing an efficient and stereoselective procedure for βmannosylation by employing 2-(hydroxycarbonyl) benzyl (HCB) mannopyranoside with
triflic anhydride. They could also access α−glucopyranoside as the major product in
glycosylation of the HCB 4, 6-O-benzylidene glucoside. Crich has also reported this
change in selectivity in corresponding glucopyranosyl and galactopyranosyl donors
which are α-selective.

35-41

While it is known that mannosylation of more reactive

primary alcohol acceptors show poor reactivity with known 4, 6-O-benzylidene-

6

substituted mannosyl donors,

42

it is noteworthy that Kim has developed an efficient β-

mannosylation of primary alcohol employing 4, 6-O-benzylidene-substituted mannosyl
pentenoate donor and PhSeOTf as a promoter.

43

4, 6-Di-tert-butylsilylene (DTBS)-

protected galactosyl donor has been employed by Kiso for α-selective galactosylation
despite the presence of C-2 participating group (Scheme 1.4c).

7

44

β/α > 25 : 1

α/β ~ 2 : 1

75%, α-only

Scheme 1.4 Benzylidene effect

8

Indirect methods for stereoselective glycosylation have been developed over the
last couple of decades. An earlier strategy first developed by Barresi and Hindsgaul,
entailed a two-step tethering-glycosylation in a process commonly referred to as
intramolecular agylcon delivery (IAD) for the synthesis of β mannoside.

45

It has proven

to be a reliable method of achieving 1,2-cis-stereocontrol. This strategy involves the
covalent attachment of an aglycon alcohol to the O-2 of the glycosyl donor by a
temporary tether. Activation of the donor leads to intramolecular delivery of the agylcon
in a concerted reaction giving a five membered ring intermediate and a complete
46

stereocontrolled formation of 1,2-cis product.
Hindsgaul,

45

Seminal studies by Barresi and

entailed the use of acetal linkages. However, this process suffers low yields

during both acetal formation and glycosylation stage with increased steric bulk of either
the enol ether or the alcohol. As an improvement to this original approach, the research
groups of Stork

47-51

utilized a silicon bridged mediated agylcon delivery to provide high

yields and excellent stereoselectivity for primary and secondary alcohols, but limitations
were found in glycosylation of an OH-4 of glucose aglycon (Scheme 1.5).

9

48

Scheme 1.5 Intramolecular aglycon delivery

10

In 1994 Ogawa and Ito introduced a p-methoxybenzyl-assisted intramolecular
aglycon delivery for the synthesis of β-mannose focusing on the core structure of Nglycans.

Tethering mediated by DDQ (2,3-dichloro-5,6-dicyanobenzoquinone) in

dichloromethane typically gave high yields for primary and secondary alcohols.

52

Using

this approach they were able to access 4-O-linked glycosides in moderate yields. A
significant increase in efficiency of glycosylation was achieved for 4-OH glycosyl
agylcon using 4, 6-O-cyclohexylidene protecting group, further proving that rigidifying
the ring system has an influence on stereoselectivity.
conjugation with glycals for β mannosides
β−glycosides followed by reduction.

54

53

Other indirect methods include

and direct formation of 2-ulosyl

55, 56

While all these strategies have resulted in considerable advances in accessing
complex oligosaccharides, a general strategy for stereoselective glycosylation is yet to
emerge. A particular strategy that we chose to exploit in an effort to address this problem
was the use of pre-activation glycosylation strategy. An area that is yet to be fully
exploited is the use of preactivation protocol to control the stereoselectivity of
glycosylation reactions. Unique stereochemical outcomes can be obtained using the preactivation strategy.

57

The one pot methods initially developed were mostly based upon reacting donors
with decreasing anomeric reactivities in a sequential manner.

12

However, these strategies

are limited by extensive protective group manipulations and/or aglycon leaving group
adjustments. These excessive manipulations limit the overall synthetic efficiency.

11

17

Our group developed a general one-pot method independent of differential
glycosyl donor reactivities, achieved by pre-activating the donor generating a reactive
intermediate in the absence of the acceptor.

18

This approach has been used to assemble
x 19

several biologically significant oligosaccharides, including Lewis ,
Globo-H,

20

and hyaluronic acid oligosaccharides.

21,22

x 19

dimeric Lewis ,

Whereas stereoselective

glycosylation using donors bearing participating ester groups on 2-O position, 1, 2-trans
glycoside products can be formed reliably under the pre-activation condition,

21-22

efficient 1,2-cis glycosylations are still a major bottleneck for this protocol (Scheme 1.6).

Scheme 1.6 Preactivation-based glycosylation strategy
Xin-Shan Ye’s research group has recently developed a highly stereoselective
pre-activation

glycosylation

protocol.

In

their

studies,

4,

6-di-O-acetyl-N-

acetyloxazolidinone protected donor afforded either β-or α- isomer in high selectivity
depending upon the presence or absence of a hindered base TTBP (2,4,6-Tri-tertbutylpyrimidine).

58

In addition, they have further developed this strategy in the synthesis

of 2-deoxysugars using 3, 4-O-carbonate glycosyl in high α-selectivity, which is quite
remarkable especially for donor without an O-2 participating neighboring group.

59

These results give a glimpse of the potential of accessing stereocontrolled
glycosylation using pre-activation protocol. With this in mind, we decided to investigate
12

the effect of triflate additives and solvents have on the pre-activation of thioglycosides
without a participating neighboring group at O-2 of the thioglycoside donor.

We

envisoned that these type of donors would allow us to establish factors that control
stereoselectivity of pre-activation glycosylation without the formation of a dioxolenium
intermediate which would lead to formation of 1,2-trans glycosides. We intended to
establish conditions that would also lead to 1,2-cis glycosides.

13

1.2 Results and Discussion
Stereochemical outcomes of glycosylation can be controlled by placing
participating protective groups at O-2 position of the donor.

However this limits

glycosylation to 1,2-trans linkage. While it is well known that solvent effect and Lewis
acid promoters play a role in stereoselectivity of glycosylation as exemplified by the
nitrile effect,

60

and insoluble silver salts,

61, 62

their effects on pre-activation based

glycosylation have not been thoroughly investigated. We set out to explore the effect of
solvent and promoter on stereoselectivity of pre-activation based glycosylation with the
aim of establishing a general method of stereoselective glycosylation using donors
without participating protective group on 2-O.
Thioglycosides were chosen as glycosyl donors because they are easily activated
by a variety of thiophilic promoters and are stable under most chemical reaction
conditions.

Moreover they can be conveniently stored on the benchtop for months

without decomposition. The thioglycoside donor and acceptor pairs were synthesized as
outlined in Scheme 1.7 resulting in donor 4 and acceptor 7, used for the initial screening
in our study.

18

Primary sugar alcohols were selected as acceptors for the initial screening

because their use in glycosylation reactions often leads to poor stereoselectivity.

14

63

Scheme 1.7 Synthesis of building blocks 4, 7, and 8. Conditions, i) 1.6 eq p-thiocresol, 1.3 eq BF3.OEt2 , DCM,
rt, 85%, ii, NaOMe/MeOH, rt, 77%, iii) 1.3 eq TBDPSCl, Pyridine, rt, 90%, iv) 3.45 eq BzCl, Pyridine, rt, 95%,
v) 6.5 eq NaH, DMF, 4.8 eq BnBr, 0 °C to rt, 80%, vi) 4.8 eq NaH, DMF, 3.6 eq BnBr, 0 °C to rt, 90%, vii)
HF/Pyridine, -20 °C to rt, 90%.

15

The straightforward synthesis of 4 and 7 started with a one step conversion of
commercially available per-acetylated glucose 1 using p-thiocresol and BF3.OEt2 to
peracetylated thioglycoside 2. This was followed by deacetylation using NaOMe/MeOH
to give compound 3. Subsequently, 3 was converted either to glycosyl donor 4 via BnBr
and NaH mediated benzylation or to 6-O-t-butyldiphenylsilyl (TBDPS) protected
thiogylcoside 5 using TBDPSCl in pyridine. Compound 5 was then benzoylated at 2-O,
3-O and 4-O position using benzoyl chloride (BzCl) in pyridine to give glycosyl donor 6.
Subsequent deprotection of the 6-O-silyl protecting group via HF/pyridine afforded
glycosyl acceptor 7. Donor 9 (Scheme 1.8) was accessed using pre-activation based
glycosylation protocol of donor 6 with acceptor 8. With thiogylcosides 4, 9 and 7 in
hand, we turned our attention to investigate the stereoselectivity of glycosylation via the
pre-activation protocol.
Our investigation started from the per-benzylated glucoside 4.
solvents can enhance α selectivity,

21,69,74-77

As ethereal

we first performed the glycosylation in

diethyl ether. Donor 4 dissolved in diethyl ether was pre-activated by 1 eq of p-TolSOTf,
21

formed in situ through the stoichiometric reaction of p-TolSCl
AgOTf was typically added as a standard protocol.

22,26,77

with AgOTf. 3 eq of

Upon the rapid complete

activation of 4 within a minute as judged by TLC, acceptor 7 was added, which led to the
formation of disaccharide 10 with close to equal amounts of α and β anomers (α:β = 1.1 :
1) in 67% total yield (Table 1.1, entry 1). While 10 eq of AgOTf produced more β
anomer (α:β = 1 : 1.5) (Table 1.1, entry 2), interestingly, decreasing the amount of
AgOTf to 1.1 eq significantly improved α selectivity (α:β = 6 : 1) (Table 1.1, entry 3).
16

The α selectivity could be further enhanced by performing the reaction under a more
dilute condition. Increasing the volume of diethyl ether by 10 folds under otherwise
identical conditions produced disaccharide 3 in a 10 : 1 α:β ratio (Table 1.2, entry 4).
As α selectivity was obtained, it would be desirable that from the same glycosyl
donor and acceptor, simple changes of the reaction condition can lead to a switch to
β products. Following formation of p-TolSOTf, the thioglycosyl donor is first converted
to a disulfonium ion (Scheme 1.9), which can evolve into other intermediates such as
glycosyl triflate. Crich and coworkers have reported that with benzylidene protected
thio-glycosides including glucosides without 2-O acyl groups, the α glycosyl triflates
were the predominant intermediates following pre-activation as observed by low
temperature NMR studies.

34,38

Similarly, in our low temperature NMR studies, we did

not observe the presence of the glycosyl disulfonium ion,
this species.

64

suggesting transient nature of

As the most likely intermediate is the glycosyl triflate and the

stereochemical outcome in glycoside formation is dependent upon the balance between
glycosyl triflates and the oxacarbenium ion,

66

we envision that addition of exogenous

triflate ion could potentially shift the equilibrium towards glycosyl triflate. This would
favor the formation of the β glycoside product through a SN2 like reaction pathway,
which could be supported by our observation that excess AgOTf led to more β products
(Table 1.1, entry 2). To test this hypothesis, we explored the effects of triflate salts on
stereoselectivity. The reaction between 4 and 7 was performed with 1.1 eq of AgOTf in
the presence of up to 10 eq of triflate salts including NaOTf, Hf(OTf)4 and the more

17

organic solvent soluble tetrabutylammonium triflate.

However, none of these salts

affected the stereoselectivity, which ruled out that the additional triflate anion could
significantly influence the reaction pathway.
Next, we tested the reactions in a variety of solvents including dichloromethane,
76

cyclopentyl methyl ether, THF, toluene, toluene/1,4-dioxane

and acetonitrile. THF,

toluene, toluene/1,4-dioxane and acetonitrile did not lead to productive coupling.
Cyclopentyl methyl ether has been reported to improve cis selectivity.

69

However, in our

reaction, it gave similar results as diethyl ether. Interestingly, a selectivity shift was
observed when the reaction between 4 and 7 was performed in dichloromethane with 3 eq
of AgOTf, which gave disaccharide 10 with 90% total yield with the β anomer becoming
the major product (α:β = 1 : 1.8) (Table 1.2, entry 2). Decreasing the amount of AgOTf
to 1.1 eq greatly enhanced the β selectivity (α:β = 1 : 8) (Table 1.2, entry 1). Therefore,
the stereochemical outcome of the reaction can be controlled by simply switching the
reaction solvent, with diethyl ether favoring α glycoside and dichloromethane generating
more β product.
With the stereoselective reaction conditions in hand, we examined their
generality. Pre-activation of donor 4 in diethyl ether by 1 eq of p-TolSCl and 1.1 eq of
AgOTf followed by addition of the electron rich glucoside acceptor 8 gave disaccharide
23 in 90% yield with the α anomer as the major isomer (α:β = 5.7 : 1) (Table 1.2, entry
15). Exchanging the reaction solvent to dichloromethane produced the β isomer as the
major product (α:β = 1 : 1.7) (Table 1.2, entry 16). The same trend held for a variety of
building blocks, including glucoside acceptor 12 without the STol aglycon, galactoside
18

acceptor 11 with a secondary hydroxyl group, electron poor glucosyl donor 14 and
disaccharide donor 9 (Table 1, entries 7-19). β-Mannoside formation is a challenging
problem for carbohydrate synthesis. The excellent methodology developed by Crich and
coworkers for stereoselective β-mannoside formation required the installation of a
benzylidene moiety on the mannosyl donor.

31

It is noteworthy that under the β selective

reaction condition, the per-benzylated electron rich mannosyl donor 19 without the
benzylidene glycosylated glucoside 12 in dichloromethane forming β-mannoside 20 as
the major product (Table 1.2, entry 18).
As the glycosyl triflate is a likely intermediate formed after pre-activation, when
the reaction is performed in diethyl ether, it is possible that it goes through a double
inversion mechanism (Scheme 1.9, pathway a).

The diethyl ether can act as a

nucleophile, displacing the triflate in an SN2 like fashion from the β-face. Subsequent
SN2 like displacement of the ether molecule by the nucleophilic acceptor can lead to α
glycoside as the major product. Under a dilute condition, the larger amount of diethyl
ether can participate more effectively thus resulting in higher α selectivity.

In the

presence of excess AgOTf, it is most likely that AgOTf coordinates with the oxygen atom
of the triflate, leading to its activation and glycosylation through a more SN1 like pathway
(Scheme 1.9, pathway b). This would result in the formation of anomeric mixtures.
When the glycosylation is performed in dichloromethane, due to the low
solubility of AgOTf in dichloromethane, a solution of AgOTf in acetonitrile was added to
the reaction. Although the amount of acetonitrile is small (2% of the final solvent
volume for the reaction), it is possible that the β selectivity observed is a result of
19

acetonitrile participating from the α face due to the known nitrile effect.

67,78,79

To test

this possibility, we replaced acetonitrile with toluene in reaction of 9 with 12. The β
linked trisaccharide was the only product isolated (Table 1.2, entry 9), which suggests
that acetonitrile does not play a significant role in determining the stereochemical
outcome of the reaction. The β selectivity observed with dichloromethane as the reaction
medium is thus likely due to the non-nucleophilic and non-polar nature of the solvent.
The reaction goes through a more SN2 like pathway with the acceptor directly displacing
the α-glycosyl triflate leading to β glycosides (Scheme 1.9, pathway c).
Table 1.1. Evaluation of effect of triflate additives on stereoselectivity

Entry
1
2
3
4
5
6
7
8
9
10
11
12
a.
b.

Donor

Acceptor

AgOTf

Product

b

(α/β)
α/β)

Yield
(%)
67
66
68
76
68
89
63
86
67
87
76

none
1.1 : 1
10
none
1 : 1.5
10
none
6:1
10
NaOTf
5:1
10
HfOTf
4:1
10
none
2:1
22
none
β
22
none
13
1.8 : 1
TBAOTf
13
1.3 : 1
TBAOTf
1 : 1.5
13
a 13
1.4 : 1
TBAOTf
3 eq
none
α
74
9
11
16
3 equivalents of TBAOTf used in the reaction.
1
Ratio determined by H-NMR intergration. α and β anomers were assigned based
3
1
1
on JH1,H2 and/or JC1,H1 obtained through gHMQC 2-D NMR (without H
decoupling).
4
4
4
4
4
9
9
4
4
4
4

7
7
7
7
7
7
7
12
12
12
12

3 eq
10 eq
1 eq
1 eq
1 eq
1 eq
3 eq
1 eq
1 eq
3 eq
1 eq

MOTf

20

Table 1.2. Solvent effects on stereoselectivity using donors with no 2-O acyl groups.

Entry

Donor

Acceptor

AgOTf

Solvent

Product

(α/β)
α/β)

1
2

4

a

7
7

1 eq
3 eq

10
10

1:8
1 : 1.8

b

CH2Cl2
CH2Cl2

Yield
(%)
90
90

7

1 eq

10

6:1

68

c

Et2O

7

1 eq

Et2O

10

10 : 1

41

1 eq
1 eq
1 eq
1 eq
1 eq

CH2Cl2
Et2O
CH2Cl2
CH2Cl2
CH2Cl2

3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
a.
b.
c.
d.

4
4

4
4
4
14
9

1:9
79
1.8 : 1
90
3.5 : 1
50
76
β
d
76
β
9
1 eq
1.5 : 1
65
9
12
18
Et2O
70
1 eq
9
8
17
α
Et2O
1 : 1.2
90
1 eq
9
8
17
CH2Cl2
1 eq
2:1
42
4
11
24
CH2Cl2
1 eq
60
4
11
24
α
Et2O
5.7 : 1
90
1 eq
4
8
23
Et2O
1 : 1.7
72
1 eq
4
8
23
CH2Cl2
1 eq
61
19
8
21
α
Et2O
1 eq
1:1
63
19
8
21
CH2Cl2
1 eq
1:3
71
19
12
20
CH2Cl2
1 eq
1:1
58
19
12
20
Et2O
1 eq
74
9
11
16
α
Et2O
Reaction was performed in dichloromethane (donor concentration was 50 mM).
Reaction was performed in diethyl ether (donor concentration was 50 mM).
Donor concentration was 5 mM.
Toluene (5% of final volume) was added to the reaction to dissolve AgOTf.
12
12
7
12
12

21

13
13
15
18
18

Scheme 1.8 Building blocks and glycosylation products
22

Scheme 1.9 Proposed mechanism of the effects of solvents and excess AgOTf on stereoselectivity

23

1.3 Conclusion
In a continuing effort to develop an efficient strategy for stereoselective synthesis
of oligosaccharides using the pre-activation protocol, effects of solvent and triflate salt
additives on stereoselectivity of pre-activation based glycosylation were investigated
using a variety of donors without a participating protective group on 2-O. We have
discovered that solvents play a significant role in determining stereochemical outcome of
glycosylation reactions. Dichloromethane favor β selectivity whereas diethyl ether leads
to α-isomers. Besides its role in generating the promoter p-TolSOTf, AgOTf can also
exert significant impact on stereoselectivity presumably due to coordination with the
glycosyl triflate intermediate.
Our findings establish that by simply changing the reaction solvent, we can bias
the stereoselectivity of pre-activation based glycosylation for a variety of building blocks
including the formation of β-mannosides. Other factors including donor and acceptor
structures and protective groups also affect the outcome. However a detailed mechanistic
study to determine the fate of reaction intermediates once the acceptor is added to the
reaction mixture and the role they play in determining anomeric stereoselectivity needs to
be undertaken.

24

1.4 Experimental Section
All reactions were carried out under nitrogen with anhydrous solvents in flamedried glassware. All glycosylation reactions were performed in the presence of molecular
sieves, which were flame dried right before the reaction under high vacuum.
Glycosylation solvents were dried using a solvent purification system and used directly
without further drying. Chemicals used were reagent grade as supplied except where
noted. Analytical thin-layer chromatography was performed using silica gel 60 F254
glass plate. Compound spots were visualized by UV light (254 nm) and by staining with
a yellow solution containing Ce (NH4)2(NO3)6 (0.5 g) and (NH4)6Mo7O24 4H2O (24.0 g)
in 6% H2SO4 (500 mL). Flash column chromatography was performed on silica gel 60
(230–400 Mesh). NMR spectra were referenced using Me4Si (0 ppm), residual CHCl3 (δ
1

H-NMR 7.24 ppm,

13

1

1

C-NMR 77.0 ppm). Peak and coupling constant assignments are
1

1

13

1

13

based on H-NMR, H– H gCOSY and (or) H– C gHMQC and H– C gHMBC
experiments. All optical rotations were measured at 25 °C using the sodium D line. ESI
mass spectra were recorded in positive ion mode. High-resolution mass spectra were
recorded on a Micromass electrospray

p-Tolyl-2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranoside (2)
Compound 1 (22 g, 57.7 mmol) was coevaporated with anhydrous toulene (2 x 50 mL)
then dried in vacuo for 1 hour then dissolved in anhydrous dichloromethane (40 mL),
followed by addition of p-thiocresol (1.6 eq, 11.5 g). BF3.OEt2 (1.3 eq, 9.4 mL) was

25

added dropwise to the reaction mixture then stirred overnight. The reaction mixture was
diluted with dichloromethane (300 mL), washed with saturated NaHCO3 (200 mL)
followed by an aqueous workup using 200ml of water. The aqueous phase was reextracted with dichloromethane (2 x 200 mL) and the organic phases recombined then
dried over anhydrous Na2SO4. The solvents were evaporated and the resulting off-white
residue was recrystallized in hexanes / ethyl acetate (10:1) giving compound 2 in 85%
1

yield (22.3 g) as a white solid.

Comparison of H-NMR with literature values

71

confirmed the identity of compound 2.
1

H-NMR (400 MHz, CDCl3) δ 1.98 (s, 3H, COCH3), 2.00(s, 3H, COCH3), 2.07 (s, 3H,

COCH3), 2.08 (s, 3H, COCH3), 2.35 (s, 3H, SPhCH3), 3.69-3.71 (m, 1H, H-5), 4.18-4.20
(m, 2H, H-6, H-6’), 4.64 (d, 1H, J
(t, 1H, J

3

3

= 10 Hz, H-1), 4.95 (t, 1H, J

= 9.6 Hz, H-4), 5.23 (t, 1H, J

3

3

= 9.6 Hz, H-2), 5.05

=9.6 Hz, H-3), 7.10-7.12 (m, 2H aromatic),

7.36-7.38 (m, 2H aromatic).

p-Tolyl-1-thio-β-D-glucopyranoside (3)
Compound 2 (7.42 g, 16.3mmol) was dissolved in anhydrous dichloromethane (10 mL)
and methanol (70 mL) followed by addition of freshly prepared 1M NaOMe solution to a
pH of 13. NaOMe solution was freshly prepared by adding Na metal (1.15 g, 0.05 mol)
in small pieces to anhydrous methanol (50 mL) at -5 °C in a 250 ml round-bottomed flask
equipped with a reflux condenser, which was swirled until the metal had been completely
consumed. This was slowly added to the reaction mixture to a pH of 13. The reaction
mixture was stirred for 4 hours. TLC confirmed complete conversion of starting material
26

to product. The reaction mixture was quenched with Amberlite resin (IR-120) to adjust
the pH to pH 7. The resin was subsequently washed with methanol until no product
could be detected by TLC. The filtrates were combined then concentrated in vacuo, and
the

resultant

residue

was

recrystallized

in

dichloromethane/

hexanes/ethyl

acetate/methanol (3:2:1:0.2) giving compound 3 in 77% yield (3.6 g). The identity of
1

compound 3 was confirmed by comparison of H-NMR with literature values.
1

71

3

H-NMR (500 MHz, CD3OD) δ 2.32(s, 3H, SPhCH3), 3.17 (dd, 1H, J = 9, 10 Hz), 3.283

3

3

3.30 (m, 2H), 3.37 (t, 1H, J = 9 Hz), 3.65(dd, 1H, J = 5.5, 12 Hz), 3.85 (dd, 1H, J =
3

2, 12 Hz), 4.51 (d, 1H, J = 10 Hz, H-1), 7.11-7.13 (m, 2H, aromatic), 7.44-7.47 (m, 2H,
aromatic).

p-Tolyl-2,3,4,6-tetra-O-benzyl-6-O-1-thio-β-D-glucopyranoside (4).
Compound 3 (1.6 g, 5.4 mmol) was dissolved in DMF (10 mL) and cooled to 0 °C. NaH
(0.84 g, 34.6mmol) was added in portions followed by addition of benzyl bromide (25.9
mmol, 3.1 mL). The reaction mixture was stirred at room temperature overnight. The
reaction mixture was concentrated using high pressure vacuum followed by
coevaporation with toluene.

The resultant residue was then dissolved in 100mL

dichloromethane and added to ice cooled water. The aqueous phase was extracted twice
with dichloromethane (50 mL).

The organic phases were combined then washed

successively with 1N HCl, sat. NaHCO3 solution, brine and dried over Na2 SO4. The
filtrates were combined then concentrated in vacuo, and the resultant residue was
recrystallized in dichloromethane/ hexanes/ethyl acetate (1.4:1) giving compound 4 in 80
27

1

% yield (2.88 g). Comparison of H-NMR with literature values

71

confirmed the identity

of compound 4.
1

H-NMR (600 MHz, CDCl3) δ 2.29 (s, 3H, SPhCH3), 3.44-3.47 (m, 2H), 3.63(t, 1H, J
3

3

3

= 9.6 Hz), 3.67-3.72 (m, 2H), 3.76 (dd, 1H, J = 1.8, 11.4 Hz), 4.53 (d, 1H, J = 12 Hz),
3

3

4.55-4.60 (m, 3H), 4.72(d, 1H, J = 10.2 Hz), 4.79-4.84 (m, 2H), 4.86 (d, 2H, J = 10.8
Hz), 7.01-7.48 (m, 24H, aromatic).

p-Tolyl-6-O-t-butyldiphenylsilyl-β-1-thio-β-D-glucopyranoside (5)
Compound 3 (1.3 g, 4.6 mmol) was dissolved in pyridine (20 mL), followed by the
addition of tert-butyldiphenylsilyl chloride (TBDPSCl) (1.3 eq, 1.6 mL ) then stirred at
room temperature for 6 hours. Pyridine was evaporated under high vacuum. The residue
was dissolved in dichloromethane, washed successively with ice cold water (60 mL), 1M
HCl (60 mL), sat. aqueous NaHCO3 (60 mL), brine (50 mL) and the organic layer was
dried over Na2SO4. The desired product 5 18 was obtained as a clear syrup in 90% yield
(2.17 g).
1

H-NMR (600 MHz, CDCl3) δ 1.12, (s, 9H, C(CH3)3Si), 2.30 (s, 3H, SPhCH3), 3.38 (t,

1H, J

3

= 8.4 Hz), 3.45-3.48 (m, 1H), 3.58-3.66 (m, 2H), 3.92 (dd, 1H, J
3

3

3

= 4.8, 10.8

Hz), 4.01 (dd, 1H, J = 3.6, 11.4 Hz), 4.52 (d, 1H, J = 9.6 Hz, H-1), 7.02-7.04 (m, 2H,
aromatic), 7.40-7.50 (m, 8H, aromatic), 7.78-7.85 (m, 4H, aromatic).

28

p-Tolyl-2,3,4-tri-O-benzoyl-6-O-t-butyldiphenylsilyl-β-D-glucopyranosyl-1-thio-β-Dglucopyranoside (6)
To a solution of compound 5 (1.7 g, 5.8 mmol) in pyridine (20 mL) was added benzoyl
chloride (3.6 eq, 2.5 mL) at 0 °C. The mixture was stirred overnight and solvents
evaporated under high vacuum.

The residue dissolved in dichloromethane, washed

successively with ice cold water (60 mL), 1M HCl (60 mL), sat. aqueous NaHCO3 (60
mL), brine (50 mL) and the organic layer was dried over Na2SO4. The desired compound
6 was obtained as white foam after column chromatography (hexanes/ ethyl acetate 4:1)
1

in 95% yield (4.6 g). The identity of compound 6 was confirmed by comparison of HNMR with literature values.
1

18

H-NMR (600 MHz, CDCl3) δ 1.03 (s, 9H, C (CH3)3Si), 2.30 (s, 3H, SPhCH3), 3.83 –

3.88 (m, 3H, H-5,H-6, H-6’), 4.96 (d, 1H, J
3

3

= 9.6 Hz, H-1), 5.45 (t, 1H, J 3 = 9.6 Hz, H3

2), 5.61 (t, 1H, J = 9.6 Hz, H-4), 5.82 (t, 1H J = 9 Hz, H-3), 7.02 – 8.20 (m, 29 H,
aromatic).

p-Tolyl-2,3,4-tri-O-benzoyl-1-thio-β-D-glucopyranoside (7)
To a solution of compound 6 (1.2 g, 1.43 mmol) in pyridine (30 mL) was added dropwise
hydrogen fluoride in pyridine (65%), (25 mL) at-20 °C and the reaction mixture was
stirred to room temperature over 2 hours. The reaction mixture was washed with aqueous
CuSO4 solution (2 x 40 mL), 1M HCl (40 mL), sat. aqueous NaHCO3 (40 mL), brine (50
mL) and the organic layer dried over Na2SO4. The desired compound 7 was obtained in
29

90% yield (0.77 g) after flash column chromatography (hexanes/ ethyl acetate 2:1). The
1

identity of compound 7 was confirmed by comparison of H-NMR with literature
values.
1

18

H-NMR (600 MHz, CDCl3) δ 2.34 (s, 3H, SPhCH3), 2.42(dd, 1H J
3

3

= 5.4, 8.4 Hz),

3.70-3.84 (m, 3H, H-5, H-6, H-6’), 4.97 (d, 1H, J = 10.2 Hz, H-1), 5.44 (t, 2H, J

3

=

3

10.2 Hz, H-2, H-4), 5.92(t. 1H, J = 9.6 Hz, H-3), 7.12-7.13 (m, 2H, aromatic), 7.22-7.26
(m, 2H, aromatic), 7.35-7.42 (m, 7H, aromatic), 7.50-7.53 (m, 2H, aromatic), 7.78-7.82
(m, 2H, aromatic), 7.90–7.96 (m, 4 H, aromatic).

p-Tolyl-2,3,4-tri-O-benzyl-1-thio-β-D-glucopyranoside (8)
A solution of compound 5 (1 g, 1.91 mmol) in DMF was cooled down to 0 °C followed
by addition of NaH (4.8 eq, 0.7 g) and benzyl bromide (3.6 eq, 2.46 mL) then stirred
overnight at room temperature.

The mixture was stirred overnight and solvents

evaporated under high vacuum. The residue was dissolved in dichloromethane, washed
successively with ice cold water (60 mL), 1M HCl (60 mL), sat. aqueous NaHCO3 (60
mL), brine (50 mL) and the organic layer dried over Na2SO4. The residue obtained was
dissolved in pyridine (30 mL) followed by dropwise addition of hydrogen fluoride in
pyridine (65 %) (25 mL) at-20 °C and the reaction mixture stirred to room temperature
over 2 hours. The reaction mixture was washed with aqueous CuSO4 solution (2 x 40
mL), 1M HCl (40 mL), sat. aqueous NaHCO3 (40 mL), brine (50 mL) and the organic
layer dried over Na2SO4. The desired compound 8 was obtained in 90% (0.96 g) yield
30

after flash column chromatography (hexanes/ ethyl acetate 2:1). Comparison of 1H-NMR
with literature values
1

71

confirmed the identity of compound 8.

H-NMR (600 MHz, CDCl3) δ 2.32 (s, 3H, SPhCH3), 3.34-3.43, (m, 1H), 3.48 (t, 1H, J =
3

9.2 Hz), 3.58(t, 1H, J = 9.2 Hz), 3.68-3.78 (m, 2H), 3.84 – 3.91 (m, 1H), 4.63-4.70 (m,
3

2H), 4.78 (d, 1H, J = 10.4 Hz), 4.85-4.97 (m, 4H), 7.10-7.17 (m, 2H, aromatic), 7.22 –
7.48 (m, 17 H, aromatic).

p-Tolyl-2,3,4-tri-O-benzyl-6-O-(2,3,4-tri-O-benzoyl-6-O-t-butyldiphenylsilyl-β-Dglucopyranosyl)-1-thio-β-D-glucopyranoside (9)
A mixture of donor 6 ( 40 mg, 0.050 mmol) and freshly activated molecular sieves 4 Å
(150 mg) in CH2Cl2 (2 mL) was stirred at room temperature for 30 minutes, cooled to
−60 °C, and AgOTf (39 mg, 0.15 mmol) dissolved in Et2O (1 mL) was added without the
solution touching the wall of the flask. After 5 min, orange colored p-TolSCl (7.9 µL,
0.060 mmol) was added through a microsyringe. Since the reaction temperature was
lower than the freezing point of p-TolSCl, the reagent 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 within a few seconds, indicating
depletion of p-TolSCl. When the donor was completely consumed (TLC, ~ 5 minutes at
−60 °C), a solution of acceptor 8 (28 mg, 0.050 mmol) in CH2Cl2 (0.2 mL) was added
slowly and dropwise along the flask wall with the aid of a syringe. The reaction mixture
was stirred and allowed to warm to −10°C within 2 hours. CH2Cl2 (20 mL) was added,
31

and the mixture was filtered though a Celite pad. The Celite was washed with CH2Cl2
until no organic compounds were present in the filtrate (TLC). The combined CH2Cl2
solutions were washed successively with a saturated aqueous solution of NaHCO3 (2 x
20 mL) and water (2 x 10 mL). The organic phase was dried (Na2SO4), concentrated and
purified by chromatography resulting in the desired amorphous oligosaccharide 9 (42 mg,
1

69 % yield). The identity of compound 9 was confirmed by comparison of HNMR with
literature values.

18

[α]20 +10.0 (c = 1, CH2Cl2); H-NMR (500 MHz, CDCl3) 1.04 (s, 9H,C(CH3)3Si), 2.34
D
1

3

(s, 3H, SPhCH3), 3.39 (d, 1H, J = 9.6 Hz, H-4), 3.41 (d, 1H, J
3

3

= 9.6 Hz, H-2), 3.45

3

(dd, 1H, J = 3.6, 9.6 Hz, H-5), 3.61 (t, 1H, J = 9.6 Hz, H-3), 3.72 - 3.77 (m, 1H, H-5’),
3

3.78 (dd, 1H, J = 4.2, 11.2 Hz, H-6b), 3.82 – 3.88 (m, 2H, H-6’a, H-6’b), 4.18 (d, 1H, J
3

3

3

= 11.2 Hz, H-6a), 4.41 (d, 1H, J = 11.0 Hz, CH2Ph), 4.52 (d, 1H, J = 9.6 Hz, H-1),
3

3

3

4.54 (d, 1H, J = 10.4 Hz, CH2Ph), 4.65 (d, 1H, J = 10.4 Hz, CH2Ph), 4.72 (d, 1H, J =
10.4 Hz, CH2Ph), 4.82 (d, 1H, J
CH2Ph), 4.89 (d, 1H, J
3

3

3

= 10.4 Hz, CH2Ph), 4.85 (d, 1H, J

3

= 10.4 Hz,

3

= 8.0 Hz, H-1’), 5.56 (dd, 1H, J = 8.0, 9.6 Hz, H-2’), 5.65 (t,
3

1H, J = 9.6 Hz, H-4’), 5.78 (t, 1H, J = 9.6 Hz, H-3’), 7.08 – 7.90 (m, 44 H, aromatic) ;
13

C-NMR (125 MHz, CDCl3) δ 19.41 (SiC(CH3)3), 21.83 (SPhCH3), 26.92 (C(CH3)3),

63.07 (C-6’), 67.88 (C-6), 69.58 (C-4’), 72.41 (C-2’), 73.73 (C-3’), 75.00 (CH2Ph), 75.42
(C-5), 75.52 (CH2Ph), 75.84 (CH2Ph), 77.64 (C-2), 79.19 (C-5’), 80.67 (C-4), 86.87 (C-

32

3), 87.88 (JC-1,H-1 = 157.7 Hz, C-1), 101.21 (JC1,H1 = 163.0 Hz, C-1’), 127.88, 127.96,
128.04, 128.09, 128.43, 128.49, 128.53, 128.59, 128.64, 129.31, 129.53, 129.66, 129.82,
129.87, 129.90, 129.99, 130.01, 130.03, 130.13, 133.04, 133.22, 133.30, 133.38, 133.41,
135.74,

135.92,

138.15,

138.18,

138.40,

138.63,

165.26,

166.15;

HRMS

C77H76NaO13SSi [M + Na]+ calc. 1291.4674 found 1291.4651; Anal. calc.
C77H76NaO13SSi: C, 72.85; H, 6.03; found C, 72.78, H, 5.85

General procedures for pre-activation based glycosylation.
Method A: Donor (50 mg) was dissolved in CH2Cl2 (5 mL) and stirred at -78 °C with
freshly activated molecular sieves MS 4 Å (100 mg) under nitrogen atmosphere for 30
minutes. AgOTf (1eq) dissolved in acetonitrile/ CH2Cl2 (v: v = 0.025:1) was added to the
reaction mixture. After 5 minutes p-TolSCl (1 eq) was added to the reaction mixture.
The low temperature of the reaction mixture requires for the direct addition of p-TolSCl
which prevents it from freezing on the flask wall. The characteristic yellow color of pTolSCl dissipated within a few seconds. The donor was completely consumed after 5
minutes as confirmed by TLC analysis. Glycosyl acceptor (0.9 eq) dissolved in CH2Cl2
(2 ml) was then added dropwise to the reaction mixture. This was stirred for 20 minutes
at -78 °C under N2 at which point, the acceptor was completely consumed as confirmed
by TLC. The reaction mixture was warmed up to -20 °C then quenched with Et3N. This
was followed by dilution with CH2Cl2 (20 mL) and filtration over Celite. The Celite was
further washed with CH2Cl2 until no organic compounds was present in the filtrate. The

33

fractions were combined then concentrated to dryness. The residue was purified by silica
gel flash chromatography.
Method B: Donor (50 mg) was dissolved in Et2O (5 mL) and stirred at -78 °C with
freshly activated molecular sieves MS 4 Å (100 mg) under nitrogen atmosphere for 30
minutes. AgOTf (1 eq) dissolved in Et2O (1 mL) was added to the reaction mixture.
After 5 minutes p-TolSCl (1 eq) was added to the reaction mixture. The low temperature
of the reaction mixture allows for direct addition of p-TolSCl which prevents it from
freezing on the flask wall. The characteristic yellow color of p-TolSCl dissipated within
a few seconds. The donor was completely consumed after 5 minutes as confirmed by
TLC analysis. Glycosyl acceptor (0.9 eq) dissolved in Et2O (2 mL) was then added
dropwise to the reaction mixture. This was stirred for 20 minutes at -78 °C under N2 at
which point, the acceptor was completely consumed as confirmed by TLC. The reaction
mixture was warmed up to -20 °C, quenched with Et3N then concentrated to dryness. The
residue was diluted with CH2Cl2 (20 mL) and filtrated over Celite. The Celite was
further washed with CH2Cl2 until no organic compounds was present in the filtrate as
determined by TLC. The fractions were combined, concentrated to dryness and the
residue purified by silica gel flash chromatography.

34

p-Tolyl-2,3,4-tri-O-benzyl-D-glucopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-1-thio-β-Dβ
glucopyranoside (10)
Using method B of general procedure for glycosylation, donor 4 (40 mg, 0.06 mmol) was
preactivated using p-TolSCl (9 µL, 0.06 mmol) and reacted with acceptor 7 (30 mg, 0.05
mmol) to give desired product (10) in 69 % yield (46 mg), (α/β 6:1) after column
1

purification (hexanes/ ethyl acetate 3:1). Comparison of H-NMR with literature values
18
1

confirmed the identity of compound 10.
3

H-NMR (600 MHz, CDCl3) α anomer δ 2.15 (s, 3H), 3.21 (dd, 1H, J = 3.6, 10.2 Hz),
3

3.45-3.53 (m, 2H), 3.57 (dd, 1H, J = 3.6, 10.8 Hz), 3.77 (d, 1H, J

3

= 12.6 Hz), 3.79-

3

3

3.83 (m, 1H), 3.88 (t, 1H, J = 9.6 Hz), 3.89 – 3.93 (m, 1H), 4.06 (t, 1H, J = 9.6 Hz),
3

3

3

4.11 (d, 1H, J = 12.6 Hz), 4.30 (d, 1H, J = 12.0 Hz), 4.36 (d, 1H, J = 10.8 Hz), 4.47
3

3

(d, 1H, J = 12.0 Hz), 4.54 (dd, 1H, J = 4.8, 12.0 Hz), 4.66 – 4.71 (m, 2H), 4.72 (d, 1H,
3

3

3

3

J = 10.8 Hz), 4.79 (d, 1H, J = 3.0 Hz), 4.86 (d, 1H, J = 10.2 Hz), 4.96 (dd, 1H, J =
3

3

1.8, 12.0 Hz), 5.39 (t, 1H, J = 9.6 Hz), 5.82 (t, 1H, J = 9.0 Hz), 6.84 – 6.88 (m, 2 H,
aromatic), 7.04 – 7.52 (m, 30 H, aromatic), 7.61 – 7.65 (m, 1 H, aromatic), 7.92 – 7.97
(m, 4 H, aromatic), 8.01 – 8.04 (m, 2 H, aromatic);

13

C-NMR (100.5 MHz, CDCl3)

δ 21.31, 67.52, 68.63, 69.88, 70.42, 70.89, 73.62, 73.70, 74.64, 75.23, 75.98, 77.54,
77.96, 80.40, 82.23, 87.34, 97.54, 127.69, 127.78, 127.85, 128.02, 128.06, 128.12,
128.35, 128.44, 128.49, 128.62, 128.63, 128.67, 129.12, 129.58, 129.97, 130.13, 133.39,

35

133.49, 133.70, 133.93, 138.22, 138.48, 138.68, 138.74, 139.24, 165.32, 165.39, 166.00;
HRMS C68H64NaO13S [M + Na+] calc. 1143.3965 found 1143.3999.

Methyl-2,3,4-tri-O-benzyl-D-glucopyranosyl-(1→6)-2,3,4-tri-O-benzyl-α-Dα
glucopyranoside (13)
Using method A of general procedure for glycosylation, donor 4 (40 mg, 0.06 mmol) was
preactivated using p-TolSCl (13.4µL, 0.06 mmol) and reacted with acceptor 12 (25.6 mg,
0.05 mmol) to give the desired product (13) in 79 % yield (60.4 mg), (α/β 1 : 9) after
column purification (hexanes/ ethyl acetate 3:1). Comparison of 1H-NMR spectra with
literature values
1

72

confirmed the identity of compound 13.

H-NMR (500 MHz, CDCl3) δ 3.36 (s, 3H, OCH3), 3.44-3.47 (m, 2H), 3.51-3.71(m,
3

3

14H), 3.74 (d, 1H, J = 1.5 Hz), 3.76 (d, 1H, J = 2 Hz), 3.84-3.87 (m, 1H), 4.0-4.04 (m,
2H), 4.22 (dd, 1H, J

3

= 2, 11 Hz), 4.39 (d, 1H, J

3

= 8 Hz), 4.50-4.85 (m, 18H), 4.92-

4.95 (m, 1H), 4.98-5.02 (m, 3H), 7.14-7.38 (m, 64H, aromatic), 7.81-7.86 (m, 6H,
aromatic).

p-Tolyl-2,3-di-O-benzyl-4,6-di-O-benzoyl-D-glucopyranosyl-(1→6)-2,3,4-tri-Obenzoyl-1-thio-β-D-glucopyranoside (15)
β
Using method B of general procedure for glycosylation, donor14 (40 mg, 0.06 mmol)
was preactivated using p-TolSCl (15 µL, 0.1 mmol) and reacted with acceptor 7 (33 mg,
0.05 mmol) to give the desired product (15) in 69 % yield (43.6 mg), (α/β 6:1 α as

36

determined from SPhCH3 singlet and H-3’ ratios) after column purification (hexanes/
ethyl acetate 3:1)
[α]20 +33 (c =1.0, CH2Cl2);
D

1

H-NMR (600 MHz, CDCl3) δ 2.18 (s, 3H, SPhCH3, α),

2.27(s, SPhCH3 β), 3.56 (m, 1H), 3.72 (dd, 1H, J

3

= 3.6, 9.6 Hz), 3.95-4.00 (m, 1H),
3

4.14-4.24 (m, 3H), 4.32-4.36 (m, 1H), 4.42 (dd, 1H, J = 2.4, 12 Hz), 4.64-4.68 (m, 2H),
3

3

3

4.74 (d, 1H, J = 3 Hz, H-1), 4.81 (d, 1H, J = 12 Hz, CH2Ph), 4.89 (d, 1H, J = 12 Hz,
3

CH2Ph), 4.99 (d, 1H, J = 9.6 Hz, H-1), 5.38-5.45 (m, 3H), 5.84 (t, 1H, J
7.07-7.56 (m, 32H, aromatic), 7.77-8.04 (m, 11H, aromatic).

13

3

= 12 Hz),

C-NMR (150 MHz,

CDCl3), δ 21.07, 62.99,66.76, 68.01, 69.42, 70.54, 70.76, 73.62, 74.41, 75.69, 76.79,
a

77.0, 77.21, 79.42, 79.98, 87.07 (JC-1,H-1 = 159.07 Hz, C-1 ), 97.01 (JC-1,H-1 = 169.76 Hz,
b

C-1 ) 114.77, 127.49, 127.79, 127.98, 128.13, 128.2, 128.26, 128.32, 128.37, 128.47,
128.52, 128.81, 128.89, 129.31, 129.54, 129.74, 129.78, 129.82, 129.83, 129.87, 132.44,
132.94,133.1,133.2, 133.3, 133.5, 138.06, 138.17, 138.2, 165.02, 165.08, 165.31, 165.77,
166.04. HRMS C68H60NaO15S [M + Na] + calc. 1171.3551 found 1171.3600.

p-Tolyl-2,3,4-tri-O-benzoyl-6-O-tert-butyldiphenylsilyl-β-D-glucopyranosyl-(1→6)β
2,3,4-tri-O-benzyl-α-D-glucopyranosyl-(1→4)-2,3-di-O-benzoyl-6-O-benzyl-1-thio-βα
β
D-glactopyronoside (16)

Using method B of general procedure for glycosylation, donor 9 (60 mg, 0.05 mmol) was
preactivated using p-TolSCl (15µL, 0.05 mmol) and reacted with acceptor 11 (20.9 mg,
37

0.04 mmol) to give the desired product (16) in 74 % yield (46 mg) exclusively as α
isomer after column purification (hexanes/ ethyl acetate/CH2Cl2 6:1:0.5).
[α]20 +41 (c = 0.55, CH2Cl2); H-NMR (500 MHz, CDCl3) δ 0.94 (s, 9H, (CH3)3CSi),
D
1

3

2.10 (s, 3H, SPhCH3), 3.29 (dd, 1H, J = 3, 10.2 Hz), 3.35-3.42 (m, 2H), 3.46-3.50 (m,
1H), 3.66-3.70 (m, 1H), 3.73-3.74 (m, 2H), 3.82-3.91 (m, 4H), 4.17 (d, 1H, J
3

c

3

= 11.5

3

Hz), 4.22 (d, 1H, J = 8 Hz, H-1 ), 4.28-4.38 (m, 4H), 4.48 (d, 1H, J = 12 Hz, CH2Ph),
4.66 (d, 1H, J 3 = 10.2 Hz), 4.67 (d, 1H, J 3 = 9 Hz) 4.80 (d, 1H, J 3 = 11 Hz), 4.82 (d, 1H,
J

3

a

= 10 Hz, H-1 ), 4.90 (d, 1H, J

3

b

= 3.5 Hz, H-1 ), 5.34-5.43 (m, 2H), 5.50-5.69 (m,

3H), 6.95-7.03 (m, 4H, aromatic), 7.06-7.40 (m, 41H, aromatic), 7.44-7.51 (m, 4H
aromatic), 7.64-7.65 (m, 2H, aromatic), 7.77-7.79 (m, 3H, aromatic), 7.82-7.85 (m, 4H,
aromatic), 7.87-7.89 (m, 2H, aromatic);

13

C-NMR (125 MHz, CDCl3), δ 29.9, 26.9, 21.3

19.3, 21.3, 26.9, 29.9, 63.1, 67.8, 68.17, 68.2, 69.5, 70.5, 72.2, 73.5, 73.6, 74.1, 74.34,
b

74.39, 75.0, 75.34, 77.0, 77.3, 77.5, 78.5, 80.3, 81.6, 86.3 (JC-1,H-1 = 170.04 Hz, C-1 ),
c

a

98.97 (JC-1,H-1 = 163.06 Hz, C-1 ), 101.7 (JC-1,H-1 = 159.71Hz, C-1 ), 127.55, 127.57,
127.64, 127.83, 127.84, 127.95, 127.97, 128.0, 128.02, 128.04, 128.40, 128.45, 128.48,
128.53, 128.57, 128.60, 128.64, 128.7, 129.24, 129.38, 129.6, 129.63, 129.8, 129.84,
129.88, 129.95, 129.99, 130.3, 133.0, 133.2, 133.24, 133.3, 133.34, 133.37, 133.4,
135.8,138.06, 138.1,138.7, 139.0, 139.2, 165.1, 165.2, 165.9, 166.2.
C104H104NO20SSi [M + NH4]+ calc. 1746.6636 found 1746.6638.

38

HRMS

p-Tolyl-2,3,4-tri-O-benzoyl-6-O-tert-butyldiphenylsilyl-β-D-glucopyranosyl-(1→6)β
2,3,4-tri-O-benzyl-α-D-glucopyranosyl-(1→6)-2,3,4-tri-O-benzyl-1-thio-β-Dα
β
glucopyronoside (17)
Using method B of general procedure for glycosylation, donor 9 (60 mg, 0.05 mmol) was
preactivated using p-TolSCl (15µL, 0.1 mmol), and reacted with acceptor 8 (20.9 mg,
0.04 mmol) to give desired product (17) in 70 % yield (62 mg) exclusively as α-isomer
after column purification (hexanes/ethyl acetate/CH2Cl2 6:1:0.5).
[α]20 +22 (c =0.35, CH2Cl2); H-NMR (600 MHz, CDCl3) δ 1.00 (s, 9H, (CH3)3CSi),
D
1

2.19 (s, 3H, SPhCH3), 3.18 (t, 1H, J

3

= 9.6 Hz), 3.27-3.37 (m, 1H), 3.40-3.44 (m, 2H),

3.56-3.63 (m, 2H), 3.69-3.72 (m, 2H), 3.76-3.88 (m, 5H), 4.17 (d, 1H, J
3

3

3

= 9 Hz), 4.26
c

3

(d, 1H, J = 11.4 Hz), 4.45 (d, 1H, J = 11.4 Hz), 4.50 (d, 1H, J = 9 Hz, H1 ), 4.56 (d,
1H, J

3

= 10.8 Hz), 4.59-4.64 (m, 4H), 4.70 (d, 1H, J
3

3

b

a

= 8.4 Hz, H-1 ), 4.71-4.90 (m,
3

5H), 5.01 (d, 1H, J = 3.6 Hz, H-1 ), 5.54-5.58 (m, 2H), 5.81 (t, 1H, J = 10.2 Hz), 6.967.65 (m, 49H, aromatic), 7.67-7.71 (m, 2H, aromatic), 7.81-7.89 (m, 7H, aromatic);

13

C-

NMR (125 MHz, CDCl3), δ 19.4, 21.3, 24.9, 26.86, 26.9, 29.9, 36.9, 63.1, 69.6, 69.9,
72.4, 72.6, 73.6, 74.8, 75.1, 75.6, 75.7, 75.8, 77.0, 77.3, 77.46, 77.5, 77.9, 78.0, 79.0,
c

80.3, 81.4, 81.7, 86.9, 89.0 (JC-1,H-1 = 170.04 Hz, C-1 ), 97.5 (JC-1,H-1 = 170.72 Hz, Cb

a

1 ), 101.5 (JC-1,H-1 = 161.68 Hz, C-1 ), 127.5, 127.56, 127.6, 127.7, 127.8,127.83,
127.89, 127.92, 128.03, 128.06, 128.1, 128.43, 128.49, 128.53, 128.54, 128.58, 128.6,
128.65,128.7, 129.2, 129.5, 129.58, 129.86, 129.89, 129.9, , 130.0,130.1, 130.5, 133.1,
39

133.2, 133.22, 133.28, 133.3, 133.4, 135.8, 135.9, 138.0, 138.4, 138.5, 138.7, 138.8,
HRMS C104H108NO18SSi [M + NH4]+ calc.

138.9, 139.2, 165.16, 165.26, 166.19.
1718.7135, found 1718.7137.

Methyl-2,3,4-tri-O-benzoyl-6-O-tert-butyldiphenylsilyl-β-D-glucopyranosyl-(1→6)β
2,3,4-tri-O-benzyl-β-D-glucopyranosyl-(1→6)-2,3,4-tri-O-benzyl-1-methyl-α-Dβ
α
glucopyronoside (18)
Compound 18 was synthesized from donor 9 (56.5 mg, 0.04 mmol) and acceptor 12 (20.5
mg, 0.04 mmol) in 76 % yield (54.2 mg) as β isomer using method A of general
procedure for preactivation based glycosylation and purified by flush column
chromatography (hexanes/ethyl acetate/ CH2Cl2, 4:1:0.5).
[α]20 +12 (c = 0.2, CH2Cl2); H-NMR (500 MHz, CDCl3) δ 1.00 (s, 9H, (CH3)3CSi),
D
1

3.33 (d, 1H, J

3

= 8.5 Hz), 3.35 (s, 3H, OCH3), 3.39-3.41 (m, 2H), 3.43-3.49 (m, 2H),

3.52-3.56 (m, 2H), 3.61-3.65 (m, 1H), 3.70-3.72 (m, 1H), 3.76-3.79 (m, 1H), 3.83-3.85
3

3

3

(m, 2H), 3.96 (t, 1H, J = 9.5 Hz), 4.00 (dd, 1H, J = 1.8, 10.8 Hz), 4.17 (d, 1H, J = 7.5
b

Hz, H-1 ), 4.20 (dd, 1H J

3

= 1.8, 11.4 Hz), 4.39 (d, 1H, J

3

1H, J = 11 Hz, CH2Ph), 4 59 (d, 1H, J
a

3

3

3

= 11 Hz, CH2Ph), 4.44 (d,
3

= 11 Hz, CH2Ph), 4.61 (d, 1H, J = 6 Hz, H3

1 ), 4.64 (d, 1H, J = 11 Hz, CH2Ph), 4.67 (d, 1H, J = 11 Hz, CH2Ph), 4.71 (d, 1H, J

3

3

= 11 Hz, CH2Ph), 4.75-4.77 (m, 2H, CH2Ph), 4.82 (d, 1H, J = 11 Hz, CH2Ph), 4.85 (d,
1H, J

3

c

= 8Hz, H-1 ), 4.91 (d, 1H, J

3

= 11 Hz, CH2Ph), 4.95 (d, 1H, J

40

3

= 11 Hz,

c

3

c

3

CH2Ph), 5.50-5.53 (m, 1H, H-2 ), 5.61 (t, 1H, J = 9.5 Hz, H-4 ), 5.78 (t, 1H, J = 9.5
c

Hz, H-3 ), 7.08-7.40 (m, 49H aromatic), 7.49-7.52 (m, 1H, aromatic), 7.66-7.68 (m, 2H,
aromatic), 7.79-7.81 (m, 2H, aromatic), 7.81-7.86 (m, 6H, aromatic);

13

C-NMR (125

MHz, CDCl3), δ 19.4, 26.9, 55.65, 55.7, 63.1, 68.1, 69.5, 69.9, 72.4, 73.5, 73.6, 74.9,
75.08, 75.1, 75.4, 75.77, 75.8, 77.0, 77.3, 77.46, 77.5, 77.9, 78.0, 79.95, 82.1, 82.2, 85.0,
98.4 (JC-1,H-1 = 168.69 Hz, C-1a), 101.7 (JC-1,H-1 = 159.14 Hz, C-1c), 103.7 (JC-1,H-1 =
159.32 Hz, C-1b), 127.68, 127.71, 127.74, 127.8, 127.9, 128.0, 128.13, 128.16, 128.38,
128.49, 128.53, 128.54, 128.56, 128.58, 128.61, 128.7, 129.2, 129.5, 129.7, 129.84,
129.88, 129.96, 130.0, 133.2, 133.31, 133.34, 133.4, 135.7, 135.9, 138.3, 138.4, 138.7,
HRMS C98H104NO19SSi [M + NH4]+ calc.

138.8, 139.2, 165.20, 165.24, 166.1.
1626.6972, found 1626.6976.

Methyl-2,3,4-tri-O-benzyl-α-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzyl-1-Methylα
α-D-glucopyranoside (20)
Using method B of general procedure for glycosylation, donor 19 (47 mg, 0.06 mmol)
was preactivated using p-TolSCl (12 µL, 0.06 mmol) and reacted with acceptor 12 (23.2
mg, 0.05 mmol) to give the desired product (20) in 79 % yield (46.8 mg) after column
purification (hexanes/ ethyl acetate 3:1). α/β = 1:3 as determined by integration of OCH3
peaks and JC1-H1 coupling constants. Comparison with literature data confirmed its
identity.

6

41

1

H-NMR (600 MHz, CDCl3) δ 3.30 (s,OCH3-α), 3.31 (s, 3H, OCH3-β), 3.36 -3.46 (m,

6H), 3.48 (dd, 1H, J

3

= 3.6, 9.6 Hz), 3.59-3.87 (m, 10H), 3.96-4.02 (m, 2H), 4.11 (s,

3

1H), 4.13 (d, 1H, J = 10 Hz, H-1), 4.19-4.71 (m, 15H), 4.76-4.88 (m, 8H), 4.91(d, 1H, J
3

3

3

= 12 Hz), 4.46 (d, 1H, J = 3 Hz, H-1), 4.51 (d, 1H, J = 12 Hz), 7.06-7.40 (m, 49H,

aromatic).

p-Tolyl-2,3,4-tri-O-benzyl-α-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-1-thio-ββ
D-glucopyranoside (21)
Using method B of general procedure for glycosylation, donor 19 (43.6 mg, 0.07 mmol)
was preactivated using p-TolSCl (10.7 µL, 0.06 mmol) and reacted with acceptor 8 (30
mg, 0.06 mmol) to give the desired product (21) in 61 % yield (46.1 mg) after column
purification (hexanes/ ethyl acetate / CH2Cl2 5.5:1:0.5)
[α]20 +27 (c = 0.15, CH2Cl2);
D

1

H-NMR (600 MHz, CDCl3) δ 2.20 (s, 3H, SPhCH3),

3.36-3.46 (m, 3H), 3.65-3.69 (m, 2H), 3.71-3.74 (m, 2H), 3.77-3.80 (m, 1H), 3.84-3.89
3

3

(m, 3H), 4.04 (t, 1H, J = 9.5 Hz), 4.47-4.53 (m, 3H), 4.58 (dd, 1H, J = 1.8, 10.2 Hz),
4.61-4.62 (m, 2H), 4.66 (d, 1H, J

3

= 12 Hz), 4.73-4.84 (m, 5H), 4.89-4.95 (m, 3H),

5.05(s, 1H), 7.01-7.03 (m, 2H, aromatic), 7.14-7.20 (m, 2H, aromatic), 7.23-7.35 (m,
29H, aromatic), 7.41-7.43 (m, 6H, aromatic);

13

C-NMR (125 MHz, CDCl3), δ 21.3, 29.9,

66.6, 69.4, 72.2, 72.6, 73.5, 74.8, 75.1, 75.23, 75.3, 75.7, 76.1, 77.0, 77.3, 77.5, 77.8,
b

a

78.6, 79.9, 81.3, 86.9, 88.3 (JC-1,H-1 = 169.9 Hz, C-1 ), 98.8, (JC-1,H-1 = 157.7 Hz, C-1 )
127.96, 127.63, 127.78, 127.81, 127.88, 127.9, 127.93, 127.95, 127.99, 128.01, 128.04,
42

128.05, 128.43, 128.45, 128.48, 128.49, 128.57, 128.60, 128.63, 128.64, 128.67, 128.69,
128.7, 129.89, 129.96, 130.1, 131.96, 133.1, 138.1, 138.27, 138.59, 138.62, 138.67,
138.95. HRMS C68H74NO10S [M + NH4]+ calc. 1096.5033 found 1096.5031.

p-Tolyl-2,3,4-tri-O-benzoyl-6-O-tert-butyldiphenylsilyl-β-D-glucopyranosyl-(1→6)β
2,3,4-tri-O-benzyl-β-D-glucopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-1-thio-β-Dβ
β
glucopyronoside (22)
Using method B of general procedure for glycosylation, donor 9 (60.8 mg, 0.05 mmol)
was preactivated using p-TolSCl (7.7 µL, 0.05 mmol), 3eq AgOTf (37.9 mg, 0.15 mmol)
and reacted with acceptor 7 (26.4 mg, 0.09 mmol) to give the desired product (22)
exclusively as β isomer in 63 % yield (51.5 mg) after column purification (hexanes/ethyl
acetate/ CH2Cl2, 4:1:0.5)
1
[α]20 + 18 (c = 0.2, CH2Cl2); H-NMR (600 MHz, CDCl3) δ 1.00 (s, 9H, (CH3)3CSi),
D
3

2.27 (s, 3H, SPhCH3), 3.24 (m, 1H), 3.32 (t, 1H, J = 9 Hz), ), 3.40-3.42 (m, 1H), 3.52 (t,
3

1H, J = 9 Hz), 3.59-3.62 (m, 1H), 3.70-3.72 (m, 1H), 3.79-3.81 (m, 2H), 3.93-4.01 (m,
2H), 4.09 (dd, 1H, J
b

3

= 2.4, 11.4 Hz), 4.18-4.21 (m, 1H), 4.29 (d, 1H, J

3

= 7.8 Hz, H-

3

1 ), 4.42 (d, 1H, J = 10.8 Hz, CH2Ph), 4.61-4.66 (m, 2H), 4.83-4.89 (m, 2H), 4.97 (d,
3

c

a

3

3

1H, J = 7.8 Hz, H-1 ), 5.03 (d, 1H, J = 9.6 Hz, H-1 ), 5.45 (t, 1H, J = 10.2 Hz), 5.46c

3

3

c

5.52 (m, 2H), 5.87 (t, 1H, J = 9.6 Hz, H-4 ), 5.99 (t, 1H, J = 9.6 Hz, H-3 ), 7.14-7.42
(m, 38H, aromatic), 7.45-7.55 (m, 6H aromatic), 7.63-7.64 (m, 2H, aromatic), 7.76-7.78
43

(m, 2H, aromatic), 7.85-7.89 (m, 9H, aromatic), 7.96-7.97 (m, 2H, aromatic);

13

C (125

MHz, CDCl3), δ 26.9, 63.2, 68.3, 69.4, 69.6, 69.9, 70.7, 73.4, 74.7, 74.90, 74.95, 75.0,
75.7, 77.0, 77.1, 77.2, 77.3, 77.4, 77.6, 77.9, 78.1, 82.4, 84.7, 85.96 (JC-1,H-1 = 156.64 Hz,
a

c

b

C-1 ), 101.7(JC-1,H-1 = 160.43 Hz, C-1 ), 103.6 (JC-1,H-1 = 155.84 Hz, C-1 ), 127.68,
127.71, 127.79, 127.82, 127.84, 127.9, 127.99, 128.41, 128.44, 128.49, 128.51, 128.55,
128.61, 128.65, 129.2, 129.26, 129.3, 129.5, 129.7, 129.77, 129.79, 129.82, 129.89,
129.95, 129.97, 130.0, 130.05, 130.1, 133.2, 133.3, 133.35, 133.6, 133.9, 135.7, 135.8,
138.3, 138.7, 138.82, 138.8, 165.2, 165.3, 165.37, 165.4, 166.0, 166.2; HRMS
C104H102NO21SSi [M + NH4]+ calc. 1760.6434 found 1760.6335.

p-Tolyl-2,3,4-tri-O-benzyl-D-glucopyranosyl-(1→6)-2,3,4-tri-O-benzyl-1-thio-β-Dβ
glucopyranoside (23)
Using method B of general procedure for glycosylation, donor 4 (43.6 mg, 0.07 mmol)
was preactivated using p-TolSCl (10.7 µL, 0.06 mmol) and reacted with acceptor 8 (30
mg, 0.06 mmol) to give the desired product (9) in 80% yield (60.4 mg), (α/β 5.7 : 1) after
1

column purification (hexanes/ ethyl acetate / CH2Cl2, 5.5:1:0.5). Comparison of H-NMR
spectra with literature values
1

71

confirmed the identity of compound 23.
3

H-NMR (400 MHz, CDCl3) δ α anomer 2.23 (s, 3H), 3.27 (t, 1H, J = 9.2 Hz), 3.42 –
3

3

3

3.92 (m, 10 H), 4.01 (t, 1H, J = 9.2 Hz), 4.47 (d, 1H, J = 12.0 Hz), 4.52 (d, 1H, J =
3

3

3

11.2 Hz), 4.58 (d, 1H, J = 9.6 Hz), 4.63 (d, 1H, J = 10.0 Hz), 4.64 (d, 1H, J = 12.0

44

3

3

Hz), 4.68 (d, 1H, J = 11.2 Hz), 4.76 – 4.95 (m, 8 H), 5.00 (d, 1H, J = 10.8 Hz), 5.04
3

(d, 1H, J = 3.2 Hz), 7.06 – 7.12 (m, 2H), 7.13 – 7.52 (m, 37H); β anomer 2.30 (s, 3H,
3

SPhCH3), 3.46–3.56 (m, 4H), 3.63–3.70 (m, 3H), 3.73–3.81 (m, 3H), 4.24 (dd, 1H, J =
3

1.8 Hz, 11.2 Hz,), 4.47 (d, 1H, J = 7.6 Hz), 4.57–4.67 (m, 4H, CH2Ph), 4.70 (d, 1H, J

3

= 9.8 Hz), 4.75–5.02 (m, 10H, CH2Ph), 7.06–7.52 (m, 39H, aromatic).

p-Tolyl-2,3,4-tri-O-benzyl-1-thio-β-D-glucopyranosyl-(1→4)-2,3-di-O-benzoyl-6-Oβ
benzyl-1-thio-β-D-galatcopyranoside (24)
β
Using method B of general procedure for glycosylation, donor 4 (40 mg, 0.06 mmol) was
preactivated using p-TolSCl (9 µL, 0.06 mmol) and reacted with acceptor 11 (29 mg,
0.05 mmol) to give the desired product (24) in 60 % yield (40 mg) after column
purification (hexanes / ethyl acetate 3:1). Comparison of 1HNMR with literature values18
confirmed the identity of compound 24.
1

H-NMR (600 MHz, CDCl3) δ 2.14 (s, 3H SPhCH3), 2.88 (dd, 1H, J

3.13 (dd, 1H, J

3

= 1.8, 10.8 Hz), 3.49 (dd, 1H, J

3

3

= 1.8, 11.4 Hz),

= 3.0, 9.6 Hz), 3.65(t, 1H, J

Hz), 3.76 (m, 1H), 3.74-3.77. (m, 1H), 3.89-3.96 (m, 4H), 4.04 (d, 1H, J

3

3

= 9.6

= 12 Hz,

3

CH2Ph), 4.34-4.41 (m, 5H), 4.61 (d, 1H, J = 11.4 Hz, CH2Ph), 4.74-4.92 (m, 6H), 5.29
(dd, 1H, J

3

= 3.0, 10.2 Hz), 5.65 (t, 1H, J

3

= 10.2 Hz), 6.99-7.00 (m, 2H, aromatic),

7.10-7.14 (m, 4H, aromatic), 7.20-7.19 (m, 29H, aromatic), 7.89-7.94 (m, 4H, aromatic).

45

APPENDIX

46

APPENDIX 1

Spectral Data

47

1

Figure 1.1 (500 MHz, CDCl3) H-NMR compound 3

8

7

6

5

4

48

3

2

1

0

1

Figure 1.2 (600 MHz, CDCl3) H-NMR compound 4

8

7

6

5

4

49

3

2

1

0

1

Figure 1.3 (600 MHz, CDCl3) H-NMR compound 5

9

8

7

6

5

4

50

3

2

1

0

1

Figure 1.4 (600 MHz, CDCl3) H-NMR compound 6

8

7

6

5

4

3

51

2

1

0

1

Figure 1.5 (600 MHz, CDCl3) H-NMR compound 7

9

8

7

6

5

4

52

3

2

1

0

1

Figure 1.6 (600 MHz, CDCl3) H-NMR compound 8

9

8

7

6

5

4

53

3

2

1

0

1

Figure 1.7 (500 MHz, CDCl3) H-NMR compound 9

9

8

7

6

5

4

54

3

2

1

0

1

Figure 1.8 (600 MHz, CDCl3) H-NMR compound 10

9

8

7

6

5

4

55

3

2

1

0

1

Figure 1.9 (500 MHz, CDCl3) H-NMR compound 13

9

8

7

6

5

4

56

3

2

1

0

1

Figure 1.10 (600 MHz, CDCl3) H-NMR compound 15

9

8

7

6

5

4

57

3

2

1

0

13

Figure 1.11 (150 MHz, CDCl3) C-NMR compound 15

220

200

180

160

140

120

100

58

80

60

40

20

0

1

Figure 1.12 (500 MHz, CDCl3) H-NMR compound 16

9

8

7

6

5

4

59

3

2

1

0

13

Figure 1.13 (125 MHz, CDCl3) C-NMR compound 16

220

200

180

160

140

120

60

100

80

60

40

20

0

Figure 1.14 (500 MHz, CDCl3) gCOSY compound 16

1
2
3

4

5

6
7

8

8

7

6

5

61

4

3

2

1

0

Figure 1.15 (500 MHz, CDCl3) gHMQC-coupled compound 16

1
2
3
4
5
6
7
8
9
160

140

120

100

80

62

60

40

20

0

Figure 1.16 (500 MHz, CDCl3) gHMQC-coupled (anomeric region expansion) compound 16

4.2
1

J = 162.85 Hz

4.3
4.4
4.5
4.6
4.7
4.8

1

J = 155.02 Hz

1

J = 169.062 Hz

4.9
5.0
5.1
5.2
102

100

98

96

63

94

92

90

88

86

Figure 1.17 (500 MHz, CDCl3) gHMQC-decoupled compoound 16

1
2
3
4
4
5
6
7
8
140

130

120

110

100

90

80

64

70

60

50

40

30

20

Figure 1.18 (500 MHz, CDCl3) gHMBC compound 16

1
2
3
4
5
6
7
8
9
200

180

160

140

120

65

100

80

60

40

20

1

Figure 1.19 (600 MHz, CDCl3) H-NMR compound 17

10

9

8

7

6

5

66

4

3

2

1

0

13

Figure 1.20 (125 MHz, CDCl3) C-NMR compound 17

200

180

160

140

120

100

67

80

60

40

20

0

Figure 1.21 (600 MHz, CDCl3) gCOSY compound 17
TBDPSO
BzO
BzO

O

O
BzO BnO
BnO
17

O
BnO
O

BnO
BnO

O STol
BnO

1
2
3

4
5
6
7

8
8

7

6

5

68

4

3

2

1

0

Figure 1.22 (600 MHz, CDCl3) gHMQC-coupled compound 17
TBDPSO
BzO
BzO

O

O
BzO BnO
BnO
17

O
BnO
O

BnO
BnO

O STol
BnO

1
2
3
4
5
6
7
8
130

120

110

100

90
69

80

70

60

50

40

30

20

Figure 1.23 gHMQC-coupled (anomeric reagion expansion) compound 17

4.5

J 1 156.36 Hz

4.6
4.7

1

J = 161.68 Hz

4.8
4.9
1

J = 169.72 Hz

5.0
5.1
5.2
102 101 100 99

98

70

97

96

95

94

93

92

91

90

89 88

Figure 1.24 (600 MHz, CDCl3) gHMQC-decoupled compound 17

1
2
3
4
5
6
7
8
140

130

120

110

100

90

80

71

70

60

50

40

30

20

10

Figure 1.25 (600 MHz, CDCl3) gHMBC compound 17

1
2
3
4
5
6
7
8
9
180

160

140

120

100

72

80

60

40

20

1

Figure 1.26 (600 MHz, CDCl3) H-NMR compound 18

10

9

8

7

6

5

73

4

3

2

1

0

13

Figure 1.27 (150 MHz, CDCl3) C-NMR compound 18

200

180

160

140

120

100

74

80

60

40

20

0

Figure 1.28 (600 MHz, CDCl3) gCOSY compound 18

1
2
3
4
5
6
7
8
9
9

8

7

6

5

75

4

3

2

1

Figure 1.29 (600 MHz, CDCl3) gHMQC-coupled compound 18

1
2
3
4
5
6
7
8
9
160

140

120

100

80
76

60

40

20

0

Figure 1.30 (600 MHz, CDCl3) gHMQC-coupled (anomeric region expansion) compound 18

4.1

1

J =159.32 Hz

4.2
4.3
4.4
4.5

1

4.6

J =168.69 Hz

4.7
4.8

1

J =164.80 Hz

4.9
5.0
5.1

104.0

103.0

102.0

77

101.0

100.0 99.5

99.0

98.5 98.0

Figure 1.31 (600 MHz, CDCl3) gHMQC-decoupled compound 18

1
2
3
4
5
6
7
8
140

130

120

110

100

90
78

80

70

60

50

40

30

Figure 1.32 (600 MHz, CDCl3) gHMBC compound 18

1
2
3
4
5
6
7
8

200

180

160

140

79

120

100

80

60

40

20

0

1

Figure 1.33 (600 MHz, CDCl3) H-NMR compound 20

9

8

7

6

5

4

80

3

2

1

0

1

Figure 1.34 (600 MHz, CDCl3) H-NMR compound 21

9

8

7

6

5

4

81

3

2

1

0

13

Figure 1.35 (125 MHz, CDCl3) C-NMR compound 21

200

180

160

140

120

100

82

80

60

40

20

0

Figure 1.36 (600 MHz, CDCl3) gHMQC-coupled compound 21

2
3
4
5
6
7
8
130

120

110

100
83

90

80

70

60

50

40

30

20

Figure 1.37 (600 MHz, CDCl3) gHMQC-coupled (anomeric region expansion) compound 21

4.5

1

J = 169.9 Hz

4.6
4.7
4.8
4.9
5.0

1

J = 157.7 Hz

5.1
5.2

99

98

97

84

96

95

94

93

92

91

90

89

88

Figure 1.38 (600 MHz, CDCl3) gHMQC-decoupled compound 21

0
1
2
3
4
5
6
7
8
140

130

120

110

100

90
85

80

70

60

50

40

30

20

Figure 1.39 (600 MHz, CDCl3) gHMBC compound 21

1
2
3
4
5
6
7
8
9
180

160

140

120

100
86

80

60

40

20

1

Figure 1.40 (600 MHz, CDCl3) H-NMR compound 22

9

8

7

6

5

4

87

3

2

1

0

13

Figure 1.41 (125 MHz, CDCl3) C-NMR compound 22

200

180

160

140

120

100

88

80

60

40

20

Figure 1.42 (600 MHz, CDCl3) gCOSY compound 22

1
2
3
4
5
6
7
8
9
9

8

7

6

5

89

4

3

2

1

0

Figure 1.43 (600 MHz, CDCl3) gHMQC-coupled compound 22

1
2
3
4
5
6
7
8
130

120

110

100

90

90

80

70

60

50

40

30

20

Figure 1.44 (600 MHz, CDCl3) gHMQC-coupled (anomeric region expansion) compound 22

1

J = 155.84 Hz
4.3
4.4
4.5
4.6
4.7
4.8
1

J = 160.43 Hz

4.9

1

5.0

J = 156.64 Hz

5.1

104

102

100

98

96

91

94

92

90

88

86

Figure 1.45 (600 MHz, CDCl3) gHMQC-decoupled compound 22

1
2
3
4
5
6
7
8
140

130

120

110

100

90
92

80

70

60

50

40

30

20

Figure 1.46 (600 MHz, CDCl3) gHMBC compound 22

1
2
3
4
5
6
7
8
220

200

180

160

140

120

93

100

80

60

40

20

1

Figure 1.47 (400 MHz, CDCl3) H-NMR compound 23

9

8

7

6

5

4

94

3

2

1

0

1

Figure 1.48 (600 MHz, CDCl3) H-NMR compound 24

9

8

7

6

5

4

95

3

2

1

0

REFERENCE

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Crich, D.; Sun, S., Direct Formation of β-Mannopyranosides and Other Hindered
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Crich, D.; Sun, S., Direct chemical synthesis of β-mannopyranosides and other
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33.

Crich,
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Smith,
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S-(4-Methoxyphenyl)
Benzenethiosulfinate
(MPBT)/Trifluoromethanesulfonic Anhydride: A Convenient System for the
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4067-4069.

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Crich, D.; Sun, S., Are Glycosyl Triflates Intermediates in the Sulfoxide
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35.

Kim, K. S.; Kim, J. H.; Lee, Y. J.; Lee, Y. J.; Park, J., 2-(Hydroxycarbonyl)benzyl
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36.

Crich, D.; Smith, M., 1-Benzenesulfinyl Piperidine/Trifluoromethanesulfonic
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Formation of Diverse Glycosidic Linkages. J. Am. Chem. Soc. 2001, 123, (37),
9015-9020.

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Crich, D.; de la Mora, M.; Vinod, A. U., Influence of the 4,6-O-benzylidene, 4,6O-phenylboronate, and 4,6-O-polystyrylboronate protecting groups on the
stereochemical outcome of thioglycoside-based glycosylations mediated by 1benzenesulfinyl piperidine/triflic anhydride and N-iodosuccinimide/trimethylsilyl
triflate. J. Org. Chem. 2003, 68, (21), 8142-8148.

38.

Crich, D.; Cai, W., Chemistry of 4,6-O-Benzylidene-D-glycopyranosyl Triflates:
Contrasting Behavior between the Gluco and Manno Series. J. Org. Chem. 1999,
64, (13), 4926-4930.

39.

Bousquet, E.; Khitri, M.; Lay, L.; Nicotra, F.; Panza, L.; Russo, G., Capsular
polysaccharide of Streptococcus pneumoniae type 19F: synthesis of the repeating
unit. Carbohydr. Res. 1998, 311, (4), 171-181.

40.

Kim, K. S.; Kang, S. S.; Seo, Y. S.; Kim, H. J.; Lee, Y. J.; Jeong, K.-S.,
Glycosylation with 2'-carboxybenzyl glycosides as glycosyl donors: Scope and
application to the synthesis of a tetrasaccharide. Synlett 2003, (9), 1311-1314.

41.

Crich, D.; Vinogradova, O., On the Influence of the C2-O2 and C3-O3 Bonds in
4,6-O-Benzylidene-Directed β-Mannopyranosylation and a-Glucopyranosylation.
J. Org. Chem. 2006, 71, (22), 8473-8480.

42.

Codee, J. D. C.; Hossain, L. H.; Seeberger, P. H., Efficient Installation of βMannosides Using a Dehydrative Coupling Strategy. Org. Lett. 2005, 7, (15),
3251-3254.

43.

Baek, J. Y.; Choi, T. J.; Jeon, H. B.; Kim, K. S., A highly reactive and
stereoselective β-mannopyranosylation system: mannosyl 4-pentenoate/PhSeOTf.
Angew. Chem., Int. Ed. 2006, 45, (44), 7436-7440.

44.

Imamura, A. A., H.; Korogi, S.; Tanabe, G.; Muraoka, O.; Ishida, H.; Kiso, M.,
Di-tert-butylsilylene (DTBS) group-directed α-selective galactosylation
unaffected by C-2 participating functionalities. Tetrahedron Lett. 2003, 44, (35),
6725-6728.

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Barresi, F.; Hindsgaul, O., Synthesis of β-mannopyranosides by intramolecular
aglycon delivery. J. Am. Chem. Soc. 1991, 113, (24), 9376-7.

46.

Barresi, F.; Hindsgaul, O., Improved synthesis of β-mannopyranosides by
intramolecular aglycon delivery. Synlett 1992, (9), 759-61.

47.

Stork, G.; Kim, G., Stereocontrolled synthesis of disaccharides via the temporary
silicon connection. J. Am. Chem. Soc. 1992, 114, (3), 1087-8.

48.

Stork, G.; La Clair, J. J., Stereoselective Synthesis of β-Mannopyranosides via the
Temporary Silicon Connection Method. J. Am. Chem. Soc. 1996, 118, (1), 247-8.

49.

Bols, M., Stereocontrolled synthesis of α-glucosides by intramolecular
glycosidation. J. Chem. Soc., Chem. Commun. 1992, (12), 913-14.

50.

Bols, M., Application of intramolecular glycosidation to the stereocontrolled
synthesis of disaccharides containing α-gluco and α-galacto linkages. J. Chem.
Soc., Chem. Commun. 1993, (9), 791-2.

51.

Bols, M., Efficient stereocontrolled glycosidation of secondary sugar hydroxyls
by silicon tethered intramolecular glycosidation. Tetrahedron 1993, 49, (44),
10049-60.

52.

Ito, Y.; Ogawa, T., A Novel Approach to the Stereoselective Synthesis of βMannosides. Angew. Chem., Int. Ed. 1994, 33, (17), 1765-1767.

53.

Ito, Y.; Ohnishi, Y.; Ogawa, T.; Nakahara, Y., Highly Optimized βMannosylation via β-Methoxybenzyl Assisted Intramolecular Aglycon Delivery.
Synlett 1998, (10), 1102-1104.

54.

Danishefsky, S. J.; Hu, S.; Cirillo, P. F.; Eckhardt, M.; Seeberger, P. H., A highly
convergent total synthetic route to glycopeptides carrying a high-mannose core
pentasaccharide domain N-linked to a natural peptide motif. Chem.--Eur. J. 1997,
3, (10), 1617-1628.

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Lichtenthaler, F. W.; Schneider-Adams, T., 3,4,6-Tri-O-benzyl-α-D-arabinohexopyranos-2-ulosyl Bromide: A Versatile Glycosyl Donor for the Efficient
Generation of β-D-Mannopyranosidic Linkages. J. Org. Chem. 1994, 59, (22),
6728-34.

56.

Frieder, L. W.; Ulrich, K.; Zoltán, S.; Bernd, W., 4,6-Di-O-benzoyl-3-O-benzylα-D-arabino-hexopyranos-2-ulosyl bromide: A conveniently accessible glycosyl
donor for the expedient construction of diantennary /3-D-mannosides branched at
0-3 and 0-6. Carbohydr. Res. 1998, 305, (2), 293-303.

57.

Kim, J.-H.; Yang, H.; Park, J.; Boons, G.-J., A General Strategy for
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58.

Geng, Y.; Zhang, L.-H.; Ye, X.-S., Pre-activation protocol leading to highly
stereoselectivity-controllable glycosylations of oxazolidinone protected
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59.

Lu, Y.-S.; Li, Q.; Zhang, L.-H.; Ye, X.-S., Highly Direct α-Selective
Glycosylations of 3,4-O-Carbonate-Protected 2-Deoxy- and 2,6Dideoxythioglycosides by Preactivation Protocol. Org. Lett. 2008, 10, (16), 34453448.

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Can. J. Chem. 1979, 57, (10), 1244-51.

61.

Crich, D.; Vinod, A. U.; Picione, J., The 3,4-O-Carbonate Protecting Group as a
β-Directing Group in Rhamnopyranosylation in Both Homogeneous and
Heterogeneous Glycosylations As Compared to the Chameleon-like 2,3-OCarbonates. J. Org. Chem. 2003, 68, (22), 8453-8458.

62.

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2-Azido-2-deoxy-glucosides Using Intermediate Sulfonium Ions. Org. Lett. 2007,
9, (10), 1959-1962.

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Zeng, Y.; Wang, Z.; Whitfield, D.; Huang, X., Installation of Electron-Donating
Protective Groups, a Strategy for Glycosylating Unreactive Thioglycosyl
Acceptors using the Preactivation-Based Glycosylation Method. J. Org. Chem.
2008, 73, (20), 7952-7962.

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Crich, D.; Dudkin, V., Why are the hydroxy groups of partially protected Nacetylglucosamine derivatives such poor glycosyl acceptors, and what can be
done about it? A comparative study of the reactivity of N-acetyl-, N-phthalimido-,
and 2-azido-2-deoxy-glucosamine derivatives in glycosylation. 2-Picolinyl ethers
as reactivity-enhancing replacements for benzyl ethers. J. Am. Chem. Soc. 2001,
123, (28), 6819-6825.

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Crich, D.; Chandrasekera, N. S., Mechanism of 4,6-O-benzylidene-directed βmannosylation as determined by a-deuterium kinetic isotope effects. Angew.
Chem., Int. Ed. 2004, 43, (40), 5386-5389.

67.

Crich, D.; Patel, M., On the nitrile effect in L-rhamnopyranosylation. Carbohydr.
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Unusual Stereochemical Observation. J. Org. Chem. 2007, 72, (23), 8976-8979.

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long-range
effect
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[2-(4phenylbenzyl)oxycarbonyl]benzoyl group. Tetrahedron: Asymmetry 2005, 16, (2),
441-447.

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Org. Chem. 1976, 41, (26), 4110-12.

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France, R. R.; Compton, R. G.; Davis, B. G.; Fairbanks, A. J.; Rees, N. V.;
Wadhawan, J. D., Selective electrochemical glycosylation by reactivity tuning.
Org. Biomol. Chem. 2004, 2, (15), 2195-2202.

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Kim, W.-S.; Hosono, S.; Sasai, H.; Shibasaki, M., Rare earth salts promoted
glycosidation of glycosyl fluorides. Heterocycles 1996, 42, (2), 795-809.

73.

Chiba, H.; Funasaka, S.; Mukaiyama, T., Catalytic and stereoselective
glycosylation with glucosyl thioformimidates. Bull. Chem. Soc. Jpn. 2003, 76,
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Adinolfi, M.; Barone, G.; Iadonisi, A.; Schiattarella, M., Efficient activation of
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glycosylation reaction. Tetrahedron Lett. 2002, 43, 5573-5577.

75.

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catalyzed stereoselective glycosylation using glycosyl fluorides. Bull. Chem. Soc.
Jpn. 2002, 75, 291-309.

76.

Demchenko, A.; Stauch, T.; Boons, G.-J., Solvent and other effects on the
stereoselectivity of thioglycoside glycosidations. Synlett 1997, 818-819.

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Huang, L.; Wang, Z.; Li, X.; Ye, X.-S.; Huang, X., Iterative one-pot syntheses of
chitotetroses Carbohydr. Res. 2006, 341, 1669-1679.

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Schmidt, R. R.; Behrendt, M.; Toepfer, A., Nitriles as solvents in glycosylation
reactions: highly selective β-glycoside synthesis. Synlett 1990, 694-696.

106

Chapter 2: Stereoselective Glycosylation of Azido Glucosides
2.1 Introduction
Another aspect of stereoselective glycosylation we became interested in was a
strategy entailing trapping of the oxacarbenium ion intermediate formed upon activation
of glycosyl donors with suitable additives that would allow for subsequent stereoselective
introduction of sugar hydroxyl nucleophiles. Boons and coworkers have established an
auxiliary based method for stereoselective introduction of 1,2-cis glycosides. They were
able to achieve this using (S)-phenylthiomethyl benzyl ether at C-2 which formed an
intermediate sulfonium ion upon activation of glycosyl donor as a trans-decalin ring
system (Scheme 1.3). Subsequent displacement of the sulfonium ion by a hydroxyl leads
to stereoselective formation of α-glycosides.

1

However, the implementation of this

methodology to 2-deoxy sugars is hampered by the lack of a C-2 hydroxyl group which is
required for anchoring the chiral auxiliary.
Boons and coworkers have developed a new methodology for 1,2-cis
glycosylation based on the use of exogenous sulfides in glycosylation reactions of 2azido-2-deoxy donors which upon activation are transformed to oxacarbenium ions that
can react with the thioether additive resulting in a sulfonium ion intermediate, typically
formed as a β-isomer due to steric factors.

2

This allows for the stereoselective

introduction of sugar hydroxyl from the α-face leading to α-glycosides. Enhancement in
α-selectivity of their reactions was achieved using thiophene as the thioether and
performing the reaction at relatively high temperature.

While this is a significant

contribution to the continuing quest for a general method for 1, 2-cis glycosylation, it still
107

suffers loss of anomeric α-selectivity when highly reactive 2-azido-2-deoxy
perbenzylated glucopyranosyl donors are used for glycosylation (Scheme 2.1).

Scheme 2.1 Stereoselective glycosylation using intermediate sulfonium ions
To better understand the effects thioether additives had in determining the
stereochemical outcome of glycosylation reactions, we initiated a study entailing
glycosylation reactions of 2,3-O-dibenzyl-4,6-O-dibenzoyl galactosyl donor without a
participating protective group on 2-O.

3

We became intrigued as to whether we could

observe enhanced α selectivity using thioether additives. Moreover, this would allow us
to determine stereoselectivity trends in glycosylation reactions using donors without
neighboring group participation with the hope of improving 1,2-cis selectivity. With this
donor, we anticipated that we could tune the reactivity of the glycosyl donor by the
presence of benzoyl protecting groups as opposed to using per-benzylated glycoside
donor, which had been reported to have low anomeric selectivity due to their high
reactivity.

2, 4

108

2.2 Results and Discussion
14

N-Phenyl trifluoroacetimidate donor

was chosen as donor for the initial

screening because of their lower tendency to undergo side reactions.

Its has been

reported that reactions with trichloroacetimidate donors are often accompanied by
formation of N-glycoside by-products.

5

This could be ascribed to the presence of a
6, 7

phenyl substituent on the nitrogen which could minimize undesired rearrangements.

The desired donor was easily accessed from thiogalactopyranoside 1 obtained from Dr.
Youlin Zeng.
Compound 1 was benzylated at C-2 and C-3 hydroxyls via reaction with BnBr
and NaH in anhydrous DMF to provide compound 2. Subsequent deprotection of the 4,
6-benzylidene protecting group via in situ generated HCl using AcCl and anhydrous
MeOH in DCM resulted in diol 3 which was benzoylated using benzoyl chloride in
pyridine to provide glycosyl donor 4.

To access hemiacetal 5, compound 4 was

hydrolyzed using NBS (N-bromosuccinimide), acetone/H2O mixture at room temperature
in 20 minutes.

8

This set the stage for the synthesis of our desired N- phenyl

trifluoroacetimidate donor 6, which was accomplished by reacting trifluoroacetimidoyl
chloride with anomeric hemiactal 5. The desired compound 6 was difficult to recover
from the reaction mixture due to generation of equimolar amounts of salt which not only
complicated its characterization but also led to relatively low yield (28%) (Scheme 2.2).
With glycosyl donor 6 in hand, we turned our attention to glycosylation reactions using
commercially available di-isopropylidene galactopyranosyl and glucofuranosyl acceptors
7 and 8. A typical reaction would entail glycosylation reactions in the presence or
109

absence of exogenous thioether additives, which were expected to enhance α-selectivity as reported by Boons.

Scheme 2.2 Synthesis of donor 6. Conditions, i) 2.4 eq BnBr, 2 eq NaH, DMF, 0 °C to rt, 75%, ii) 6 eq AcCl,
MeOH/DCM, rt, 94%, iii) 2.6 eq BzCl, Pyridine, rt, 75%, iv) 6 eq NBS, Acetone/H2O, rt, 58%, v) 1.5 eq
PhNCClCF3, DBU, DCM, 28%.

110

2

Glycosyl donor 6, glycosyl acceptor 7 or 8 and activated molecular sieves (4Å) in
DCM in the presence or absence of thioether were mixed together followed by stirring for
20 min under an atmosphere of nitrogen at room temperature then the reaction mixture
was cooled to -20 °C or 0 °C. TMSOTf (0.1 eq) was then added to the reaction mixture
and stirred at -20 °C or 0 °C. Upon completion of the reaction as detected by TLC
analysis, the reaction mixture was quenched with Et3N, concentrated in vacuo followed
by purification by column chromatography.
1

The anomeric stereoselectivity was

1

determined by H-NMR and J C1-H1 coupling of the anomeric carbon and hydrogen as
outlined in Table 2.1.
Table 2.1. Glycosylations of donor 6
BzO OBz
O
OC(NPh)CF3 +
BnO
OBn

O OH
O
O
or
O
O

6

Entry
1
2
3

O O
HO

O

7

Donor
(1 eq)
6
6
6

Acceptor
(0.9 eq)
7
7
8

O
O

MS 3Å,
0.1eq TMSOTf
DCM

9 or 10

8

Thioether
(10 eq)
none
thiophene
none

Temp
( °C )
-20
0
-20

Conc
(mM)
60
60
60

Product
9
9
10

α/β
(Yield)
5/1 (95%)
7/1 (81%)
α-only
(82%)

The stereochemical outcomes of glycosylations of donor 6 with acceptors 7 and 8
were investigated by varying the reaction temperature and carrying out the reaction in
presence of thiophene with the intention of enhancing the stereoselectivity. The reaction
of donor 6 with acceptor 7 in dichloromethane at -20 °C gave disaccharide 9 with an
anomeric selectivity of α/β = 5/1 in excellent yield (entry 1). Probing this further, by
111

increasing the reaction temperature to 0 °C with the addition of thiophene, we expected a
2

significant enhancement in stereoselectivity as reported by Boons and coworkers. Under
these conditions, only marginal increase in α stereoselectivity to α/β = 7/1 (entry 2) was
observed. Using secondary sugar alcohol 8 as acceptor obtained glycosyl product 10 that
was exclusively formed as α-isomer in good yield (entry 3). The exclusive α-selectivity
of the secondary alcohol can be attributed to the sterically hindered nature of the glycosyl
acceptor 8 which could plausibly favor approach of the oxacarbenium ion intermediate
exclusively from the α-face.
To better understand factors controlling the stereochemical outcomes of our
experimental results which were contrary to what we had expected for reactions
involving thioether additives as report by Boons and coworkers, we carried out
glycosylation reaction using 2-azido-2-deoxy-glucoside donor 14 to reproduce the results
reported in literature.

2

To access desired donor we began our synthesis from

commercially available glucosamine hydrochloride 11 which was reacted with TfN3 in
the presence of catalytic amount of CuSO4.5H2O in a H2O/MeOH/DCM mixed solvent
system for 42 hours followed by acetylation of the resultant azido sugar intermediate to
access compound 12.

9

Subsequent selective deactylation of the anomeric acetate using

NH4OAc in DMF gave compound 13 in 85 % yield. Compound 13 was reacted with
trichloroacetonitrile in DBU for 3 hours resulting in the desired 2-azido-2-deoxy-glucosyl
acetaimidate donor 14 (Scheme 2.3).

112

Scheme 2.3 Conditions i) TfN3, CuSO4, NaHCO3, DCM/MeOH, ii) Ac2O, DMAP, Pyridine,
+-

88%, iii) 2.5 eq NH4 OAc, DMF, 85%, iv) 4 eq CCl3CN, 6.5 eq DBU, 75%.

With compound 14 in hand, we screen various reaction conditions for
glycosylation involving donor 14 and acceptor 7 as outlined in Table 2.2.

Table 2.2. Glycosylation of donor 14
Entry

1
2
3
4
5
6
7
8
9
10
a.

14 (mM)

7 (mM)

Temperature
°C

21
19
-20
21
19
0
21
19
0
21
19
rt
2.1
1.9
rt
21
19
-78
21
19
-78
21
19
0
21
19
0
21
19
0
2
Entries 6-10 are results reported in literature

113

Thioether
(10 eq)

none
thiophene
PhSEt
thiophene
none
none
PhESt
none
PhSEt
thiophene

α/β Yield (%)

4/1 (74)
2/1 (82)
3/1 (92)
11/1 (89)
10/1 (90)
1/1 (87)
1/1 (93)
2/1 (87)
5/1 (92)
14/1 (95)

The glycosylation reactions with donor 14 and acceptor 7 were performed at
2

different temperatures in the presence of different thioethers.

Setting the reaction

temperature at -20 °C resulting in relatively modest anomeric selectivity (entry 1).
Raising the reaction temperature to 0 °C with addition of thioether surprisingly lead to
decrease in selectivity in the presence of either thiophene (entry 2, α/β = 2/1) or phenylthioethyl ether (entry 3, α/β = 3/1) contrary to what was reported in literature (entries 910).

2

Interestingly, when the reaction was performed at room temperature under more

dilute conditions in the presence of thiophene, there was significant improvement in αselectivity (entry 4). Based on the observation that running the glycosylation reaction
under diluted conditions at room temperature resulted in enhanced α-selectivity, we
speculated that the observed α-selectivity could also be attributed to other factors such as
solvent effect by diethyl ether used in the reaction.

To prove this hypothesis, we

performed the reaction in the absence of thiophene under diluted conditions and were
pleasantly surprised by the α-selectivity (entry 5, α/β = 10/1) we obtained which was
comparable to the selectivity obtained in the presence of thiophene (entry 4, α/β = 11/1).
This indicated that high α-selectivity could be obtained without exogenous thioether
2

additives. Our study suggests that the reported literature results were most likely due to
dilution rather than assistance from thioethers.

114

2.3 Experimental Section
All reactions were carried out under nitrogen with anhydrous solvents in flamedried glassware. All glycosylation reactions were performed in the presence of molecular
sieves, which were flame dried right before the reaction under high vacuum.
Glycosylation solvents were dried using a solvent purification system and used directly
without further drying. Chemicals used were reagent grade as supplied except where
noted. Analytical thin-layer chromatography was performed using silica gel 60 F254
glass plate. Compound spots were visualized by UV light (254 nm) and by staining with
a yellow solution containing Ce (NH4)2(NO3)6 (0.5 g) and (NH4)6Mo7O24 4H2O (24.0 g)
in 6% H2SO4 (500 mL). Flash column chromatography was performed on silica gel 60
(230–400 Mesh). NMR spectra were referenced using Me4Si (0 ppm), residual CHCl3 (δ
1

H-NMR 7.24 ppm,

13

1

1

C-NMR 77.0 ppm). Peak and coupling constant assignments are
1

1

13

1

13

based on H-NMR, H– H gCOSY and (or) H– C gHMQC and H– C gHMBC
experiments. All optical rotations were measured at 25 °C using the sodium D line. ESI
mass spectra were recorded in positive ion mode. High-resolution mass spectra were
recorded on a Micromass electrospray

Phenyl-2,3-Di-O-benzyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside (2)
Phenyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside 1 (8 g, 21.4 mmol) and NaH
(0.72 g, 29.9 mmol) were stirred at 0 °C for 10 minutes followed by dropwise addition of
benzyl bromide (6.1 mL, 5.13 mmol). The reaction mixture was stirred overnight at
room temperature then poured into ice water (200 mL) and extracted with DCM (3 x
115

50 mL ). The combined organic layers were dried over Na2SO4, concentrated then
purified by column chromatography (hexanes/ EtOAc 3:1) to provide compound 2 in 75
% yield (9 g) as an amorphous solid. Comparison of 1HNMR with reported literature
values confirmed the identity of compound 2.
1

10
3

H-NMR (600MHz, CDCl3) δ 2.29 (s, SPhCH3), 3.39 (bs, 1H, H-5), 3.60 (dd, 1H, J
3

3.6, 9.6 Hz, H-3), 3.81 (t, 1H, J = 9 Hz, H-2), 3.96 (dd, 1H, J
3

3

=

= 1.2, 12 Hz, H-6), 4.12

3

3

(d, 1H, J = 3.0 Hz, H-4), 4.35 (dd, 1H, J = 1.2, 12 Hz, H-6), 4.55 (d, 1H, J = 9.6 Hz,
H-1), 4.67-4.72 (m, 4H,CH2Ph), 5.47 (s, 1H, CHPh), 6.99-7.00 (m, 2H, aromatic), 7.247.43 (m, 7H, aromatic), 7.50-7.52 (m, 4H, aromatic), 7.59-7.61 (m, 2H, aromatic).

2,3-Di-O-benzyl-1-thio-β-D-galactopyranoside (3)
Compound 2 (5.27 g, 9.5mmol) was dissolved in anhydrous methanol (40 mL) and
CH2Cl2 (60 mL), cooled to 0 °C followed by dropwise addition of AcCl (4.05 mL, 57
mmol). The reaction mixture was stirred for 2 hours, quenched with Et3N to pH 7 then
concentrated to dryness. The residue was passed through a short silica gel column
(hexanes/EtOAc 2:1) to give compound 3 as a white solid in 94 % yield (4.2 g).
1

Comparison of H-NMR with literature values confirmed the identity of compound 3.
1

16

H-NMR (600MHz, CDCl3) δ 2.24 (bs, 1H, OH), 2.33 (s, 3H, SPhCH3), 2.64 (bs, 1H,

OH), 3.46-3.48 (m, 1H, H-5), 3.56 (dd, 1H, J

3

= 3.6, 10.8 Hz, H-3), 3.69 (t, 1H, J

3

=

11.4 Hz, H-2), 3.79-3.81 (m, 1H, H-6), 3.95-3.98 (m, 1H, H-6’), 4.05 (bs, 1H, H-4), 4.56
(d, 1H, J

3

= 11.4 Hz, H-1), 4.69 (bs, 2H, CH2Ph), 4.72(d, 1H, J
116

3

= 12 Hz, CH2Ph),

4.82(d, 1H, J

3

= 12 Hz, CH2Ph), 7.10-7.11 (m, 2H, aromatic), 7.31-7.37 (m, 8H,

aromatic), 7.41-7.43 (m, 2H, aromatic), 7.45-7.47 (m, 2H, aromatic).

2,3-Di-O-benzyl-4, 6-di-O-benzoyl-1-thio-β-D-galactopyranoside (4)
Compound 3 (7.31 g, 15.7 mmol) was dissolved in pyridine (30 mL) followed by
addition of BzCl (6 mL, 50.13 mmol) and the resulting solution was stirred overnight at
room temperature. Solvents were evaporated under high vacuum. The residue was
dissolved in dichloromethane, washed successively with, 1M HCl (60 mL), sat. aqueous
NaHCO3 (60 mL), brine (50 mL) and the organic layer was dried over Na2SO4. The
desired compound 4 was obtained in 75 % yield (7.93 g) after column purification
1

(hexanes/EtOAc 2:1). Comparison of H-NMR with literature values confirmed the
identity of compound 4.
1

15

H-NMR (600MHz, CDCl3) δ 2.32 (s, 3H, SCH3Ph), 3.70 (t, 1H, J

1H, J

3

= 3.0, 9 Hz), 4.02 (t, 1H, J

(m, 2H), 4.65 (d, 1H, J
Hz), 4.81 (d, 1H, J

3

3

3

= 6.6 Hz), 4.38 (dd, J

= 9.6 Hz), 4.72 (d,1H, J

= 11.4 Hz), 5.87 (d 1H, J

3

3

3

3

= 9.0 Hz), 3.76 (dd,

= 5.4, 11.4 Hz), 4.52-4.58
3

= 10.2 Hz), 4.77 (d, 1H, J = 10.2

= 2.4 Hz), 7.02-7.04 (m, 2H, aromatic),

7.20-7.68 (m, 16H, aromatic), 8.01-8.03 (m, 4H, aromatic), 8.15-8.16 (m, 2H, aromatic).

2, 3-Di-O-benzyl-4, 6-di-O-benzoyl-1-D-galactopyranoside (5)
To a vigorously stirred suspension of compound 4 (2.0 g, 2.96 mmol) in acetone/water
(9:1, 200 mL) was added NBS (1.13 g, 5.9 mmol). After disappearance of starting

117

material, the reaction mixture was quenched with NaHCO3, concentrated and the residue
dissolved in CH2Cl2, washed with brine, dried over Na2SO4 then purified by column
chromatography (hexanes/ EtOAc 3:1) to afford 0.98 g (58 %, α/β 1:0.43) of compound
1

5. Comparison of H-NMR with literature values confirmed the identity of compound
5.
1

15
3

H-NMR (400MHz, CDCl3) δ 3.72-3.76 (m, 1H), 3.91 (dd, 1H, J = 3.6, 10.2 Hz), 4.13-

4.94 (m, 14H), 5.40 (d, 1H, J

3

= 3.6 Hz H-1 α), 5.95-5.96 (m, 1H), 7.23-8.15 (m, 29H,

aromatic)
2,3-Di-O-benzyl-4,6-di-O-benzoyl-1-D-galactopyranosyl-(N-Phenyl)trifluoroacetimidate (6)
A 200 mL two-necked flask equipped with a septum cap, a condenser, and a Tefloncoated magnetic stir bar was charged with Ph3P (34.5 g, 132 mmol), Et3N (7.3 mL, 53
mmol), CCl4 (21.1 mL, 220 mmol) and TFA (3.4 mL, 44 mmol). After the solution was
stirred for about 10 minutes at 0 °C, aniline (5.80 mL, 53 mmol) dissolved in CCl4 (21.1
mL, 220 mmol) was added. The mixture was then refluxed under stirring for 3 hours.
Solvent were removed under reduced pressure, and the residue was diluted with hexane
and filtered.

Residual solid Ph3PO, Ph3P and Et3N-HCl were washed with hexane

several times. The filtrate was concentrated under reduced pressure, and the residue was
distilled to afford N-(phenyl) trifluoroacetimidoyl chloride as yellow oil. To compound 5
(1.24 g, 2.18 mmol) dissolved in CH2Cl2 (45 mL) was added N-(phenyl)

118

trifluoroacetimidoyl chloride (0.9 mL, 3.27 mmol), DBU (0.07 mL, 0.52 mmol) and the
reaction mixture was stirred overnight, concentrated and purified by column
chromatography ( hexanes/EtOAc 3:1) to afford compound 6 in 29 % yield (0.47 g).
[α]20 + 36.8 (c = 0.1, CH2Cl2);
D

1

H-NMR (600MHz, CDCl3) δ 3.80 (bs, 1H), 3.98 (bs,
3

1H) 4.41-4.43 (m, 1H), 4.51-4.54 (m, 1H), 4.60 (d, 1H, J = 11.4 Hz), 4.80-4.83 (m, 2H),
3

4.85 (d, 1H, J = 11.4Hz), 5.86 (bs, 1H), 6.73 (d, 1H, J

3

= 6.0 Hz), 7.07-7.63 (m, 21H,

aromatic), 7.97-7.99 (m, 2H, aromatic), 8.13-8.14 (m, 1H, aromatic). HRMS
C42H36N2O8F [M + Na+] calc. 762.2285 found 726.2290.

General procedures for glycosylation
Method A: A mixture of glycosyl donor and acceptor, activated molecular sieves (4Å)
in CH2Cl2 (3 mL) was stirred for 10 minutes under N2 atmosphere at rt then cooled to -20
C. After addition of TMSOTf (0.1 eq), the reaction mixture was stirred at -20 °C, 0 °C or

°

at rt. When the donor was completely consumed as detected by TLC analysis, the
reaction mixture was quenched with Et3N, concentrated in vacuo then purified by column
chromatography (hexanes/EtOAc 5:1-3:1)

Method B: A mixture of glycosyl donor and acceptor, activated molecular sieves (4Å)
and thiophene in CH2Cl2 (3 mL) was stirred for 10 minutes under N2 atmosphere at rt
then cooled to -20 °C. After addition of TMSOTf (0.1 eq), the reaction mixture was
stirred at -20 °C, 0 °C or rt. When the donor was completely consumed as detected by

119

TLC analysis, the reaction mixture was quenched with Et3N, concentrated in vacuo then
purified by column chromatography (hexanes/EtOAc 5:1-3:1)

2,3-Di-O-benzyl-4,6-di-O-benzoyl-1-D-galactopyranosyl-(1→6)-1,2:3,4-di-Oisopropylidene-α-D-galactopyranoside (9)
A mixture of glycosyl donor 6 (117 mg, 0.179 mmol) and acceptor 7 (42 mg, 0.161
mmol), activated molecular sieves (4Å) in CH2Cl2 (3 mL) was stirred for 10 minutes
under N2 atmosphere at rt then cooled to -20 °C . After addition of TMSOTf (4 µL, 0.018
mmol), the reaction mixture was stirred at -20 °C or 0 °C.

When the donor was

completely consumed as detected by TLC analysis, the reaction mixture was quenched
with Et3N, concentrated in vacuo then purified by column chromatography
(hexanes/EtOAc 5:1-3:1) to afford disaccharide 9 in 95 % yield (126.2 mg, α/β 5:1).
1

Comparison of H-NMR with literature values confirmed the identity of compound 9.
1

3

H-NMR (600MHz, CDCl3) δ 1.29 (s, 3H, CH3), 1.32 (s, 3H, CH3), 1.42 (s, 3H, CH3),
3

1.51 (s, 3H, CH3), 3.80-3.85 (m, 2H), 3.93 (dd, 1H, J = 3.6, 9.6 Hz), 4.02-4.13 (m, 3H),
3

3

4.46-4.57 (m, 3H), 4.59 (d, 1H, J = 10.8 Hz), 4.69 (d, 1H, J = 12 Hz, CHPh), 4.78 (d,
3

3

3

1H, J = 12 Hz), 4.82 (d, 1H, J = 11.4 Hz), 5.04 (d, 1H, J 3 = 3.6 Hz), 5.49 (d, 1H, J =
4.8 Hz), 5.90 (d, 1H, J

3

= 3.0 Hz), 7.18-7.57 (m, 20 H, aromatic), 8.00-8.02 (m, 4 H,

aromatic).

120

2,3-Di-O-benzyl-4,6-di-O-benzoyl-1-D-galactopyranosyl-(1→4)-1,2:5,6-di-Oisopropylidene-α-D-glucofuranoside (10)
Using method A of general procedures for glycosylation, donor 6 (135 mg, 0.182 mmol)
and acceptor 8 (43 mg, 0.164 mmol) were reacted using TMSOTf (4.7 µL, 0.02 mmol) at
1

-20 °C to afford compound 10 in 82 % (120 mg) yield as α-isomer. Comparison of HNMR with literature values confirmed the identity of compound 10.
1

3

H-NMR (600MHz, CDCl3) δ 1.13 (s, 3H, CH3), 1.27 (s, 3H, CH3), 1.41 (s, 3H, CH3),
3

1.43 (s, 3H, CH3), 3.95-4.09 (m, 5H), 4.29 (d, J = 2.4 Hz), 4.38-4.53 (m, 4H), 4.56 (d,
1H, J

3

= 11.4Hz, CHPh), 4.59 (d, 1H, J

3

3

= 3.6 Hz), 4.71(d, 1H, J

3

3

= 12 Hz), 4.77 (d,
3

b

1H, J = 11.4 Hz, CHPh), 4.81 (d, 1H, J = 11.4 Hz), 5.37 (d, 1H, J = 3.6 Hz, H-1 ),
5.88 (d, 1H, J

3

a

= 3.6 Hz, H-1 ), 5.89 (d, 1H, J

3

= 3 Hz), 7.15-7.17 (m, 2H, aromatic),

7.23-7.31 (m, 12H, aromatic), 7.39-7.46 (m, 4H, aromatic), 7.54-7.59 (m, 2H, aromatic),
8.02-8.06 (m, 4H, aromatic);

13

C-NMR (150 MHz, CDCl3) δ 25.7, 26.3, 27.1, 27.3, 64.0,

67.3, 68.2, 68.8, 72.2, 72.4, 74.1, 75.4, 76.2, 80.7, 81.5, 84.2, 98.99 (JC-1,H-1 = 170.84 Hz,
b

a

C-1 ), 105.4 (JC-1,H-1 = 169.61 Hz, C-1 ), 109.4, 112.1, 125.6, 127.8, 127.9, 128.03,
128.09, 128.48, 128.5, 128.7, 129.3, 129.8, 130.2, 133.5, 133.6, 138.1, 138.5, 166.02,
166.6.

121

1,3,4,6-Tetra-O-acetyl-2-azido-2-deoxy-α-D-glucopyranoside (12)
NaN3 (5.94 g, 91.6 mmol) was dissolved in H2O (15 mL), CH2Cl2 (20 mL) then cooled to
0 °C with vigorous stirring. Tf2O (17.8 mmol, 3.2 mL) was added dropwise and the
reaction mixture stirred for 2 hours at 0 °C. The organic phase was extracted twice using
CH2Cl2 (10 mL) and saturated NaHCO3 (10 mL) to afford TfN3 then kept at 0 °C for use
in the next step. Compound 11 (2 g, 9.27 mmol) was dissolved in H2O (30 mL) and
MeOH (60 mL) followed by addition of NaHCO3 (0.44 g, 5.19 mmol), CuSO4.H2O (0.02
g, 0.093 mmol) and TfN3 then stirred at rt for 42 hours. Upon completion of reaction as
detected by TLC, the reaction mixture was concentrated in vacuo and the resultant
residue dried under vacuum for 1 hour. The residue was dissolved in pyridine (24 mL)
followed by addition of Ac2O (8 mL, 80 mmol) and DMAP (1 g, 8.19 mmol) and stirred
at until completion of reaction as detected by TLC. The solvents were removed and the
residue was coevaporated 3 times with toluene, dissolved in CH2Cl2, washed with H2O,
dried over Na2SO4, filtered, concentrated and purified by silica column (hexanes/ EtOAc
2:1) to afford compound 12 in 88 % yield (3 g, α/β 1.4: 1). Comparison of 1H-NMR with
literature values confirmed the identity of compound 12.
1

13

H-NMR (600MHz, CDCl3) δ 2.01-2.19 (m, 20H, COCH3), 3.64-3.67 (m, 2H, H-2α, H-

2β), 3.78-3.80 (m, 1H, H-5), 4.03-4.12 (m, 3H, H-5,H-6,H-6’), 5.02-5.12 (m, 2H, H-3, H4), 5.43 (t, 1H, J

3

= 9.6 Hz, H-3α), 5.53 (d, J

Hz, H-1α).

122

3

= 8.4 Hz, H-1β), 6.28 (d, 1H, J

3

= 3.6

3,4,6-Tri-O-acetyl-2-azido-2-deoxy-α-D-glucopyranoside (13)
Compound 12 (2.9 g, 7.78 mmol), was dissolved in DMF (11 mL) and NH4OAc (1.49 g,
19.4 mmol) added followed by stirring at rt for 16 hours. The reaction mixture was
concentrated

and

the

resultant

residue

purified

by

column

chromatography

(Hexanes/EtOAc 2:1) to afford compound 13 in 85 % yield (2.18 g, α/β 1.7:1).
1

Comparison of H-NMR with literature values confirmed the identity of compound 13.
1

11

3

H-NMR (600MHz, CDCl3) δ 2.00-2.08 (m, 14H, CH3CO), 3.40 (dd, 1H, J = 3.6, 10.2

Hz, H-2α), 3.78 (bs, 1H, OH), 4.09-4.32 (m, 5H, H-5α, H-6, H-6’, H-6, H-6’), 4.72 (d, J
= 7.8 Hz, H-1β), 4.99-5.06 (m, 2H, H-3,H-4), 5.38 (d, 1H, J

3

3

=3.6 Hz, H-1α), 5.50 (t,

3

1H, J = 10.8 Hz, H-3).

3,4,6-Tri-O-acetyl-2-azido-2-deoxy-1-α-D-glucopyranosyl-trichlororoacetimidate
(14)
Compound 13 (2.18 g, 6.58 mmol) was dissolved in CH2Cl2 (20 mL) followed by
addition CCl3CN (3.64 mL, 26.3 mmol) and DBU (0.5 mL, 3.29 mmol). The reaction
mixture was stirred for 3 hours at room temperature, concentrated then purified by
column chromatography (hexanes/EtOAc 3:1) to afford compound 14 in 75 % yield (2.36
g).

literature values.
1

1

The identity of compound 14 was confirmed by comparison of H-NMR with
12
3

H-NMR (600MHz, CDCl3) δ 2.04-2.16 (m, 9H, CH3), 3.75 (dd, 1H, J = 3.6, 10.2 Hz,
3

H-6), 4.08-4.10 (m, 1H, H-6), 4.19-4.21 (m, 1H, H-5), 4.25 (d, 1H, J = 4.2, 12.6 Hz, H123

3

3

3

2), 5.13 (t, 1H, J = 9.6 Hz, H-4), 5.49 (t, 1H, J = 10.8 Hz, H-3), 6.47 (d,1H, J = 3.6
Hz, H-1).

3,4,6-Tri-O-acetyl-2-azido-2-deoxy-D-glucopyranosyl-(1→6)-1,2:3,4-di-Oisopropylidene-α-D-galactopyranoside (15)
Using method B of general procedures for glycosylation, donor 14 (20 mg, 0.04 mmol) in
CH2Cl2 (10 mL) reacted with acceptor 7 (11 mg, 0.04 mmol) in the presence of thiophene
(3.45 µL, 0.421 mmol) and TMSOTf (0.8 µL, 0.004mmol) at rt to afford compound 15 in
1

91 % yield (α/β = 11:1). Compound 15 was identified by comparison of H-NMR with
literature values.

2

Using method B of general procedures for glycosylation, donor 14 (20 mg, 0.04 mmol) in
CH2Cl2 (10 mL) was reacted with acceptor 7 (11 mg, 0.04 mmol) and TMSOTf (0.8 µL,
0.004mmol) at rt to afford compound 15 in 90% yield (α/β = 10:1).
1

H-NMR (600MHz, CDCl3) δ 1.30-1.53 (m,12H, CH3), 2.01-2.06 (m, 9H,CH3), 3.26 (dd,
3

1H, J = 3.6, 10.8 Hz, H-2), 3.73-3.81 (m, 2H, H-6’a, H-6’b), 3.98-4.11 (m, 4H, H-5, H5’, H-6a, H-6b ), 4.26-4.30 (m, 2H, H-2’, H-4’), 4.58 (dd, 1H, J
3

3

= 2.4, 7.8 Hz, H-3’),
3

5.00-5.04 (m, 2H, H-4, H-1), 5.42 (t, 1H, J = 9.6 Hz, H-3), 5.48 (d, 1H, J = 4.8 Hz, H1’ α).

124

APPENDIX

125

APPENDIX 2

Spectral Data

126

1

Figure 2.1 (600MHz, CDCl3) H-NMR compound 2

8

7

6

5

4

127

3

2

1

0

1

Figure 2.2 (500MHz, CDCl3) H-NMR compound 3

9

8

7

6

5

4

128

3

2

1

0

1

Figure 2.3 (600MHz, CDCl3) H-NMR compound 4

8

7

6

5

4

129

3

2

1

0

Figure 2.4 (600MHz, CDCl3) compound 6

9

8

7

6

5

4

130

3

2

1

0

1

Figure 2.5 (600MHz, CDCl3) H-NMR compound 9

8

7

6

5

4

131

3

2

1

0

1

Figure 2.6 (600MHz, CDCl3) H-NMR compound 10

8

7

6

5

4

132

3

2

1

0

1

Figure 2.7 (600MHz, CDCl3) H-NMR compound 12

8

7

6

5

4

133

3

2

1

0

1

Figure 2.8 (600MHz, CDCl3) H-NMR compound 13

8

7

6

5

4

134

3

2

1

0

1

Figure 2.9 (600MHz, CDCl3) H-NMR compound 14

8

7

6

5

4

135

3

2

1

0

1

Figure 2.10 (600MHz, CDCl3) H-NMR compound 15

8

7

6

5

4

136

3

2

1

0

REFERENCE

137

REFERENCE

1.

Kim, J.-H.; Yang, H.; Boons, G.-J., Stereoselective glycosylation reactions with
chiral auxiliaries. Angew. Chem., Int. Ed. 2005, 44, (6), 947-949.

2.

Park, J.; Kawatkar, S.; Kim, J.-H.; Boons, G.-J., Stereoselective glycosylations of
2-azido-2-deoxy-glucosides using intermediate sulfonium Ions. Org. Lett. 2007,
9, (10), 1959-1962.

3.

Cheng, Y.-P.; Chen, H.-T.; Lin, C.-C., A convenient and highly stereoselective
approach for α-galactosylation performed by galactopyranosyl dibenzyl phosphite
with remote participating groups. Tetrahedron Lett. 2002, 43, (43), 7721-7723.

4.

Douglas, N. L.; Ley, S. V.; Lucking, U.; Warriner, S. L., Tuning glycoside
reactivity: new tool for efficient oligosaccharide synthesis. J. Chem. Soc., Perkin
Trans. 1 1998, (1), 51-66.

5.

Schmidt, R. R.; Gaden, H.; Jatzke, H., Glycosylimidates. 45. New catalysts for the
glycosyl transfer with O-glycosyl trichloroacetimidates. Tetrahedron Lett. 1990,
31, (3), 327-30.

6.

Unverzagt, C.; Seifert, J., Chemoenzymatic synthesis of deca and
dodecasaccharide N-glycans of the "bisecting" type. Tetrahedron Lett. 2000, 41,
(23), 4549-4553.

7.

Zhu, T.; Boons, G.-J., Intermolecular aglycon transfer of ethyl thioglycosides can
be prevented by judicious choice of protecting groups. Carbohydr. Res. 2000,
329, (4), 709-715.

8.

Lin, C.-C.; Hsu, T.-S.; Lu, K.-C.; Huang, I. T., Synthesis of β-Dglucopyranosyl(1→3)-1-thiol-β-glucosamine disaccharide derivative as building
block for the synthesis of hyaluronic acid. J. Chin. Chem. Soc. (Taipei) 2000, 47,
(4B), 921-928.

138

9.

Alper, P. B.; Hung, S.-C.; Wong, C.-H., Metal catalyzed diazo transfer for the
synthesis of azides from amines. Tetrahedron Lett. 1996, 37, (34), 6029-6032.

10.

Zhang, Z.; Ollmann, I. R.; Ye, X.-S.; Wischnat, R.; Baasov, T.; Wong, C.-H.,
Programmable onepot oligosaccharide synthesis. J. Am. Chem. Soc. 1999, 121,
(4), 734-753.

11.

Ishida, H.; Imai, Y.; Kiso, M.; Hasegawa, A.; Sakurai, T.; Azuma, I., Studies on
immunoadjuvant-active compounds. 41. Synthesis and immunoadjuvant activity of
2,2'-O-[2,2'-diacetamido-2,3,2',3'-tetradeoxy-6,6'-di-O (2tetradecylhexadecanoyl)α,α-trehalose-3,3'-diyl]bis(N-D-lactoyl-L-alanyl-D-isoglutamine). Carbohydr. Res.
1989, 195, (1), 59-66

12.

Grundler, G.; Schmidt, R. R., Glycosyl imidates, 13. Application of the
trichloroacetimidate procedure to 2-azidoglucose and 2-azidogalactose
derivatives. Liebigs Ann. Chem. 1984, (11), 1826-47.

13.

Vasella, A.; Witzig, C.; Chiara, J. L.; Martin-Lomas, M., Convenient synthesis of
2-azido-2-deoxy-aldoses by diazo transfer. Helv. Chim. Acta 1991, 74, (8), 20737.

14.

Yu, B.; Tao, H., Glycosyl trifluoroacetimidates. Part 1: Preparation and
application as new glycosyl donors. Tetrahedron Lett. 2001, 42, (12), 2405-2407.

15.

Fan, G.-T.; Pan, Y.-s.; Lu, K.-C.; Cheng, Y.-P.; Lin, W.-C.; Lin, S.; Lin, C.-H.;
Wong, C.-H.; Fang, J.-M.; Lin, C.-C., Synthesis of a-galactosyl ceramide and the
related glycolipids for evaluation of their activities on mouse splenocytes.
Tetrahedron 2005, 61, (7), 1855-1862.

16.

Yan, M.-C.; Chen, Y.-N.; Wu, H.-T.; Lin, C.-C.; Chen, C.-T.; Lin, C.-C.,
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139

CHAPTER 3: Synthesis of Hyaluronan Biosynthesis Inhibitors
3.1 Introduction
Oligosaccharide sequences frequently serves as important components that
mediate complex cellular events such as cellular signaling pathways and can act as
ligands in cell-cell adhesion events as diverse as fertilization, tumor metastasis,
Alzheimer’s disease and inflammation.

1

They have also been implicated in host-

pathogen interactions critical for bacterial and viral infections.

2, 3

Investigations into

pathogenesis in cancer arthritis Alzheimer’s disease and diabetes implicate the
extracellular matrix in progression of major human diseases.

4-7

Hyaluronan is a major

component of the extracellular matrix and alterations of its metabolism, distribution and
function have been implicated in tumor progression; being recognized as a key
8

component of the unique stroma that surrounds and probably supports the tumor.

In normal tissues, hyaluronan provides an environment that facilitates cellular
proliferation and migration and is necessary for various physiological processes such as
embryonic development and wound healing. In cancer, however, these properties appear
to enhance tumor invasion, growth, angiogenesis and metastasis

9-11

Hyaluronan is a high molecular weight glycosaminoglycan (GAG) composed of
repeating disaccharide units of N-acetyl-glucosamine (GlcNAc) and glucuronic acid
(GlcUA). It is synthesized from the precursors UDP-GlcUA and UDP-GlcNAc at the
inner leaflet of the plasma membrane by a membrane associated hyaluronan synthase
(HAS).

12

By developing chemical tools which can control GAG biosynthesis we can
140

develop a new class of therapeutic agents that can help to understand the role of
hyaluronan biosynthesis in tumor progression.

13

Inhibition or interference of correct GAG metabolism presents an opportunity to
develop a new class of therapeutic agents that can control GAG biosynthesis. One
potential strategy would be to invoke chain termination of oligosaccharide biosynthesis.

13

Analogues of GlcNAc could be exploited as potential chain terminators with the
requirement that glycosyl transferases can process activate donor substrates with minimal
modifications.

14

Interest in the synthesis of these GlcNAc analogs is augmented by the

fact that fully acetylated derivatives could act as suitable prodrugs that are able to
penetrate into the cell without relying on carbohydrate-transport mechanism.

13

Endogenous carboxyesterases present in the cytoplasm remove the acetyl groups and the
resulting deactylated derivatives can act as acceptors for the enzymes involved glycan
biosynthesis.15-17
Mono-, di-, and oligo-saccharides containing fluorine have been developed to
study enzymes involved in carbohydrate metabolism, and some have been shown to be
inhibitors. The atomic size of fluorine is comparable to that of oxygen; with the van der
Waal’s radius of fluorine (135pm) only slightly smaller than that of oxygen at 140 pm.
Moreover, the C-F bond has a higher energy (485 kJ/mol) compared with that of oxygen
18

(370 kJ/mol).

141

As mentioned above, many researchers have attempted to discover specific
inhibitors of glycosaminoglycan biosynthesis. In the early nineties, Korytnyk developed
a series of fluorinated N-acetylglucosamine and N-acetylgalactosamine (GalNAc) as part
of a program aimed at developing potential inhibitors of cell growth for treatment of
leukemia. In their evaluation of the effect of fluorinated analogs on cell growth of L210
leukemia cells, 4-F-GlcNAc and 4-F-GalNAc were shown to be active in inhibiting
leukemia cell proliferation at IC 50 values of 34 and 35 µM respectively.

15

Kisilevsky and coworkers have proposed that 4-deoxy analogs of GlcNAc are
effective anti-amyloid agents both in vitro and in vivo by truncating the growth of the
linear polysaccharide portion of heparan sulfate proteoglycan that has been identified as
essential for amyloidogenesis. When they interact with their respective amyloidogenic
proteins, they have the ability to alter their conformations so that they can take on a
secondary fibrillar structural characteristic typical of an amyloid.

19-21

GlcNAc analogues have found applications in studies related to the ability of
glycosylation inhibitors to modulate the structure and selectin-binding function of
cutaneous lymphocyte-associated antigen (CLA) as natively expressed by human CLA+ T
cells.

Sackstein and coworkers have demonstrated that 4-F-GlcNAc exhibits anti-

inflammatory effects by blocking polylactosamine synthesis necessary for selectin ligand
production. 4-F-GlcNAc was directly incorporated into native cutaneous lymphocyteassociated antigen expressed on human T cells, thus reducing leukocyte homing to areas
of contact allergic hypersensitivity in mice in vivo at concentrations that do not interfere
142

with homeostatic pathways of protein synthesis and growth.22 Moreover, reducing the
formation of sialyl Lewis X glycan using disaccharide with 4-deoxy GlcNAc residue at
the terminal position was effective in diminishing tumor metastasis by Lewis lung
carcinoma in vivo.

23

Although the effects of GlcNAc analogs on heparan sulfate and chondroitin/
dermatan sulfate have been described their effects on hyaluronan biosynthesis have not
been described.

3, 23

With this in mind, we embarked on a project aimed at synthesizing

GlcNAc analogs modified at the C-3 position which would be used as HA synthesis chain
terminators potentially retarding tumor progression.

143

3.2: Results and Discussion
Hyaluronan is synthesized in mammals by a family of three hyaluronan synthases:
HAS1, HAS2 and HAS3.24,

25

All three isozymes catalyze the same reaction by

successively adding glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) from
the respective UDP esterified sugar precursors, in a repeating GlcNAc-β (1→4)-GlcUA-β
(1→3)-O-linked disaccharide motif to a growing oligomeric chain. Faced with a choice
between making GlcUA and GlcNAc analogs, we opted for the glucosamine precursors
envisioning minimal synthetic modification to get to our desired product.
We envision that these analogs would be processed by HAS and subsequently be
transferred to the terminus of a growing hyaluronan polymer resulting in truncation by
preventing the formation of the normal 1,3 glycosidic linkage between GlcUA on the
nonreducing end of the growing chain. In our initial design for the synthesis of these
GlcNAc derivatives, we opted to use furanose oxazoline 2 as a key intermediate. This
was an attractive route because we envisioned that it would limit the number of
protecting group manipulations necessary to get to our target compound. Moreover,
compound 2 can be conveniently prepared in large quantities from commercially
available GlcNAc 1 using acetone and anhydrous FeCl3 as a catalyst.

26

Methylation of

oxazoline 2 was carried out using MeI and NaH in pyridine proceeding smoothly in
quantitative yield to compound 3. Purification of compound 3 was unnecessary as
aqueous work up using ice cold water and brine proved to be sufficient. Subsequent acid
catalyzed hydrolysis

27

of compound 3 furnished the desired 3-O-methyl-D-glucosamine

144

which was acetylated using acetic anhydride in pyridine to give the desired product 4 in
50 % overall yield over two steps (Scheme 3.1).

27

Scheme 3.1 Synthesis of compound 8. Conditions, i) Acetone/ 2 eq FeCl3, reflux, 60
°C, 65%, ii) 2.5 eq NaH, 1.5 eq MeI, DMF, 0 °C to rt, quantitative, iii) 0.4 eq PSA,
THF/H2O 2:1, iv) Ac2O/Pyridine, rt, 50%.
With our first desired analog in hand, we focused our attention to synthesis of the
3-oxo-glucosamine analog, N-acetyl glucosamine 5 was selectively benzylated at the
anomeric position using concentrated HCl leading to compound 6 which was followed by
benzylidenation of the 4,6-hydroxyls to access compound 7. With compound 7 in hand,
subsequent oxidation via Swern oxidation protocol step which provided ulose derivative
8 in excellent yield (Scheme 3.2). When submitted to catalytic hydrogenation to remove
benzyl and benzylidene groups compound 8 was transformed to multiple side products.

145

One of the side products was compound 9 resulting from a retro-Claisen reaction
(Scheme 3.3). This cleavage could not be avoided using other solvents (EtOAc or THF).

Scheme 3.2 Synthesis of compound 8. Conditions, i) BnOH, HCl, 90 °C, 95%, ii) 5 eq
PhCH(OMe)2, 0.6 eq CSA, DMF, rt, 95%, iii) 1.4 eq COCl2, 2.5 eq DMSO, 5eq Et3N,75 °C to rt, 90%.

Scheme 3.3 Proposed mechanism for synthesis of compound 9

146

While it was apparent that introducing carbonyl functionality at C-3 had a great
effect on the overall stability of the sugar moiety especially upon deprotection, we had no
feasible way of circumventing this problem. We abandoned attempts to synthesize 3-oxo
GlcNAc analog and shifted our attention to synthesis of the 3F-GlcNAc derivative. We
began by exploring reactions of compound 2; compound 7 and its corresponding N-acetyl
allosamine with DAST but all these efforts were futile resulting in complex product
mixtures. A search through literature indicated much precedence for fluorination at C-3
would proceed successfully only when the NH2 functional group was protected with a
phthaloyl moiety; therefore synthesis of N-phthalamide glycoside was employed in the
synthesis of 3-F-GlcNAc.

28

Glucosamine hydrochloride 10 was neutralized with of freshly prepared NaOMe
then treated directly with freshly powdered phthalic anhydride, triethylamine and
29

methanol as established by Wei and coworkers.

Upon conversion to the intermediate

phthalamate, the crude product was cooled to -20 oC and collected by filtration and dried
under reduced pressure, then resuspended in pyridine and treated with Ac2O to access
compound 11 predominantly as the β isomer. The glycosyl tetraacetate 11was efficiently
converted to β-benzyl glycoside 12 using BF3.Et2O in CH2Cl2.

Subsequent

chemoselective saponification with 0.3 M NaOMe in a 3:2 mixture of MeOH-CH2Cl2 at 10 °C to 0 °C produced triol 13. The O-4 and O-6 free hydroxyl groups were protected as
their benzylidene acetal by treatment with benzaldehyde dimethyl acetal in the presence
of a catalytic amount of camphorsulfonic acid to yield benzyildene glycoside 14.

147

Inversion of hydroxyl at C-3 position was anticipated to be accomplished via a two step
sequence.
To achieve this, we employed Lattrell-Dax method which has been shown to be
efficient in inverse hydroxyl configuration under very mild conditions but not extensively
adopted probably due to difficulties in predicting the outcome for specific structures.30-32
Ramstrom and coworkers have established that protecting group pattern is an essential
element in the reaction, both with regard to configuration and functionality. Good yields
were obtained when esters were chosen as protecting groups on the carbon adjacent to the
carbon atom carrying the triflate group. In contrast, no efficient reaction occurred when
benzyl protecting groups were employed suggesting that a neighboring group ester is able
to induce the inversion reaction. Good inversion yields depended mainly on the relative
configurations between the two groups. When the ester and the trilfate leaving group
have a trans diaxial relationship, this lead to products where configuration is retained.

31

On the other hand, only equatorially oriented neighboring ester groups induced the
nucleophilic displacement reaction.

32

Testing the efficiency of this method, compound

14 was treated with Tf2O in CH2Cl2 in the presence of pyridine to generate the
intermediate triflate. When TBAOAc in toluene was employed to displace the OTf
group, a straightforward SN2 reaction was achieved yielding 3-OAc glycoside 15.
Subsequent treatment of compound 15 with 0.3M NaOMe at -10 oC revealed the
corresponding allosamine glycoside 16. Fluorination of compound 16 was accomplished
using DAST which resulted in the inversion of configuration at C-3 to access the fluoro
analog and a side product which could not be separated from the desired product.
148

Pleasantly, upon removal of benzylidene protecting group, we were able to access the
desired product 16 and identify the side product from the previous step as elimination
product 17. To access our desired product, compound 16 was initially treated with
methylamine with heating but this attempt only resulted in degradation product.
Conducting the reaction at room temperature over 36 hours resulted in mixed amides.
Treatment of the mixed amides with ethylene diamine exposed the free amine
intermediate at C-2 which was acetylated to provide compound 18. Treating 16 with
excess ethylene diamine at 90 oC, allowed the access of free amine derivative which on
subsequent acetylation provided compound 18. Pd-catalyzed hydrogenolysis of 18 and
subsequent acetylation revealed the desired product 19 with the α isomer as the major
product. (Scheme 3.4).
Compound 4 and 19 are currently being tested in Japan by our collaborator Dr.
Ikuko Kakizaki for their ability to inhibit hyaluronan synthesis and the formation of
pericellular hyaluronan matrix in human pancreatic cancer cell line KP1-NL. Based on
the efficacy of these compounds in the reduction of pericellular hyaluronan matrix in
cancer cells, we will determine wheter they have a syngetic anticancer effect when used
in combination with anticancer drug gemcitabine in vitro. We intend to investigate in
vivo whether administation of compound 4 or 19 and gemcitabine influence primary
tumor growth and inhibit liver metastasis as compared with the administering
gemcitabine alone in animal models implated with pancreatic cancer cells KP1-NL.

149

Scheme 3.4 Synthesis of compound 19. Conditions i) 1M NaOMe, MeOH, Ph(CO)2O, Et3N, ii) Ac2O, Pyridine 60.2%,

iii) 2 eq BnOH, 3 eq BF3.OEt, DCM, 79%, iv) 0.3M NaOMe, MeOH/DCM, -10 °C, v) 2 eq PhCH(OCH3)2, 0.08 eq
CSA, CAN, 77%, vi) 2 eq Tf2O,0.5 eq Pyridine, DCM, -20 °C to 10 °C, vii) 6 eq TBAOAc, Toluene, 60 °C, 74 %,
viii) 0.3M NaOMe, MeOH/DCM, -10 °C, ix) 6 eq DAST, DCM, -5°C to rt, x) 6 eq AcCl, MeOH/DCM, 80 %, xi)
H2N(CH)2NH2, n-butanol, 90 °C, xii) Ac2O, Pyridine, rt, 80%, xiii) H2, Pd/C, MeOH, rt, xiv) Ac2O, Py, rt, 76%.
150

3.3 Conclusion
In conclusion, 3-methoxy GlcNAc glycoside has been efficiently prepared using a
furanose oxazoline intermediate which is well suited for large scale synthesis without the
need for extensive column chromatography. In addition, we have developed a robust and
rapid procedure for synthesis of 3F-GlcNAc derivative required for mechanistic study of
hyaluronan biosynthesis. In the course of our synthesis, we have shown the expanded
utility of Lattrell-Dax method for carbohydrate epimerization reactions. These analogs
are currently under biological evaluation as potential hyaluronan synthesis chain
terminators.

151

3.4 Experimental Section
2-Methyl-(1,2-dideoxy-5,6-O-isopropylidene-α-D-glucofurano)-[2,1-d]-2-oxazoline
(2)
Anhydrous FeCl3 (15 g, 0.092 mol) was added to a suspension of 2-acetamido-2-deoxy-D
glucopyranose (10 g, 0.068 mol) in dry acetone (200 mL), and the mixture was stirred
and boiled under reflux for 20 min with exclusion of moisture. The solution was cooled
to 0 °C, followed by addition of diethylamine (35.7 mL), acetone (135 mL) and a solution
of sodium bicarbonate (21.3 g) in water (135 mL) with continuous stirring. Acetone,
diethylamine, and some water were then removed in vacuo at < 30 °C. The mixture was
then extracted with diethyl ether (5 x 200 mL), the combined extracts dried over MgSO4
and concentrated at room temperature to give compound 2 as brownish syrup (7.15 g, 65
1

%). The H-NMR was identical to what was reported in literature.
1

26

H-NMR (600MHz, CDCl3) δ 1.33 (s, 3H, CH3), 1.39 (s, 3H, CH3), 2.00 (s, 3H,
3

3

N=COCH3), 2.92 (s, 1H, OH-3), 3.72 (dd, 1H, J = 3.6, 9.6 Hz, H-4), 3.97 (dd, 1H, J =
3

4.8, 8.4 Hz, H-6), 4.11 (dd, 1H, J = 5.4, 7.8 Hz, H-6’), 4.28-4.32 (m, 1H, H-5), 4.39 (d,
3

3

3

1H, J = 3 Hz, H-3), 4.44 (dd, 1H, J = 1.2, 4.8 Hz, H-2), 6.14 (d, J = 4.8 Hz, H-1).

152

2-Methyl-(1,2-di-deoxy-3-methoxy-5,6-O-isopropylidene-α-D-glucofurano)-[2,1-d]-2oxazoline (3)
Compound 2 (0.4 g, 1.64 mmol) was dissolved in DMF (15 mL) and cooled to 0 °C. NaH
(0.08 g, 3.29 mmol) was added in three portions with vigorous stirring followed by
dropwise addition of MeI (0.15 mL, 2.47 mmol). The reaction mixture was allowed to
warm up to rt and was confirmed as complete after 3 hours by TLC.

DMF was

evaporated in vacuo and the residue coevaporated twice with toluene. The resultant
residue was dissolved in CH2Cl2 (20 mL) and washed with ice cold water and the
aqueous phase extracted twice with CH2Cl2 (2 x 20 mL). The organic phases were
combined, washed with brine (30 mL) and dried with Na2SO4. Upon filtration and
concentration in vacuo, compound 3 was accessed in 96 % yield (0.4 g).
[α]20 -15.5 (c = 1, CH2Cl2); H-NMR (600MHz, CDCl3) δ 1.27 (s, 3H, CH3), 1.33 (s,
D
1

3

3H, CH3).1.94 (s, 3H, N=CĈH3), 3.40 (s, 3H, OCH3), 3.71 (dd, 1H, J = 3.6, 9 Hz, H-4),
3.77 (d, 1H, J

3

= 4.2 Hz, H-3), 3.91-3.94 (m, 1H, H-6), 3.97-3.99 (m, 1H, H-6’), 4.21

(m, 1H, H-5), 4.42 (dd, 1H, J

3

= 1.8, 6 Hz, H-4), 6.01 (d, 1H, J

3

= 6.6 Hz, H-1);

13

C-

NMR (150 Hz, CDCl3) δ 14.3. 25.5, 26.99, 57.98, 67.0, 72.7, 74.8, 81.5, 83.7, 107.1,
109.2, 167.2. HRMS C12H20NO5 [M + H+] calc. 258.1341 found 258.1333.

1,4,6-Tri-O-acetyl-2-acetamido-2-deoxy-3-methoxy-β-D-glucopyranoside (4)
Compound 3 (0.204 g, 0.793 mmol) was dissolved in 1:2 H2O/THF mixture (10 mL: 20
mL) and p-toluenesulfonic acid (0.06 g, 0.264 mmol) was added to the reaction mixture
153

then stirred overnight. The reaction was quenched with Et3N to a neutral pH and the
solvents evaporated in vacuo. The residue was washed several times with CH2Cl2 and the
resultant white powder dried under vacuum. The residue was then dissolved in pyridine
(8 mL), Ac2O (0.5 mL) added and the reaction mixture stirred at rt overnight. The
reaction mixture was concentrated, residue dissolved in CH2Cl2, washed with H2O, brine,
dried over Na2SO4 and purified by column chromatography (CH2Cl2/MeOH, 8:1) to
reveal compound 4 in 50 % (0.143 g) yield over two steps.
[α]20 + 10.5 (c = 1, CH2Cl2);
D

1

H-NMR (600MHz, CDCl3) δ 1.99 (s, 3H, NHCOCH3),

2.07 (s, 3H, COCH3), 2.10 (s, 3H, COCH3), 2.11 (s, 3H, COCH3), 3.40 (s, 3H, OCH3),
3

3.75-3.84 (m, 3H, H-2, H-4, H-5), 4.09 (dd, 1H, J = 2.4, 10.8 Hz, H-6), 4.23 (dd, 1H, J
3

3

3

= 4.5, 12Hz, H-6’), 5.02 (t, 1H, J = 8.4 Hz, H-3), 5.70 (d, 1H, J = 7.2 Hz, H-1), 5.89

(d, 1H, J

3

= 6.6 Hz, CONH);

13

C-NMR (125 Hz, CDCl3) 20.8, 20.84, 20.9, 23.4, 53.8,

58.0, 62.0, 68.5, 72.9, 79.2, 91.97, 169.4, 169.41, 170.2, 170.7. HRMS C15H23NO9 Na[M
+ Na+] calc. 384.1271 found 384.1259.

Benzyl-2-acetamido-2-deoxy-α-D-glucopyranoside (6)
N-Acetyl glucosamine 5 (6.00 g, 27.12 mmol) was dissolved in benzyl alcohol (50 mL)
and conc. HCl (2.9 mL) added. The mixture was heated to 90 °C for 3 h, cooled to room
temperature and then poured onto 500 mL Et2O and stored overnight at -20 °C. The

154

resulting precipitate was recovered by filtration and rinsed with Et2O and hexanes to
yield 17.64 g of crude material, which was purified by silica gel chromatography (8-15 %
1

MeOH/CH2Cl2) to provide 5 (5.98 g, 71 %) as white foam. Comparison of H-NMR
with literature values confirmed the identity of compound 6.
1

33

H-NMR (500 MHz, CD3OD), δ 1.95 (s, 3H, COCH3), 3.37-3.39 (m, 1H ), 3.69-3.73 (m,
3

3

3

3H), 3.82 (d, 1H, J = 9.5 Hz), 3.89 (dd, 1H, J = 3.5, 11 Hz), 4.49 (d, 1H, J = 12 Hz),
3

4.74 (d, 1H, J = 11.5 Hz), 7.28-7.40 (m, 5H, aromatic).

Benzyl-2-acetamido-2-deoxy-4,6-O-benzylidene-α-D-glucopyranoside (7)
Compound 6 (0.412 g, 1.32 mmol) was dissolved in DMF (20 mL) followed by the
addition of benzaldehyde dimethyl acetal (0.6 mL, 3.97 mmol) and catalytic amount of ptoluenesulfonic acid (0.123 g, 0.53 mmol). The reaction mixture was stirred for 3 hours,
DMF was removed in vacuo and the resultant white residue suspended in saturated
sodium bicarbonate solution. Upon filtration, the residue was washed several time with
hexanes/EtOAc/CH2Cl2 (4/1/0.5) solvent system then dried in under vacuum to afford
1

compound 7 in 95 % yield (0.5 g). Comparison of H-NMR with reported literature
values confirmed the identity of compound 7.
1

36

H-NMR (600MHz, CDCl3) δ 1.82 (s, 3H, CH3), 3.59 (t, 1H, J

3

= 9 Hz, H-6), 3.75 (t,

3

1H, J = 10.2 Hz, H-6’), 3.84-3.91 (m, 1H, H-5), 3.92-3.95 (m, 1H, H-4), 4.23-4.26 (m,
3

3

2H, H-2, H-3), 4.93 (d, 1H, J = 12 Hz, CH2Ph), 4.74 (d, 1H, J = 12 Hz, CH2Ph), 4.93

155

3

3

(d, 1H, J = 3.5 Hz, H-1), 5.57 (s, 1H, CHPh), 5.82 (d, 1H, J = 8.4 Hz, CONH), 7.337.51 (m,10H, aromatic).

Benzyl-2-acetamido-2-deoxy-4,6-O-benzylidene-3-oxo-α-D-glucopyranoside (8)
To a solution of COCl2 (0.11 mL, 1.25 mmol) and DMSO (0.16mL, 2.23 mmol) in
CH2Cl2 (30 mL) cooled to -78 °C and stirred for 20 minutes was added compound 7 (0.36
g, 0.894 mmol) dissolved in CH2Cl2 (20 mL). After 20 minutes, Et3N (0.63 mL, 4.47
mmol) was added and the reaction mixture allowed to warm to rt then quenched with
H2O (100 mL). The aqueous layer was extracted with CH2Cl2 (50 mL), the organic
layers combined and washed with brine and dried over MgSO4. Compound 8 was
obtained in 90 % yield (0.32 g) by recrystallization from hexanes/EtOAc/CH2Cl2
(5/1/0.5).
[α]20 + 38.8 (c = 0.1, CH2Cl2);
D

1

H-NMR (500MHz, CDCl3) δ 2.00 (s, 3H, CH3), 3.90

3

3

(t, 1H, J = 10.5 Hz, H-6), 4.11-4.16 (m, 1H), 4.29 (dd,1H, J = 5, 10.5 Hz, H-6’), 4.37(d,
3

3

3

1H, J = 10.5 Hz, H-4), 4.50 (d, 1H, J = 12 Hz, CH2Ph), 4.69 (d, 1H, J = 12 Hz,
3

CH2Ph), 4.95-4.97 (m, 1H, H-2), 5.41 (d, 1H, J = 4.5 Hz, H-1), 5.56 (s, 1H, CHPh), 6.22
3

(d, 1H, J = 8.0Hz, CONH) 7.25-7.49 (m, 10H, aromatic);

13

C-NMR (125 Hz, CDCl3) δ

23.2, 59.1, 66.6, 69.6, 70.8, 77.0, 77.2, 77.5, 82.9, 100.8, 102.2, 126.6, 128.2, 128.6,
128.9, 129.6, 136.5, 170.1, 195.2. HRMS C22H24NO6 [M + H+] calc. 398.1580 found
398.1577.
156

1,3,4,6-Tetra-O-acetyl-2-deoxy-2-phthalamido-β-D-glucopyranoside (11)
A 1 M NaOMe solution was prepared by adding Na metal (1.99 g) in small pieces to
anyhdrous MeOH (84 mL) at -5 °C in a 250 mL round-bottemed flask. Upon complete
consumption of Na metal, this was slowly added to a flask containing glucosamine
hydrochloride 10 (9.4 g, 46.4 mmol) and stirred for 45 min at rt. The reaction mixture
was treated with finely ground phthalic anhydride (7.02 g, 47.4 mmol) added in two
portions, followed by addition of Et3N (6.7 mL, 47.9 mmol) and MeOH (44 mL) and
stirred for 24 hrs during which the solution slowly turned from milky white to a thick
yellow paste. The intermediate pthalamate was precipitated as a white solid by cooling
the reaction mixture to -20 °C for 1 hr, filtered, washed several times with cold MeOH
then dried overnight in vacuo. The solid was resuspended in pyridine (70 mL) and
treated with Ac2O (84 mL) then stirred at rt for 24 hrs. Cold EtOH was added to the
reaction mixture to quench excess Ac2O followed by evaporation of solvents.

The

resultant yellow slurry was resuspended in toluene (3 x 20 mL) and concentrated several
times for removal of pyridine. The resultant residue was redissolved in CH2Cl2 ( 250
mL) and washed with water (4 x 50 mL), brine (50 mL), dried over Na2SO4 and
evaporated to dryness. The crude product was dissolved in hot EtOAc (20 mL), then
diluted with hexanes (150 mL) and left to cool at -5 °C. The recrystallization product was
collected by filtration, washed with cold hexanes and dried to yield compound 11 (12.67

157

1

g, 60.2 %) as the major β isomer.

Comparison of H-NMR with literature values

confirmed the identity of compound 11.

34

1

H-NMR (500MHz, CDCl3) δ 1.88 (s, 3H, COCH3), 1.99 (s, 3H, COCH3), 2.04 (s, 3H,
3

COCH3), 2.11 (s, 3H, COCH3), 4.02-4.05 (m, 1H, H-5), 4.15 (dd, 1H, J = 2.5, 12.5 Hz,
3

3

H-6), 4.36 (dd, 1H, J = 4.0, 12.5 Hz, H-6’), 4.46 (dd, 1H, J = 8.5, 10.5 Hz, H-2), 5.20
3

3

3

(t, 1H, J = 9.5 Hz, H-4), 5.88 (dd, 1H, J = 9.0, 10.5 Hz, H-3), 6.52 (d,1H, J = 9 Hz,
H-1), 7.76-7.89 (m, 4H, aromatic).

Benzyl-3,4,6-tri-O-acetyl-2-deoxy 2-phthalamido-β-D-glucopyranoside (12)
Compound 11 (2.2 g, 4.61 mmol) was dissolved in CH2Cl2 (30 mL), followed by addition
of BnOH (1.1 mL, 9.22 mmol). Then BF3.OEt2 (1.75 mL, 11.4 mmol) was added
dropwise and the reaction mixture was stirred at rt for 24 hours. The mixture was diluted
with CH2Cl2 (20 mL), washed with saturated NaHCO3 (3 x 20 mL). The aqueous layer
was extracted with CH2Cl2 (60 mL) and the combined organic phases were dried over
Na2SO4, filtered, concentrated and purified by column purification (hexanes/ EtOAc 3:2)
1

to yield compound 12 (1.92 g, 79 %). Comparison of H-NMR with reported literature
values confirmed the identity of compound 12.
1

35

H-NMR (600MHz, CDCl3) δ 1.86 (s, 3H, COCH3), 2.02 (s, 3H, COCH3), 2.13 (s, 3H,
3

COCH3), 3.85-3.88 (m, 1H, H-5), 4.19 (dd, 1H, J = 2.4, 12 Hz, H-6), 4.33-4.39 (m, 2H,
3

3

H-2, H-6’), 4.53 (d, 1H, J = 12 Hz, CHPh), 4.84 (d, 1H, J = 12.6 Hz, CHPh), 5.17 (t,
158

3

3

3

1H, J = 9.6 Hz, H-4) 5.37 (d, 1H, J = 8.4 Hz, H-1), 5.77(dd, 1H, J = 9.0, 10.2 Hz, H3), 7.08-7.79 (m, 9H, aromatic).

Benzyl-4,6-O-benzylidene-2-deoxy-2-phthalamido-β-D-glucopyranoside (13)
Compound 12 (1.88 g, 3.79 mmol) was dissolved in MeOH/ DCM solvent mixture (3:2,
20 mL) and cooled to -10 °C followed by dropwise addition of 0.3M NaOMe in MeOH (6
mL) and stirred for 2hrs. Upon completion of the reaction as established by TLC, the
reaction mixture was neutralized with Amberlite ion exchange resin to pH 7. The crude
triol was concentrated to evaporate solvents and the residue coevaporated with toluene
twice followed by drying under vaccum overnight. The reisdue was then suspended in
ACN (15 mL), PhCH(OCH3)2 (0.7 mL, 4.55 mmol) was added, followed by addition of
CSA (0.2 g, 0.87 mmol). The mixture was stirred for 3 hours then queched with Et3N
upon completion of reaction as confirmed by TLC. Solvents were evaporated and the
residue purified by column chromatography (hexanes/EtOAc 3:1) to yield compound 13
(1.42 g, 77 %).

The identity of compound 13 was confirmed by comparison with

reported literature values.
1

35

H-NMR (600MHz, CDCl3) δ 3.62-3.67 (m, 2H, H-4, H-6), 3.85-3.88 (m, 1H, H-6’),

4.29-4.32 (m, 1H, H-2), 4.42 (dd, 1 H, J

3

= 4.8, 10.8 Hz, H-5), 4.52 (d, 1H, J

3

= 12.6
3

Hz, CHPh), 4.63-4.67 (m, 1H, H-3), 4.84 (d, 1H, J 3 = 12 Hz, CHPh), 5.28 (d, 1H, J =
8.4 Hz, H-1), 5.58 (s, 1H CHPh) 7.03-7.79 (m, 14H, aromatic).

159

Benzyl-3-O-acetyl-2-deoxy-4,6-O-benzylidene-2-phthalamido-β-D-allopyranoside
(14)
To a solution of compound 13 (0.833 g, 1.71 mmol) dissolved in CH2Cl2 (30 mL),
pyridine (0.7 mL, 8.54 mmol) was added and the mixture was cooled to -20 0C. Tf2O
(0.6 mL, 3.42 mmol) dissolved in CH2Cl2 (1 mL) was added dropwise and the reaction
mixture was allowed to warm up to rt within 1.5 hrs. The reaction mixture was dilute
with CH2Cl2 (20 mL) and then quenched with saturated NaCHO3, washed with water,
brine and dried over Na2SO4 and concentrated under reduced pressure at ambient
temperature (25-30 °C). The resultant yellow syrup was dissolved in toluene, TBAOAc
(3.09 g, 10.26 mmol) added and the mixture stirred at 65 °C overnight. The mixture was
allowed to cool to rt then diluted with EtOAc. Solvents were evaporated and the resultant
residue was purified by column chromatography ( hexanes/ EtOAc 3.5:1) to yield
compound 14 (0.72 g, 80 %).
[α]20 - 47.0 (c = 0.5, CH2Cl2);
D

1

H-NMR (600MHz, CDCl3) δ 2.07 (s, 3H, COCH3),

3.86-3.93 (m, 2H, H-4, H-6), 4.18-4.22 (m, 1H, H-6’), 4.44-4.49 (m, 2H, H-2, H-4), 4.64
3

3

(d, 1H, J = 10.8 Hz, CHPh), 4.93 (d, 1H, J = 12 Hz, CHPh), 5.61 (s, 1H, CHPh), 5.775.78 (m, 1H, H-3), 6.15 (d, 1H, J

3

= 8.4, H-1), 7.21-7.84 (m, 14H, aromatic). HRMS

C30H27NO8Na [M + Na+] calc. 552.1634 found 552.1595.

160

Benzyl-2-deoxy-3-Fluoro-2-phthalamido-β-D-glucopyranoside (16)
Compound 14 (0.6 g, 1.13 mmol) was dissolved in MeOH/ CH2Cl2 (3:2, 10 mL), cooled
to -10 °C and treated with 0.3M NaOMe solution in methanol (3 mL). After 1hr, the
reaction was complete as confirmed by TLC and subsequently quenched with Amberlite
ion exchange resin (IR 120) to pH 7. Solvents were evaporated and the resultant white
solid was coevaporated with toluene (3 x 10 mL) then dried under vaccum for 2 hours.
The intermediate allosamine was dissolved in CH2Cl2 (10 mL) in a 50 mL falcon tube
and cooled to -5 °C. This was followed by dropwise addition of DAST (0.27 mL, 6.78
mmol) and the reaction mixture was allowed to warm to rt over 3 hours. The reaction
mixture was cooled to -5 °C followed by dropwise addition of MeOH to destroy the
excess DAST, concentrated and passed through a short silica column. The resultant
mixture of compound was dissolved in MeOH/CH2Cl2 (3:2; 10 mL) followed by addition
of AcCl (0.16 mL, 2.26 mmol) then stirred overnight at rt. The reaction mixture was
quenched with Et3N to pH 7. Solvents were evaporated in vacuo and the resultant
residue was purified by column chromatography (hexanes/ EtOAc/MeOH 3:1:0.1) to
acquire the desired compound 16 in 37.3 % yield (0.17 g) over three steps and
elimination product 17 (0.07 g, 14.5 %).
[α]20 - 64.0 (c = 0.5, CH2Cl2);
D

1

H-NMR (600MHz, CDCl3) δ 2.29 (s, 1H, OH), 3.11

(s,1H, OH), 3.52-3.54 (m, 1H, H-5), 3.89-3.99 (m, 3H, H-4, H-6, H-6’), 4.34-4.39 (m,
1H, H-2), 4.53 (d, 1H, J

3

= 12 Hz, CHPh), 4.79 (d, 1H, J

3

= 12 Hz, CHPh), 5.13-5.25

(m, 3H), 7.05-7.26 (m, 5H, aromatic), 7.22-7.80 (m, 4H, aromatic);

161

19

F-NMR (282 MHz,

CDCl3) -194.44 (dt, 1F, J

3

= 51.9, 13.8 Hz). HRMS C21H20NaO6NF [M + Na+] calc.

424.1167 found 424.1172.
Benzyl-3-deoxy-2-(methylcarbamoyl)benzoate-6-methoxy-4-oxo-β-glucopyranoside
(17)
1

[α]20 - 51.4 (c = 0.5, CH2Cl2); H-NMR (600MHz, CDCl3) 2.24 (dd, 1H, J
D

3

= 4.2, 13.2

3

Hz, H-3), 2.48 (t, 1H, J = 13.2 Hz, H-3), 3.32 (s, 3H, OCH3), 3.36 (s, 3H, OCH3), 3.733.74 (m, 1H, H-5), 3.88-3.96 (m, 2H, H-6, H-6’), 4.43-4.48 (m, 1H, H-2), 4.58 (d, 1H, J
3

3

3

= 12 Hz, CHPh), 4.79 (d, 1H, J = 12 Hz, CHPh), 5.33 (d, 1H, J = 8.4 Hz, H-1), 7.087.16 (m, 5H, aromatic), 7.70-7.84 (m, 4H, aromatic) ;

13

C-NMR (150 Hz, CDCl3) 32.6,

49.4, 49.95, 50.6, 60.9, 71.2, 80.1, 98.7, 99.4, 123.3, 123.6, 127.6, 127.8, 127.9, 128.1,
128.3, 128.5, 131.4, 131.5, 131.7, 134.0, 167.6, 167.8, 204.4, 204.7. HRMS C23H29N2O7
[M + NH4+] calc. 445.1975 found 445.1994.

Benzyl-4,6-tri-O-acetyl-2-acetamido-2-deoxy-3-fluoro-β-D-glucopyranoside (18)
Compound 16 (0.34 g, 0.847 mmol) was dissolved in n-butanol (10 mL) and treated with
ethylene diamine (6 mL). The reaction mixture was then heated at 90 0C for 23hrs.
Solvents were then removed under reduced pressure and the resultant residue was
coevaporated with toluene (3 x 10 mL), which was followed by drying in vacuo for 3 hrs.
The intermediate free amine was dissolved in Pyridine (10 mL), followed by addition of
Ac2O (6 mL) and stirred overnight at rt. The mixture was treated with EtOH to react
with excess Ac2O, concentrated, dissolved in CH2Cl2, washed with water, brine and dried
162

over Na2SO4 followed by purification by column chromatography (hexanes/ EtOAc/
CH2Cl2 / MeOH, 2:1:1:0.2) to afford compound 18. (0.27 g, 80 %).
1

H-NMR (600MHz, CDCl3) δ 1.94 (s, 3H, NHCOCH3), 2.09 (s, 3H, COCH3), 2.10 (s,

3H, COCH3), 3.33-3.38 (m, 1H, H-2), 3.66-3.67 (m, 1H, H-5), 4.14-4.17 (m, 1H, H-6),
3

3

3

4.27 (dd, 1H, J = 5, 12.5 Hz, H-6’) 4.57 (d, 1H, J = 12.5 Hz, CHPh), 4.88 (d, 1H, J =
3

12 Hz, CHPh), 5.10-5.24 (m, 3H, H-1, H-3, H-4), 5.78 (d, 1H, J = 7 Hz, CONH);

19

F-

3

NMR (282.2 MHz, CDCl3) -193.26 (dt, 1F, J = 53.3, 12.4 Hz). HRMS C19H25FNO7 [M
+ H+] calc. 398.1615 found 398.1595.

1,4,6-Tri-O-acetyl-2-acetamido-2-deoxy-3-fluoro-α-D-glucopyranoside (19)
Compound 18 (20 mg, 0.05 mmol) was dissolved in MeOH (6 mL) and treated with Pd/C
(30 mg). The reaction mixture was the stirred under hyrdogen for 36 hours and the
reaction confirmed as complete by TLC. The mixture was filtered, concentrated and the
residue coevaporated with toluene (3 x 10 mL) followed by drying under vaccum. The
resultant residue was dissolved in pyridine (3 mL), treated with Ac2O (0.5 mL) and
stirred overnight at rt. The mixture was concentrated, washed with 1M HCl, saturated
NaHCO3, brine and dried over Na2SO4. Column purification (hexanes/ EtOAc, 1.5:1)
afforded compound 19 in 76 % (15 mg) yield over two steps.
[α]20 + 10.1 (c = 1, CH2Cl2);
D

1

H-NMR (600MHz, CDCl3) δ 2.03 (s, 3H, NHCOCH3),

2.09 (s, 3H, COCH3), 2.11 (s, 3H, COCH3), 2.16 (s, 3H, COCH3), 3.93-3.96 (m, 1H, H-

163

5), 4.06-4.09 (m, 1H, H-6), 4.22 (d, 1H, J 3 = 4.2, 12.6 Hz, H-6’), 4.58-4.69 (m, 2H, H-2,
H-3), 5.25-5.30 (m, 1H, H-4), 5.52 (d, 1H, J 3 = 7.8 Hz, NH), 6.20 (t, 1H, J 3 = 3 Hz, H1); 13C-NMR (150 Hz, CDCl3) 20.6, 20.7, 20.8, 23.2, 50.9, 50.96, 61.3, 68.1, 68.2, 69.5,
69.6, 88.97, 90.2, 90.9, 91.0, 168.4, 169.0,170.0, 170.7; 19F-NMR (282.2 MHz, CDCl3) 194.24 (dt, 1F, J 3 = 50.8, 13.5 Hz, ). HRMS C14H24N2O8F [M + NH4+] calc. 367.1517
found 367.1512.

164

APPENDIX

165

APPENDIX 3

Spectral Data

166

1

Figure 3.1 (600MHz, CDCl3) H-NMR compound 2
O O
HO

O

2

8

N

O

7

6

5

4

167

3

2

1

1

Figure 3.2 (600MHz, CDCl3) H-NMR compound 3

8

7

6

5

4

168

3

2

1

0

1

Figure 3.3 (600MHz, CDCl3) H-NMR compound 4

8

7

6

5

4

169

3

2

1

0

13

Figure 3.4 (150 MHz, CDCl3) C-NMR compound 4

200

180

160

140

120

100

170

80

60

40

20

1

Figure 3.5 (500 MHz, CD3OD) H-NMR compound 6

8

7

6

5

171

4

3

2

1

Figure 3.6 (600MHz, CDCl3) H-NMR compound 7

8

7

6

5

4

172

3

2

1

0

1

Figure 3.7 (500MHz, CDCl3) H-NMR compound 8

8

7

6

5

4

173

3

2

1

13

Figure 3.8 (150MHz, CDCl3) C-NMR compound 8

174

1

Figure 3.9 (600MHz, CDCl3) H-NMR compound 11

8

7

6

5

4

175

3

2

1

0

1

Figure 3.10 (500MHz, CDCl3) H-NMR compound 12

8

7

6

5

4

176

3

2

1

1

Figure 3.11 (600MHz, CDCl3) H-NMR compound 13

8

7

6

5

4

177

3

2

1

1

Figure 3.12 (600MHz, CDCl3) H-NMR compound 14

8

7

6

5

4

178

3

2

1

1

Figure 3.13 (600MHz, CDCl3) H-NMR compound 16

8

7

6

5

4

179

3

2

1

1

Figure 3.14 (600MHz, CDCl3) H-NMR compound 17

8

7

6

5

4

180

3

2

1

1

Figure 3.15 (600MHz, CDCl3) H-NMR compound 18

8

7

6

5

4

181

3

2

1

0

1

Figure 3.16 (600MHz, CDCl3) H-NMR compound 19

8

7

6

5

4

182

3

2

1

13

Figure 3.17 (150MHz, CDCl3) C-NMR compound 19

180

160

140

120

100

183

80

60

40

20

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