STEREOSELECTIVE GLYCOSYLATIONS and SYNTHESIS of HYALURONAN BIOSYNTHESIS INHIBITORS by Gilbert Ochieng Wasonga a THESIS Submitted

STEREOSELECTIVE GLYCOSYLATIONS AND SYNTHESIS OF HYALURONAN BIOSYNTHESIS INHIBITORS

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

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.

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

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

List of Figures

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

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

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

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

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

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

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

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

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

Figure 1.10 1H-NMR compound 15 ...... 57

Figure 1.11 13 C-NMR compound 15 ...... 58

Figure 1.12 1H-NMR compound 15 ...... 59

Figure 1.13 13 C-NMR compound 16 ...... 60

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

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

Figure 1.19 1H-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

Figure 1.26 1H-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

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

Figure 1.34 1H-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

Figure 1.40 1H-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

Figure 1.47 1H-NMR compound 23 ...... 94

viii

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

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

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

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

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

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

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

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

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

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

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

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

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

Figure 3.3 1H-NMR compound 4 ...... 169

Figure 3.4 13 C-NMR compound 4 ...... 170

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

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

Figure 3.7 1H-NMR compound 8 ...... 173

Figure 3.8 13 C-NMR compound 8 ...... 174

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

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

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

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

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

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

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

Figure 3.16 1H-NMR compound 19 ...... 182

Figure 3.17 13 C-NMR compound 19 ...... 183

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

List of Abbreviations and Symbols

Å Angstrom

α alpha

Ac Acetyl

AcCl Acetyl chloride

Ac 2O Acetic anhydride

AgOTf Silver trifluoromethanesulfonate

β beta

BF 3.OEt 2 Boron trifluoride etherate

Bn Benzyl

BnBr Benzyl bromide

BnOH Benzyl alcohol

Bz Benzoyl

BzCl Benzoyl chloride

C Carbon

C Concentration

CSA Camphorsulfonic acid

CCl 4 Carbon tetrachloride

CH 2Cl 2 Dichloromethane

CD 3OD Deuterated methanol

CDCl 3 Deuterated chloroform

CCl3CN Trichloroacetonitrile

xii

Ce Cerium

COCl 2 Oxalyl chloride conc. Concentration

CuSO 4 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

Et 3N Triethylamine

Et 4NBr Tetraethylammonium bromide

Et 2O 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

H2SO 4 Sulfuric acid

L Litre m Multi

M Mega

Me Methyl

Me 4Si 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

NaHCO 3 Sodium bicarbonate

NaOMe Sodium methoxide

NH 4OAc Ammonium acetate

NMR Nuclear magnetic resonance

NaN 3 Sodium azide

Ph Phenyl

PhSEt Ethyl phenyl thioether

PPh 3 Triphenyl phosphine ppm Part per million

PhSOTf Benzenesulfenyl trifluoromethanesulfonate

PhSeOTf Benzeneselenenyl trifluoromethanesulfonate p-TolSCl para -Toluenesulfenyl chloride rt room temperature

s Singlet

t Triplet

Temp Temperature

TFA Trifluoroacetic acid

Tf 2O 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 orthogonal glycosylation 5, 6, chemoselective glycosylation 7, 8 ,iterative glycosylation 9, 10,11 and one-pot multistep glycosylations 8, 12-22 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-tran s 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.

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 (1 S)-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-cis -

glycoside. 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

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

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 py ranosyl systems resist the formation of oxacarbenium ion. 28, 29 Taking advantage of this benzylidene effect, Crich made a significant breakthrough in β-

mannoside synthesis by developing a direct approach for the formation of β-

mannopyranosides. 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 S N2 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 β-

mannosylation 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). 44

β/α > 25 : 1

α/β ~ 2 : 1

75%, α -only

Scheme 1.4 Benzylidene effect

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

stereocontrolled formation of 1,2-cis product .46 Seminal studies by Barresi and

Hindsgaul,45 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 ).48

Scheme 1.5 Intramolecular aglycon delivery

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 N- glycans. 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.53 Other indirect methods include conjugation with glycals for β mannosides 54 and direct formation of 2-ulosyl

β− glycosides followed by reduction. 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 pre- activation 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. 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 several biologically significant oligosaccharides, including Lewis x,19 dimeric Lewis x,19

Globo-H, 20 and hyaluronic acid oligosaccharides. 21,22 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-tert- butylpyrimidine).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.

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. 63

Scheme 1.7 Synthesis of building blocks 4, 7, and 8. Conditions, i) 1.6 eq p-thiocresol, 1.3 eq BF 3.OEt 2 , 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%.

The straightforward synthesis of 4 and 7 started with a one step conversion of commercially available per-acetylated glucose 1 using p-thiocresol and BF 3.OEt 2 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. As ethereal solvents can enhance α selectivity, 21,69,74-77 we first performed the glycosylation in diethyl ether. Donor 4 dissolved in diethyl ether was pre-activated by 1 eq of p-TolSOTf, formed in situ through the stoichiometric reaction of p-TolSCl 21 with AgOTf. 3 eq of

AgOTf was typically added as a standard protocol. 22,26,77 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).

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, 64 suggesting transient nature of this species. 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 S N2 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, cyclopentyl methyl ether, THF, toluene, toluene/1,4-dioxane 76 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 S N2 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 S N1 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 S N2 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

b Entry Donor Acceptor AgOTf MOTf Product (α/β) Yield (%) 1 4 7 3 eq none 10 1.1 : 1 67 2 4 7 10 eq none 10 1 : 1.5 66 3 4 7 1 eq none 10 6 : 1 68 4 4 7 1 eq NaOTf 10 5 : 1 76 5 4 7 1 eq HfOTf 10 4 : 1 68 6 9 7 1 eq none 22 2 : 1 89 7 9 7 3 eq none 22 β 63 8 4 12 1 eq none 13 1.8 : 1 86

9 4 12 1 eq TBAOTf 13 1.3 : 1 67 10 4 12 3 eq TBAOTf 13 1 : 1.5 87 a 11 4 12 1 eq TBAOTf 13 1.4 : 1 76 12 9 11 3 eq none 16 α 74 a. 3 equivalents of TBAOTf used in the reaction. b. Ratio determined by 1H-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).

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

Entry Donor Acceptor AgOTf Solvent Product (α/β) Yield (%)

1 4 7 1 eq CH 2Cl 2 10 1 : 8 90 a 2 4 7 3 eq CH 2Cl 2 10 1 : 1.8 90 b 3 4 7 1 eq Et 2O 10 6 : 1 68 c 4 4 7 1 eq Et 2O 10 10 : 1 41

5 4 12 1 eq CH 2Cl 2 13 1 : 9 79 6 4 12 1 eq Et 2O 13 1.8 : 1 90 7 14 7 1 eq CH 2Cl 2 15 3.5 : 1 50 8 9 12 1 eq CH 2Cl 2 18 β 76 d 9 9 12 1 eq CH 2Cl 2 18 β 76

10 9 12 1 eq Et 2O 18 1.5 : 1 65 11 9 8 1 eq Et 2O 17 α 70 12 9 8 1 eq CH 2Cl 2 17 1 : 1.2 90 13 4 11 1 eq CH 2Cl 2 24 2 : 1 42 14 4 11 1 eq Et 2O 24 α 60 15 4 8 1 eq Et 2O 23 5.7 : 1 90 16 4 8 1 eq CH 2Cl 2 23 1 : 1.7 72 17 19 8 1 eq Et 2O 21 α 61 18 19 8 1 eq CH 2Cl 2 21 1 : 1 63 19 19 12 1 eq CH 2Cl 2 20 1 : 3 71 20 19 12 1 eq Et 2O 20 1 : 1 58 21 9 11 1 eq Et 2O 16 α 74 a. Reaction was performed in dichloromethane (donor concentration was 50 mM). b. Reaction was performed in diethyl ether (donor concentration was 50 mM). c. Donor concentration was 5 mM. d. Toluene (5% of final volume) was added to the reaction to dissolve AgOTf.

Scheme 1.8 Building blocks and glycosylation products

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

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.

1.4 Experimental Section

All reactions were carried out under nitrogen with anhydrous solvents in flame- dried 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 (NH 4)2(NO 3)6 (0.5 g) and (NH 4)6Mo 7O24 4H 2O (24.0 g) in 6% H 2SO 4 (500 mL). Flash column chromatography was performed on silica gel 60

(230–400 Mesh). NMR spectra were referenced using Me 4Si (0 ppm), residual CHCl 3 (δ

1H-NMR 7.24 ppm, 13 C-NMR 77.0 ppm). Peak and coupling constant assignments are based on 1H-NMR, 1H–1H gCOSY and (or) 1H–13 C gHMQC and 1H–13 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). BF 3.OEt 2 (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 NaHCO 3 (200 mL)

followed by an aqueous workup using 200ml of water. The aqueous phase was re-

extracted with dichloromethane (2 x 200 mL) and the organic phases recombined then

dried over anhydrous Na 2SO 4. The solvents were evaporated and the resulting off-white residue was recrystallized in hexanes / ethyl acetate (10:1) giving compound 2 in 85% yield (22.3 g) as a white solid. Comparison of 1H-NMR with literature values 71 confirmed the identity of compound 2.

1 H-NMR (400 MHz, CDCl 3) δ 1.98 (s, 3H, COCH 3), 2.00(s, 3H, COCH 3), 2.07 (s, 3H,

COCH 3), 2.08 (s, 3H, COCH 3), 2.35 (s, 3H, SPhCH 3), 3.69-3.71 (m, 1H, H-5), 4.18-4.20

(m, 2H, H-6, H-6’), 4.64 (d, 1H, J 3 = 10 Hz, H-1), 4.95 (t, 1H, J 3 = 9.6 Hz, H-2), 5.05

(t, 1H, J 3 = 9.6 Hz, H-4), 5.23 (t, 1H, J 3 =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

compound 3 was confirmed by comparison of 1H-NMR with literature values. 71

1 3 H-NMR (500 MHz, CD 3OD) δ 2.32(s, 3H, SPhCH 3), 3.17 (dd, 1H, J = 9, 10 Hz), 3.28-

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

2, 12 Hz), 4.51 (d, 1H, J 3= 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. NaHCO 3 solution, brine and dried over Na 2 SO 4. 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

% yield (2.88 g). Comparison of 1H-NMR with literature values 71 confirmed the identity of compound 4.

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

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

4.55-4.60 (m, 3H), 4.72(d, 1H, J 3 = 10.2 Hz), 4.79-4.84 (m, 2H), 4.86 (d, 2H, J 3 = 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 NaHCO 3 (60 mL), brine (50 mL) and the organic layer was

18 dried over Na 2SO 4. The desired product 5 was obtained as a clear syrup in 90% yield

(2.17 g).

1 H-NMR (600 MHz, CDCl 3) δ 1.12, (s, 9H, C(CH 3)3Si) , 2.30 (s, 3H, SPhCH 3), 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 = 4.8, 10.8

Hz), 4.01 (dd, 1H, J 3 = 3.6, 11.4 Hz), 4.52 (d, 1H, J 3 = 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-D-

glucopyranoside (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 NaHCO 3 (60

mL), brine (50 mL) and the organic layer was dried over Na 2SO 4. The desired compound

6 was obtained as white foam after column chromatography (hexanes/ ethyl acetate 4:1)

in 95% yield (4.6 g). The identity of compound 6 was confirmed by comparison of 1H-

NMR with literature values. 18

1 H-NMR (600 MHz, CDCl 3) δ 1.03 (s, 9H, C (CH 3)3Si), 2.30 (s, 3H, SPhCH 3), 3.83 –

3.88 (m, 3H, H-5,H-6, H-6’), 4.96 (d, 1H, J 3 = 9.6 Hz, H-1), 5.45 (t, 1H, J 3 = 9.6 Hz, H-

2), 5.61 (t, 1H, J 3 = 9.6 Hz, H-4), 5.82 (t, 1H J 3 = 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

CuSO 4 solution (2 x 40 mL), 1M HCl (40 mL), sat. aqueous NaHCO 3 (40 mL), brine (50

mL) and the organic layer dried over Na 2SO 4. The desired compound 7 was obtained in

90% yield (0.77 g) after flash column chromatography (hexanes/ ethyl acetate 2:1). The identity of compound 7 was confirmed by comparison of 1H-NMR with literature values. 18

1 3 H-NMR (600 MHz, CDCl 3) δ 2.34 (s, 3H, SPhCH 3), 2.42(dd, 1H J = 5.4, 8.4 Hz),

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

10.2 Hz, H-2, H-4), 5.92(t. 1H, J 3 = 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 NaHCO 3 (60

mL), brine (50 mL) and the organic layer dried over Na 2SO 4. 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 CuSO 4 solution (2 x 40

mL), 1M HCl (40 mL), sat. aqueous NaHCO 3 (40 mL), brine (50 mL) and the organic

layer dried over Na 2SO 4. 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 71 confirmed the identity of compound 8.

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

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

2H), 4.78 (d, 1H, J 3 = 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-D-

glucopyranosyl)-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 CH 2Cl 2 (2 mL) was stirred at room temperature for 30 minutes, cooled to

−60 °C, and AgOTf (39 mg, 0.15 mmol) dissolved in Et 2O (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 CH 2Cl 2 (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. CH 2Cl 2 (20 mL) was added,

31 and the mixture was filtered though a Celite pad. The Celite was washed with CH 2Cl 2 until no organic compounds were present in the filtrate (TLC). The combined CH 2Cl 2 solutions were washed successively with a saturated aqueous solution of NaHCO 3 (2 x

20 mL) and water (2 x 10 mL). The organic phase was dried (Na 2SO 4), concentrated and purified by chromatography resulting in the desired amorphous oligosaccharide 9 (42 mg,

69 % yield). The identity of compound 9 was confirmed by comparison of 1HNMR with

literature values. 18

20 1 [α] D +10.0 ( c = 1, CH 2Cl 2); H-NMR (500 MHz, CDCl 3) 1.04 (s, 9H,C(CH 3)3Si) , 2.34

3 3 (s, 3H, SPhCH 3), 3.39 (d, 1H, J = 9.6 Hz, H-4), 3.41 (d, 1H, J = 9.6 Hz, H-2), 3.45

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

3.78 (dd, 1H, J 3 = 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, CH 2Ph), 4.52 (d, 1H, J = 9.6 Hz, H-1),

3 3 3 4.54 (d, 1H, J = 10.4 Hz, CH 2Ph), 4.65 (d, 1H, J = 10.4 Hz, CH 2Ph), 4.72 (d, 1H, J =

3 3 10.4 Hz, CH 2Ph), 4.82 (d, 1H, J = 10.4 Hz, CH 2Ph), 4.85 (d, 1H, J = 10.4 Hz,

3 3 CH 2Ph), 4.89 (d, 1H, J = 8.0 Hz, H-1’), 5.56 (dd, 1H, J = 8.0, 9.6 Hz, H-2’), 5.65 (t,

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

13 C-NMR (125 MHz, CDCl 3) δ 19.41 (Si C(CH 3)3), 21.83 (SPh CH3), 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-

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

+ C77 H76 NaO 13 SSi [M + Na] calc. 1291.4674 found 1291.4651; Anal. calc.

C77 H76 NaO 13 SSi: 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 CH 2Cl 2 (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/ CH 2Cl 2 (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 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 CH 2Cl 2

(2 ml) was then added dropwise to the reaction mixture. This was stirred for 20 minutes at -78 °C under N 2 at which point, the acceptor was completely consumed as confirmed

° by TLC. The reaction mixture was warmed up to -20 C then quenched with Et 3N. This was followed by dilution with CH 2Cl 2 (20 mL) and filtration over Celite. The Celite was further washed with CH 2Cl 2 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 Et 2O (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 Et 2O (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 Et 2O (2 mL) was then added dropwise to the reaction mixture. This was stirred for 20 minutes at -78 °C under N 2 at which point, the acceptor was completely consumed as confirmed by TLC. The reaction mixture was warmed up to -20 °C, quenched with Et 3N then concentrated to dryness. The

residue was diluted with CH 2Cl 2 (20 mL) and filtrated over Celite. The Celite was further washed with CH 2Cl 2 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-βββ-D- 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 purification (hexanes/ ethyl acetate 3:1). Comparison of 1H-NMR with literature values

18 confirmed the identity of compound 10.

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

3.45-3.53 (m, 2H), 3.57 (dd, 1H, J 3 = 3.6, 10.8 Hz), 3.77 (d, 1H, J 3 = 12.6 Hz), 3.79-

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

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

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

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

1.8, 12.0 Hz), 5.39 (t, 1H, J 3 = 9.6 Hz), 5.82 (t, 1H, J 3 = 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

13 (m, 4 H, aromatic), 8.01 – 8.04 (m, 2 H, aromatic); C-NMR (100.5 MHz, CDCl 3)

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,

133.49, 133.70, 133.93, 138.22, 138.48, 138.68, 138.74, 139.24, 165.32, 165.39, 166.00;

+ HRMS C 68 H64 NaO 13 S [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-ααα-D- glucopyranoside (13)

Using method A of general procedure for glycosylation, donor 4 (40 mg, 0.06 mmol) was preactivated using p-TolSCl (13.4 µL, 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 72 confirmed the identity of compound 13 .

1 H-NMR (500 MHz, CDCl 3) δ 3.36 (s, 3H, OCH 3), 3.44-3.47 (m, 2H), 3.51-3.71(m,

14H), 3.74 (d, 1H, J 3 = 1.5 Hz), 3.76 (d, 1H, J 3 = 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 -O- benzoyl-1-thio-βββ-D-glucopyranoside (15)

Using method B of general procedure for glycosylation, donor 14 (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

’ determined from SPhCH 3 singlet and H-3 ratios ) after column purification (hexanes/

ethyl acetate 3:1)

20 1 [α] D +33 ( c =1.0, CH 2Cl 2); H-NMR (600 MHz, CDCl 3) δ 2.18 (s, 3H, SPhCH 3, α),

3 2.27(s, SPhCH 3 β), 3.56 (m, 1H), 3.72 (dd, 1H, J = 3.6, 9.6 Hz), 3.95-4.00 (m, 1H),

4.14-4.24 (m, 3H), 4.32-4.36 (m, 1H), 4.42 (dd, 1H, J 3 = 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, CH 2Ph), 4.89 (d, 1H, J = 12 Hz,

3 3 CH 2Ph), 4.99 (d, 1H, J = 9.6 Hz, H-1), 5.38-5.45 (m, 3H), 5.84 (t, 1H, J = 12 Hz),

7.07-7.56 (m, 32H, aromatic), 7.77-8.04 (m, 11H, aromatic). 13 C-NMR (150 MHz,

CDCl 3), 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 C 68 H60 NaO15 S [M + Na] calc. 1171.3551 found 1171.3600.

p-Tolyl-2,3,4-tri-O-benzoyl-6-O-tert-butyldiphenylsilyl-βββ-D-glucopyranosyl-(1 →6)-

2,3,4-tri-O-benzyl-ααα-D-glucopyranosyl-(1 →4)-2,3-di -O-benzoyl-6-O-benzyl-1-thio-βββ-

D-glactopyronoside (16)

Using method B of general procedure for glycosylation, donor 9 ( 60 mg, 0.05 mmol) was

preactivated using p-TolSCl (15 µL, 0.05 mmol) and reacted with acceptor 11 (20.9 mg,

0.04 mmol) to give the desired product (16 ) in 74 % yield (46 mg) exclusively as α isomer after column purification (hexanes/ ethyl acetate/CH 2Cl 2 6:1:0.5).

20 1 [α] D +41 (c = 0.55, CH 2Cl 2); H-NMR (500 MHz, CDCl 3) δ 0.94 (s, 9H, (CH 3)3CSi),

3 2.10 (s, 3H, SPhCH 3), 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 = 11.5

3 c 3 Hz), 4.22 (d, 1H, J = 8 Hz, H-1 ), 4.28-4.38 (m, 4H), 4.48 (d, 1H, J = 12 Hz, CH 2Ph),

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,

a b J 3 = 10 Hz, H-1 ), 4.90 (d, 1H, J 3 = 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,

13 aromatic), 7.87-7.89 (m, 2H, aromatic); C-NMR (125 MHz, CDCl 3), 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. HRMS

+ C104 H104 NO 20 SSi [M + NH 4] calc. 1746.6636 found 1746.6638.

38 p-Tolyl-2,3,4-tri-O-benzoyl-6-O-tert-butyldiphenylsilyl-βββ-D-glucopyranosyl-(1 →6)-

2,3,4-tri-O-benzyl-ααα-D-glucopyranosyl-(1 →6)-2,3,4-tri -O-benzyl-1-thio-βββ-D- glucopyronoside (17)

Using method B of general procedure for glycosylation, donor 9 ( 60 mg, 0.05 mmol) was

preactivated using p-TolSCl (15 µL, 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/CH2Cl 2 6:1:0.5) .

20 1 [α] D +22 (c =0.35, CH 2Cl 2); H-NMR (600 MHz, CDCl 3) δ 1.00 (s, 9H, (CH 3)3CSi),

3 2.19 (s, 3H, SPhCH 3), 3.18 (t, 1H, J = 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 = 9 Hz), 4.26

c (d, 1H, J 3 = 11.4 Hz), 4.45 (d, 1H, J 3 = 11.4 Hz), 4.50 (d, 1H, J 3 = 9 Hz, H1 ), 4.56 (d,

a 1H, J 3 = 10.8 Hz), 4.59-4.64 (m, 4H), 4.70 (d, 1H, J 3 = 8.4 Hz, H-1 ), 4.71-4.90 (m,

b 5H), 5.01 (d, 1H, J 3 = 3.6 Hz, H-1 ), 5.54-5.58 (m, 2H), 5.81 (t, 1H, J 3 = 10.2 Hz), 6.96-

7.65 (m, 49H, aromatic), 7.67-7.71 (m, 2H, aromatic), 7.81-7.89 (m, 7H, aromatic); 13C-

NMR (125 MHz, CDCl 3), 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, C-

b 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,

133.2, 133.22, 133.28, 133.3, 133.4, 135.8, 135.9, 138.0, 138.4, 138.5, 138.7, 138.8,

+ 138.9, 139.2, 165.16, 165.26, 166.19. HRMS C 104 H108 NO 18 SSi [M + NH 4] calc.

1718.7135, found 1718.7137.

Methyl-2,3,4-tri-O-benzoyl-6-O-tert-butyldiphenylsilyl-βββ-D-glucopyranosyl-(1 →6)-

2,3,4-tri-O-benzyl-βββ-D-glucopyranosyl-(1 →6)-2,3,4-tri -O-benzyl-1-methyl-ααα-D- 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/ CH 2Cl 2, 4:1:0.5).

20 1 [α] D +12 (c = 0.2, CH 2Cl 2); H-NMR (500 MHz, CDCl 3) δ 1.00 (s, 9H, (CH 3)3CSi),

3 3.33 (d, 1H, J = 8.5 Hz), 3.35 (s, 3H, OCH 3), 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

(m, 2H), 3.96 (t, 1H, J 3 = 9.5 Hz), 4.00 (dd, 1H, J 3 = 1.8, 10.8 Hz), 4.17 (d, 1H, J 3 = 7.5

b 3 3 Hz, H-1 ), 4.20 (dd, 1H J = 1.8, 11.4 Hz), 4.39 (d, 1H, J = 11 Hz, CH 2Ph), 4.44 (d,

3 3 3 1H, J = 11 Hz, CH 2Ph), 4 59 (d, 1H, J = 11 Hz, CH 2Ph), 4.61 (d, 1H, J = 6 Hz, H-

a 3 3 3 1 ), 4.64 (d, 1H, J = 11 Hz, CH 2Ph), 4.67 (d, 1H, J = 11 Hz, CH 2Ph), 4.71 (d, 1H, J

3 = 11 Hz, CH 2Ph), 4.75-4.77 (m, 2H, CH 2Ph), 4.82 (d, 1H, J = 11 Hz, CH 2Ph), 4.85 (d,

3 c 3 3 1H, J = 8Hz, H-1 ), 4.91 (d, 1H, J = 11 Hz, CH 2Ph), 4.95 (d, 1H, J = 11 Hz,

c 3 c 3 CH 2Ph), 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, CDCl 3), 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,

a c 98.4 ( JC-1,H-1 = 168.69 Hz, C-1 ), 101.7 ( JC-1,H-1 = 159.14 Hz, C-1 ), 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,

+ 138.8, 139.2, 165.20, 165.24, 166.1. HRMS C 98 H104 NO 19 SSi [M + NH 4] calc.

1626.6972, found 1626.6976.

Methyl-2,3,4-tri-O-benzyl-ααα-D-mannopyranosyl-(1 →6)-2,3,4-tri -O-benzyl-1-Methyl-

ααα-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 OCH 3 peaks and JC1-H1 coupling constants. Comparison with literature data confirmed its

identity .6

1 H-NMR (600 MHz, CDCl 3) δ 3.30 (s,OCH 3-α), 3.31 (s, 3H, OCH 3-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,

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

3 = 12 Hz), 4.46 (d, 1H, J 3 = 3 Hz, H-1), 4.51 (d, 1H, J 3 = 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-βββ-

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 / CH 2Cl 2 5.5:1:0.5)

20 1 [α] D +27 ( c = 0.15, CH 2Cl 2); H-NMR (600 MHz, CDCl 3) δ 2.20 (s, 3H, SPhCH 3),

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

(m, 3H), 4.04 (t, 1H, J 3 = 9.5 Hz), 4.47-4.53 (m, 3H), 4.58 (dd, 1H, J 3 = 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,

13 29H, aromatic), 7.41-7.43 (m, 6H, aromatic); C-NMR (125 MHz, CDCl 3), 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,

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 C 68 H74 NO 10 S [M + NH 4] calc. 1096.5033 found 1096.5031.

p-Tolyl-2,3,4-tri-O-benzoyl-6-O-tert-butyldiphenylsilyl-βββ-D-glucopyranosyl-(1 →6)-

2,3,4-tri-O-benzyl-βββ-D-glucopyranosyl-(1 →6)-2,3,4-tri -O-benzoyl-1-thio-βββ-D-

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/ CH 2Cl 2, 4:1:0.5)

20 1 [α] D + 18 (c = 0.2, CH 2Cl 2); H-NMR (600 MHz, CDCl 3) δ 1.00 (s, 9H, (CH 3)3CSi),

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

1H, J 3= 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 3 = 2.4, 11.4 Hz), 4.18-4.21 (m, 1H), 4.29 (d, 1H, J 3 = 7.8 Hz, H-

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

c a 1H, J 3 = 7.8 Hz, H-1 ), 5.03 (d, 1H, J 3 = 9.6 Hz, H-1 ), 5.45 (t, 1H, J 3 = 10.2 Hz), 5.46-

c c 5.52 (m, 2H), 5.87 (t, 1H, J 3 = 9.6 Hz, H-4 ), 5.99 (t, 1H, J 3 = 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

(m, 2H, aromatic), 7.85-7.89 (m, 9H, aromatic), 7.96-7.97 (m, 2H, aromatic); 13 C (125

MHz, CDCl 3), 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

+ C104 H102 NO21 SSi [M + NH 4] 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-βββ-D-

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 / CH 2Cl 2, 5.5:1:0.5). Comparison of H-NMR spectra with literature values 71 confirmed the identity of compound 23 .

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

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

11.2 Hz), 4.58 (d, 1H, J 3 = 9.6 Hz), 4.63 (d, 1H, J 3 = 10.0 Hz), 4.64 (d, 1H, J 3 = 12.0

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

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

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

3 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

= 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-βββ-D-glucopyranosyl-(1 →4)-2,3-di -O-benzoyl-6-O- benzyl-1-thio-βββ-D-galatcopyranoside (24)

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 values 18 confirmed the identity of compound 24 .

1 3 H-NMR (600 MHz, CDCl 3) δ 2.14 (s, 3H SPhCH 3), 2.88 (dd, 1H, J = 1.8, 11.4 Hz),

3.13 (dd, 1H, J 3 = 1.8, 10.8 Hz), 3.49 (dd, 1H, J 3 = 3.0, 9.6 Hz), 3.65(t, 1H, J 3 = 9.6

Hz), 3.76 (m, 1H), 3.74-3.77. (m, 1H), 3.89-3.96 (m, 4H), 4.04 (d, 1H, J 3 = 12 Hz,

3 CH 2Ph), 4.34-4.41 (m, 5H), 4.61 (d, 1H, J = 11.4 Hz, CH 2Ph), 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).

APPENDIX

APPENDIX 1

Spectral Data

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

8 7 6 5 4 3 2 1 0

1 Figure 1.2 (600 MHz, CDCl 3) H-NMR compound 4

8 7 6 5 4 3 2 1 0

1 Figure 1.3 (600 MHz, CDCl 3) H-NMR compound 5

9 8 7 6 5 4 3 2 1 0

1 Figure 1.4 (600 MHz, CDCl 3) H-NMR compound 6

8 7 6 5 4 3 2 1 0

1 Figure 1.5 (600 MHz, CDCl 3) H-NMR compound 7

9 8 7 6 5 4 3 2 1 0

1 Figure 1.6 (600 MHz, CDCl 3) H-NMR compound 8

9 8 7 6 5 4 3 2 1 0

1 Figure 1.7 (500 MHz, CDCl 3) H-NMR compound 9

9 8 7 6 5 4 3 2 1 0

1 Figure 1.8 (600 MHz, CDCl 3) H-NMR compound 10

9 8 7 6 5 4 3 2 1 0

1 Figure 1.9 (500 MHz, CDCl 3) H-NMR compound 13

9 8 7 6 5 4 3 2 1 0

1 Figure 1.10 (600 MHz, CDCl 3) H-NMR compound 15

9 8 7 6 5 4 3 2 1 0

13 Figure 1.11 (150 MHz, CDCl 3) C-NMR compound 15

220 200 180 160 140 120 100 80 60 40 20 0

1 Figure 1.12 (500 MHz, CDCl 3) H-NMR compound 16

9 8 7 6 5 4 3 2 1 0

13 Figure 1.13 (125 MHz, CDCl 3) C-NMR compound 16

220 200 180 160 140 120 100 80 60 40 20 0

Figure 1.14 (500 MHz, CDCl 3) gCOSY compound 16

8 7 6 5 4 3 2 1 0

Figure 1.15 (500 MHz, CDCl 3) gHMQC-coupled compound 16

9 160 140 120 100 80 60 40 20 0

Figure 1.16 (500 MHz, CDCl 3) gHMQC-coupled (anomeric region expansion) compound 16

4.2 1 4.3 J = 162.85 Hz 4.4 4.5 4.6 4.7

4.8 1 1 J = 155.02 Hz 4.9 J = 169.062 Hz 5.0 5.1 5.2

102 100 98 96 94 92 90 88 86

Figure 1.17 (500 MHz, CDCl 3) gHMQC-decoupled compoound 16

4 4 5

140 130 120 110 100 90 80 70 60 50 40 30 20

Figure 1.18 (500 MHz, CDCl 3) gHMBC compound 16

200 180 160 140 120 100 80 60 40 20

1 Figure 1.19 (600 MHz, CDCl 3) H-NMR compound 17

10 9 8 7 6 5 4 3 2 1 0

13 Figure 1.20 (125 MHz, CDCl 3) C-NMR compound 17

200 180 160 140 120 100 80 60 40 20 0

Figure 1.21 (600 MHz, CDCl 3) gCOSY compound 17

TBDPSO BzO O O BzO BzOBnO O BnO BnO O 17 BnO O STol BnO BnO

8 7 6 5 4 3 2 1 0

Figure 1.22 (600 MHz, CDCl 3) gHMQC-coupled compound 17

TBDPSO BzO O O BzO BzOBnO O BnO BnO O 17 BnO O STol BnO BnO

130 120 110 100 90 80 70 60 50 40 30 20

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

4.5 1 4.6 J 156.36 Hz 1 4.7 J = 161.68 Hz 4.8 4.9 5.0 J 1 = 169.72 Hz 5.1 5.2

102 101 100 99 98 97 96 95 94 93 92 91 90 89 88

Figure 1.24 (600 MHz, CDCl 3) gHMQC-decoupled compound 17

140 130 120 110 100 90 80 70 60 50 40 30 20 10

Figure 1.25 (600 MHz, CDCl 3) gHMBC compound 17

9 180 160 140 120 100 80 60 40 2 0

1 Figure 1.26 (600 MHz, CDCl 3) H-NMR compound 18

10 9 8 7 6 5 4 3 2 1 0

13 Figure 1.27 (150 MHz, CDCl 3) C-NMR compound 18

200 180 160 140 120 100 80 60 40 20 0

Figure 1.28 (600 MHz, CDCl 3) gCOSY compound 18

9 8 7 6 5 4 3 2 1

Figure 1.29 (600 MHz, CDCl 3) gHMQC-coupled compound 18

8 9

160 140 120 100 80 60 40 20 0

Figure 1.30 (600 MHz, CDCl 3) gHMQC-coupled (anomeric region expansion) compound 18

4.1 J 1=159.32 Hz 4.2 4.3 4.4 4.5 1 4.6 J =168.69 Hz 4.7 4.8 J 1=164.80 Hz 4.9 5.0 5.1 104.0 103.0 102.0 101.0 100.0 99.5 99.0 98.5 98.0

Figure 1.31 (600 MHz, CDCl 3) gHMQC-decoupled compound 18

140 130 120 110 100 90 80 70 60 50 40 30

Figure 1.32 (600 MHz, CDCl 3) gHMBC compound 18

200 180 160 140 120 100 80 60 40 20 0

1 Figure 1.33 (600 MHz, CDCl 3) H-NMR compound 20

9 8 7 6 5 4 3 2 1 0

1 Figure 1.34 (600 MHz, CDCl 3) H-NMR compound 21

9 8 7 6 5 4 3 2 1 0

13 Figure 1.35 (125 MHz, CDCl 3) C-NMR compound 21

200 180 160 140 120 100 80 60 40 20 0

Figure 1.36 (600 MHz, CDCl 3) gHMQC-coupled compound 21

130 120 110 100 90 80 70 60 50 40 30 20

Figure 1.37 (600 MHz, CDCl 3) gHMQC-coupled (anomeric region expansion) compound 21

4.5 J 1 = 169.9 Hz 4.6 4.7 4.8

4.9

5.0 J 1 = 157.7 Hz 5.1

5.2

99 98 97 96 95 94 93 92 91 90 89 88

Figure 1.38 (600 MHz, CDCl 3) gHMQC-decoupled compound 21

140 130 120 110 100 90 80 70 60 50 40 30 20 85

Figure 1.39 (600 MHz, CDCl 3) gHMBC compound 21

9 180 160 140 120 100 80 60 40 20

1 Figure 1.40 (600 MHz, CDCl 3) H-NMR compound 22

9 8 7 6 5 4 3 2 1 0

13 Figure 1.41 (125 MHz, CDCl 3) C-NMR compound 22

200 180 160 140 120 100 80 60 40 20

Figure 1.42 (600 MHz, CDCl 3) gCOSY compound 22

9 8 7 6 5 4 3 2 1 0

Figure 1.43 (600 MHz, CDCl 3) gHMQC-coupled compound 22

130 120 110 100 90 80 70 60 50 40 30 20

Figure 1.44 (600 MHz, CDCl 3) gHMQC-coupled (anomeric region expansion) compound 22

J 1 = 155.84 Hz 4.3 4.4

4.5

4.6 4.7

4.8 1 4.9 J = 160.43 Hz 5.0 J 1 = 156.64 Hz

5.1

104 102 100 98 96 94 92 90 88 86

Figure 1.45 (600 MHz, CDCl 3) gHMQC-decoupled compound 22

140 130 120 110 100 90 80 70 60 50 40 30 20

Figure 1.46 (600 MHz, CDCl 3) gHMBC compound 22

220 200 180 160 140 120 100 80 60 40 20

1 Figure 1.47 (400 MHz, CDCl 3) H-NMR compound 23

9 8 7 6 5 4 3 2 1 0

1 Figure 1.48 (600 MHz, CDCl 3) H-NMR compound 24

9 8 7 6 5 4 3 2 1 0

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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 2-

azido-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

N-Phenyl trifluoroacetimidate donor 14 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

phenyl substituent on the nitrogen which could minimize undesired rearrangements .6, 7

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/H 2O 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. 2

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/H 2O, rt, 58%, v) 1.5 eq PhNCClCF 3, DBU, DCM, 28%.

110

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. The anomeric stereoselectivity was

1 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

OBz BzO O OH O O O MS 3Å, O + O OC(NPh)CF3 0.1eq TMSOTf BnO O or HO 9 or 10 OBn O O DCM O O

6 7 8

Entry Donor Acceptor Thioether Temp Conc Product α / β (1 eq) (0.9 eq) (10 eq) (°C ) (mM) (Yield) 1 6 7 none -20 60 9 5/1 (95%) 2 6 7 thiophene 0 60 9 7/1 (81%) 3 6 8 none -20 60 10 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 significant enhancement in stereoselectivity as reported by Boons and coworkers. 2 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 TfN 3 in the presence of catalytic amount of CuSO 4.5H 2O in a H 2O/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

NH 4OAc 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) TfN 3, CuSO 4, NaHCO 3, DCM/MeOH, ii) Ac 2O, DMAP, Pyridine, + - 88%, iii) 2.5 eq NH 4 OAc, DMF, 85%, iv) 4 eq CCl 3CN, 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 14 (mM) 7 (mM) Temperature Thioether α/β Yield (%) °C (10 eq) 1 21 19 -20 none 4/1 (74) 2 21 19 0 thiophene 2/1 (82) 3 21 19 0 PhSEt 3/1 (92) 4 21 19 rt thiophene 11/1 (89) 5 2.1 1.9 rt none 10/1 (90) 6 21 19 -78 none 1/1 (87) 7 21 19 -78 PhESt 1/1 (93) 8 21 19 0 none 2/1 (87) 9 21 19 0 PhSEt 5/1 (92) 10 21 19 0 thiophene 14/1 (95) a. Entries 6-10 are results reported in literature 2

113

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

different temperatures in the presence of different thioethers .2 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 phenyl-

thioethyl ether (entry 3, α/β = 3/1) contrary to what was reported in literature (entries 9-

10). 2 Interestingly, when the reaction was performed at room temperature under more dilute conditions in the presence of thiophene, there was significant improvement in α-

selectivity (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

additives. Our study suggests that the reported literature results 2 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 flame-

dried 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 (NH 4)2(NO 3)6 (0.5 g) and (NH 4)6Mo 7O24 4H 2O (24.0 g) in 6% H 2SO 4 (500 mL). Flash column chromatography was performed on silica gel 60

(230–400 Mesh). NMR spectra were referenced using Me 4Si (0 ppm), residual CHCl 3 (δ

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 Na 2SO 4, 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 2.10

1 3 H-NMR (600MHz, CDCl 3) δ 2.29 (s, SPhCH 3), 3.39 (bs, 1H, H-5), 3.60 (dd, 1H, J =

3.6, 9.6 Hz, H-3), 3.81 (t, 1H, J 3 = 9 Hz, H-2), 3.96 (dd, 1H, J 3 = 1.2, 12 Hz, H-6), 4.12

(d, 1H, J 3 = 3.0 Hz, H-4), 4.35 (dd, 1H, J 3 = 1.2, 12 Hz, H-6), 4.55 (d, 1H, J 3 = 9.6 Hz,

H-1), 4.67-4.72 (m, 4H,CH 2Ph), 5.47 (s, 1H, CHPh), 6.99-7.00 (m, 2H, aromatic), 7.24-

7.43 (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

° CH 2Cl 2 (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 Et 3N 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).

Comparison of 1H-NMR with literature values confirmed the identity of compound 3.16

1 H-NMR (600MHz, CDCl 3) δ 2.24 (bs, 1H, OH), 2.33 (s, 3H, SPhCH 3), 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

3 3 (d, 1H, J = 11.4 Hz, H-1), 4.69 (bs, 2H, CH 2Ph), 4.72(d, 1H, J = 12 Hz, CH 2Ph),

116

3 4.82(d, 1H, J = 12 Hz, CH 2Ph), 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

NaHCO 3 (60 mL), brine (50 mL) and the organic layer was dried over Na 2SO 4. The

desired compound 4 was obtained in 75 % yield (7.93 g) after column purification

(hexanes/EtOAc 2:1). Comparison of 1H-NMR with literature values confirmed the identity of compound 4.15

1 3 H-NMR (600MHz, CDCl 3) δ 2.32 (s, 3H, SCH 3Ph), 3.70 (t, 1H, J = 9.0 Hz), 3.76 (dd,

1H, J 3 = 3.0, 9 Hz), 4.02 (t, 1H, J 3 = 6.6 Hz), 4.38 (dd, J 3 = 5.4, 11.4 Hz), 4.52-4.58

(m, 2H), 4.65 (d, 1H, J 3 = 9.6 Hz), 4.72 (d,1H, J 3 = 10.2 Hz), 4.77 (d, 1H, J 3 = 10.2

Hz), 4.81 (d, 1H, J 3 = 11.4 Hz), 5.87 (d 1H, J 3 = 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 NaHCO 3, concentrated and the residue dissolved in CH 2Cl 2, washed with brine, dried over Na 2SO 4 then purified by column

chromatography (hexanes/ EtOAc 3:1) to afford 0.98 g (58 %, α/β 1:0.43) of compound

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

5.15

1 3 H-NMR (400MHz, CDCl 3) 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 Teflon-

coated magnetic stir bar was charged with Ph 3P (34.5 g, 132 mmol), Et 3N (7.3 mL, 53 mmol), CCl 4 (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 CCl 4 (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 Ph 3PO, Ph 3P and Et 3N-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 CH 2Cl 2 (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).

20 1 [α] D + 36.8 (c = 0.1, CH 2Cl 2); H-NMR (600MHz, CDCl 3) δ 3.80 (bs, 1H), 3.98 (bs,

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

4.85 (d, 1H, J 3 = 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

+ C42 H36 N2O8F [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 CH 2Cl 2 (3 mL) was stirred for 10 minutes under N 2 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 Et 3N, 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 CH 2Cl 2 (3 mL) was stirred for 10 minutes under N 2 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 Et 3N, 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-O- isopropylidene-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 CH 2Cl 2 (3 mL) was stirred for 10 minutes

° under N 2 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 Et 3N, 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).

Comparison of 1H-NMR with literature values confirmed the identity of compound 9. 3

1 H-NMR (600MHz, CDCl 3) δ 1.29 (s, 3H, CH 3), 1.32 (s, 3H, CH 3), 1.42 (s, 3H, CH 3),

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

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

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

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-O- isopropylidene-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

-20 °C to afford compound 10 in 82 % (120 mg) yield as α-isomer. Comparison of 1H-

NMR with literature values confirmed the identity of compound 10 . 3

1 H-NMR (600MHz, CDCl 3) δ 1.13 (s, 3H, CH 3), 1.27 (s, 3H, CH 3), 1.41 (s, 3H, CH 3),

3 1.43 (s, 3H, CH 3), 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 = 3.6 Hz), 4.71(d, 1H, J 3 = 12 Hz), 4.77 (d,

b 1H, J 3 = 11.4 Hz, CHPh), 4.81 (d, 1H, J 3 = 11.4 Hz), 5.37 (d, 1H, J 3 = 3.6 Hz, H-1 ),

5.88 (d, 1H, J 3 = 3.6 Hz, H-1a), 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),

13 8.02-8.06 (m, 4H, aromatic); C-NMR (150 MHz, CDCl 3) 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)

NaN 3 (5.94 g, 91.6 mmol) was dissolved in H 2O (15 mL), CH 2Cl 2 (20 mL) then cooled to

° 0 C with vigorous stirring. Tf 2O (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

° CH 2Cl 2 (10 mL) and saturated NaHCO 3 (10 mL) to afford TfN 3 then kept at 0 C for use in the next step. Compound 11 (2 g, 9.27 mmol) was dissolved in H 2O (30 mL) and

MeOH (60 mL) followed by addition of NaHCO 3 (0.44 g, 5.19 mmol), CuSO 4.H 2O (0.02

g, 0.093 mmol) and TfN 3 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 Ac 2O (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 CH 2Cl 2, washed with H 2O,

dried over Na 2SO 4, 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 .13

1 H-NMR (600MHz, CDCl 3) δ 2.01-2.19 (m, 20H, COCH 3), 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, H-

4), 5.43 (t, 1H, J 3 = 9.6 Hz, H-3α), 5.53 (d, J 3 = 8.4 Hz, H-1β), 6.28 (d, 1H, J 3 = 3.6

Hz, H-1α).

122

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 NH 4OAc (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).

Comparison of 1H-NMR with literature values confirmed the identity of compound 13 .11

1 3 H-NMR (600MHz, CDCl 3) δ 2.00-2.08 (m, 14H, CH 3CO), 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 3

= 7.8 Hz, H-1β), 4.99-5.06 (m, 2H, H-3,H-4), 5.38 (d, 1H, J 3 =3.6 Hz, H-1α), 5.50 (t,

1H, J 3 = 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 CH 2Cl 2 (20 mL) followed by addition CCl 3CN (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). The identity of compound 14 was confirmed by comparison of 1H-NMR with literature values. 12

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

H-6), 4.08-4.10 (m, 1H, H-6), 4.19-4.21 (m, 1H, H-5), 4.25 (d, 1H, J 3 = 4.2, 12.6 Hz, H-

123

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

Hz, H-1).

3,4,6-Tri-O-acetyl-2-azido-2-deoxy-D-glucopyranosyl-(1 →6)-1,2:3,4-di-O- isopropylidene-α-D-galactopyranoside (15)

Using method B of general procedures for glycosylation, donor 14 (20 mg, 0.04 mmol) in

CH 2Cl 2 (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

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

literature values. 2

Using method B of general procedures for glycosylation, donor 14 (20 mg, 0.04 mmol) in

CH 2Cl 2 (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, CDCl 3) δ 1.30-1.53 (m,12H, CH 3), 2.01-2.06 (m, 9H,CH 3), 3.26 (dd,

1H, J 3 = 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, H-

5’, H-6a, H-6b ), 4.26-4.30 (m, 2H, H-2’, H-4’), 4.58 (dd, 1H, J 3 = 2.4, 7.8 Hz, H-3’),

5.00-5.04 (m, 2H, H-4, H-1), 5.42 (t, 1H, J 3 = 9.6 Hz, H-3), 5.48 (d, 1H, J 3 = 4.8 Hz, H-

1’ α).

124

APPENDIX

125

APPENDIX 2

Spectral Data

126

1 Figure 2.1 (600MHz, CDCl 3) H-NMR compound 2

8 7 6 5 4 3 2 1 0

127

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

9 8 7 6 5 4 3 2 1 0

128

1 Figure 2.3 (600MHz, CDCl 3) H-NMR compound 4

8 7 6 5 4 3 2 1 0

129

Figure 2.4 (600MHz, CDCl 3) compound 6

9 8 7 6 5 4 3 2 1 0

130

1 Figure 2.5 (600MHz, CDCl 3) H-NMR compound 9

8 7 6 5 4 3 2 1 0

131

1 Figure 2.6 (600MHz, CDCl 3) H-NMR compound 10

8 7 6 5 4 3 2 1 0

132

1 Figure 2.7 (600MHz, CDCl 3) H-NMR compound 12

8 7 6 5 4 3 2 1 0

133

1 Figure 2.8 (600MHz, CDCl 3) H-NMR compound 13

8 7 6 5 4 3 2 1 0

134

1 Figure 2.9 (600MHz, CDCl 3) H-NMR compound 14

8 7 6 5 4 3 2 1 0

135

1 Figure 2.10 (600MHz, CDCl 3) H-NMR compound 15

8 7 6 5 4 3 2 1 0

136

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-D- glucopyranosyl(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), 2073- 7.

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., Removal of Acid-Labile Protecting Groups on Carbohydrates Using Water- Tolerant and Recoverable Vanadyl Triflate Catalyst. J. Org. Chem. 2007, 72, (1), 299-302.

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 component of the unique stroma that surrounds and probably supports the tumor .8

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

(370 kJ/mol). 18

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

15 leukemia cell proliferation at IC 50 values of 34 and 35 µM respectively.

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 lymphocyte- associated 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

26 available GlcNAc 1 using acetone and anhydrous FeCl 3 as a catalyst. 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 FeCl 3, 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/H 2O 2:1, iv) Ac 2O/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 COCl 2, 2.5 eq DMSO, 5eq Et 3N,- 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 NH 2 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 methanol as established by Wei and coworkers .29 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 Ac 2O to access compound 11 predominantly as the β isomer. The glycosyl tetraacetate 11 was efficiently converted to β-benzyl glycoside 12 using BF 3.Et 2O in CH 2Cl 2. Subsequent

chemoselective saponification with 0.3 M NaOMe in a 3:2 mixture of MeOH-CH 2Cl 2 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 Tf 2O in CH 2Cl 2 in the presence of pyridine to generate the

intermediate triflate. When TBAOAc in toluene was employed to displace the OTf

group, a straightforward S N2 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, Et 3N, ii) Ac 2O, Pyridine 60.2%, iii) 2 eq BnOH, 3 eq BF 3.OEt, DCM, 79%, iv) 0.3M NaOMe, MeOH/DCM, -10 °C, v) 2 eq PhCH(OCH 3)2, 0.08 eq

CSA, CAN, 77%, vi) 2 eq Tf 2O,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) 2NH 2, n-butanol, 90 °C, xii) Ac 2O, Pyridine, rt, 80%, xiii) H 2, Pd/C, MeOH, rt, xiv) Ac 2O, 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 FeCl 3 (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 MgSO 4 and concentrated at room temperature to give compound 2 as brownish syrup (7.15 g, 65

%). The 1H-NMR was identical to what was reported in literature. 26

1 H-NMR (600MHz, CDCl 3) δ 1.33 (s, 3H, CH 3), 1.39 (s, 3H, CH 3), 2.00 (s, 3H,

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

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

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

152

2-Methyl-(1,2-di-deoxy-3-methoxy-5,6-O-isopropylidene-α-D-glucofurano)-[2,1-d]-2-

oxazoline (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 CH 2Cl 2 (20 mL) and washed with ice cold water and the

aqueous phase extracted twice with CH 2Cl 2 (2 x 20 mL). The organic phases were combined, washed with brine (30 mL) and dried with Na 2SO 4. Upon filtration and

concentration in vacuo, compound 3 was accessed in 96 % yield (0.4 g).

20 1 [α] D -15.5 (c = 1, CH 2Cl 2); H-NMR (600MHz, CDCl 3) δ 1.27 (s, 3H, CH 3), 1.33 (s,

3 3H, CH 3).1.94 (s, 3H, N=C ĈH3), 3.40 (s, 3H, OCH 3), 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, CDCl 3) 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 C 12 H20 NO5 [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 H 2O/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 Et 3N to a neutral pH and the

solvents evaporated in vacuo. The residue was washed several times with CH 2Cl 2 and the resultant white powder dried under vacuum. The residue was then dissolved in pyridine

(8 mL), Ac 2O (0.5 mL) added and the reaction mixture stirred at rt overnight. The reaction mixture was concentrated, residue dissolved in CH 2Cl 2, washed with H 2O, brine, dried over Na 2SO 4 and purified by column chromatography (CH 2Cl 2/MeOH, 8:1) to

reveal compound 4 in 50 % (0.143 g) yield over two steps.

20 1 [α] D + 10.5 (c = 1, CH 2Cl 2); H-NMR (600MHz, CDCl 3) δ 1.99 (s, 3H, NHCOCH 3),

2.07 (s, 3H, COCH 3), 2.10 (s, 3H, COCH 3), 2.11 (s, 3H, COCH 3), 3.40 (s, 3H, OCH 3),

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

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

3 13 (d, 1H, J = 6.6 Hz, CONH); C-NMR (125 Hz, CDCl 3) 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 C 15 H23 NO9 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 Et 2O and stored overnight at -20 °C. The

154 resulting precipitate was recovered by filtration and rinsed with Et 2O and hexanes to

yield 17.64 g of crude material, which was purified by silica gel chromatography (8-15 %

1 MeOH/CH 2Cl 2) to provide 5 (5.98 g, 71 %) as white foam. Comparison of H-NMR with literature values confirmed the identity of compound 6.33

1 H-NMR (500 MHz, CD 3OD), δ 1.95 (s, 3H, COCH 3), 3.37-3.39 (m, 1H ), 3.69-3.73 (m,

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

4.74 (d, 1H, J 3 = 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 p-

toluenesulfonic 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/CH 2Cl 2 (4/1/0.5) solvent system then dried in under vacuum to afford

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

values confirmed the identity of compound 7.36

1 3 H-NMR (600MHz, CDCl 3) δ 1.82 (s, 3H, CH 3), 3.59 (t, 1H, J = 9 Hz, H-6), 3.75 (t,

1H, J 3 = 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, CH 2Ph), 4.74 (d, 1H, J = 12 Hz, CH 2Ph), 4.93

155

(d, 1H, J 3 = 3.5 Hz, H-1), 5.57 (s, 1H, CHPh), 5.82 (d, 1H, J 3 = 8.4 Hz, CONH), 7.33-

7.51 (m,10H, aromatic).

Benzyl-2-acetamido-2-deoxy-4,6-O-benzylidene-3-oxo-α-D-glucopyranoside (8)

To a solution of COCl 2 (0.11 mL, 1.25 mmol) and DMSO (0.16mL, 2.23 mmol) in

° CH 2Cl 2 (30 mL) cooled to -78 C and stirred for 20 minutes was added compound 7 (0.36 g, 0.894 mmol) dissolved in CH 2Cl 2 (20 mL). After 20 minutes, Et 3N (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 CH 2Cl 2 (50 mL), the organic layers combined and washed with brine and dried over MgSO 4. Compound 8 was

obtained in 90 % yield (0.32 g) by recrystallization from hexanes/EtOAc/CH 2Cl 2

(5/1/0.5).

20 1 [α] D + 38.8 (c = 0.1, CH 2Cl 2); H-NMR (500MHz, CDCl 3) δ 2.00 (s, 3H, CH 3), 3.90

(t, 1H, J 3= 10.5 Hz, H-6), 4.11-4.16 (m, 1H), 4.29 (dd,1H, J 3= 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, CH 2Ph), 4.69 (d, 1H, J = 12 Hz,

3 CH 2Ph), 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 13 (d, 1H, J = 8.0Hz, CONH) 7.25-7.49 (m, 10H, aromatic); C-NMR (125 Hz, CDCl 3) 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 C 22 H24 NO6 [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 Et 3N (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 Ac 2O (84 mL) then stirred at rt for 24 hrs. Cold EtOH was added to the reaction mixture to quench excess Ac 2O 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 CH 2Cl 2 ( 250

mL) and washed with water (4 x 50 mL), brine (50 mL), dried over Na 2SO 4 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 g, 60.2 %) as the major β isomer. Comparison of 1H-NMR with literature values confirmed the identity of compound 11 .34

1 H-NMR (500MHz, CDCl 3) δ 1.88 (s, 3H, COCH 3), 1.99 (s, 3H, COCH 3), 2.04 (s, 3H,

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

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

(t, 1H, J 3 = 9.5 Hz, H-4), 5.88 (dd, 1H, J 3 = 9.0, 10.5 Hz, H-3), 6.52 (d,1H, J 3 = 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 CH 2Cl 2 (30 mL), followed by addition

of BnOH (1.1 mL, 9.22 mmol). Then BF 3.OEt 2 (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 CH 2Cl 2 (20 mL), washed with saturated NaHCO 3 (3 x 20 mL). The aqueous layer was extracted with CH 2Cl 2 (60 mL) and the combined organic phases were dried over

Na 2SO 4, filtered, concentrated and purified by column purification (hexanes/ EtOAc 3:2) to yield compound 12 (1.92 g, 79 %). Comparison of 1H-NMR with reported literature

values confirmed the identity of compound 12 .35

1 H-NMR (600MHz, CDCl 3) δ 1.86 (s, 3H, COCH 3), 2.02 (s, 3H, COCH 3), 2.13 (s, 3H,

3 COCH 3), 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,

H-2, H-6’), 4.53 (d, 1H, J 3 = 12 Hz, CHPh), 4.84 (d, 1H, J 3 = 12.6 Hz, CHPh), 5.17 (t,

158

1H, J 3 = 9.6 Hz, H-4) 5.37 (d, 1H, J 3 = 8.4 Hz, H-1), 5.77(dd, 1H, J 3 = 9.0, 10.2 Hz, H-

3), 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(OCH 3)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 Et 3N

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. 35

1 H-NMR (600MHz, CDCl 3) 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

Hz, CHPh), 4.63-4.67 (m, 1H, H-3), 4.84 (d, 1H, J 3 = 12 Hz, CHPh), 5.28 (d, 1H, J 3 =

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 CH 2Cl 2 (30 mL),

0 pyridine (0.7 mL, 8.54 mmol) was added and the mixture was cooled to -20 C. Tf 2O

(0.6 mL, 3.42 mmol) dissolved in CH 2Cl 2 (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 CH 2Cl 2 (20 mL) and then quenched with saturated NaCHO 3, washed with water, brine and dried over Na 2SO 4 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 %).

20 1 [α] D - 47.0 (c = 0.5, CH 2Cl 2); H-NMR (600MHz, CDCl 3) δ 2.07 (s, 3H, COCH 3),

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

(d, 1H, J 3 = 10.8 Hz, CHPh), 4.93 (d, 1H, J 3 = 12 Hz, CHPh), 5.61 (s, 1H, CHPh), 5.77-

5.78 (m, 1H, H-3), 6.15 (d, 1H, J 3 = 8.4, H-1), 7.21-7.84 (m, 14H, aromatic). HRMS

+ C30 H27 NO8Na [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/ CH 2Cl 2 (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 CH 2Cl 2 (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/CH 2Cl 2 (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 Et 3N 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 %).

20 1 [α] D - 64.0 (c = 0.5, CH 2Cl 2); H-NMR (600MHz, CDCl 3) 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); 19 F-NMR (282 MHz,

161

3 + CDCl 3) -194.44 (dt, 1F, J = 51.9, 13.8 Hz). HRMS C 21 H20 NaO 6NF [M + Na ] calc.

424.1167 found 424.1172.

Benzyl-3-deoxy-2-(methylcarbamoyl)benzoate-6-methoxy-4-oxo-β-glucopyranoside

(17)

20 1 3 [α] D - 51.4 (c = 0.5, CH 2Cl 2); H-NMR (600MHz, CDCl 3) 2.24 (dd, 1H, J = 4.2, 13.2

3 Hz, H-3), 2.48 (t, 1H, J = 13.2 Hz, H-3), 3.32 (s, 3H, OCH 3), 3.36 (s, 3H, OCH 3), 3.73-

3.74 (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

= 12 Hz, CHPh), 4.79 (d, 1H, J 3 = 12 Hz, CHPh), 5.33 (d, 1H, J 3 = 8.4 Hz, H-1), 7.08-

13 7.16 (m, 5H, aromatic), 7.70-7.84 (m, 4H, aromatic) ; C-NMR (150 Hz, CDCl 3) 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 C 23 H29 N2O7

+ [M + NH 4 ] 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

Ac 2O (6 mL) and stirred overnight at rt. The mixture was treated with EtOH to react with excess Ac2O, concentrated, dissolved in CH 2Cl 2, washed with water, brine and dried

162 over Na 2SO 4 followed by purification by column chromatography (hexanes/ EtOAc/

CH 2Cl 2 / MeOH, 2:1:1:0.2) to afford compound 18 . (0.27 g, 80 %).

1 H-NMR (600MHz, CDCl 3) δ 1.94 (s, 3H, NHCOCH 3), 2.09 (s, 3H, COCH 3), 2.10 (s,

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

4.27 (dd, 1H, J 3 = 5, 12.5 Hz, H-6’) 4.57 (d, 1H, J 3 = 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 3 = 7 Hz, CONH); 19 F-

3 NMR (282.2 MHz, CDCl 3) -193.26 (dt, 1F, J = 53.3, 12.4 Hz). HRMS C 19 H25 FNO7 [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 Ac 2O (0.5 mL) and stirred overnight at rt. The mixture was concentrated, washed with 1M HCl, saturated

NaHCO 3, brine and dried over Na 2SO 4. Column purification (hexanes/ EtOAc, 1.5:1) afforded compound 19 in 76 % (15 mg) yield over two steps.

20 1 [α] D + 10.1 (c = 1, CH 2Cl 2); H-NMR (600MHz, CDCl 3) δ 2.03 (s, 3H, NHCOCH 3),

2.09 (s, 3H, COCH 3), 2.11 (s, 3H, COCH 3), 2.16 (s, 3H, COCH 3), 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, H-

13 1); C-NMR (150 Hz, CDCl 3) 20.6, 20.7, 20.8, 23.2, 50.9, 50.96, 61.3, 68.1, 68.2, 69.5,

19 69.6, 88.97, 90.2, 90.9, 91.0, 168.4, 169.0,170.0, 170.7; F-NMR (282.2 MHz, CDCl 3) -

3 + 194.24 (dt, 1F, J = 50.8, 13.5 Hz, ). HRMS C 14 H24 N2O8F [M + NH 4 ] calc. 367.1517 found 367.1512.

164

APPENDIX

165

APPENDIX 3

Spectral Data

166

1 Figure 3.1 (600MHz, CDCl 3) H-NMR compound 2

O O O HO O N 2

8 7 6 5 4 3 2 1

167

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

8 7 6 5 4 3 2 1 0

168

1 Figure 3.3 (600MHz, CDCl 3) H-NMR compound 4

8 7 6 5 4 3 2 1 0

169

13 Figure 3.4 (150 MHz, CDCl 3) C-NMR compound 4

200 180 160 140 120 100 80 60 40 20

170

1 Figure 3.5 (500 MHz, CD 3OD) H-NMR compound 6

8 7 6 5 4 3 2

171

1 Figure 3.6 (600MHz, CDCl 3) H-NMR compound 7

8 7 6 5 4 3 2 1 0

172

1 Figure 3.7 (500MHz, CDCl 3) H-NMR compound 8

8 7 6 5 4 3 2 1

173

13 Figure 3.8 (150MHz, CDCl 3) C-NMR compound 8

174

1 Figure 3.9 (600MHz, CDCl 3) H-NMR compound 11

8 7 6 5 4 3 2 1 0

175

1 Figure 3.10 (500MHz, CDCl 3) H-NMR compound 12

8 7 6 5 4 3 2 1

176

1 Figure 3.11 (600MHz, CDCl 3) H-NMR compound 13

8 7 6 5 4 3 2 1

177

1 Figure 3.12 (600MHz, CDCl 3) H-NMR compound 14

8 7 6 5 4 3 2 1

178

1 Figure 3.13 (600MHz, CDCl 3) H-NMR compound 16

8 7 6 5 4 3 2 1

179

1 Figure 3.14 (600MHz, CDCl 3) H-NMR compound 17

8 7 6 5 4 3 2 1

180

1 Figure 3.15 (600MHz, CDCl 3) H-NMR compound 18

8 7 6 5 4 3 2 1 0

181

1 Figure 3.16 (600MHz, CDCl 3) H-NMR compound 19

8 7 6 5 4 3 2 1

182

13 Figure 3.17 (150MHz, CDCl 3) C-NMR compound 19

180 160 140 120 100 80 60 40 20

183

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184

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