Protecting Group-Free Chemical Modifications on Carbohydrates

by

Anna Valborg Guðmundsdóttir

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy

Graduate Department of Chemistry

University of Toronto

© Copyright by Anna Valborg Guðmundsdóttir 2009

ii

 

Abstract

Protecting Group-Free Chemical Modifications on Carbohydrates

Doctor of Philosophy, 2009

Graduate Department of Chemistry

University of Toronto

The synthesis of glycoconjugates has facilitated a wide variety of techniques for the

detailed study of carbohydrates and their interactions in biological systems. However,

when only small amounts of the isolated oligosaccharide are available, multistep synthetic

approaches are not possible. This thesis explores new synthetic methods for the

preparation of glycoconjugates without protecting group manipulations.

A new glycosidation method was developed which introduces N-

glycopyranosylsulfonohydrazides as glycosyl donors for the protecting group-free synthesis

of O-glycosides, glycosyl azides and oxazolines. The glycosyl donors were synthesized in

a single chemical step by condensing p-toluenesulfonylhydrazide with the corresponding

mono- and disaccharides. The N-glycopyranosylsulfonohydrazides were activated with

NBS and subsequently glycosylated with the desired alcohol or transformed to the

oxazoline or glycosyl azide.

iii

 

Recent advances in chemoselective ligation methods for the functionalization of

unprotected carbohydrates have provided new routes towards complex glycoconjugates.

Despite the wide use of those chemoselective methods, the properties of these linkages

have not been thoroughly investigated. Characterization of a series of glycoconjugates

formed by chemoselective ligation of xylose, glucose and N-acetylglucosamine with either

an acyl hydrazide, a p-toluenesulfonylhydrazide or an N-methylhydroxylamine were carried

out to gain further insight into the optimal conditions for the formation and the stability of

these useful conjugates. Their apparent association constants (9-74 M-1) at pD 4.5, as well

as rate constants for hydrolysis were determined at pH 4.0, 5.0 and 6.0. The half-lives of

the conjugates varied between 1 h and 300 days. All the compounds were increasingly

stable as the pH approached neutrality.

Finally, selective chemical modification of a glycosaminoglycan chondroitin sulfate was

attempted at the non-reducing end by utilizing the Δ4-uronic acid functional group formed

upon cleavage of the glycosaminoglycan with a bacterial lyase enzyme. The captodative

double bond of the unique Δ4-uronic acid functionality was unreactive towards Michael

addition, even if the carboxylate was methylated. Trials towards radical addition using

thiyl radicals were unsuccessful, although a synthesized model phenyl Δ4-uronic acid

monosaccharide was successfully functionalized under the same conditions.

iv

 

Heilræði

Oft er sá í orðum nýtur, sem iðkar menntun kæra,

en þursinn heimskur þegja hlýtur, sem þrjóskast við að læra.

Hallgrímur Pétursson (1614-1674)

v

 

Acknowledgements

A person does not obtain a Ph.D. by themselves. This journey has had its ups and downs,

lefts and rights which would not have been completed without the endless support,

motivation, inspiration and love from the extraordinary people that surrounded me every

day. There are thus quite a few individuals that I wish to thank sincerely for making this

dissertation possible.

First, I would like to thank my supervisor, Professor Mark Nitz for guiding me through this

rewarding experience. Since I arrived to UofT he has provided me with nothing but

endless support, encouragement and patience which have made this journey possible. Mark

is an incredible scientist with a brilliant mind which has an infinite passion for scientific

discoveries which he passes on to his students. Thank you Mark, I am forever grateful.

Secondly, I would like to thank the members of my advisory committee, Andrew Woolley

and Jik Chin for providing advice and suggestions towards my research. Deborah Zamble

also deserves special acknowledgments for her invaluable contributions and somewhat

being a fourth member of my advisory committee.

I would also like to acknowledge the past and present Nitzers that have contributed to this

thesis in one way or another. Joanna and our adopted member Svetlana, thank you for

being supportive through my laughter, my celebrations and my tears. You truly are great

friends that I am especially grateful for being able to get to know you and we will remain

close friends forever. Carmen, we have gotten to be extremely good friends during our

studies together. The lab just would not have been the same without you. Our common

clumsiness has brought many invaluable riots of laughter necessary to survive during our

studies, “mer you”. I was extremely lucky to get Caroline to work with me on my

chemoselective ligation project during her fourth year undergraduate project. She is a truly

genuine and smart girl who loves chocolate and preferably more chocolate. Thank you for

all your help with the project as well as proofreading my thesis, you are fabulous and I will

miss you. Urja, my brilliant little beauty, thanks for all the support and help. Grace and

Heather, thank you for everything. On to the boys, Rullo your insights and contributions

vi

 

have been invaluable as well as your great company and our common coffee breaks while

analyzing problems. Rodolfo, your mind is always on full speed and I have been grateful

to have access to it during my studies. Remember to enjoy life while you do your

chemistry, it is important for us to smile and laugh as we have discussed many times.

Chibba, my nitrogen boy, thanks for everything you have contributed towards my thesis.

Your establishment of Wednesday sandwich club really brought the group closer together

which was amazing. Mike, thanks for all your helpful discussions and suggestions. Past

members, thank you for your contributions, they are greatly acknowledged.

To all my Nitzers, thank you for your helpful discussions, advice and friendship which I

will remember, always. Thank you for everything, I am forever in debt. It is not often

during a lifetime that a person is so lucky to meet so many inspired and brilliant people that

love science and life. I will miss you all.

Elsku mamma og pabbi, takk fyrir að styðja við bakið á mér sama hvaða vitleysu ég hef

ákveðið að taka mér fyrir hendur. Ég hefði aldrei getað öðlast doktorsgráðu mína án ykkar

og ég vildi að þið vissuð hvað mér finnst þið frábær, æðisleg og bestust í heimi. Takk fyrir

að vera æðisleg amma og afi líka. Ég veit að þið bíðið spennt eftir að fá okkur öll heim

aftur til að geta snúist í kringum litla strákinn ykkar hann Gumma Leo. Elska ykkur, alltaf.

Elsku Rafn minn, ég hefði aldrei getað gert þetta án þín. Takk fyrir að vaka með mér þegar

ég hef þurft að vinna lengi fram á nótt, takk fyrir að styðja mig í gegnum súrt og sætt og

mest af öllu takk fyrir að elska mig eins og ég er. Þú hefur hugsað svo vel um litla

drenginn okkar síðustu tvö árin alveg frá því hann var 8 vikna á meðan ég var í skólanum.

Ég elska þig ástin mín.

Elsku litli drengurinn minn, Guðmundur Leo. Þakka þér fyrir að vera alltaf yndislegur og

elska mömmu þína. Eftir erfiðan vinnudag, var aldrei eins gott að koma heim og fá knús og

kossa og þetta ljómandi bros sem lét öll vandamál og áhyggjuefni gleymast á svipstundu.

Ég elska þig meira en allt í heiminum og geiminum.

Þakka verð ég bræðrum mínum tveimur, Tómasi og Jóa Baldri. Ef allir væru jafn heppnir

og ég að eiga tvo yndislega bræður. Einnig mágkonu minni henni Örnu ásamt æðislegu

vii

 

frændum mínum þeim Baldri Erni, Óðni Erni og Hreiðari Erni. Sigga Björg frænka, takk

fyrir allar heimsóknirnar og stuðninginn.

Amma og afi í Bólstaðarhlíðinni, þið eruð yndisleg. Amma einn minn besti vinur, takk

fyrir að stappa í mig stálinu þegar ég hef þurft þess. Amma Valborg fyrir að fylgja mér í

anda og vernda okkur hvern einasta dag. Einar frændi, takk fyrir stuðninginn hann hefur

reynst okkur ómetanlegur.

Einnig má ég nú ekki gleyma hinum fjölskyldumeðlimunum sem hafa stutt við bakið á

okkur meðan á námi mínu stóð. Guðrún og Siggi, Júlli og Gunna, Greipur, Karó, Hemmi,

Júlli og Guðrún Ósk takk fyrir heimsóknirnar, stuðninginn og góðu stundirnar sem við

höfum átt saman, þið eruð frábær, takk fyrir allt.

Inga Dóra, takk fyrir að vera yndislegur vinur í raun, get ekki beðið eftir að geta kíkt í

heimsókn og eytt tíma með þér, Sverri og Rúnu Björg. Elsku Elfa og Eyrún takk fyrir að

vera alltaf til staðar. Anna Guðný, takk fyrir stuðninginn, vinskapinn og alla hjálpina sem

þú hefur veitt mér meðan á námi mínu stóð. Aðrir vinir og vandamenn, takk fyrir að vera

til staðar fyrir mig og fjölskyldu mina.

viii

 

Doktorsritgerð mín er tileinkuð ömmum mínum, Önnu Jónínu

Þórarinsdóttur og Valborgu Hjartardóttur (04.06.1918 - 20.12.2002).

-- Tvær einstakar konur sem ég mun ávallt elska og heiðra --

ix

 

Table of Contents

Abstract ...................................................................................................................... ii 

Acknowledgements .................................................................................................... v 

Table of Contents ..................................................................................................... ix 

List of Abbreviations ............................................................................................... xii 

List of Tables .......................................................................................................... xvi 

List of Figures ........................................................................................................ xvii 

List of Schemes ........................................................................................................ xx 

List of Appendices ................................................................................................ xxiii 

Chapter 1. Protecting Group-Free Modifications on Carbohydrates .................. 1 

1.1 Biological importance of carbohydrates ....................................................................... 1 

1.2 Preparation of carbohydrates for biological studies ..................................................... 6 

1.3 Protecting group-free O-glycosidations ........................................................................ 8 1.3.1 Fischer-type glycosidations ............................................................................................... 8 

1.4 Glycosidations using unprotected donors ................................................................... 11 

1.4.1 Unprotected 3-methoxypyridyl glycosyl donors ............................................................. 11 

1.4.2 Miscellaneous methods ................................................................................................... 14 

1.5 Formation of N-glycosides using unprotected donors ................................................ 14 

1.6 Protecting group-free chemoselective ligation ........................................................... 16 

1.7 Chemical modifications on unprotected glycosaminoglycans .................................... 19 

1.8 Scope of projects ......................................................................................................... 21 

x

 

Chapter 2. Stability Studies of Hydrazide- and Hydroxylamine-Based

Glycoconjugates in Aqueous Solution ................................................................... 23 

2.1 Introduction ................................................................................................................. 23 

2.2 Results and discussion ................................................................................................ 24 

2.2.1 Glycoconjugates investigated .......................................................................................... 24 

2.2.2 Synthesis of N-methylhydroxylamine nucleophiles ........................................................ 25 

2.2.3 Synthesis of glycoconjugates .......................................................................................... 27 

2.2.4 Glycoconjugate hydrolysis .............................................................................................. 28 

2.2.5 Hydrolysis rates ............................................................................................................... 30 

2.2.6 Factors affecting hydrolysis rates .................................................................................... 33 

2.3 Conclusions ................................................................................................................. 39 

2.4 Future directions ......................................................................................................... 40 

Chapter 3. Protecting Group-Free Glycosidations using p-

Toluenesulfonohydrazide Donors .......................................................................... 45 

3.1 Introduction ................................................................................................................. 45 

3.2 Results and discussion ................................................................................................ 46 

3.2.1 Glycosyl donor formation ............................................................................................... 46 

3.2.2 Method optimization ....................................................................................................... 47 

3.2.3 Mechanistic considerations ............................................................................................. 50 

3.2.4 Formation of O-glycosides .............................................................................................. 51 

3.2.5 Formation of other glycosides ......................................................................................... 53 

3.2.6 Glycosidation on oligosaccharides .................................................................................. 56 

3.3 Conclusion .................................................................................................................. 57 

3.4 Future directions ......................................................................................................... 58 

Chapter 4. Chemical Modifications on Unprotected Glycosaminoglycans ....... 62 

4.1 Introduction ................................................................................................................. 62 

4.2 Results and discussion ................................................................................................ 62 

xi

 

4.2.1 Preparation and purification of Δ4-uronic acid chondroitin sulfate disaccharide ............ 62 

4.2.2 Synthesis of a model Δ4-uronic acid ................................................................................ 67 

4.2.3 Radical addition to the synthesized model Δ4-uronic acid .............................................. 69 

4.2.4 Effort towards the radical addition onto isolated Δ4-chondroitin sulfate disaccharide free

hemiacetal ................................................................................................................................. 70 

4.2.5 Effort towards the radical addition onto Δ4-chondroitin sulfate disaccharide protected at

the anomeric center .................................................................................................................. 72 

4.2.6 Effort towards a Michael addition to methyl ester of N-methyl-O-octyl hydroxylamine

Δ4-chondroitin sulfate disaccharide .......................................................................................... 75 

4.2.7 Efforts towards modifying the Δ4-chondroitin sulfate disaccharide using electrophiles . 75 

4.3 Conclusion .................................................................................................................. 77 

4.4 Future directions ......................................................................................................... 78 

Chapter 5. Experimental ........................................................................................ 81 

5.1 General methods ......................................................................................................... 81 5.1.1 Hydrolysis method for glycoconjugates 1-4 .................................................................... 81 

5.1.2 Hydrolysis method for glycoconjugates 13-21 ................................................................ 82 

5.1.3 Determination of equilibrium constants, Ka, for glycoconjugates 13-21 ........................ 82 

5.2 Procedures ................................................................................................................... 83 

References .............................................................................................................. 109 

Appendix A ............................................................................................................ 117 

Appendix B ............................................................................................................ 125 

xii

 

List of Abbreviations

AcOH Acetic acid

4-BBA 4-Benzoylbenzoic acid

Boc tert-Butyloxycarbonyl

ºC Degrees Celsius

d Day(s)

DBU Diazabicyclo(5.4.0)undec-7-ene

DCC Dicyclohexylcarbodiimide

DIPEA Diisopropylethylamine

DMF Dimethylformamide

DMSO Dimethylsulfoxide

DNA Deoxyribonucleic acid

E. coli Escherichia coli

EtOAc Ethyl acetate

FRET Fluorescence resonance energy transfer

Fuc L-Fucose

GAG Glycosaminoglycan

Gal D-Galactose

GalNAc N-Acetyl-D-galactosamine

gCOSY Gradient correlation spectroscopy

xiii

 

Glc D-Glucose

GlcN D-Glucosamine

GlcNAc N-Acetyl-D-glucosamine

GPI Glucosylphosphatidylinositol

GSH N-glycosylsulfonohydrazide

1H/13C NMR Proton/Carbon nuclear magnetic resonance

h Hour(s)

HAase Hyaluronidase

HIV Human immunodeficiency virus

HMPA Hexamethylphosphoramide

HOBt N-hydroxybenzotriazole

HPLC High pressure liquid chromatography

HRMS High resolution mass spectrometry

Hz Hertz

IdoUA L-Iduronic acid

IPTG Isopropyl-β-D-thiogalactopyranoside

m/z Mass per charge

Ka Equilibrium association constant

KLH Keyhole limpet hemocyanin

Man D-Mannose

xiv

 

MeOH Methanol

MeOTf Methyl trifluoromethanesulfonate

MHz Mega hertz

min Minute(s)

MOP 3-Methoxypyridyl

MWCO Molecular weight cut off

NBS N-Bromosuccinimide

NeuNAc N-Acetylneuraminic acid

NIS N-Iodosuccinimide

NOE Nuclear overhauser effect

NOESY Nuclear overhauser enhancement spectroscopy

O-GSH N’-(2-acetamido-2-deoxy-β-D-glucopyranosyl)octylsulfonohydrazide

OSH Octylsulfonylhydrazide

p-TSH p-Toluenesulfonylhydrazide

p-TSOH p-Toluenesulfonic acid

PI Phosphatidylinositol

PNAG Poly-β-(1 6)-N-acetylglucosamine

Pbu3 Tributylphosphine

ppm Parts per million

Pyr Pyridine

xv

 

RNA Ribonucleic acid

rt Room temperature

SAX Strong anion exchange

t1/2 Half-life

TBA-Cl Tetrabutylammonium chloride

TBAN3 Tetrabutylammonium azide

TEA Tetraethylammonium

TEMPO 2,2,6,6-Tetramethylpiperidine-1-oxyl

T-GSH N’-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-p-

toluenesulfonohydrazide

TFA Trifluoroacetic acid

THF Tetrahydrofuran

TMSN3 Trimethylsilyl azide

TMSOTf Trimethylsilyl trifluoromethanesulfonate

UA D-Glucuronic acid

V-50 2,2’-Azobis(2-methylpropionamidine)

V-501 4,4’-Azobis(4-cyanovaleric acid)

Xyl D-Xylose

xvi

 

List of Tables

Table 2.1 Equilibrium constants for the formation of glycoconjugates 13-21......... 28 

Table 2.2 Half-lives in aqueous solution of glycoconjugates 1-4 and 13-21 ........... 33 

Table 3.1 Formation of methyl glycoside using GSH donors 19 and 23 under different conditions ................................................................................................... 49 

Table 3.2 Yields and selectivities for O-glycosidations ........................................... 52 

Table 4.1 Conditions for radical reaction attempts using a mixture of chondroitin sulfate Δ4-uronic acid disaccharides 48 and 49 ......................................................... 71 

xvii

 

List of Figures

Figure 1.1 Structure of the N-linked core pentasaccharide connected to the peptide consensus sequence. .................................................................................................... 2 

Figure 1.2 Structure of the tetra-antennary core pentasaccharide isolated from human granulocytes. The box indicates a sialyl Lewisx tetrasaccharide. ................... 2 

Figure 1.3 Structure of a mucin type O-linked glycan linked to Ser/Thr residue on protein.......................................................................................................................... 3 

Figure 1.4 Structure of the four major classes of GAGs. ........................................... 4 

Figure 1.5 Structure of the GPI anchor. ..................................................................... 5 

Figure 1.6 Structure of the N-linked glycan Man9GlcNAc2 which can be isolated from soybean agglutinin. ............................................................................................. 7 

Figure 1.7 Remote activation concept. ..................................................................... 12 

Figure 1.8 Proposed mechanism for the three component Staudinger ligation using unprotected glycosyl azide. ....................................................................................... 16 

Figure 1.9 Maltotriose conjugated to aminooxy somatostatin analogue RC-160. ... 18 

Figure 2.1 Glycoconjugates synthesized using different para-substituted benzoylhydrazides. .................................................................................................... 24 

Figure 2.2 Glycoconjugates synthesized using p-toluenesulfonylhydrazide and N-methylhydroxylamines. ............................................................................................. 25 

Figure 2.3 1H NMR (400 MHz, D2O) spectra showing degradation of glycoconjugate 16 to p-toluenesulfonylhydrazide 5, p-toluenesulfinic and p-toluenesulfonic acid. ................................................................................................. 29 

Figure 2.4 Hydrolysis of p-toluenesulfonylhydrazide 5 at 37 °C at pH 4.0-6.0. ..... 30 

Figure 2.5 Hydrolysis of 5 mM N-(β-D-xylopyranosyl)-p-toluenesulfonohydrazide 13 in 20 mM NaOAc (0.5% DMSO) pH 4.0 at 37 °C. ............................................. 31 

Figure 2.6 Hydrolysis of 2 mM N-(β-D-glucopyranosyl)benzoylhydrazide 1 in 50 mM NaOAc (5% MeOH) pH 4.0 at 50 °C. ............................................................... 32 

xviii

 

Figure 2.7 pH rate profile for glycoconjugates 1-4 and 13-21. ................................ 36 

Figure 2.8 Hydrolysis of N-methyl-O-benzyl-N-(β-D-glucopyranosyl)hydroxylamine 17 at pH 4.0 and 37 °C using different buffer concentrations. .......................................................................................................... 37 

Figure 2.9 Hydrolysis of N-methyl-O-benzyl-N-(β-D-glucopyranosyl)hydroxylamine 17 at pH 6.0 and 37 °C using different buffer concentrations. .......................................................................................................... 38 

Figure 2.10 Hydrolysis of N-(β-D-glucopyranosyl)benzoylhydrazide 1 at pH 4.0 and 50 °C using different buffer concentrations. ...................................................... 39 

Figure 2.11 The antibody 2G12 recognizes the terminal high mannose structure of its epitope structure Man9GlcNAc2. .......................................................................... 41 

Figure 3.1 1H NMR (400 MHz, D2O) of first glycosidation attempt using T-GSH donor 19 to form methyl 2-acetamido-2-deoxy-D-glucopyranoside. ....................... 48 

Figure 3.2 Lactone hydrazone structure of GSH donors 19 and 23. ........................ 50 

Figure 3.3 Support for chloride 2-acetamido-2-deoxy-α-D-glucopyranoside 39b .. 54 

Figure 3.4 Support for formation of oxazoline 40. .................................................. 55 

Figure 3.5 Oligosaccharide GSH donors 42-44 and methyl oligosaccharide glycosides 45-47. ....................................................................................................... 56 

Figure 3.6 Aliphatic region of the 1H NMR (400 MHz, D2O) of methylated PNAG oligosaccharides. ....................................................................................................... 57 

Figure 4.1 Two chondroitin sulfate disaccharide isomers, 48 and 49, formed upon cleavage of chondroitin sulfate polysaccharide with Chondroitinase AC. ............... 63 

Figure 4.2 HPLC chromatogram of separation of the two Δ4-uronic acid chondroitin sulfate isomers using a SAX column. ....................................................................... 64 

Figure 4.3 1H NMR (400 MHz, D2O) of Δ4-uronic acid chondroitin sulfate disaccharide 4-SO3

- 48. ............................................................................................. 65 

Figure 4.4 1H NMR (400 MHz, D2O) of Δ4-uronic acid chondroitin sulfate disaccharide 6-SO3

- 49. ............................................................................................. 65 

xix

 

Figure 4.5 gCOSY (400 MHz, D2O) of Δ4-uronic acid chondroitin sulfate disaccharide 4-SO3

- isomer 48. ................................................................................. 66 

Figure 4.6 gCOSY (400 MHz, D2O) of Δ4-uronic acid chondroitin sulfate disaccharide 6-SO3

- isomer 49. ................................................................................. 67 

Figure 4.7 Aliphatic region of the 1H NMR spectrum (500 MHz, d4-MeOH) of phenyl 4-(2-N-acetylethylthio)-β-D-galactopyranuronic acid 58. ............................ 70 

Figure 4.8 Proposed mechanism for the degradation of bromohydrin derivatized lyase-cleaved GAG. .................................................................................................. 76 

Figure 4.9 Internally quenched fluorescent hyaluronidase substrates. .................. 80 

xx

 

List of Schemes

Scheme 1.1 Fischer glycosidation of D-glucose to produce methyl glucoside. ......... 9 

Scheme 1.2 Key features of the Fischer glycosidation. .............................................. 9 

Scheme 1.3 Modified Fischer glycosidation methods. ............................................. 10 

Scheme 1.4 Glycosylation of unprotected and unactivated glycosyl donors in ionic liquids. ....................................................................................................................... 11 

Scheme 1.5 Glycosylation using unprotected MOP glycosyl donor. ....................... 12 

Scheme 1.6 Preparation of unprotected α-MOP glycosyl donors. ........................... 13 

Scheme 1.7 Electrochemical glycosylation method. ................................................ 14 

Scheme 1.8 The Kochetkov reaction. ....................................................................... 15 

Scheme 1.9 Synthesis of unprotected β-D-glycosyl azide through 1,2-cyclic sulfite intermediate. .............................................................................................................. 15 

Scheme 1.10 Reductive amination between a free hemiacetal and an amine. ......... 17 

Scheme 1.11 Formation of glycoconjugates using chemoselective methods........... 19 

Scheme 1.12 Formation of the Δ4-uronic acid functional group at the non-reducing terminus upon cleavage of chondroitin sulfate with Chondroitin AC lyase. ............ 20 

Scheme 1.13 Functionalization of the Δ4-uronic acid by thiols on gold surface using oxymercuration. ........................................................................................................ 21 

Scheme 2.1 Synthesis of O-benzyl-N-methylhydroxylamine 6. .............................. 26 

Scheme 2.2 Synthesis of N-methyl-O-(N’-benzylacetamide)hydroxylamine 7. ...... 26 

Scheme 2.3 Equilibrium association constant determined for glycoconjugates 13-21.......................................................................................................................... 27 

Scheme 2.4 Hydrolysis products observed in 1H NMR from hydrolysis of p-toluenesulfonohydrazide conjugates. ........................................................................ 29 

xxi

 

Scheme 2.5 Putative mechanism for the hydrolysis of N-alkylhydroxylamine glycoconjugates. ........................................................................................................ 36 

Scheme 2.6 Proposed synthesis of a N-methylhydroxylamine β-alanine based-linker to use in development of an HIV vaccine. ................................................................ 42 

Scheme 2.7 Simplified preparation of a carbohydrate-based HIV vaccine utilizing a N-methylhydroxylamine β-alanine linker. ................................................................ 43 

Scheme 3.1 Formation and structure of the GSH donors. ........................................ 46 

Scheme 3.2 Proposed mechanism of GSH activation. ............................................. 51 

Scheme 3.3 Formation of glycosyl azide 41 via oxazoline 40. ................................ 53 

Scheme 3.4 Using GSH donors in protecting group-free glycosidation to provide glycosyl azide and oxazoline needed in the synthesis of homogeneous glycopeptides or glycoproteins. ........................................................................................................ 59 

Scheme 3.5 Synthesis of a modified PNAG using the T-GSH donor and a modified N-acetylglucosamine residue. ................................................................................... 60 

Scheme 4.1 Proposed radical addition of a thiol to a Δ4-uronic acid chondroitin sulfate disaccharide. .................................................................................................. 62 

Scheme 4.2 Synthesis of the model Δ4-uronic acid 57. ............................................ 68 

Scheme 4.3 Radical additions to phenyl Δ4-uronic acid 57 using N-acetylcysteamine and cysteamine. ......................................................................................................... 69 

Scheme 4.4 Synthesis of N-methyl-O-octylhydroxylamine 62. ............................... 73 

Scheme 4.5 Synthesis of octylsulfonohydrazide and N-methyl-O-octylhydroxylamine chondroitin sulfate Δ4-uronic acid 60 and 63, respectively. .... 73 

Scheme 4.6 Products observed in mass spectrometry following reverse phase purification after radical addition on Δ4-chondroitin sulfate disaccharide octylsulfonohydrazide 60 with cysteamine. .............................................................. 74 

Scheme 4.7 Synthesis of N-methyl-O-octylhydroxylamine functionalized chondroitin sulfate disaccharide methyl ester 64. ..................................................... 75 

xxii

 

Scheme 4.8 Iodoalkoxylation of the N-methyl-O-octylhydroxylamine chondroitin sulfate disaccharide 65. ............................................................................................. 76 

Scheme 4.9 Chemoenzymatically functionalizing the Δ4-uronic acid with a thiol. . 78 

xxiii

 

List of Appendices

Appendix A Hydrolysis data for glycoconjugates in Chapter 2………………… 117 

Appendix B Selected NMR spectra……………………………………………... 125

1

 

Chapter 1. Protecting Group-Free Modifications on

Carbohydrates

1.1 Biological importance of carbohydrates

Nucleic acids, proteins and carbohydrates are the three major classes of naturally occurring

biopolymers which are responsible for the majority of biological processes. The DNA

contains the genetic code which stores information from generation to generation

functioning as the recipe for life.1,2 This information is then translated via RNA into

proteins,3 which carry out most of the duties inside the cell. The proteins can be post-

translationally modified by truncation or by the covalent addition of groups like phosphates

or carbohydrates.4 Glycosylation of proteins located at the cell’s surface has been shown to

mediate intercellular communication important for biological processes such as

immunological response, metastasis and fertilization.5,6,7

Generally, glycans are present on the cell surface in the form of glycoproteins and

glycolipids. There are four major classes of glycans in mammals: N-linked glycans, O-

linked glycans, glycolipids and glycosylphosphatidylinositol (GPI) anchors.

The N-linked glycans contain a reducing terminal 2-acetamido-2-deoxy-β-D-

glucopyranosyl (GlcNAc) residue which is covalently linked to the side chain amide

nitrogen of an asparagine amino acid. This asparagine residue is part of a consensus

sequence which consists of Asn-Xaa-Ser/Thr, where Xaa can be any amino acid other than

proline.8,9 Since this sequence occurs commonly in proteins which lack sugars, its presence

is not sufficient for glycosylation.10 The reducing terminal N-acetylglucosamine residue is

a component of the core pentasaccharide structure, Man3GlcNAc2, found in all N-linked

glycans (Figure 1.1).

2

 

O

NHAc

OHOHO

HN

CO

HN

C OHN Xaa Ser/ThrOH

OHOO

HOO

OOHHO

HOHO

OHO

HOHO

O

NHAc

OHOHO

Figure 1.1 Structure of the N-linked core pentasaccharide connected to the peptide consensus sequence.

This core is commonly elaborated further to tetra-antennary structures and, rarely, to penta-

antennary structures.11 Illustrating the diversity of these glycan structures is an

oligosaccharide which is present in human granulocytes (Figure 1.2).12 This glycan is

capped with the sialyl Lewisx structure, which has been implicated in leukocyte

recruitment.13

NeuNAcα2 3Galβ1 4GlcNAcβ1

L-Fuc 1

33Galβ1 4GlcNAcβ1 3Galβ1 4GlcNAcβ1

Galβ1 4GlcNAcβ1 3Galβ1 4GlcNAcβ1 3Galβ1 4GlcNAcβ1

6 Manα12

2 Manα14

3

1L-Fuc

6 Manβ13

4GlcNAcβ1 4GlcNAcβ1 Asn

NeuNAcα2 6Galβ1 4GlcNAcβ1 3Galβ1 4GlcNAcβ1 3Galβ1 4GlcNAcβ1

Galβ1 4GlcNAcβ1 3Galβ1 4GlcNAcβ1 3Galβ1 4GlcNAcβ1

α

α 3

1L-Fuc

α 3

1L-Fuc

α

L-Fuc 1

6α

Figure 1.2 Structure of the tetra-antennary core pentasaccharide isolated from human granulocytes. The box

indicates a sialyl Lewisx tetrasaccharide.

In contrast to N-linked glycans, O-linked glycans are based on a number of different cores,

depending on the nature of the sugar and amino acid residues involved in the linkage

between the carbohydrate and the protein. The most abundant form of O-linked glycans in

higher eukaryotes are termed "mucin-type" which is characterized by an α-N-

3

 

acetylgalactosamine (GalNAc) attached to the hydroxyl group of Ser/Thr side chains

(Figure 1.3).14

O

AcHN

O

HO

OO H (CH3)

HN

NH O

NHAc

OOH

OHO

O

OH

OHOH

OOHO

AcHN

CO2-OH OH

HO

OHO

AcHN

CO2-OH OH

HO O

OH

OHOH

O

Figure 1.3 Structure of a mucin type O-linked glycan linked to Ser/Thr residue on protein.

Mucin-type O-linked glycans are located on membranes or as secreted mucins. The sugar

component of mucins typically comprises more than 50% of their molecular weight. The

O-linked glycans have been receiving increasing attention for their potential use in

immunotherapy for certain cancers, since it is believed they express many tumour-

associated epitopes.15

The glycosaminoglycans (GAGs) are a family of mucin-type O-linked glycans which are

characteristically long, unbranched, highly charged acidic polysaccharides commonly

found in the extracellular matrices. Four major classes of GAGs have been identified:

heparan sulfate/heparin, chondroitin sulfate/dermatan sulfate, hyaluronan and keratan

sulfate. These high-molecular weight polysaccharides consist of a backbone of repeating

disaccharide units of an amino sugar and uronic/iduronic acids, with varying sulfation

patterns (Figure 1.4).16

4

 

O

OR

-OOC

O HO NHX

OOR

ORO O

OH O

RO

-OOC

O

O

XHNRO

OR

O

O

OR

-OOC

O HOO

AcHN

OR

OOR

O

OH

-OOC

O HO

O

NHAc

HOO

OHO

O O

NHAcO HO

O

OH

OH

OOR

OOR

Heparan sulfate/Heparin[-4)-β-GlcUA/α-IdoUA-(1 4)-α-GlcNX-(1-]n

R = SO3- or H

X = SO3-, COCH3 or H

Chondroitin/Dermatan sulfate[-4)-β-GlcUA/α-IdoUA-(1 3)-β-GalNAc-(1-]n

Keratan sulfate[-4)-β-GlcNAc-(1 3)-β-Gal-(1-]n

Hyaluronan[-4)-β-GlcUA-(1 3)-β-GlcNAc-(1-]n

n

n n

n

Figure 1.4 Structure of the four major classes of GAGs.

Chondroitin, dermatan and heparan sulfate are most commonly O-linked to serine or

threonine side chains through a trisaccharide: GAG-(1 3)-α/β-Gal-(1 3)-β-Gal-(1 3)-β-

Xyl, where xylose is attached to the side chain of the amino acid at the reducing terminus,

while the non-reducing terminal galactose is linked to the corresponding GAG.

The GAGs interact with a wide variety of proteins, including growth factors and

chemokines which regulate many important physiological processes, as well as being

exploited by infectious pathogens to gain access and entry into animal cells.16

Glycolipids13,16 are amphipathic in nature, given that they have a hydrophilic mono- or

oligosaccharide covalently attached to a hydrophobic component. The major core structure

of glycolipids is a ceramide unit which is glycosylated with either a β-D-gluco- or a β-D-

galactopyranoside. Galactosylceramide have a tissue-specific distribution and are

predominantly found in myelin in the nervous system while the glucosylceramides are

present in all mammalian tissues and cells. The glucosylceramides usually have a galactose

residue added on, giving lactosylceramide, which is further elaborated to give more

complex structures such as the gangliosides, in addition to the blood or the Lewis group

5

 

determinants. The gangliosides are more complex glycosphingolipids which contain one or

more residues of N-acetylneuraminic acid (NeuNAc) and have been identified as targets for

cancer immunotherapy.17

The GPI anchors however are characterized by a common core structure (Figure 1.5). They

contain an unusual non-acetylated glucosamine residue which is glycosidated with

phosphatidylinositol through C-6 on the inositol ring. The glucosamine residue is

glycosylated with three mannose residues giving rise to the phosphatidylinositol (PI)

tetrasaccharide Man-(1 2)-α-Man-(1 6)-α-Man-(1 4)-β-GlcN-(1 6)-PI. The non-

reducing terminal mannose residue has a phosphoethanolamine at the C-6 position which

links the GPI to a wide variety of proteins. These GPI-linked proteins are extremely

diverse and carry out different tasks; however, the physiological function of the GPI anchor

itself is still under investigation.16

PO

OR3

HOHO

R2O

OR3R3

O

OR2

O

R3R3 OHO

H2NO

HOO

HOHO

O

OH

P OO

O

6

1

OOO

R1

Phosphatidylinositol

Phosphoethanolamine

R1 = Fatty acid or OHR2 = Phosphoethanolamine or OHR3 = Carbohydrate substituents or OH

HNProtein

O

H2N

R1

R1

Figure 1.5 Structure of the GPI anchor.

6

 

1.2 Preparation of carbohydrates for biological studies

Investigations into the biological roles of carbohydrates have led to an increased demand

for pure oligosaccharides and glycoconjugates for further study. The biosynthesis of

carbohydrates is not a template driven process, as is the case for nucleic acids and

polypeptides, but rather a common co- or post-translational modification. Thus, no

amplification system is available for their preparation and the oligosaccharides must be

obtained from either natural or synthetic sources.

Immense progress has been made in the synthetic methods available to generate complex

carbohydrates. These methods have enabled the stereocontrolled synthesis of glycans as

large as the recently reported docosanasaccharide.18 However, chemical synthesis of

complex glycans is still a difficult task as it involves extensive protecting group

manipulations and lengthy synthetic schemes. In order to simplify the synthetic scheme,

enzymes have become an attractive alternative for the construction of complex glycans.

Glycosyltransferases and glycosidases can be utilized to form stereo- and regiospecific

glycosidic linkages in oligosaccharide structures giving rise to homogeneous glycan

structures.

Isolating glycans from biological sources circumvents the tedious and time-consuming

chemical synthesis of the glycans. However, in biological systems glycoproteins are

produced as heterogeneous mixtures of glycoforms and isoforms which must be separated

from one another chromatographically. Procedures for the isolation of homogeneous N-

linked glycans have been developed using highly N-glycosylated glycoproteins from

soybeans19 and hen egg yolk.20 The asparagine-linked glycan structure of soybean

agglutinin is homogeneous, and has been used as a source of the mannose glycan

Man9GlcNAc2 (Figure 1.6).21

7

 

OOHOHOHO

OOHOHOHO

O

OHOHOHOHO

O

NHAc

OHOHO

HN

OOHO

HO

OOH

O

HO

O

NHAc

OHOHO

OOHO

HOHO

OOHHO

HOHO

OO

HOHO

HO

OHO

HOHO

HO

O

O

Figure 1.6 Structure of the N-linked glycan Man9GlcNAc2 which can be isolated from soybean agglutinin.

Isolation of homogeneous O-glycans from natural sources is more challenging than for N-

glycans since they show a higher degree of structural diversity and do not share a single

common core structure. Sufficient quantities of these native homogeneous O-glycans for

both structural and functional studies are therefore generally obtained through chemical

and/or enzymatic synthesis.22

GAGs are abundant biomolecules located in the extracellular matrix of animal cells as

proteoglycans. They can be isolated in large quantities from various sources such as the

mucous and connective tissues of mammals.23 Chondroitin sulfate, heparan sulfate,

dermatan sulfate and hyaluronan are easily obtained and are commercially available in

semi-purified forms of the major biologically represented structures.

Given that these glycan structures are accessible from natural sources, new methods are

required for their manipulations to obtain functionalized carbohydrate moieties for

biological studies. The general strategies for their functionalization include O- and N-

glycoside formation. Most methods involve multiple protection and deprotection steps

which are often not applicable to the minute amounts of glycans acquired from natural or

chemoenzymatic sources. Therefore, access to a novel synthetic method to functionalize

mono- or oligosaccharides without the use of protecting groups would be extremely

valuable. This would facilitate access to the glycoconjugates needed for structural

determination and biological investigations. Chemoselective ligation provides an alternate

8

 

route to functionalize unprotected glycans, where a non-natural glycoconjugate is formed.

This method is extremely useful although information about the stability of the non-natural

linkage must be obtained before allowing the discovery of new targets for therapeutics,

diagnostics and vaccines.

1.3 Protecting group-free O-glycosidations

Obtaining pure glycans of defined structure is crucial for investigating biological functions.

Glycosidations on oligosaccharides which have been obtained in small quantities can be

difficult since protection-deprotection strategies may not always be feasible. It is important

to have access to glycosidation methods which can successfully form glycosides

stereospecifically under mild conditions from simple as well as complex glycans without

protecting group manipulations.

The preparation of O-glycosides, without the use of protecting groups, has its advantages as

well as its shortcomings which have to be addressed.24 The benefits include fewer

synthetic steps and the prospect for milder reaction conditions since unprotected donors are

more reactive than donors with electron-withdrawing protecting groups. However, using

unprotected glycosyl donors also brings forward challenges such as controlling selectivity

so that the glycosyl donor only reacts with the desired hydroxyl group on the acceptor

while avoiding self-condensation. In addition, obtaining good stereoselectivity is more

difficult due to the absence of neighboring participating groups.

1.3.1 Fischer-type glycosidations

The first report of a protecting group-free glycosidation was published in 1893, when

Fischer’s group reported the acid-catalyzed condensation reaction between glucose and

methanol to yield methyl α-D-glucopyranoside in what is now commonly known as the

Fischer glycosidation (Scheme 1.1).25

9

 

O

OH

HO

OHHOHO

D-Glucopyranoside

MeOH

HClO

HO

HO

OCH3HO

HO+O

HO

HO

OCH3

HOHO

Methyl-β-D-glucopyranosideMethyl-α-D-glucopyranoside66% 33%

Scheme 1.1 Fischer glycosidation of D-glucose to produce methyl glucoside.

The reaction is an equilibrium process and can lead to a mixture of furanose and pyranose

isomers in addition to α‐ and β-glycosides, plus, in some cases, small amounts of acyclic

forms (Scheme 1.2).26 With hexoaldoses, short reaction times usually yielded furanosides

while longer reaction times led to pyranosides. With longer reaction times the most

thermodynamically stable product will result, which owing to the glycoside effect, is

usually the alpha glycoside for common hexoaldoses.

OHO

HO OH

OH

OH

OHHO

HO OH

O

OH

MeOH OHHO

HO OHOH

OH

OMe

OHO

HO OH

OMe

OH

O

HO

HOHO

OH

OMekf

kp

All reactions are reversible: kf >> kp

Scheme 1.2 Key features of the Fischer glycosidation.

It was not until 1991 that Lewis acids were introduced as catalysts for the Fischer

glycosidation. When D-glucose and D-galactose were treated with ferric chloride (FeCl3) in

MeOH, the corresponding methyl glycofuranosides were formed exclusively, in

approximately 75% yield (Scheme 1.3).27 The ferric ions lead to the kinetic furanosides

products instead of the thermodynamically favoured pyranosides.

10

 

O

OH

HO

OHHOHO MeOH

FeCl3 OHO

HOHO

OH

OMe

O

OH

R'

OHHOHO ROH (1.5 equiv)

FeCl3 (3 equiv)O

HO

HOR'

OH

ORDioxane or THF

R = alkylR' = CH2OH or CO2H

O

OH

R'

OHHOHO

ROH (1.5 equiv)BF3 OEt2 (3 equiv)

R = alkylR' = CH2OH or CO2H

THF

.O

OH

R'

ORHOHO

75%α:β, 1:2

67-73%α:β, 1:1.4-1:9

61-93%α:β, 10:1-24:1

Scheme 1.3 Modified Fischer glycosidation methods.

Improvements to the previously mentioned method were made a few years later by

Plusquellec and co-workers,28 which introduced dioxane and THF as solvents with near

stoichiometric amounts of alcohol (1.5 equiv) that could generate O-glycosides of D-

glucose, D-galactose, D-mannose and even D-galacturonic acid. The methyl furanosides

were isolated when ferric chloride was used as the promoter in the reaction. However,

substitution of the ferric chloride with BF3⋅OEt2 in THF yielded predominantly the α-

glycopyranoside (Scheme 1.3).29

Recently Linhardt and co-workers30 achieved glycosylation of unprotected and unactivated

glycosyl donors at 50 °C using ionic liquids. Benzyl glycosides, disaccharides as well as

oligosaccharides were synthesized using D-glucose, D-mannose, D-galactose and D-N-

acetylgalactosamine. Stereoselective α-glycosylation was observed for all glycosylations

using the ionic liquid 1-ethyl-3-methylimidazolium benzoate ([emIm][ba]), with either

Amberlite (H+) resin or p-toluenesulfonic acid (p-TsOH) as promoters (Scheme 1.4).

11

 

OOH

OMe

OO

O

OH

Amberlite (H+)[emIm][ba]

HO

O

O

HO

OOHHO

O

OMe

ROp-TsOH

[emIm][ba]

RO

HOOH

+

+

R = BnR = Allyl

61-64%

20-41%

Scheme 1.4 Glycosylation of unprotected and unactivated glycosyl donors in ionic liquids.

The yields for the benzyl glycosylation were good for all donors except for D-N-

acetylgalactosamine, which was fairly low at 27%. The formation of disaccharides using

protected glycosyl acceptors gave fair yields of the α-glycosidic product. The authors

nonetheless comment on the difficulty of stirring the reaction mixture as well as product

recovery due to the viscosity of the ionic liquid employed. Although yields were low, it

must be kept in mind that overall yield for synthesis of similar disaccharides using

protection-deprotection steps would likely be in the same range.

1.4 Glycosidations using unprotected donors

Formation of O-glycosides using unprotected activated glycosyl donors has received

increased attention in recent years. These glycosyl donors can successfully be activated

and glycosidated without protection of the hydroxyl groups on the carbohydrate donor.

However, significant improvements are needed for these methods since the preparation of

the glycosyl donors requires protection-deprotection manipulations. A more useful

approach would be the formation of the glycosyl donor in a single chemical step allowing

subsequent glycosidation of the activated unprotected donor.

1.4.1 Unprotected 3-methoxypyridyl glycosyl donors

In 1981, Hanessian et al. introduced the concept of remote activation, a method based on

leaving groups that are activated at an atom that is not directly attached to the anomeric

center of the glycosyl donor.31 As can be seen in Figure 1.7, a leaving group containing

12

 

two heteroatoms, X and Y, can be activated at the remote atom Y by an electrophilic

species or a metal cation.

O

OH

X Y

OH

R

O

OH

X Y

OH

R

M

M = Metal cation

ORO

OH

OR+

HY

X

HY

X

ORActivation

M

Figure 1.7 Remote activation concept.

This may lead to the formation of reactive intermediates such as an ion pair or a loose

complex, which can undergo an attack by the acceptor’s hydroxyl group, resulting in the

inversion of stereochemistry at the anomeric center.

After investigating a series of substituted heterocycles, Hanessian et. al found that a 3-

methoxy-2-pyridyloxy (MOP) group was the most effective leaving group, which, upon

activation with MeOTf, afforded the 2-propyl-α-D-glucopyranoside in 79% yield and

excellent α:β selectivity (Scheme 1.5).32

CH3NO2-2-PrOH (1:1)

O

OH

HO

OHOHO

O

OH

HO

OHOHO

69%

MeOTf (1 equiv)

N

OO

HO

HO

O

HOHO

10%

+

Scheme 1.5 Glycosylation using unprotected MOP glycosyl donor.

Using the same conditions, various unprotected MOP glycosyl donors were glycosidated

with different alcohols in good to excellent yields and excellent stereoselectivity. For all

13

 

the reactions, an excess of alcohol was used to ensure high coupling efficiency as well as to

avoid undesirable side reactions. These included 1,6-anhydro formation which is self-

condensation due to the primary hydroxyl group at C-6, as well as hydrolysis. However,

when a 2-acetamido group was present in the MOP glycosyl donor, only β-glycoside was

obtained via an oxazoline intermediate.

In the presence of excess protected glycosyl acceptor (7-10 equiv), disaccharides and

trisaccharides could be prepared in good yield (42-77%) but anomeric selectivity ranged

from good to fair. It is also important to mention that the unprotected MOP glycosyl

donors could also be reacted successfully to form glycosyl esters,24 azides,32 phosphates33

and nucleotides.24

Although glycosidation could be achieved efficiently in a single step, the preparation of the

unprotected MOP glycosyl donors required four synthetic steps from the unprotected

hemiacetals: i) peracetylation, ii) formation of halide, iii) coupling to MOP and iv) O-acetyl

deprotection.24 Depending on the glycosyl halide, two methods were developed for the

formation of the α‐acetyl protected MOP glycosyl donor (Scheme 1.6).

O

Y

AcOOAc

X

O OAcOOAc

X

NaOMeMeOH

X = OAc, N3, NHAc, Y = Br or Cl

Ag(MOP), Toluene110 °C

3-methoxy-2-(1H)-pyridoneBu4NBr or (Hex)4NHSO4

CH2Cl2 - NaOHaq

A

B N

OO OHO

OH

X N

O

Scheme 1.6 Preparation of unprotected α-MOP glycosyl donors.

The formation of α-MOP donors of common monosaccharides from their peracetylated

halide precursors using method A or B was achieved in 45-77% yield, although 2-

acetamido glycosyl MOP donors were obtained in only 30% yield.

14

 

1.4.2 Miscellaneous methods

An electrochemical glycosylation method using unprotected phenolic-D-glucopyranosides

as glycosyl donors was developed by Noyori and co-workers in 1986 (Scheme 1.7).34 The

aromatic glycosides react under mild electrolytic conditions with simple alcohols to give

the corresponding O-alkyl-D-glucopyranosides in good yield for simple alcohols, but very

low anomeric selectivity. The method was later adapted using unprotected aryl

thioglycosides which required less oxidative power, but yields and anomeric selectivities

remained similar.35

CH3CN, LiClO4

<2.3 V vs Ag/AgCl+

O

OH

HO

OArHOHO ROH O

OH

HO

ORHOHO

CH3CN, LiClO4

1.8 V vs Ag/AgCl+O

OH

HO

SArHOHO ROH O

OH

HO

ORHOHO

(1.0-1.4 equiv)

(10 equiv)

59-93%α:β, 1:1-3:1

46-89%α:β, 4:6-3:7

Scheme 1.7 Electrochemical glycosylation method.

The phenol and thiophenol glycosides can be prepared in three steps which include

protection and deprotection strategies.36,37

1.5 Formation of N-glycosides using unprotected donors

The formation of an amide bond between glycosyl and asparagine moieties was first

reported in 1961.38 Generally, the amide bond formation is achieved by coupling

glycosylamines with an aspartate carboxylate using a peptide coupling reagent. The

preparation of glycosylamines using unprotected aldoses can be accomplished using the

Kochetkov amination.39 This involves the treatment of the corresponding unprotected

aldoses with 50-fold excess of ammonium bicarbonate for 6 days (Scheme 1.8).

15

 

O

NHAcOH

HOHO

NH4HCO3 (50 equiv)

H2O

.OH

O

NHAcNH2

HOHO

OH

50-60%

Scheme 1.8 The Kochetkov reaction.

The glycosyl amines are unstable species which are prone to hydrolysis as well as

mutarotation which results in mixtures of anomers making their stereocontrol extremely

difficult.40 Acceleration of the Kochetkov reaction to a 2 h reaction time can be achieved

using microwave irradiation, low pressure and anhydrous DMSO as the solvent.41

Another route to glycosylamines and the glycosyl-Asn amide bond is through the reduction

of an unprotected glycosyl azide.42 The reduction of the azide to the amine is achieved

with palladium on carbon under anhydrous conditions. This prevents the undesirable

anomerization and subsequent coupling with the corresponding aspartate providing the

desired amide bond without mutorotation.

Few examples of the synthesis of glycosyl azides without the use of protecting groups can

be found in the literature. Activation of the anomeric hydroxyl group on D-glucose using

triphenylphosphine and N-chlorosuccinimide yields an alkoxyphosphonium salt

intermediate which is then attacked by the azide anion on either the α- or the β-face of the

sugar, to give a mixture of anomers in moderate to good yield.43 A similar route which

involves activation of the anomeric hydroxyl group through a 1,2-cyclic sulphite using

SO(Im)2 at low temperature following attack by the azide anion gave predominantly the β-

anomer in fair to good yield (Scheme 1.9).44

O

OHOHHO

HO

OHO

OOS

O

N3-

O

OHN3

HOHO

OHSO(Im)2

LiN3

Scheme 1.9 Synthesis of unprotected β-D-glycosyl azide through 1,2-cyclic sulfite intermediate.

16

 

Indirect methods, such as the Staudinger ligation, towards the glycosyl-Asn amide bond are

becoming more popular since they do not involve the unstable glycosylamine. However,

many of these methods only use protected sugars45,46 which, upon deprotection, can lead to

the anomerization of the glycopeptide bond.47,48 Bertozzi et al.49 reported a traceless

Staudinger ligation using an unprotected methyl glycoside with the azide group located on

C-2, which was then followed by a report by Davis and co-workers using an unprotected

glycosyl azide.50

The direct deprotected glycosyl-asparagine ligation executed by Davis is a one-pot, three-

component Staudinger ligation, with the proposed mechanism shown in Figure 1.8.

O N3X

X = OH or NHAc

Bu3P O NX

NNP

BuBuBu

-N2 O NX

PBu Bu

Bu Aza-ylid

R'O

OR

O NX

PBu Bu

Bu

OR

H2OO H

NX O

R

Figure 1.8 Proposed mechanism for the three component Staudinger ligation using unprotected glycosyl

azide.

First, preactivated N-α-Fmoc-protected-L-aspartic acid  α-tert butyl ester was stirred with

DCC and HOBt in CH3CN. The glycosyl azide was then added followed by

tributylphosphine (PBu3). This gave the desired Asn-linked glycoamino acid in 87% yield,

and only in the β-configuration.

1.6 Protecting group-free chemoselective ligation

Access to sufficient quantities of well defined homogeneous glycoconjugates is essential

for structural and functional analysis. However, the coupling of the glycan to the peptide or

protein of interest requires multiple protection-deprotection steps which are often not

compatible or lead to low yields of the glycoconjugate. As a result, considerable effort has

17

 

been directed towards creating new synthetic methods for the de novo assembly of

homogeneous and chemically defined glycoconjugates in order to elucidate their structure-

function relationship. These alternative approaches to glycoconjugate formation have been

directed towards functionalizing the reducing terminal hemiacetal of the oligosaccharides

without the use of protecting groups. That is also especially useful when functionalizing

minute quantities of oligosaccharides which have been isolated from natural sources or

generated enzymatically.

Classically, reductive amination has been used, where the free reducing end is condensed

with an amine on the protein or peptide of interest to form a Schiff base, which is

subsequently reduced to form a secondary amine (Scheme 1.10). 51

O OHOH

H2N ROH OOH

OH NOH

ROH H

NOH

RNaCNBH3

H

Scheme 1.10 Reductive amination between a free hemiacetal and an amine.

Unfortunately, reductive amination gives an acyclic structure at the reducing terminus of

the oligosaccharide, which can have consequences on the glycoconjugate, potentially

causing the loss of its biological activity.

Another direction has involved the condensation of a hemiacetal with a strong α-effect

nucleophile in an aqueous environment without the need for protecting groups or activation

procedures. The first examples of this method, later referred to as chemoselective ligation,

were published in the late 19th century when glycosyl phenylhydrazones52 and oximes53

were synthesized by Emil Fischer and Rischbieth, respectively, proving the presence of

aldehydes in molecules.

Now, over a century later, chemoselective ligation has become an established labelling

method for carbohydrates. These chemoselective conjugations were first applied to

synthesize glycopeptide mimics by Mutter and co-workers54 in 1996, where a series of

unprotected carbohydrates were conjugated to the somatostatin analog RC-160

functionalized with an oxyamino group that showed increased bioavailability (Figure 1.9).

18

 

The main shortcoming of this method is that the first attached sugar is not presented in its

natural pyranose form, as the oxime linkage results in the acyclic reducing terminal residue.

O

HO

HOHO

OH

O

HO

OHO

OH

OH

OHNO

HO

OH

O O

HN

NHO

OHN

NHO

HN

O

O

HN

HNO

ONH

OH2N

OH

HN

NH2

NH S S

Figure 1.9 Maltotriose conjugated to aminooxy somatostatin analogue RC-160.

This problem was solved two years later with N-methylation of the hydroxylamine.55 This

led to a cyclic β-pyranoside which is structurally similar to the parent N-linked

glycopeptides.

The glycoconjugate formation can also be achieved using other α-effect nucleophiles such

as acylhydrazides,56,57,58 sulfonylhydrazides,57 semicarbazides59,60 or semithiocarbazides61

(Scheme 1.11). These condensation reactions produce a thermodynamic mixture of

glycosides, predominantly in the cyclic β-pyranoside form for reducing terminal gluco-

configured monosaccharides.62,63

19

 

O OHOH

OH NOH

O R

O NOH

O R

O HN

OHNH

R

O

O HN

OHNH

NH2

S

O HN

OHNH

S R

O

O

O HN

OHNH NH2

O

H2N OR

HN O R

H2NNH

R

O

H2NNH

NH2

Sa)

b)

c)d)

H2N NH

S R

O

O

H2N NH

NH2

Oe)

f)

Scheme 1.11 Formation of glycoconjugates using chemoselective methods. a) hydroxylamine, b) N-

methylhydroxylamine, c) acylhydrazide, d) sulfonylhydrazide, e) semicarbazide, f) semithiocarbazide.

The conditions for the chemoselective conjugations are mild and can often be carried out in

aqueous solution, making them efficient procedures amenable to work with small quantities

of oligosaccharide. The simplicity and efficiency of the conjugations have made them

excellent methods for the formation of glycoconjugates for applications such as biotin

labelling,64,65 labelling for mass spectrometry,66,67 labelling for chromatography,68,69

formation of glycoarrays,70,71,72 the creation of glycopeptide analogues73,74,75,76,77 or

neoglycoproteins78 and to ‘sweeten’ the structures of therapeutics.56,79,80,81

1.7 Chemical modifications on unprotected glycosaminoglycans

Due to the structural heterogeneity and polyanionic character of GAGs (Figure 1.4)

chemical functionalization of these highly sulfated carbohydrates is extremely challenging.

Derivatization of their reducing end involves the same challenges as with other

carbohydrates which have been discussed earlier in this chapter. The selective introduction

of functional groups at the non-reducing end would provide a route towards doubly-

labelled GAGs thus making preparation of substrates for the mammalian chondroitinase,

heparinase and hyaluronidase enzymes a possibility.

20

 

When aiming towards selective and single functionalization at the non-reducing end,

bacterially derived GAG lyases can be utilized to cleave the common β‐(1 4) glycosidic

bond located between alternating disaccharide units. The product formed upon cleavage by

these GAG lyases contains a free hemiacetal at the reducing end and a Δ4-uronic acid at the

non-reducing terminus (Scheme 1.12).82

O

OR

-OOC

O HOO

AcHN

RO

OOR

O O

OR

-OOC

HOO

AcHN

RO

OOR

O

O

AcHN

RO

OOR

O

OR

-OOCO

HOO

AcHN

RO

OOR

OHO

Free hemiacetal Δ4-Uronic acid

n

+

R = SO3- or H

Chondroitinase ACLyase

O

OR

-OOC

HO

Scheme 1.12 Formation of the Δ4-uronic acid functional group at the non-reducing terminus upon cleavage of

chondroitin sulfate with Chondroitin AC lyase.

The Δ4-uronic acid is an attractive target for selective functionalization at the non-reducing

end comparing to the carboxylic acids which are located on every other sugar unit.

However, only a few examples of functionalization at the captodative Δ4-uronic acid have

been reported to date,83 emphasizing the challenges of this functional group due to its

acrylic acid and enol ether electronic character.84

Oxymercuration of unprotected Δ4-uronic acid with Hg(OAc)2 results in a cyclic

mercurinium intermediate and, upon subsequent reduction, eliminates Hg to produce ethers

or alcohols and selectively removes the terminal non-reducing end sugar unit from GAGs.85

Nonetheless, avoiding the reduction step and trapping the mercurinium intermediate allow

modifications by thiols on gold surfaces in THF-H2O solvents (Scheme 1.13).86

21

 

R = SO3- or H

Hg(OAc)2

H2O-THF (1:1)

SHAu

O

OH

-OOC

HOO

NHAc

OHHOO OH O

NHAc

OHHOO OHO

OH-OOCHO

HgAcO

O

NHAc

OHHOO OHO

OH-OOCHO

HgSAu

Scheme 1.13 Functionalization of the Δ4-uronic acid by thiols on gold surface using oxymercuration.

An oxidative addition of the Δ4-uronic acid double bond through dihydroxylation is also

known to result in the cleavage of the non-reducing terminal sugar unit.87 Additionally,

reaction of GAGs with NBS in aqueous THF has been shown to form a mixture of

bromohydrin stereoisomers in good yields.88 These methods have nevertheless been

observed to cause extensive decomposition in lyase-cleaved heparin-derived GAG

disaccharides, most likely through glycosidic bond cleavage.83

So far, no other chemical functionalizations have been attempted at the Δ4-uronic acid,

which is unfortunate since selective functionalization at the non-reducing end would result

in a wide variety of new chemically defined GAGs.

1.8 Scope of projects

Developments in the synthesis of glycoconjugates have facilitated a wide variety of

techniques for the detailed study of carbohydrates and their interactions in biological

systems. However, when only small amounts of the isolated oligosaccharide are available,

multistep synthetic approaches are usually not possible.

The scope of this study has two main goals, firstly, to develop new alternative routes for

functionalizing carbohydrates without the use of protecting groups and secondly, to

investigate the stability of glycoconjugate mimics generated by chemoselective ligation.

Starting with the reducing terminus, our approach was to develop a simple glycosidation

method applicable to monosaccharides as well as to oligosaccharides isolated from natural

22

 

sources or those generated by chemoenzymatic synthesis. The new glycosidation method

would provide an attractive alternative to current methods which require multistep

protection-deprotection strategies and extensive purification steps. It should also provide

access to both O- and N-glycosides which can be used for structural analysis and/or provide

building blocks for the preparation of more complex glycans and glycopeptides.

Our strategy also encompasses the development of a new methodology which would enable

the functionalization of a GAG oligosaccharide at its non-reducing terminus. Currently, no

selective functionalization methods for the non-reducing terminus of GAGs exist. Our

approach was to cleave GAG polymers with a bacterial lyase enzyme which produces

oligosaccharides containing Δ4-uronic acid at the non-reducing terminus (Scheme 1.12).

This unique functional group provides an attractive handle for selective functionalization

which would facilitate the preparation of chemically defined doubly-labelled GAG

substrates. These substrates can then be employed in the investigation of hyaluronidases

which have been implicated in tumour growth and metastasis.89

Despite the wide use of chemoselective methods, the properties of the non-natural linkages

have not been thoroughly investigated with regards to their stability and rates of hydrolysis

under aqueous conditions. The results of stability studies would then provide guidelines for

conditions under which a glycoconjugate may be useful, as in the development of drugs and

vaccines.

23

 

Chapter 2. Stability Studies of Hydrazide- and

Hydroxylamine-Based Glycoconjugates in Aqueous

Solution

Sections of this chapter have been reproduced in part with full permission from:

Gudmundsdottir, A. V.; Nitz, M. Carbohydr. Res. 2007, 342, 749-752. © 2008 Elsevier.

Sections of this chapter have been reproduced in part with full permission from:

Gudmundsdottir, A. V.; Paul, C. E.; Nitz, M. Carbohydr. Res. 2009, 344, 278-284 © 2009

Elsevier.

2.1 Introduction

Chemoselective ligation techniques take advantage of the reaction between the aldehydes,

which are in equilibrium with the hemiacetals of carbohydrates, and strong α-effect

nucleophiles such as hydrazides or hydroxylamines, to form non-natural glycosidic

linkages under mildly acidic aqueous conditions. Chemoselective ligation provides a

simple protecting group-free route to assemble complex neoglycoconjugates. Despite the

wide use of these chemoselective methods, the properties of the linkages formed have not

been thoroughly investigated with regard to their stability and rates of hydrolysis under

aqueous conditions.65,79 A more complete understanding of these properties will allow for

the optimization of condensation conditions for glycoconjugate formation. This

investigation will also provide guidance as to under which conditions the glycoconjugates

are stable and when they can best be used in glycoconjugate studies.

24

 

2.2 Results and discussion

2.2.1 Glycoconjugates investigated

The conjugates synthesized are derived from three different monosaccharides: xylose,

glucose and N-acetylglucosamine. These monosaccharides were chosen for their biological

relevance and for the wealth of information that is available about their O-glycoside

hydrolysis. N-Acetylglucosamine and xylose are the reducing terminal sugars found in N-

linked glycoproteins and GAG, respectively, thus knowledge of the properties of

conjugates derived from these monosaccharides will be directly applicable to conjugates

formed from these oligosaccharides. The hydrolysis mechanisms for the corresponding

methyl glycosides of these monosaccharides have been studied in detail. This allows

parallels to be drawn for the rates of hydrolysis determined for the glycoconjugates studied

here, allowing the results obtained to be potentially extrapolated to other glycoconjugates.

Initially four N-(β-D-glucopyranosyl)benzoylhydrazide derivatives were synthesized using

acid-catalyzed condensation of glucose with the respective hydrazide in ethanol. The

chosen nucleophilic benzoylhydrazides have varying electronic properties, ranging from

electron-withdrawing to electron-donating, at the para position on the phenyl ring (Figure

2.1).

R =1) H2) OCH33) Cl4) NO2

O

OH

HOHO

HOHN

NH

O

R

Figure 2.1 Glycoconjugates synthesized using different para-substituted benzoylhydrazides.

The condensations proceeded smoothly under reflux. After 3 h the reactions were complete

and the glycoconjugates precipitated out of the solution. After hot ethanol wash of the

precipitates, the products were obtained pure in 60-83% yield.

25

 

The investigation was expanded to include additional α-effect nucleophiles such as p-

toluenesulfonylhydrazide and N-methylhydroxylamines as well as other monosaccharides.

Nine other conjugates were synthesized and are derived from three different

monosaccharides: xylose, glucose and N-acetylglucosamine. These monosaccharides were

chosen to evaluate the effect of a variety of substituents around the pyranose ring, which in

turn affects the electronic properties of the monosaccharide. These monosaccharides were

condensed with p-toluenesulfonylhydrazide (p-TSH) 5, O-benzyl-N-methylhydroxylamine

6 and N-methyl-O-(N’-benzylacetamide)hydroxylamine 7 (Figure 2.2). The p-

toluenesulfonylhydrazide 5 is commercially available while the two N-

methylhydroxylamines, 6 and 7, were synthesized.

13 14 15

16 17 18

19 20 21

5 6 7

O

OH

HOHO

O

OH

HOHO

HO

O

NHAc

HOHO

HO

SOO NH

NHON

NH

OON

H

Figure 2.2 Glycoconjugates synthesized using p-toluenesulfonylhydrazide and N-methylhydroxylamines.

2.2.2 Synthesis of N-methylhydroxylamine nucleophiles

The O-benzyl-N-methylhydroxylamine 6 was prepared from t-butyl N-methyl-N-

hydroxycarbamate,90 which was alkylated at the hydroxylamine oxygen using benzyl

bromide to give 8. The Boc protecting group was removed with TFA in CH2Cl2 (1:1) to

obtain the O-benzyl-N-methylhydroxylamine 6 with an overall yield of 73% (Scheme 2.1).

26

 

NON

OH

OOOO H2

NO

O

F3C O- 68

NaH, BnBrDMF

85%90%

TFA-CH2Cl2 (1:1)

Scheme 2.1 Synthesis of O-benzyl-N-methylhydroxylamine 6.

N-Methyl-O-(N’-benzylacetamide)hydroxylamine 7 was also prepared from t-butyl N-

methyl-N-hydroxycarbamate, as shown in Scheme 2.2, following the method of Niikura et

al.91 Compound 9 was obtained by alkylating the oxygen of the hydroxylamine with ethyl

bromoacetate. The ester was hydrolyzed with sodium hydroxide in THF to form 10. The

acid 10 was then activated with DCC and reacted with N-hydroxysuccinimide to afford the

actived ester 11. This compound is a versatile intermediate for forming other N-

alkylhydroxylamine-based glycoconjugates. Benzyl amine was condensed with the

succinimide ester to give 12, which was then deprotected using TFA in CH2Cl2 (1:1) to

afford N-methyl-O-(N’-benzylacetamide)hydroxylamine 7 as the TFA salt.

NOH

Boc

H2NO

HN

O

OBr

O

NO

O

OEt

NO

O

ON

O

O

NO

O

HN

O

F3C O-

7

+9

1112

NaH, THF

10

BocBoc

BocN

OO

OHBoc

95%

NaOHTHF

86%

DCC, N-hydroxysuccinimideEtOAc

87%

Benzyl amineCH3CN

73%

TFA-CH2Cl2 (1:1)

87%

Scheme 2.2 Synthesis of N-methyl-O-(N’-benzylacetamide)hydroxylamine 7.

27

 

2.2.3 Synthesis of glycoconjugates

Four N-(β-D-glucopyranosyl)benzoylhydrazide derivatives were synthesized using acid-

catalyzed condensation of glucose with the respective hydrazide in ethanol. Successful

formation of glycoconjugates under aqueous conditions rather than under organic

conditions is of significant value, since with larger and more complex unprotected

carbohydrates, solubility in organic solvents is decreased significantly. Before synthesizing

glycoconjugates 13-21, a synthetic method involving an aqueous buffer was developed and

conditions to drive the reaction to completion were evaluated. To determine the optimal

conditions for glycoconjugate synthesis under aqueous conditions, the equilibrium

association constants for formation of glycoconjugates 13-21 were determined using 1H

NMR spectroscopy with equimolar concentrations of hydrazide or hydroxylamine at four

different concentrations: 50, 75, 100 and 125 mM, in deuterated sodium acetate buffer (pD

4.5). The relative amounts of glycoconjugate and corresponding free monosaccharide

were determined by integration of the 1H NMR peaks. After 4 days of equilibration at 37

°C, no further change was observed in the relative amounts of species present, and the

integrated values were used to determine the apparent equilibrium constants under these

conditions (Scheme 2.3).

O NO

RO OH HNO

+HOHO

H2OR +Ka

Scheme 2.3 Equilibrium association constant determined for glycoconjugates 13-21.

As seen from the apparent association constants in Table 2.1, the p-

toluenesulfonohydrazide glycoconjugates (13, 16, 19) are moderately more stable than the

N-methylhydroxylamine conjugates. Larger association constants were observed with

more electron-rich monosaccharides (xylose > glucose > N-acetylglucosamine). All of the

equilibrium constants are small and within the same order of magnitude. Thus, only under

concentrated conditions do the conjugation reactions proceed to near completion. It has

been reported that the formation of some N-methylhydroxylamine conjugates does not

proceed to completion, and that the products of the condensation reaction with less reactive

monosaccharides (i.e. N-acetylglucosamine) are produced in low yields.55,92 These

28

 

observations may be the result of the small equilibrium constant for the condensation in

aqueous solution.

Table 2.1 Equilibrium constants for the formation of glycoconjugates 13-21

Compound 13b 14 15 16b 17 18 19b 20 21

Ka (M-1)a 74 21 20 65 18 19 18 16 9

a Ka = [glycoconjugate] / ([free nucleophile] [free hemiacetal]). Apparent association constants determined in D2O (pD 4.5, 37 ºC); values are the average of 4 determinations at different compound concentrations. Standard deviations were between 6% and 9%. b 3% d6-DMSO was used to solubilize the p-toluenesulfonylhydrazide.

Given the small equilibrium constants for the formation of the glycoconjugates, the

condensation reactions for glycoconjugates 13-21 were carried out under concentrated

conditions (0.75 M, equimolar) at pH 4.5 (2 M NH4OAc). The mixtures were incubated at

37 °C for 72 h and glycoconjugates 13, 16 and 19 were crystallized from isopropanol, while

the remaining compounds were purified using column chromatography. The isolated yields

from the conjugation reactions were between 80% and 90%, and only β-pyranosides were

isolated in all cases.

2.2.4 Glycoconjugate hydrolysis

Hydrolysis products of the conjugates were analyzed by 1H NMR (3% d6-DMSO in 10 mM

Na2DPO4 buffer at pD 6.0, with t-butanol as internal standard) to confirm the formation of

the hemiacetal and hydrazide or hydroxylamine. The N-methylhydroxylamine and

benzoylhydrazide conjugates hydrolyzed to the expected products. However, the p-

toluenesulfonohydrazide conjugates 13, 16 and 19 gave not only the aldose and p-

toluenesulfonylhydrazide but also p-toluenesulfinic and p-toluenesulfonic acids (Scheme

2.4 and Figure 2.3).

29

 

O HN

NH

SO O H+

O OH

+ HOSO

HOS

O O+

H2NNH

SO O

+

p-toluenesulfonic acid

p-toluenesulfinic acid

HO

HO

Scheme 2.4 Hydrolysis products observed in 1H NMR from hydrolysis of p-toluenesulfonohydrazide

conjugates.

Figure 2.3 1H NMR (400 MHz, D2O) spectra showing degradation of glycoconjugate 16 to p-toluenesulfonylhydrazide 5, p-toluenesulfinic and p-toluenesulfonic acid. N-(β-D-glucopyranosyl)-p-toluenesulfonohydrazide 16 in 10 mM NaDPO4 pD 6.0 (3% d6-DMSO) at 37 °C (I) time 0 h. (II) time 48 h. Peaks identifying reaction products are as marked: (a) N-(β-D-glucopyranosyl)-p-toluenesulfonohydrazide 16 (b) p-toluenesulfonylhydrazide 5 (c) p-toluenesulfinic acid (d) p-toluenesulfonic acid.

The degradation of p-toluenesulfonylhydrazide to p-toluenesulfinic and p-toluenesulfonic

acid has previously been observed.93,94 In addition to the oxidation of N,N’-

II

I

30

 

ditoluenesulfonylhydrazide acetate to its corresponding diazene, subsequent elimination of

two p-toluenesulfinic acid molecules forms diazacetate.95 Attempts were made to

determine the rate of degradation of p-toluenesulfonylhydrazide under the hydrolysis

conditions but the data obtained does not fit to a simple first-order process (Figure 2.4).

0 5 10 15 20 25 30 350

20

40

60

80

100

% p

-tolu

enes

ulfo

nylh

ydra

zide

5 re

mai

ning

Time (h)

Figure 2.4 Hydrolysis of p-toluenesulfonylhydrazide 5 at 37 °C at pH 4.0-6.0. ( ) 20 mM NaOAc pH 4.0;

(●) 20 mM NaOAc pH 5.0; ( ) 20 mM Na2HPO4 pH 6.0. Lines indicate best fit of data to a first-order rate

law.

Qualitatively, the rate of decomposition of p-toluenesulfonylhydrazide is faster than

hydrolysis of the glycoconjugate at pH 6.0 but slower than hydrolysis of the glycoconjugate

at pH 4.0. Thus, with the analyses carried out here, it is not possible to determine if the p-

toluenesulfonohydrazide conjugates are hydrolyzing exclusively by an initial C-N bond

cleavage, or by competitive pathways involving both C-N and N-S bond cleavage.

2.2.5 Hydrolysis rates

To obtain hydrolysis rates, glycoconjugates were incubated at the physiologically relevant

pH values of 4.0, 5.0 and 6.0, with the glycoconjugates 13-21 observed at 37 ºC and the

31

 

benzoylhydrazide glycoconjugates 1-4 observed at 50 °C. The hydrolysis reactions were

followed by reversed-phase HPLC (Figure 2.5).

4 5 6 7 8 9 10 11 12 13

20

40

60

80

100

120

140

160

300220

15070

0

Hydro

lysis

time (

min)

Retention Time (min)

mA

U

Figure 2.5 Hydrolysis of 5 mM N-(β-D-xylopyranosyl)-p-toluenesulfonohydrazide 13 in 20 mM NaOAc

(0.5% DMSO) pH 4.0 at 37 °C. Peaks correspond to glycoconjugate 13 (6.8 min), benzyl alcohol (9.2 min,

internal standard), and p-toluenesulfonylhydrazide (10.5 min). The corresponding salts of p-toluenesulfinic

and p-toluenesulfonic acids eluted at the solvent front.

The p-toluenesulfonohydrazide derivatives 16 and 19, as well as the benzoylhydrazide

derivatives 1-4 eluted as two peaks (Figure 2.6). This may be due to cis-trans isomerisation

about the hydrazide bond. Cis-trans isomerism in glycosylhydrazides has been observed

for N-(β-D-glucopyranosyl)acetylhydrazide.58 When the two peaks were collected, and re-

analyzed separately, two peaks were again observed corresponding to re-equilibration of

the isomers.

32

 

10 11 12 13 14 15 16 17 18 19 20

20

40

60

80

100

120

140

180

120

60

1

Hydro

lysis

time (

min)

Retention Time (min)

mAU

Figure 2.6 Hydrolysis of 2 mM N-(β-D-glucopyranosyl)benzoylhydrazide 1 in 50 mM NaOAc (5% MeOH)

pH 4.0 at 50 °C. The peaks at 13 min corresponds to benzoylhydrazide, while glycoconjugate 1 appears as

two peaks in the form of cis-trans isomer at 14 and 16 min.

In the calculation of the hydrolysis rates, the integrated areas under the peaks derived from

both the isomers were combined. Benzyl alcohol was used as an internal standard in all

cases for glycoconjugates 13-21, and control experiments showed no glycoconjugate

formation upon incubating the nucleophile and hemiacetal at equivalent concentrations (2

mM and 5 mM) under the hydrolysis conditions for time frames consistent with those used

in the hydrolysis experiments.

The observed rate constants for hydrolysis (pseudo-first-order conditions) were determined

by directly fitting the integrated areas indicative of the glycoconjugates remaining

compared to an internal standard for conjugates 13-21 and to the total integration for both

the benzoylhydrazide and glycoconjugates for conjugates 1-4. The observed half-lives for

hydrolysis of conjugates 13-21 and 1-4 are presented in Table 2.2. The hydrolysis of all the

conjugates fit well to a first-order rate law (Figures A.1-A.13; Appendix A) despite the

possibility of two hydrolysis pathways for the p-toluenesulfonohydrazide conjugates (vide

supra).

33

 

Table 2.2 Half-lives in aqueous solution of glycoconjugates 1-4 and 13-21

t1/2 (h) t1/2 (h) t1/2 (h) Temperature Glycoconjugate pH 4.0 pH 5.0 pH 6.0 (°C)

1a 0.9 6.0 51 50

2 a 0.8 4.5 39 50

3 a 1.0 7.2 52 50

4 a 1.3 8.9 59 50

13 b 2.9 13 29 37

14 b 7.8 26 59 37

15 b 13 140 540 37

16 b 19 48 71 37

17 b 11 210 890 37

18 b 100 990 5100 37

19 b 72 230 1100 37

20 b 76 660 2600 37

21 b 350 3100 7500 37

a 5 mM sample in 50 mM NaOAc pH 4.0, 50 mM NaOAc pH 5.0 or 50 mM Na2HPO4 pH 6.0 incubated at 50 °C, 5% MeOH added to increase solubility. b 2 mM sample in 20 mM NaOAc pH 4.0, 20 mM NaOAc pH 5.0 or 20 mM Na2HPO4 pH 6.0 incubated at 37 °C, 0.5% DMSO was added for solubility of glycoconjugates 13, 16 and 19. 200 μL samples were taken out at the corresponding time intervals and quenched with the addition of 400 μL of 4 °C 200 mM Na2HPO4 buffer at pH 7.0 and immediately analyzed by HPLC. Standard deviation between replicate runs was between 3% and 5%.

2.2.6 Factors affecting hydrolysis rates

Methyl-α-D-xylopyranoside undergoes specific acid-catalyzed hydrolysis approximately

five times faster than methyl-α-D-glucopyranoside.96 The difference in the rate of

hydrolysis between these species correlates well with differences in electron affinity of the

substituent at C-5, as glucose is less electron-rich than xylose.97 The rate-limiting step in

the hydrolysis reaction is formation of the oxocarbenium ion and because xylose lacks a C-

34

 

5 substituent, the xylosyl oxocarbenium ion is of lower energy, leading to faster

hydrolysis.26 Similar effects have been measured for a range of glycosides with deoxy- and

deoxyfluoro- substituents of differing stereochemistry and, in general, the field effect of the

substitution on the electron-deficient transition state directly influences the hydrolysis

rate.98 In contrast, the rate of hydrolysis of methyl-β-D-2-acetamido-2-

deoxyglucopyranoside is faster than methyl-β-D-glucopyranoside, despite the greater

electron affinity of the N-acetamido group than that of the hydroxyl substituent at C-2.

This observation is most likely due to neighbouring group participation of the N-acetamido

group during hydrolysis.99 Thus, by analogy, comparison of the rates of hydrolysis of the

xylo- and glucopyranosides conjugates 13-18 synthesized here provides insight into the

importance of the electronic properties of the monosaccharides with respect to the rate of

hydrolysis. The comparison of glucose glycoconjugates 16-18 with N-acetylglucosamine

derived conjugates 19-21 indicates the possible influence of neighbouring group

participation on the rate of hydrolysis.

From analysis of the kinetic data, it is apparent that the rates of hydrolysis for all the

conjugates investigated are strongly pH dependent (Table 2.2, Figure 2.7). The p-

toluenesulfonohydrazide conjugates 13, 16 and 19 hydrolyze more quickly than the N-

methylhydroxylamine conjugates at pH 5.0 and 6.0 by a factor of 2-71.

The N-(β-D-glucopyranosyl)-p-toluenesulfonohydrazide 16 has a half-life of 19 h and

hydrolyzes significantly more slowly than all of the benzoylhydrazide conjugates. The

hydrolysis rates of the benzoylhydrazide conjugates follow the pKa values for the parent

hydrazides with the most stable conjugate being formed with p-nitrobenzoylhydrazide (pKa

11.28) followed by p-chlorobenzoylhydrazide (pKa 12.09), benzoylhydrazide (pKa 12.52)

and finally the p-methoxybenzoylhydrazide (pKa 12.83),100 which forms the least stable

conjugate.12 This suggests that more electron-poor hydrazides may form even more stable

conjugates.

Comparison of hydrolysis rates of the p-toluenesulfonohydrazide glycoconjugates formed

from different monosaccharides 13, 16 and 19 indicates that hydrolysis is slower when the

monosaccharide has more electron-withdrawing groups on the pyranose ring, where xylose

35

 

conjugates are fastest, followed by glucose conjugates and finally N-acetylglucosamine

conjugates, which were determined to be the slowest.

From the analysis performed here, it cannot be determined whether the pathway of

hydrolysis for the p-toluenesulfonohydrazides conjugates proceeds exclusively via initial

C-N bond cleavage or by two competing pathways involving initial C-N or S-N bond

cleavage. The observation that the hydrolysis reaction does fit well to a first-order rate law

makes the analysis useful for determining experimental conditions under which these

conjugates can be applied without completing hydrolysis.

The conjugates formed with N-methyl-O-benzylhydroxylamine 6 and N-methyl-O-(N’-

benzylacetamide)hydroxylamine 7 display 4- to 9-fold differences in hydrolysis rates for all

of the individual monosaccharides assessed. Those differences may be explained by the

electronic properties of the N-methylhydroxylamines, where 7 is more electron-deficient

than 6 due to the inductive effect of the amide in comparison to the phenyl ring. As was

observed with the p-toluenesulfonohydrazide glycoconjugates, the N-methylhydroxylamine

conjugates formed from the more electron-poor monosaccharides hydrolyze more slowly.

In all of the hydrolysis experiments presented, the less basic conjugates formed from a less

basic nitrogen nucleophile or a more electron-poor pyranose ring, hydrolyze more slowly

than glycoconjugates that are more electron-rich. It is interesting to note that in the

hydrolysis of methyl glycosides, methyl-β-D-2-deoxy-2-acetamidoglucopyranoside

hydrolyzes 5.3 times faster than the analogous glucopyranoside.26 This has been proposed

to be caused by direct participation of the acetamido carbonyl oxygen at the reaction center.

In the glycoconjugates investigated here, the greater electron-withdrawing nature of the

acetamido group in comparison a hydroxyl group, rather than the possible anchimeric

assistance, appears to be the major factor influencing the hydrolysis rate of the conjugates.

From the results shown in Table 2.2, a pH rate profile (Figure 2.7) was obtained by plotting

the logarithm of the pseudo-first-order rate constant of hydrolysis of the glycoconjugates in

aqueous solution against the pH values.

36

 

4 5 6

-7.5

-7.0

-6.5

-6.0

-5.5

-5.0

-4.5

-4.0

-3.5

Log

k (s

-1)

pH value

Figure 2.7 pH rate profile for glycoconjugates 1-4 and 13-21. ( ) 1, ( ) 2, ( ) 3, ( ) 4, ( ), (□) 13, ( ) 14, ( ) 15, ( ) 16, ( ) 17, ( ) 18, (■) 19, (●) 20, ( ) 21. Each value represents an average of two experiments; standard deviation was between 3% and 5%. Glycoconjugates (5 mM) 1-4 buffers: 50 mM NaOAc pH 4.0, 50 mM NaOAc pH 5.0, 50 mM Na2HPO4 pH 6.0; 5% MeOH. Glycoconjugates (2 mM) 13-21 buffers: 20 mM NaOAc pH 4.0, 20 mM NaOAc pH 5.0, 20 mM Na2HPO4 pH 6.0; 0.5% DMSO was added to provide solubility for glycoconjugates 13, 16, 19.

Extensive studies have supported a mechanism of hydrolysis of oximes, and hydrazones

over a pH range of ~4.0 to 6.0 that involves a rate-limiting addition of water at the sp2

hybridized carbon. Over this pH range general base catalysis is observed. It is proposed

that the base aids in deprotonating a nucleophilic water molecule upon attack.101 We

hypothesize that the mechanism of hydrolysis of the N-methylhydroxylamine

glycoconjugates follows a similar mechanism.

O NO

RHO

HAA-

OH NO

RHO

±H2O

OH NO

RHO

OH

O OH HNO

R+

I II

III

±H+

HO

Scheme 2.5 Putative mechanism for the hydrolysis of N-alkylhydroxylamine glycoconjugates.

37

 

The expected steps in hydrolysis of the hydroxylamine-based glycoconjugates are shown in

Scheme 2.5. The effect of pH on the rate of hydrolysis is reflected in the position of the

equilibrium between the conjugate I and the ring-opened oxyiminium species II in

solution; at lower pH more of species II will be present. This is consistent with the

observation that more electron-rich conjugates, formed from either electron-rich

monosaccharides (i.e. xylose) or more electron-rich hydroxylamines (ie. 6), hydrolyze more

rapidly. It is less favourable for electron-poor glycoconjugates to form species II. The

rate-limiting step is thus attack of water onto the oxyiminium ion II. Buffer catalysis was

observed at pH 4.0 and pH 6.0 during the hydrolysis of 17, which would be expected for a

mechanism involving rate-limiting attack of water on the oxyiminium species II (Figure 2.8

and Figure 2.9).

0 5 10 15 20 250

20

40

60

80

100

% G

lyco

conj

ugat

e 17

rem

aini

ng

Time (h)

Figure 2.8 Hydrolysis of N-methyl-O-benzyl-N-(β-D-glucopyranosyl)hydroxylamine 17 at pH 4.0 and 37 °C using different buffer concentrations. ( ) 20 mM NaOAc, 480 mM NaCl pH 4.0; (●) 250 mM NaOAc, 250 mM NaCl pH 4.0; ( ) 500 mM NaOAc pH 4.0. Each value represents the average of two experiments; standard deviation was between 3-5%. Lines indicate best fit of data to a first-order rate law.

38

 

0 25 50 75 100 125 15060

70

80

90

100

% G

lyco

conj

ugat

e 17

rem

aini

ng

Time (h)

Figure 2.9 Hydrolysis of N-methyl-O-benzyl-N-(β-D-glucopyranosyl)hydroxylamine 17 at pH 6.0 and 37 °C using different buffer concentrations. ( ) 20 mM NaOAc, 480 mM NaCl pH 6.0; (●) 250 mM NaOAc, 250 mM NaCl pH 6.0; ( ) 500 mM NaOAc pH 6.0. Each value represents the average of two experiments; standard deviation was between 3-5%. Lines indicate best fit of data to a first-order rate law.

After the attack of water on II, the carbanolamine III would rapidly eliminate the N-

methylhydroxylamine, as the rate of attack of hydroxylamine on aldehydes is rapid and

reversible over the pH range investigated.102,103

The same results were observed with benzoylhydrazide glycoconjugate 1, where

acceleration in rate observed at the highest buffer concentration suggests that the hydrolysis

is also buffer catalyzed (Figure 2.10).

39

 

0 20 40 60 80 100 120 140 160 1800

20

40

60

80

100

% G

lyco

conj

ugat

e 1

rem

aini

ng

Time (min)

Figure 2.10 Hydrolysis of N-(β-D-glucopyranosyl)benzoylhydrazide 1 at pH 4.0 and 50 °C using different buffer concentrations. ( ) 20 mM NaOAc pH 4.0; (●) 250 mM NaOAc pH 4.0; ( ) 500 mM NaOAc pH 4.0. Each value is an average of two experiments; standard deviation was between 3-6%. Lines indicate best fit of data to a first-order rate law.

Although beyond the scope of this initial investigation, further studies to confirm that the

buffer is responsible for the catalysis, as well as an expansion of the pH range investigated

are required to support this mechanistic proposal. This mechanism is consistent with the

one proposed by Dumy et al. for the formation of N-methyl-O-methylhydroxylamine

glycoconjugates.55

2.3 Conclusions

The development of efficient methods to synthesize glycoconjugates enables the creation of

a wide variety of biophysical tools that can be used to study the properties of

carbohydrates. Here we have investigated the equilibrium constants for formation, as well

as the rates of hydrolysis of glycoconjugates made from xylose, glucose and N-

acetylglucosamine condensed with N-methylhydroxylamine or p-toluenesulfonylhydrazide.

The rates of hydrolysis of benzoylhydrazides with glucose were also determined.

The apparent association constants for the formation of conjugates 13-21 at pD 4.5 are

between 9 M-1 and 74 M-1, revealing the importance of carrying out the conjugation

40

 

reactions under concentrated conditions. In cases where only small amounts of

oligosaccharides are available this can be addressed by using excess nucleophile, provided

a facile separation method for the glycoconjugate product is available.72 In all cases the

hydrolysis rates of the conjugates accelerated under increasingly acidic conditions and the

half-lives of hydrolysis of these conjugates suggest that caution should be exercised when

working with these conjugates below pH 6.0. The hydrolysis rates are strongly affected by

the electronic properties of the monosaccharide involved as electron-rich monosaccharides

hydrolyze significantly more quickly than electron-poor monosaccharides. Despite the

wide utility of this chemoselective method, no quantitative studies have previously been

reported that examine the hydrolytic stability of hydrazide- and hydroxylamine-based

glycoconjugates under aqueous conditions.

Given the trends in hydrolysis rates of the glycoconjugates investigated, they are in parallel

with those observed for the well-studied O-glycosides. It is therefore possible to use the

results presented here as a guide to estimate the conditions under which a novel

glycoconjugate formed from a different mono- or oligosaccharide may be stable.

2.4 Future directions

The stability of these linkages under neutral conditions provides the possibility of utilizing

unnatural glycopeptides generated by chemoselective ligation to ‘sweeten’ therapeutics.

The glycan portion of these molecules has been shown to influence the solubility,

pharmacology and biological activity of glycosylated natural products,104 which indicates

the need for further exploration in this area of drug development.

Under physiological conditions the stability of these linkages provides the opportunity to

use non-natural glycoconjugates generated by chemoselective ligation in therapeutics such

as in the construction of carbohydrate-based vaccines. Carbohydrate-based vaccines have

been successfully used for many decades providing immunity against a variety of bacterial

infections such as S. pneumoniae, H. influenzae and N. meningitidis. As an example of the

utility of the chemoselective ligation methods investigated here, a facile route to a new anti-

HIV vaccine is proposed.

41

 

Since the discovery of the Human Immunodeficiency Virus (HIV) in 1981, tremendous

efforts have been directed towards the development of an effective HIV vaccine. The

difficulties in the development of a vaccine against HIV are accounted for the virus’s

multiple mechanisms to escape the host’s immunity system. The outer envelope contains

the glycoprotein gp120, which is heavily glycosylated105 by ‘self’ glycans which are

synthesized by the host cell, thereby providing a protective barrier against the antigens

located on the virus’s surface.106

Discovery of the HIV neutralizing antibody 2G12, which recognizes a conserved and

multivalent oligomannose cluster on gp120,107,108 has induced extensive exploration in the

generation of novel immunogens that would elicit 2G12-like antibodies. Studies have

suggested that 2G12 binds a novel cluster of oligomannose residues on the ‘silent face’ of

gp120, which are poor immunogens due to its dense glycan clusters. The epitope that 2G12

recognizes is a high mannose structure of Man9GlcNAc2, which can be seen in Figure 2.11.

Figure 2.11 The antibody 2G12 recognizes the terminal high mannose structure of its epitope structure

Man9GlcNAc2.

The design of a carbohydrate-based vaccine must include a strong T-helper epitope, e.g. a

carrier protein, since carbohydrate antigens alone are poorly immunogenic.109 However,

despite the enormous efforts in this area, a successful carbohydrate-based vaccine against

HIV has not yet been produced.

gp120O O

HO HO HO

O O HO HO HO

O

OHOHO HOHO

O NHAc

OHOHO

HN

CO

OOHO

HO

OOH

O

HO

O

NHAc

OHOHO

OOHO

HOHO

OOHHO

HOHO

OO HO

HO HO

O HO H OHO HO

O

D1

D2

D3

42

 

Due to the challenging multistep synthesis involved in the conjugation of the glycan

structure to the immunogen, an alternative route is needed to conjugate the two large

structures together in an efficient way. Since the half-lives of N-methylhydroxylamine

glycoconjugates at near physiological pH are on the order of few hundred days, they would

provide stable linkages for a new route towards creating a carbohydrate vaccine against

HIV.

The N-methylhydroxylamine linker which would connect the glycan with the immunogen

needs to be flexible and non-immunogenic. Thus, polypeptide linkers, such as β-alanine,

would be a convenient choice. A proposed synthesis of a N-methylhydroxylamine β-

alanine-based linker can be seen in Scheme 2.6. The synthesis of the linker utilizes cheap

and easily accessible reagents, and can be achieved in a few steps. In addition to the N-

methylhydroxylamine functionality, the linker is designed to contain a free thiol which can

provide a handle for the installation of an immunogen.

N OHBocH2N OH

O

Br

O

O

N O O

O

BocNaHN O N

HOH

O O

1. N-Hydroxysuccinimide2. Cysteamine

Boc

N O NH

NH

O O

BocSH TFA HN O N

HNH

O OSH

Scheme 2.6 Proposed synthesis of a N-methylhydroxylamine β-alanine based-linker to use in development of

an HIV vaccine.

The high mannose epitope Man9GlcNAc2, which was found to bind tightly to the

neutralizing antibody 2G12, can be isolated and purified from soybean agglutinin (Figure

1.6).19 The glycan structure can then be conjugated with the N-methylhydroxylamine β-

alanine-based linker in a mildly acidic aqueous solution. The free thiol located at the

reducing terminus chain can then be reacted with a maleiimide-activated immunogen, such

as keyhole limpet hemocyanin (KLH) which is commercially available (Scheme 2.7).

43

 

OOHOHOHO

OOHOHOHO

O

OHOHOHOHO

O

NHAc

OHOHO OH

OOHO

HO

OOH

O

HO

O

NHAc

OHOHO

OOHO

HOHO

OOHHO

HOHO

OOHO

HOHO

OHO

HOHO

HO

O

HN O NH

NH

O OSH

OOHOHOHO

OOHOHOHO

O

OHOHOHOHO

O

NHAc

OHOHO

OOHO

HO

OOH

O

HO

O

NHAc

OHOHO

OOHO

HOHO

OOHHO

HOHO

OOHO

HOHO

OHOHO

HOHO

O

N O NH

NH

O OSH

NImmunogenn

O

O

OOHOHOHO

OOHOHOHO

O

OHOHOHOHO

O

NHAc

OHOHO

OOHO

HO

OOH

O

HO

O

NHAc

OHOHO

OOHO

HOHO

OOHHO

HOHO

OOHO

HOHO

OHOHO

HOHO

O

N O NH

NH

O OS

N Immunogenn

O

O

NaOAc bufferpH 4.0

Scheme 2.7 Simplified preparation of a carbohydrate-based HIV vaccine utilizing a N-methylhydroxylamine

β-alanine linker.

The utilization of an N-methylhydroxylamine-based linker to construct a carbohydrate-

based vaccine against targets such as HIV is a promising approach. The complex glycan

does not require any lengthy protection or deprotection strategies, which makes this

44

 

approach suitable for large scale synthesis as is required if the vaccine is found to elicit

neutralizing antibodies against HIV.

45

 

Chapter 3. Protecting Group-Free Glycosidations

using p-Toluenesulfonohydrazide Donors

Sections of this chapter have been reproduced in part with full permission from:

Gudmundsdottir, A. V.; Nitz, M. Org. Lett. 2008, 10, 3461-3463. © 2008 American

Chemical Society.

3.1 Introduction

Although excellent examples of efficient protecting group-free glycosylations have been

reported, in most cases, the synthesis of the glycosyl donors for these reactions has required

protecting group chemistry.24 The use of N-glycosylsulfonohydrazide (GSH) as glycosyl

donors offers numerous advantages: no protecting group chemistry is required for its

synthesis, only a modest excess of alcohol is needed for glycosidation and activation is

achieved by readily available reagents. Furthermore, the conditions for glycosyl donor

formation and glycosidation are suitably mild, such that they can be carried out with

oligosaccharides. Protected derivatives of GSHs have been investigated by Vasella et al. as

precursors of lactone hydrazones,110 but GSHs have not previously been investigated as

glycosyl donors. Here we focus on N’-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-p-

toluenesulfonohydrazide donors (T-GSH), as reducing terminal N-acetylglucosamine

residues are found in a wide variety of biologically important oligosaccharides. The

acetamido group at C-2 of N-acetylglucosamine is also known to aid in stereochemical

control of glycosidation reactions.111 The condensation reactions between aldoses and

sulfonylhydrazides have been used extensively for the characterization and labelling of

mono- and oligosaccharides.112,113,114,115 The crystal structures of a series of N’-glycosyl-p-

toluenesulfonohydrazides have also been reported.116

46

 

3.2 Results and discussion

3.2.1 Glycosyl donor formation

Using 1H NMR, the equilibrium constant for the condensation of N-acetylglucosamine and

p-toluenesulfonylhydrazide (p-TSH) to form the T-GSH donor 19 in aqueous solution was

determined to be approximately 20 M-1; thus, concentrated conditions or excess hydrazide

is required to drive the reaction to completion.117 Under non-aqueous conditions, the

reaction proceeds to completion in the presence of only a small excess of the desired

hydrazide under mild acid catalysis (Scheme 3.1).

O

NHAc

RO

+

OH

O

NHAc

HOHO

HOO

NHAc

HOO HO

O

NHAc

HOHO

HO

O

NHAc

O

HOHO

O

NHAc

HOHO

HO

19

23

24

ROHO

H2NNH

SR'

O O

O

NHAc

RORO

HOHN

NH

S R'O OAcOH(cat)

H2O or polar organic

solvent

HN

NH

S TolO O

HN

NH

S TolO O

HN

NH

S TolO O

R = H or β-D-GlcNAc

O

NHAc

HOHO

HO

25

HN

NH

SO O

7

R' = Octyl 22 or Toluene 5

Scheme 3.1 Formation and structure of the GSH donors.

The octylsulfonylhydrazide (OSH) 22 was synthesized according to the procedure reported

by Cusack et al.,118 whereas the p-TSH 5 is commercially available. T-GSH donor 19 was

synthesized on a multigram scale in a suspension of DMF with a small excess of hydrazide

(1.2 equiv), and a catalytic amount of AcOH. The product was easily isolated via

precipitation with diethyl ether. N’-(2-acetamido-2-deoxy-β-D-glucopyranosyl)

octylsulfonohydrazide (O-GSH) donor 23 was synthesized under the same conditions as T-

47

 

GSH donor 19, except purification was achieved using reversed-phase chromatography. T-

GSH donors 24 and 25 are chitin (poly-β-(1,4)-N-acetylglucosamine)- and PNAG (poly-β-

(1,6)-N-acetylglucosamine)-based disaccharides, respectively. The chitin disaccharide was

obtained by acidic digestion119 of chitin from crab shells, while the PNAG oligosaccharides

were synthesized using N-acetylglucosamine in HF·pyridine.120 Both the chitin and the

PNAG oligomers were then semi-purified using size exclusion chromatography and finally

each oligosaccharide length was purified by HPLC, using a Prevail carbohydrate column.

Disaccharide T-GSH donors 24 and 25 were formed on milligram scale under the same

conditions as donor 23. These GSH donors can also be synthesized under aqueous

conditions, which is convenient when working with oligosaccharides of longer lengths.

Incubation of the mono- or oligosaccharides with their corresponding hydrazide, using a

mildly acidic buffer (2 M NH4OAc pH 4.5), at concentrated reagent conditions for 72 h at

37 °C also gives the desired glycoconjugates. Under both non-aqueous and aqueous

conditions only the cyclic β-D-pyranosyl donors were observed and the acyclic hydrazones

were not present in quantities sufficient to be observed by 1H NMR spectroscopy. The

GSH donors 19 and 23-25 are stable under ambient conditions and undergo slow hydrolysis

when dissolved in a neutral aqueous solution.

3.2.2 Method optimization

The first glycosidation attempt using GSH donor 19 was performed at rt in MeOH using

Br2 as the oxidizing agent. The reaction was stirred for 30 min after which the MeOH was

evaporated and the remaining residue was taken up in D2O for immediate 1H NMR

analysis. The results were very promising, as only the desired methyl glycoside was

observed in a mixture of β (43%) vs α  (57%) stereoisomers, without the presence of

starting materials and <1% hydrolysis products (Figure 3.1). These results prompted us to

investigate the reaction further in hopes of developing a new protecting group-free

glycosidation method.

48

 

Figure 3.1 1H NMR (400 MHz, D2O) of first glycosidation attempt using T-GSH donor 19 to form methyl 2-

acetamido-2-deoxy-D-glucopyranoside. The α and β-anomeric protons as well as their corresponding methyl

groups are labelled with arrows.

Initially, T-GSH donor 19 was prepared but its solubility in commonly used glycosidation

solvents, such as CH2Cl2 and CH3CN, was limited. This problem led us to synthesize O-

GSH donor 23, which is more hydrophobic in nature due to its long alkyl chain.

Unfortunately, O-GSH donor 23 demonstrated solubility properties similar to donor 19,

thus more polar and hydrophilic solvents, such as DMF and hexamethylphosphoramide

(HMPA) were evaluated.

Preliminary trials were conducted using GSH donors 19 and 23 to optimize conditions for

glycosidation by varying the solvent, temperature, oxidizing agent and the amount of

nucleophilic alcohol in the reaction (Table 3.1).

O

NHAc

HOHO

HO OMe

O

AcHN

HOHO

HOOMe

49

 

Table 3.1 Formation of methyl glycoside using GSH donors 19 and 23 under different conditions

MeOH T-GSH donor 19 O-GSH donor 23 (equiv) Yield (%), (α:β)a Yield (%), (α:β)a 0 °C 25 °C 0 °C 25 °C

CH3CNb 20 50 (1:2) 71 (1:2) 62 (1:2) 72 (1:2)

CH3CNc 10 -- 58 (1:2) -- 60 (1:2)

DMFb 20 80 (1:8) 94 (1:10) 78 (1:7) 97 (1:10)

DMFc 10 -- 57 (1:9) -- 66 (1:9)

HMPAb 20 88 (1:8) 94 (1:9) 87 (1:8) 93 (1:9)

HMPAc 10 -- 60 (1:8) -- 65 (1:9)

a Yield and selectivity was determined by 1H NMR. b Reactions were carried out on 0.05 mmol scale in 0.5 mL solvent with 2.4 equiv of NBS in the presence of 20 equiv MeOH at either 0 °C or 25 °C. Reactions were quenched with Amberlite (-OH), filtered, concentrated and taken up in d4-MeOH for 1H NMR analysis. c Same procedure as (b) above except 10 equiv MeOH were used at 25 °C.

Due to the limited solubility of GSH donors 19 and 23 in CH3CN the yields (71-72%) and

selectivities (α,β; 1:2) were lower than with the more polar solvents such as HMPA (93-

94%, α:β, 1:9) and DMF (94-97%, α:β, 1:10) at 25 °C using 20 equiv of MeOH. Other

solvents and solvent combinations were also explored; these include formamide, 5-50 %

HMPA in CH3CN, CH2Cl2 or tetramethylene sulfone. However, these attempts did not

prove as successful as DMF and HMPA. Since HMPA is mildly toxic and has been shown

to cause nasal cancer in rats,121 DMF was used as the solvent of choice for this method.

Lowering the temperature of the reaction to 0 °C was not effective. Yields were

considerably lower for reaction in all the solvents, and the observed decrease in yields

correlated with the solvent polarity: the yield in CH3CN was the lowest (50-62%) as it was

least polar, then DMF (78-80%) and finally HMPA (87-88%). At lower temperatures the

formation of the lactone hydrazone was observed (Figure 3.2). Previously, Vasella et al.

have shown that it is possible to form lactone hydrazones from protected N’-glycosyl-p-

toluenesulfonohydrazides under oxidation conditions similar to those used here, in the

presence of a strong base (e.g. DBU, DIPEA).110 This observation can be explained since

at lower temperatures the unprotected diazene intermediate (Scheme 3.2) has a longer half-

50

 

life than at rt which allows tautomerization to the lactone hydrazone before releasing

nitrogen gas and forming the oxocarbenium ion (Scheme 3.2).

O

AcHN

HOHO

HON

HN

SR'

O OR' = Octyl 26 or Toluene 27

Figure 3.2 Lactone hydrazone structure of GSH donors 19 and 23.

Originally, Br2 was used as the oxidizing agent in our preliminary experiments. Milder

oxidizing agents such as NBS and NIS were evaluated and were found to afford similar

yields and selectivities as Br2. To be able to expand this method for use with

oligosaccharides, mild conditions are of great value, and thus NBS was chosen as the

oxidizing agent for future experiments.122

Activation of the glycosyl donors 19 and 23-25 in the presence of a moderate excess (20

equiv) of MeOH led to good yields of the β-D-O-glycopyranosides 28-38. Lowering the

MeOH amount to 10 equiv decreased the yields to 58-66 % for all three solvents due to

increased hydrolysis although the selectivities remained the same in most cases (Table 3.1).

Little difference was observed between the yields and selectivities of the two GSH donors,

19 and 23, thus they both appear to be suitable choices for donors. Since p-TSH 5 is

available commercially, and it is necessary to synthesize the octylsulfonylhydrazide 22, T-

GSH donor 19 was chosen as the glycosyl donor for further experiments.

3.2.3 Mechanistic considerations

The oxidations of N’-alkylsulfonohydrazides have been proposed to proceed through

diazene intermediates.123,124 Acyl hydrazides have been used extensively in peptide

chemistry as convenient precursors to carboxylic acids, thioesters, amides, and esters via

their oxidization to form acyl diazenes.125,126,127,128 Following a similar reaction

mechanism, oxidation of the GSH donors 19 and 23-25 with NBS would lead to a glycosyl

diazene (Scheme 3.2).

51

 

O

NHAc

HOHO

HO N N SO

OTol

OHO

HOHO

HN O

O

NHAc

HOHO

HO OMe

SO

OTolBrS

O

TolHON2(g) +

NBS

O

NHAc

HOHO

HOHN

NH

SO O

MeOH

glycosyl diazene

NBS

MeOHSO

OTolMeO

DMF

Scheme 3.2 Proposed mechanism of GSH activation.

In this case, elimination of sulfinic acid and nitrogen gas would then yield an oxocarbenium

ion. The evolution of gas is clearly evident during these glycosidation reactions. The

oxocarbenium ion is then trapped by the incoming alcohol, wherein the stereochemistry of

the attack is biased by the neighboring acetamido group. The sulfinic acid generated in the

reaction undergoes further oxidation in situ to generate the sulfonyl halide, and it is

therefore necessary to use 2 equiv of oxidizing agent to achieve complete glycosidation.

Mass spectral analysis of crude reaction mixtures produced mass values consistent with the

formation of methyl toluenesulfonate likely resulting from methanol attack on the sulfonyl

halide.

Despite the neighboring acetamido group, small amounts of the α-glycosides are also

formed. The α-glycoside is likely the result of participation by the solvent, resulting in an

alternative ion pair.129 Other species produced in the reaction include the free hemiacetal,

formed from hydrolysis, and trace amounts of the glycosyl sulfone, resulting from the

reaction of the oxocarbenium ion with the generated p-toluenesulfinic acid.

3.2.4 Formation of O-glycosides

To validate the application of our new protecting group-free glycosidation method, a wide

range of alcohols were evaluated as potential acceptors for the oxidation of T-GSH donor

52

 

19 as well as T-GSH disaccharide donors 24 and 25. As can be seen in Table 3.2, primary

and secondary alcohols produced high yields (70-87%) with good selectivity (α:β, 1:6-

1:10), however tertiary alcohols gave poor yields and very low selectivity.

Table 3.2 Yields and selectivities for O-glycosidations

Glycosyl Donor Alcohol Product Yield (%), (α:β)a

19 MeOH (b) 28 87 (1:10)

19 (b) 29 72 (1:7)

19 (b) 30 75 (1:8)

19 (c) 31 75 (1:7)

19 (b)

32 74 (1:7)

19 (b)

33 80 (1:7)

19 (b)

34 72 (1:6)

24 MeOH (d) 35 71 (1:9)

24 (d) 36 70 (1:7)

25 MeOH (d) 37 73 (1:9)

25 (d) 38 71 (1:7)

a Isolated yield after column chromatography: silica gel (10% MeOH in CH2Cl2) for compounds 28-34, HPLC using Prevail carbohydrate column for compounds 35-38. b Reactions were carried out on 0.26 mmol scale in 2.0 mL DMF with 2.4 equiv of NBS in the presence of 20 equiv alcohol at 25 °C. Reactions were quenched with Amberlite (-OH), filtered, concentrated and purified. c Same procedure as (b) except 2.4 equiv of NIS were added. d Same procedure as (b) except the reactions were carried out on 0.027 mmol scale in 200 μL DMF.

Higher yields of the allyl glycoside 31 were obtained using NIS (20% with NBS vs 75%

with NIS), presumably due to competing electrophilic addition to the alkene. The desired

glycosides could be readily purified using flash chromatography on silica gel.

53

 

When this approach is compared to the facile formation of 2-acetamido-2-deoxy-β-D-

glucopyranosides recently introduced by Cai et al., the current approach gives similar

yields but requires a smaller excess of the alcohols.130 Furthermore, disaccharide donors 24

and 25 can be glycosidated with efficiency equal to that of the monosaccharides, as was

observed with glycosides 35-38.

3.2.5 Formation of other glycosides

Although the formation of oxazolines was not observed in the glycosidation reactions,

oxazolines could be formed under the conditions shown in Scheme 3.3.

O

NHAc

HOHO

HOHN

NH

SO O

Lutidine

OHO

HOHO

N O

O

AcHN

HOHO

HO N3

40

NaN3

41

O

AcHN

HOHO

HO Cl

O

AcHN

HOHO

HOCl

observedin situ

39a

39b19

TBA-Cl

Scheme 3.3 Formation of glycosyl azide 41 via oxazoline 40.

It was essential to have the chloride anion present in the reaction for clean conversion of

GSH donor 19 to the oxazoline 40. The requirement for chloride ions suggests the reaction

may proceed through an intermediary glycosyl chloride 39. Formation of the glycosyl

chloride ion 39 was achieved by using T-GSH donor 19 (1 equiv), TBA-Cl (5 equiv), and

NBS (2.4 equiv) in CH3CN. The reaction was stirred for 2 minutes, after which H2O was

added to dilute the solution. The aqueous layer was then washed quickly with CH2Cl2, put

through a benchtop pre-packed C-18 column, and the collected product was immediately

lyophilized. The product was taken up in D2O immediately prior to 1H NMR measurement.

54

 

1H NMR spectroscopy showed the transient formation of a low-field absorption at 6.3 ppm

(Figure 3.3), consistent with the formation of an α-glycopyranosyl chloride 39b.

Figure 3.3 Support for chloride 2-acetamido-2-deoxy-α-D-glucopyranoside 39b. Peaks indicated with arrows

indicate each compounds corresponding anomeric protons.

Halide exchange is rapid under the reaction conditions, and the β-glycosyl chloride 39a

then proceeds to the oxazoline 40 (Scheme 3.3).131 This oxazoline synthesis may provide a

useful route to generate reducing terminal oxazolines for use as substrates for

endoglucosaminidases to allow the preparation of homogeneous N-linked glycoproteins.132

The unprotected oxazolines are also considerably more reactive glycosyl donors than their

protected counterparts.130

O

AcHN

HOHO

HO OHO

AcHN

HOHO

HOCl

55

 

Figure 3.4 Support for formation of oxazoline 40. (A & B) GlcNAc-oxazoline 40 (5.92 ppm, J1,2 7.3 Hz, H-1) synthesized according to literature procedure133 in CD3CN, in the absence and the presence of 5 equiv of TBA-Cl, respectively. (C) Oxidation performed with: GSH donor 19 (1 equiv), TBA-Cl (5 equiv), 2,6-lutidine (5 equiv), NBS (2.4 equiv) in CD3CN. Reaction was stirred for 10 minutes before 1H NMR analysis. Broad peak at 4.0-4.5 ppm due to lutidinium ion.

Acetyl-protected oxazolines generally require a strong Lewis acid catalyst (i.e. TMSOTf)

for glycoside formation, and therefore are not used extensively in glycoside synthesis.134,135

In contrast, the unprotected oxazoline 40, formed in situ, can be glycosidated with azide,

with only lutidinium hydrochloride present as an acid catalyst, to give 41 in 73% yield. It

was not possible to form the glycosyl azide directly in the glycosylation reaction as NaN3,

TBAN3, and TMSN3 were incompatible with the conditions necessary for activation of the

glycosyl donors. Thus, this method may provide a useful route to generate glycosyl azides

from isolated oligosaccharides for the formation of novel N-linked glycoconjugates.136,137

56

 

3.2.6 Glycosidation on oligosaccharides

To evaluate the applicability of this method to oligosaccharide synthesis, unprotected

PNAG oligosaccharides (trimer, tetramer and pentamer) were investigated. These

oligosaccharides were reacted with p-TSH 5 using DMF and a catalytic amount of AcOH

to form GSH donors 42-44 (Figure 3.5).

O

NHAc

O

HOHO

O

NHAc

O

HOHO

HN

NH

S TolO O

O

NHAc

OHHO

HO

n

42 n = 0 43 n = 1 44 n = 2

O

NHAc

O

HOHO

O

NHAc

O

HOHO

OMe

O

NHAc

OHHO

HO

n

45 n = 0 46 n = 1 47 n = 2

Figure 3.5 Oligosaccharide GSH donors 42-44 and methyl oligosaccharide glycosides 45-47.

These donors were then subjected to oxidation using DMF as solvent, 2.4 equiv NBS and

20 equiv MeOH. The reaction was stirred at rt for 10 min and then quenched. Upon

purification with a Prevail carbohydrate column on HPLC methyl glycoside

oligosaccharides were obtained in reasonable yield, 40-55 %. 1H NMR analysis of the

oligosaccharides showed only the methylated β-glycosides 45-47 as can be seen in Figure

3.6.

57

 

Figure 3.6 Aliphatic region of the 1H NMR (400 MHz, D2O) of methylated PNAG oligosaccharides. (A)

Methyl PNAG trimer 45; (B) Methyl PNAG tetramer 46; (C) Methyl PNAG pentamer 47.

The low yield is most likely due to decreasing solubility as the oligosaccharides become

larger. Nonetheless, these results are quite respectable considering that the desired

methylated oligosaccharide was obtained in only two days.

3.3 Conclusion

In conclusion, GSH glycosyl donors can be formed in high yield under mild conditions and

can be readily activated to form a wide range of glycosides without the use of protecting

groups. The simplicity of the approach suggests that it can be extended to large

oligosaccharides isolated from natural sources or those generated by chemoenzymatic

synthesis.

A

B

C

58

 

3.4 Future directions

The availability of homogeneous glycoproteins or glycopeptides is essential for

understanding their roles in biological processes. N-linked glycopeptides can be prepared

by utilizing a pre-assembled glycosyl amino acid as a building block in stepwise solid

phase peptide synthesis. Recently, Davis and co-workers136 introduced an efficient method

towards the preparation of an N-linked glycosyl amino acid by applying the Staudinger

method. This direct glycosyl-asparagine ligation uses an unprotected mono- or

disaccharide glycosyl azide, activated asparagine residue and tributylphosphine to provide

the N-linked glycosyl amino acid with complete stereoselectivity.

This method can then be applied to generate glycopeptides containing complex glycan

structures using two different routes. The first one involves the direct ligation of a complex

glycosyl azide oligosaccharide to the asparagine residue in a linear or a convergent way.

The other possibility would be to elaborate the N-acetylglucosamine-tagged peptide or

protein using the glycosylation activity of endo-β-N-acetylglucosaminidase and a synthetic

oxazoline as the activated glycan donor (Scheme 3.4).138

59

 

O

NHAc

OHOHO

HN

CO

OHORO

HORO

O

NHAc

OHOHO

O

NHAc

OHHO

HOHN

CO

+OHORO

HORO

O

N

OHOHO

O

OCO

O

NHAc

OHOHO N3

OHORO

HORO

O

NHAc

OHOHO +

Direct glycosylasparagine ligation

Endo glycosidase

OHORO

HORO

O

NHAc

OHOHO

HN N

HSO

O

O

NHAc

OHOHO

HN

OHORO

HORO

O

NHAc

OHOHO N

HSO

O

R

R = activated ester(HOBt/DCC)

Scheme 3.4 Using GSH donors in protecting group-free glycosidation to provide glycosyl azide and

oxazoline needed in the synthesis of homogeneous glycopeptides or glycoproteins.

However, the synthesis of an elaborate unprotected glycosyl azide or oxazoline requires

multiple protecting group manipulations and purification steps which are labour intensive.

Applying our protecting group-free glycosidation method, which utilizes N-

glycosylsulfonohydrazides (GSH) as glycosyl donors, offers a convenient simple route

towards both glycosyl oxazolines and azides. The condensation of an isolated or

chemoenzymatically synthesized glycan with p-TSH should provide the glycosyl donor in a

single chemical step. The glycosyl donor can then be activated using NBS, converted into

the glycosyl oxazoline and finally form the glycosyl azide by nucleophilic attack of the

60

 

azide to the oxazoline. The combination of these two protecting group-free methods should

simplify the synthetic schemes for the preparation of homogeneous glycopeptides and

glycoproteins which are necessary for the investigation of various biological processes.

Another direction would be to apply this novel glycosidation method in the synthesis of

oligosaccharides without the use of protecting groups. Generally, the synthesis of

oligosaccharides requires extensive protection-deprotection strategies as well as

challenging purification steps. The generation of a glycosidation method which can be

applied to synthesize oligosaccharides would be of great use.

The T-GSH donors could be utilized to synthesize unmodified as well modified poly-β-

(1 6)-N-acetylglucosamine (PNAG) without the requirements of protecting groups.

PNAG is believed to be an essential component in biofilm formation in many bacterial

strains.139

The synthesis of PNAG from unprotected N-acetylglucosamine can be accomplished in

HF·pyridine in about 5 days.140 However, the acid-catalyzed formation of PNAG

oligosaccharides under these conditions is in equilibrium where glycosidic bonds are

generated and hydrolyzed. As a result, this method is not compatible for the synthesis of

modified PNAG where a stable N-acetylglucosamine glycoside would be introduced during

the polymerization reaction. However, due to the mild reaction conditions with the T-GSH

glycosidation method, the insertion of glycosidated N-acetylglucosamine residue at the

reducing end terminus should provide access to modified PNAG in a single chemical step

without protecting groups (Scheme 3.5).

O

NHAc

HOHO

HOHN

NH

S TolO O

O

NHAc

HOHO

HO R+NBS

DMFO

NHAc

O

HOHO

O

NHAc

O

HOHO

R

O

NHAc

OHHO

HO

n

Scheme 3.5 Synthesis of a modified PNAG using the T-GSH donor and a modified N-acetylglucosamine

residue.

61

 

The glycosidated N-acetylglucosamine residue can be designed to contain a functional

group such as an azide or a chloride which could later be utilized to couple a peptide or a

protein for the construction of a potential biofilm vaccine.

62

 

Chapter 4. Chemical Modifications on Unprotected

Glycosaminoglycans

4.1 Introduction

The installation of a functional group at the non-reducing end of glycosaminoglycans using

a chemoselective method would provide a useful tool to prepare chemically defined

glycosaminoglycans for biological studies. This could be achieved by functionalizing a Δ4-

uronic acid generated at the non-reducing end of glycosaminoglycans upon degradation by

bacterial lyases. Due to the captodative effects of the exocyclic carboxylate and the ring

oxygen on C-5 on the double bond of the Δ4-uronic acid, it should be a candidate for radical

addition reactions (Scheme 4.1).

R = SO3- or H

O

OH

-OOC

HOO

NHAc

ORRO

O OHSHR' O

NHAc

OROR

OO

OH

R'S

HO

COO-

OH

Scheme 4.1 Proposed radical addition of a thiol to a Δ4-uronic acid chondroitin sulfate disaccharide.

Since purification of glycosaminoglycan disaccharides is labour intensive and only small

amounts are obtained in each purification cycle, a model Δ4-uronic acid compound was

synthesized to explore the radical addition of thiols onto this functionality.

4.2 Results and discussion

4.2.1 Preparation and purification of Δ4-uronic acid chondroitin sulfate disaccharide

In order to obtain Δ4-uronic acid chondroitin sulfate disaccharides, E. coli cells transformed

with the pET-24(+) vector (Novagen) containing a sequence coding for Chondroitinase AC

from Flavobacterium heparinum141 fused to a C-terminal His6 tag. The cells were cultured,

63

 

and the protein was recombinantly expressed and purified according to Pojasek et. al.142

Cultures were grown to an OD600 of 0.7 and subsequently induced with 1 mM IPTG at 22

°C overnight. After lysing the cells through sonication, the recombinant Chondroitinase

AC enzyme was purified using Ni2+-affinity chromatography. The His6 tag was not cleaved

from the N-terminus before use since it had been shown not to influence the activity of the

enzyme.142

Digestion of chondroitin sulfate obtained from bovine trachea using the purified

Chondroitinase Lyase AC was achieved overnight in 30 mM NH4OAc buffer pH 7.0 in the

presence of 0.05% NaN3 at rt. Cleavage of the chondroitin sulfate into disaccharide

fragments was monitored by the increase in absorbance at 232 nm, the λmax (ε = 3800 cm-1

M-1, 25 °C)143 for the Δ4,5 double bond produced as a result of chondroitin sulfate

polysaccharide cleavage (Scheme 1.12).

The Δ4-uronic acid chondroitin sulfate disaccharides formed are a mixture of isomers where

the sulfate ester is either located at C-4 or C-6 of the reducing terminal N-

acetylgalactosamine residue (Figure 4.1).

Chondroitin 4-sulfate 48

O

OH

-OOC

HOO

NHAc

OH-O3SO

O OH

Chondroitin 6-sulfate 49

O

OH

-OOC

HOO

NHAc

OSO3-HO

O OH

Figure 4.1 Two chondroitin sulfate disaccharide isomers, 48 and 49, formed upon cleavage of chondroitin

sulfate polysaccharide with Chondroitinase AC.

Preliminary purification of the isomeric mixture of chondroitin sulfate disaccharides could

be achieved with a MonoQ strong anion exchange column, using 20 mM NaHPO4 pH 6.5

and 20 mM NaHPO4, 1M NaCl pH 6.5 as eluent. After lyophilisation the Δ4-uronic acid

chondroitin sulfate disaccharide was desalted using size exclusion chromatography on a

Bio-Gel P-2 (Bio-Rad, 2.5 x 35 cm) column or dialysis (MWCO 100 Da, Spectra/Por).

Further purification of the Δ4-uronic acid chondroitin sulfate disaccharide isomeric mixture

by sulfation position differences was achieved with a strong anion exchange (SAX)

64

 

column. Successful separation of the two isomers using the SAX column was

accomplished using an isocratic solution: 20 mM NaOAc, 40 mM NaCl at pH 3.5 (Figure

4.2). AU

0.00

0.20

0.40

0.60

0.80

1.00

Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

Figure 4.2 HPLC chromatogram of separation of the two Δ4-uronic acid chondroitin sulfate isomers using a

SAX column. 6-SO3- isomer 49 (27 min), 4-SO3

- isomer 48 (30 min), 20 mM NaOAc, 40 mM NaCl pH 3.5.

After dialysis the two isomers were separately obtained in pure form. The subsequent 1H

NMR spectras of Δ4-uronic acid chondroitin sulfate disaccharides 4-SO3- 48 and 6-SO3

- 49

isomers can be seen in Figure 4.3 and Figure 4.4, respectively. The 6-SO3- and 4-SO3

-

disaccharide isomeric forms of the Δ4-uronic acid chondroitin sulfate have previously been

isolated and characterized using a 300 MHz NMR spectrometer,144 whereas their

corresponding 1H NMR spectra were obtained here on a 400 MHz NMR spectrometer.

65

 

Figure 4.3 1H NMR (400 MHz, D2O) of Δ4-uronic acid chondroitin sulfate disaccharide 4-SO3- 48.

Figure 4.4 1H NMR (400 MHz, D2O) of Δ4-uronic acid chondroitin sulfate disaccharide 6-SO3- 49.

66

 

Assignments of protons were achieved using gradient H-H correlation spectroscopy

(gCOSY). The free hemiacetal containing both α‐ and β-configurations, two

diastereomers, gave two sets of peaks for the reducing terminal N-acetylgalactosamine

residue as well as the anomeric proton on the non-reducing end. The corresponding

gCOSY spectras for the 4-SO3- 48 and 6-SO3

- 49 Δ4-uronic acid chondroitin sulfate isomers

can be seen in Figure 4.5 and Figure 4.6.

Figure 4.5 gCOSY (400 MHz, D2O) of Δ4-uronic acid chondroitin sulfate disaccharide 4-SO3- isomer 48.

67

 

Figure 4.6 gCOSY (400 MHz, D2O) of Δ4-uronic acid chondroitin sulfate disaccharide 6-SO3- isomer 49.

4.2.2 Synthesis of a model Δ4-uronic acid

To explore radical addition on Δ4-uronic acid chondroitin sulfate disaccharides, a model

monosaccharide Δ4-uronic acid was synthesized. Originally, glucose was chosen as the

starting material; however, formation of the double bond by removal of the proton on C-5

by a strong base (i.e. DBU) could not be achieved because the leaving group at C-4 is in an

equatorial position. With galactose, the hydroxyl group is axial and anti to the hydrogen on

C-5, thus subsequent elimination and formation of the Δ4-uronic acid was facile. The

synthesis of the Δ4-uronic acid model monosaccharide was achieved in a total of 9 steps,

which included various protection and deprotection steps as can be seen in Scheme 4.2.

68

 

O

OH

OH

OH

OH

HOO

OAc

OAc

OAc

OAc

AcOO

OAc

OAc

OPh

OAc

AcO

O

OBz

O

OPh

O

BzO

Ph

O

OBz

OH

OPh

OH

BzOO

OBz

COOHOPh

HO

BzO

66% 84%

87%

90% 94%

81% 89%

50 51

53

54 55

56 57

KOAcAc2O-AcOH (1:1)

PhenolBF3 OEt2.

i) NaOMe, MeOHii) Benzaldehyde dimethylacetal, p-TsOH, CH3CN

Benzoyl chloridepyridine

80% AcOH

NaHCO3-CH2Cl2 (1:1)

TEMPO, KBr, Bu4N+Br-, Ca(OCl)2

pyridine, Δ

NaOMe, MeOH

Ac2O

O

OH

O

OPh

O

HO

Ph

89%52

O

OH

-OOC

HO OPhO

OBz

-OOC

BzO OPh

Scheme 4.2 Synthesis of the model Δ4-uronic acid 57.

Following peracetylation of the galactose to form 50, the phenyl glycoside 51 was formed

using boron trifluoride as the Lewis acid to promote the formation of the β-glycoside.

Removal of the acetyl groups, and subsequent protection of the hydroxyl groups located on

C-4 and C-6 with benzaldehyde dimethylacetal gave phenyl-4,6-benzylidene-β-D-

galactopyranose 52. Benzoylation of the hydroxyl groups on C-2 and C-3 led to 53 which

upon removal of the benzylidene ring, afforded compound 54. Selective oxidation of the

primary alcohol to form compound 55 was achieved using TEMPO, KBr and Ca(OCl)2 in a

bi-phasic system, which employed Bu4N+Br- as the phase transfer catalyst.

The double bond was formed by treatment of 55 with Ac2O in pyridine, NMR analysis

confirmed that the hydrogen on C-5 was missing and that the double bond had been

formed. The axial hydroxyl group on C-4 was acetylated, creating a good leaving group for

elimination, and thus when the pyridine extracted the proton on C-5, the desired Δ4-uronic

acid 56 was obtained in a single step. Removal of the benzoyl groups at C-2 and C-3 with

sodium methoxide gave access the Δ4-uronic acid model compound 57.

69

 

4.2.3 Radical addition to the synthesized model Δ4-uronic acid

Initially, N-acetylcysteamine was investigated as the thiol for radical addition onto the Δ4-

uronic acid 57. Optimal results were obtained using the radical initiator V-501 (4,4’-

azobis(4-cyanovaleric acid)), in the solvent mixture containing H2O/MeOH (1:1) and using

either heat (80 °C) or UV light as the promoter for 4 h. Interestingly only one isomer of the

phenyl 4-(2-N-acetylethylthio)-β-D-galactopyranuronic acid 58 was obtained in 72% yield

(Scheme 4.3).

57

H2O/MeOH (1:1)

N-acetylcysteamine,V-501

Cysteamine, BenzophenoneH2O/DMF (1:1)

NHO

CN

O

V-501 =

O

OH

COOH

OPhHO

58

O

OH

COOH

OPhHO

59

S

S

NH

H2N

O

2

72%

60%

O

OH

-OOC

HO OPh

Scheme 4.3 Radical additions to phenyl Δ4-uronic acid 57 using N-acetylcysteamine and cysteamine.

Examining the 1H NMR spectrum (Figure 4.7) of the product a small coupling constant of

4.5 Hz was observed between H-3 and H-4 indicating that the thiol group was located

axially. Since the coupling constant between H-4 and H-5 also proved small (1.8 Hz), a

two dimensional nuclear overhauser enhancement spectroscopy (2D NOESY) experiment

was carried out to determine the configuration of the thiol on the sugar. Nuclear overhauser

enhancement (NOE) was observed between H-1, H-3, H-4 and H-5 confirming the sugar’s

galacturonic acid configuration.

70

 

Figure 4.7 Aliphatic region of the 1H NMR spectrum (500 MHz, d4-MeOH) of phenyl 4-(2-N-

acetylethylthio)-β-D-galactopyranuronic acid 58.

Since deprotection of the acetyl group on the nitrogen is only achieved under harsh

conditions, direct addition of cysteamine would be a better approach affording a free

primary amine, which can be easily manipulated. Using cysteamine as the thiol source,

however, proved more challenging than the N-acetylcysteamine. Combining excess

cysteamine (0.4 M) and benzophenone (5 equiv) in H2O/DMF provided the cysteamine

galacturonic acid 59 in 60 % yield (Scheme 4.3).

These results were promising and suggested that the radical addition could potentially be

carried out on a chondroitin sulfate Δ4-uronic acid disaccharide.

4.2.4 Effort towards the radical addition onto isolated Δ4-chondroitin sulfate

disaccharide free hemiacetal

Radical additions were carried out with the chondroitin sulfate Δ4-uronic acid disaccharide,

starting with the conditions used for cysteamine addition onto the model Δ4-uronic acid

71

 

compound. The radical additions were carried out with the free hemiacetal mixture of

chondroitin sulfate Δ4-uronic acid disaccharides 48 and 49.

The free hemiacetal mixture of 48 and 49 was subjected to numerous conditions, using UV

light or heat in order to promote the radical addition onto the Δ4-uronic acid which

unfortunately proved ineffective. As can be seen in Table 4.1, both cysteamine and N-

acetylcysteamine, the two thiols successfully used in the model system, were used during

these trials. Four different radical initiators were examined, the azobis-based initators, V-

50 (water-soluble, 2,2’-azobis(2-methylpropionamidine) and V-501, in addition to

benzophenone (Ph2CO) and its more polar derivative 4-benzoylbenzoic acid (4-BBA). The

azobis radical initiators can be activated through either heat or UV light, while the

benzophenone derivatives are only activated with UV light. The reactions activated by UV

light were also carried out in the absence of a radical initiator, and solvent systems were

used which solubilized all of the reagents.

Table 4.1 Conditions for radical reaction attempts using a mixture of chondroitin sulfate Δ4-uronic acid

disaccharides 48 and 49

Radical Thiola [mM] initiator [mM] Solventb Time (h) Promoter

I 27 V-50 35 D2O 1, 5, 12 365 nm or 80 °C

I 27 V-50 35 Buffer A, B & C 5, 12 365 nm or 80 °C

I 27 4-BBA 35 Buffer A, B & C 5, 12 365 nm or 80 °C

I 108 Ph2CO 35 H2O/DMF (1:1) 3 365 nm

II 27 V-50 35 Buffer A, B & C 3 365 nm

II 108 V-50 35 Buffer D 5, 24 365 nm

II 35 V-50 35 Buffer D/MeOH (1:1) 5, 24 365 nm or 80 °C

II 35 V-501 35 Buffer D/MeOH (1:1) 5, 24 365 nm or 80 °C

Chondroitin sulfate disaccharide concentration was 7 mM and all reactions were done in glass tubes or flasks. a (I) cysteamine hydrochloride, (II) N-Acetylcysteamine b Conditions tried; Buffer A: 100 mM CD3COOD pD 5.0; Buffer B: 100 mM NaDPO4 pD 7.0; Buffer C: 100 mM NaDCO3 pD 10; Buffer D: 200 mM NaHPO4 pH 7.0.

72

 

Under the conditions shown above, no addition to the double bond was observed. Using a

stronger UV lamp source did not induce a reaction. In addition to the reactions mentioned

in Table 4.1, addition of acetic anhydride or ceric (IV) ammonium sulfate in order to

activate the double bond through the carboxyl group, by forming a mixed anhydride or by

metal coordination, did not induce a reaction.

It was not until excess cysteamine (0.5-1.0 M) was added in the presence of benzophenone

as the radical initiator and UV light as promoter, that product was observed in mass

spectrometry of the reaction mixture. HRMS supported the formation of the mono-addition

of cysteamine to the chondroitin sulfate Δ4-uronic acid mixture 48 and 49. After

purification of the reaction mixture using size exclusion chromatography (Bio-Gel P-2 Bio-

Rad, 2.5 x 35 cm), mass spectrometry showed that other major products were also present. 1H NMR supported this, as multiple products were observed in the spectra. The side

reactions observed under these radical conditions could not all be identified as the

separation of these side products for further analysis were not successful. The source of

these rearrangements and degradation products when using UV light might be due to the

formation of various radicals on the chondroitin sulfate, which would be stabilized by the

electron-withdrawing carboxylate and the sulfate group, since chondroitin sulfate

undergoes site-specific fragmentation upon radical formation with hypochlorite.145

4.2.5 Effort towards the radical addition onto Δ4-chondroitin sulfate disaccharide

protected at the anomeric center

In order to determine if the hemiacetal was the source of degradation and side products, the

anomeric center was protected by condensation with octylsulfonylhydrazide and N-methyl-

O-octylhydroxylamine to produce chondroitin sulfate disaccharides 60 and 63, respectively.

These compounds contain a convenient handle for purifying the disaccharides from the

reaction mixture before analysis using reversed-phase chromatography.

N-Methyl-O-octylhydroxylamine 62 was prepared in two steps from t-butyl N-methyl-N-

hydroxycarbamate, which was alkylated at the hydroxylamine oxygen using iodooctane to

give 61. The Boc protecting group was removed with TFA-H2O-TIS (95:2.5:2.5) to obtain

the N-methyl-O-octylhydroxylamine 62 in an overall yield of 80% (Scheme 4.4).

73

 

N O 7HN O 7

N OH

NaHIodooctane

DMF

TFA-H2O-TIS (95:2.5:2.5)

6296% 83%

O

O

O

O 61

Scheme 4.4 Synthesis of N-methyl-O-octylhydroxylamine 62.

The chondroitin sulfate Δ4-uronic acid derivatives 60 and 63 were synthesized under

concentrated conditions, in a mixture of DMF-H2O from their TEA or DIPEA salts to

increase solubility, at mildly acidic pH at 37 °C overnight (Scheme 4.5).

R = SO3- or H

7

60

63Isolated as DIPEA salt

O

OH

OOC

HOO

NHAc

OROR

O OH

octylsulfonylhydrazide AcOHcat

DMF/H2O (2:1)

N-methyl-O-octylhydroxylamine

O

OH

OOC

HOO

NHAc

OROR

OHN N

HSO

O

7

O

OH

OOC

HOO

NHAc

OROR

O N O

Isolated as TEA salt82%

40%AcOHcatDMF

Scheme 4.5 Synthesis of octylsulfonohydrazide and N-methyl-O-octylhydroxylamine chondroitin sulfate Δ4-

uronic acid 60 and 63, respectively.

Glycoconjugates 60 and 63 were subjected to the same conditions as the chondroitin sulfate

free hemiacetal, which had shown partial radical addition to the double bond. Taking

advantage of the hydrophobic octyl group, a quick C-18 plug was run at the end of each

reaction. Interestingly, 1H NMR showed that only 10-20% of the glycoconjugates were

still present in the mixture after both reactions, indicating the hydrazide and hydroxylamine

bonds to the chondroitin sulfate are not stable under the conditions applied. Mass

spectrometry of the residues purified with the C-18 plug provided information in support of

the mono addition of cysteamine to the double bond with both glycoconjugates 60 and 63.

However, another major side product of these glycoconjugates was identified as the 2-

acetamido-2-deoxy-4-sulfate-β-D-galactopyranosyl reducing terminal residue

74

 

functionalized with either the octylsulfonylhydrazide or N-methyl-O-octylhydroxylamine,

indicating that the glycosidic bond had been cleaved. Also, masses consistent with the

starting material, octylsulfonylhydrazide, octylsulfinic acid and N-methyl-O-

octylhydroxylamine were present; supporting the hypothesis that hydrolysis of the

chondroitin sulfate disaccharide glycoconjugates was occurring during the reaction

(Scheme 4.6).

7

60

O

OH

OOC

HOO

NHAc

OROR

OHN N

HSO

O

CysteaminePh2CO, UV

R = SO3- or H

C-18

7

O

OH

OOC

HOO

NHAc

OROR

OHN N

HSO

O

O

OH

COOH

HO

SH2N7

O

NHAc

OROR

OHN N

HSO

O

7O

NHAc

OROR

HOHN N

HSO

O

Only 10-20% stillas glycoconjugateafter reaction

7H2N N

HSO

O 7SO

HO

Scheme 4.6 Products observed in mass spectrometry following reverse phase purification after radical

addition on Δ4-chondroitin sulfate disaccharide octylsulfonohydrazide 60 with cysteamine.

Control reactions to test the stability of glycoconjugate 60 and 63 using radical initiator V-

50 and heat showed degradation as well as hydrolysis at 45 °C overnight even if all oxygen

had been removed from the system. These results led us to begin exploring a different

route towards the functionalization of the Δ4-uronic acid, not involving heat or UV light

due to the instability of the chondroitin sulfate glycoconjugates under these conditions.

75

 

4.2.6 Effort towards a Michael addition to methyl ester of N-methyl-O-octyl

hydroxylamine Δ4-chondroitin sulfate disaccharide

The conversion of the Δ4-uronic acid into its methyl ester yields a better Michael acceptor

which should facilitate thiol addition onto the Δ4-uronic acid double bond. The chondroitin

sulfate N-methyl-O-octylhydroxylamine conjugate 63 was methylated selectively at the

carboxylate using CH3I and K2CO3 in excellent yield (Scheme 4.7).

63

7

O

OHHO

O

NHAc

OROR

O N OCH3I, K2CO3

DMF

64

7O

OHHO

O

NHAc

OROR

O N O

O OO O-

83%R = SO3- or H

Scheme 4.7 Synthesis of N-methyl-O-octylhydroxylamine functionalized chondroitin sulfate disaccharide

methyl ester 64.

The Michael addition was carried out using either β-mercaptoethanol or cysteamine, with

DIPEA as a base and MeOH or CH3CN as solvents. No addition was observed with either

thiol, stirred at rt or 37 °C for 2 days. Conditions were also carried out in water using

Ytterbium (Yb3+) or Lanthanum (La3+) in the presence of excess cysteamine or β-

mercaptoethanol, and also did not provide any sign of product formation after stirring at rt

for 1-2 days. This observation leads to the hypothesis that the Δ4-uronic acid double bond

is not electrophilic enough, thus explaining the unreactivity towards the thiol nucleophiles.

4.2.7 Efforts towards modifying the Δ4-chondroitin sulfate disaccharide using

electrophiles

The reaction of a protected Δ4-uronic acid methyl ester monosaccharide by Linhardt and

co-workers146 with NBS in THF-water (1:1) to form trans-diaxial or trans-diequatorial

bromohydrin stereoisomers in good yield provided us with inspiration. However, the

presence of a hydroxyl group along with the carboxyl group at C-5 has been shown to

cause extensive degradation in lyase-cleaved, bromohydrin functionalized GAGs.83 This

76

 

most likely happens through glycosidic cleavage due to the hemiacetal nature of C-5, as can

be seen in Figure 4.8.

O

NHAc

OROR

O OH

R = SO3- or H

O

OH-OOC

BrHO

HOO

NHAc

OROR

O OHOH

OH

COO-

BrHO

O

O

OH

COO-

BrHO

OO

NHAc

OROR

HO OH+

Figure 4.8 Proposed mechanism for the degradation of bromohydrin derivatized lyase-cleaved GAG.

This degradation might be prevented with the presence of an alkoxy group at C-5 instead of

a hydroxyl group. The installation of a halogen at C-4 would allow functionalization at the

non-reducing end by substitution with a strong nucleophile such as an azide. Since iodides

are better leaving groups than bromides, NIS was used instead of NBS. The

iodoalkoxylation was carried out in methanol on the unprotected N-methyl-O-

octylhydroxylamine chondroitin sulfate disaccharide 63. The obtained product was

purified using reversed-phase HPLC and gave a single stereoisomer of the di-equatorial

addition product 63 in 55% yield (Scheme 4.8).

63

7

O

OH

OOC

HOO

NHAc

OROR

O N ONIS

MeOHO

NHAc

OROR

OO

OH-OOC

IHO

OCH3

7N O

65R = SO3

- or H 55% DIPEA salt

Scheme 4.8 Iodoalkoxylation of the N-methyl-O-octylhydroxylamine chondroitin sulfate disaccharide 65.

Compound 65 was found to be in the glucuronic acid configuration with the iodide and the

methoxy group in a di-equatorial position as was previously observed with the

bromohydrin functionalized protected monosaccharide analogue.146

A substitution reaction was then carried out on compound 65 using sodium azide in DMSO.

After stirring the reaction at rt for 2 days no azide addition product was observed. 1H NMR

77

 

and mass spectrometry showed mostly unreacted compound 65 some minor regeneration of

the double bond containing species 63 along with degradation.

These results prompted us to install an alcohol at C-5, which would yield a handle that

could be used for further functionalization. Compound 63 is readily soluble in various

alcohols since it was isolated as the DIPEA salt. This would allow the installation of

various functional groups at the non-reducing terminus such as allyl, chloro or azide by

utilizing functionalized alcohols.

Unfortunately, the previously discussed synthesis of compound 65 could not be reproduced.

Upon further investigation, it appears that the product could be decomposing through a

similar mechanism observed with the bromohydrin derivative shown in Figure 4.8, since

some glycosidic bond cleavage was observed. In this case however, cleavage could occur

through a possible decarboxylation, which would lead to the observed glycosidic bond

cleavage. When the same synthesis was carried out using functionalized alcohols such as

2-chloroethanol and 3-chloropropanol no product was formed and only small amounts

(<5%) of starting material 63 were recovered.

Despite many attempts at functionalizing the Δ4-uronic acid double bond located at the

non-reducing end of the chondroitin sulfate disaccharide, little success was achieved using

currently available chemical methods.

4.3 Conclusion

Chondroitin sulfate polysaccharide can successfully be cleaved down to a disaccharide

using a bacterial lyase enzyme. The Δ4-uronic acid double bond formed during the

cleavage turned out to be surprisingly unreactive. Radical addition conditions which were

successful with a Δ4-uronic acid model monosaccharide proved to be incompatible with the

chondroitin sulfate leading to glycosidic cleavage. Michael addition with thiols at the

methyl ester of Δ4-uronate did not lead to the expected product. Iodoalkoxylation of the

Δ4-uronic acid using NIS in alcohol provided a route towards functionalization of the non-

reducing terminus of chondroitin sulfate but major decomposition of the product was

observed and the reaction was not reproducible. Therefore, a successful chemical method

78

 

to functionalize the Δ4-uronic acid double bond of the chondroitin sulfate without the use of

protecting groups has not yet been developed.

4.4 Future directions

Successful functionalization at the non-reducing terminus of chondroitin sulfate would be

an extremely useful method when combined with a reducing end functionalization to

produce doubly-labelled and chemically defined GAGs. This approach could be utilized to

produce, for example, substrates for various biological studies. However, since protecting-

group free chemical methods proved unsuccessful in the functionalization of the Δ4-uronic

acid chondroitin sulfate disaccharide, a chemoenzymatic-based approach might be more

suitable.

In principle, it should be possible to reverse the normal function of the Chondroitin AC

lyase, which was used to form the Δ4-uronic acid, since all enzymatic processes are in

equilibrium. Exposing the lyase to a large excess of the Δ4-uronic acid (cleavage product)

should force the lyase to perform the reverse reaction if allowed to reach equilibrium. In

order to push the equilibrium towards the glycosylation products, a nucleophile such as a

thiol could be used to generate a thio ether linkage at C-4 of the non-reducing sugar residue

(Scheme 4.9). It has been shown that thio-linked disaccharides are poor substrates for the

Chondroitinase AC lyase as they have low affinity for the enzyme, and are cleaved more

slowly than the natural occurring ether-linked disaccharide.147 The efficiency of this

reaction will be dependent upon binding of the thiol to the enzyme. However, even a low

affinity for thiol could potentially be overcome by using large excess of the thiol during the

enzymatic reaction.

R = SO3- or H

O

OH

-OOC

HOO

NHAc

ORRO

O OHSHR'

LyaseO

NHAc

OROR

OO

OH

R'SHO

COO-

OH

Scheme 4.9 Chemoenzymatically functionalizing the Δ4-uronic acid with a thiol.

79

 

However, in order to apply this chemoenzymatic method to longer lengths of Δ4-uronic

acid chondroitin sulfates, such as tetra- or hexasaccharides, the lyase activity of the enzyme

needs to be eliminated. This can be achieved by engineering the lyase in such a way that it

carries out the glycosylation reaction but not hydrolysis, as has been successfully done for

various glycosidases.148

The mechanism of Chondroitin AC lyase cleavage is still unclear. It is believed that a

tyrosine residue, activated as part of a catalytic tetrad, is acting as a general base by

removing the proton on C-5.149 For Chondroitinase AC lyase, it has been shown that the

proton abstraction at C-5 is the rate-limiting step for the overall reaction; therefore, the

subsequent release of the C-4 alcohol is faster. The Tyr234Phe mutant lyase still binds to

but does not cleave the substrate.149 Therefore, in analogy to mutation of glycosidases to

glycosynthases, it can be hypothesized that supplying the enzyme with Δ4-uronic acid

chondroitin sulfate and a functionalized thiol could lead to the formation of a thioether

linkage in accordance with the principle of microscopic reversibility.

Once the chemoenzymatic functionalization of the Δ4-uronic acid double bond has been

established this approach could be utilized to generate for example a chemically defined

substrate for the mammalian hyaluronidase (HAse). The mammalian hyaluronidase is a

hydrolase which cleaves the β-(1 4)-N-acetylhexosaminide linkage in hyaluronan and, to

some extent, chondroitin sulfate. Their overexpression in various human tumours has

implicated them in cancer metastasis.89 Robust and defined biochemical assays are

required to evaluate the physiochemical properties of the hyaluronidases. The methods

which have been used to monitor the progress of the enzymatic reaction include viscosity

measurements and size fractionation. Due to the endolytic cleavage mechanism,

conventional chromogenic glycosides are poor substrates for the hydrolases. Therefore,

new methods are required to evaluate the hyaluronidase activity in a simple and

quantitative way.

Recently, a fluorescence resonance energy transfer (FRET) method was developed to

monitor hyaluronidase activity.150 However, the doubly-labelled fluorescent hyaluronan

80

 

substrate had fluorescein amine and rhodamine B amine introduced randomly at the

carboxylates of the glucuronic acids.

The hyaluronidase assay should involve a chemically defined substrate, where the

conjugation of the fluorophore and the quencher would be performed on a lyase-cleaved

hyaluronan or chondroitin sulfate. The FRET partners, fluorescein and

tetramethylrhodamine, should allow quenching of the fluorescent substrate prior to

cleavage for up to an octa- or a decasaccharide which are about 40-50 Å at an extended

length (Figure 4.9).

O

AcHN

RO

OOR

O

OR

-OOCOHO

O

AcHN

RO

OOR

O O

OR

-OOC

HOO

AcHN

RO

OOR

OH

O

OR

-OOC

HOO

AcHN

RO

OOR

O

OR

-OOCOHO

O

AcHN

RO

OOR

O O

OR

-OOC

HOO

AcHN

RO

OOR

N OHN

Oblabl

S

n

n

Chemoenzymatic addition of a functionalized thiol

Chemoselective ligationwith N-methylhydroxylamine

O

OR

-OOC

HO

Figure 4.9 Internally quenched fluorescent hyaluronidase substrates. F = fluorophore, Q = quencher.

The GAG would be first cleaved with Chondroitinase AC, and the different lengths would

be purified. The isolated oligosaccharides contain two orthogonal functional groups: the

hemiacetal and the Δ4-uronic acid. The hemiacetal can be functionalized with a N-

methylhydroxylamine linker that can subsequently be coupled to the fluorophore. At the

non-reducing terminus, the incorporation of a functionalized thiol using the

chemoenzymatic method would facilitate the installation of a quencher.

The proposed internally quenched fluorescent hyaluronidase substrate design should

provide a sensitive and robust route towards assessing hyaluronidase activity.

F Q

81

 

Chapter 5. Experimental

5.1 General methods

Column chromatography was performed on Silica Gel 60 (Silicycle, Ontario). Reactions

were monitored by TLC on Silica Gel 60 F254 (EMD Science), with detection by quenching

of fluorescence and/or by charring with 15% sulfuric acid in methanol or with

phosphomolybdic acid in ethanol. Hydrolysis HPLC analyses were performed on a Dionex

BioLC (PDA-100 Photodiode Array Detector, GS50 Gradient pump and A550

Autosampler), using a Waters Symmetry® C-18 5 μm (4.6 x 150 mm) reverse phase

analytical column. Size exclusion HPLC purifications were performed on a Gilson HPLC

(Gilson UV/VIS-156 detector, Gilson 321-H2 pump). Preparative HPLC purifications were

performed on a Waters HPLC (Waters 2487 dual λ absorbance detector, Waters 1525

binary pump). Preparative HPLC was performed using a preparative Prevail Carbohydrate

ES column from Grace or on a preparative C-18 Grace Vydac column, using acetonitrile

and water as eluents.

NMR spectra were recorded at 25 °C with either a Varian 400 MHz (AutoX8308-400

probe), Mercury 400 MHz (ATB8123-400 probe) or a Varian Unity 500 MHz (Nalorac-500

probe) spectrometer. Chemical shifts were reported in ppm (δ scale) using the solvent

residue signals as reference, and assignments were determined by gCOSY spectroscopy.

Data are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t =

triplet, q = quartet, m = multiplet), integration and coupling constant. High resolution mass

spectra were obtained on an ABI/Sciex QStar mass spectrometer with an ESI source.

5.1.1 Hydrolysis method for glycoconjugates 1-4

Each glycoconjugate sample was prepared in duplicate at 5 mM (50 mM NaOAc pH 4.0,

50 mM NaOAc pH 5.0 or 50 mM Na2HPO4 pH 6.0) and incubated at 37 °C. All

glycoconjugates required 5% MeOH for solubility. 200 μL samples were taken out at

82

 

shown time intervals, and quenched through the addition of 400 μL of 4 °C 200 mM

Na2HPO4 buffer at pH 7.0 and immediately analyzed by HPLC (15-25% CH3CN in H2O, 4

min-15 min gradient). The observed pseudo-first-order rate constants for hydrolysis were

determined by directly fitting the areas of each glycoconjugate remaining as a percentage of

the total hydrazide concentration, to an exponential decay using Origin 7.0, defined by the

equation: v = kobs· [% glycoconjugate remaining].

5.1.2 Hydrolysis method for glycoconjugates 13-21

Each glycoconjugate sample was prepared in duplicate at 2 mM (20 mM NaOAc pH 4.0,

20 mM NaOAc pH 5.0 or 20 mM Na2HPO4 pH 6.0) and incubated at 37 °C.

Glycoconjugates 13, 16, 19 required 0.5% DMSO for solubility. Benzyl alcohol (1 mM)

was used as an internal standard for all runs. 200 μL samples were taken out at time

intervals and quenched with the addition of 400 μL of 4 °C 200 mM Na2HPO4 buffer at pH

7.0 and immediately analyzed by HPLC (15-25% CH3CN in H2O, 4 min-15 min gradient).

The observed pseudo-first-order rate constants for the hydrolysis were determined by

directly fitting the areas of each glycoconjugate remaining as a percentage to an

exponential decay using Origin 7.0, defined by the equation: v = kobs· [% glycoconjugate

remaining].

5.1.3 Determination of equilibrium constants, Ka, for glycoconjugates 13-21

The Ka values were determined using 1H NMR spectroscopy by integrating the area under

the methyl peak corresponding to the N-methylhydroxylamine or the methyl peak

corresponding to the p-toluenesulfonylhydrazide. The reactions were set up using four

different solutions, each containing the corresponding sugar: xylose, glucose or N-

acetylglucosamine, and the corresponding nucleophile: 1, 2 or 3, in equimolar amounts at

50, 75, 100 and 125 mM using 500 mM deuterated sodium acetate buffer, pD 4.5. 3% d6-

DMSO was added for solubility when using p-toluenesulfonylhydrazide. The samples

were incubated at 37 °C for 4 days and their 1H NMR spectra were measured. Product and

remaining reagent concentrations were then determined using integration values.

Degradation of the p-toluenesulfonylhydrazide into p-toluenesulfinic and p-toluenesulfonic

83

 

acid was taken into account in the calculations. The represented Ka value is an average of

the four samples.

5.2 Procedures

N-(β-D-Glucopyranosyl)benzoylhydrazide (1)

To a suspension of glucose (477 mg, 2.7 mmol) in 2 mL of

ethanol and 2 drops of AcOH was added benzoylhydrazide

(540 mg, 4.0 mmol). The reaction mixture was stirred and

refluxed for 3 h. The product precipitated and was purified by hot ethanol filtration and to

give 654 mg (83%) of 1. 1H NMR (400 MHz, D2O): δ 7.75 (m, 2H, Ar), 7.63 (m, 1H, Ar),

7.53 (m, 2H, Ar), 4.22 (d, 1H, J1,2 9.0 Hz, H-1), 3.92 (dd, 1H, J6a,6b 12.2, J5,6a 2.1 Hz, H-

6a), 3.73 (dd, 1H, J6a,6b 12.2, J5,6b 5.7 Hz, H-6b), 3.57 (dd, 1H, J2,3 9.3, J3,4 9.0 Hz, H-3),

3.46 (ddd, 1H, J4,5 9.8, J5,6b 5.7, J5,6a 2.1 Hz, H-5), 3.40 (m, 2H, H-4, H-2); 13C NMR (100

MHz, D2O): δ 170.8, 132.5, 132.0, 128.9 (2), 127.4 (2), 90.0, 77.0, 76.4, 70.8, 69.7, 61.0;

HRMS m/z calcd. for C13H18N2O6Na (M+Na+) 321.1064, found 321.1057.

N-(β-D-Glucopyranosyl)-p-methoxybenzoylhydrazide (2)

Using the same procedure as for compound 1, glucose

(300 mg, 1.7 mmol) and p-methoxybenzoylhydrazide

(415 mg, 2.5 mmol) produced 363 mg (66%) of 2. 1H

NMR (100 MHz, D2O): δ 7.79 (d, 2H, J 8.9 Hz, Ar), 7.11 (d, 2H, J 8.9 Hz, Ar), 4.25 (d,

1H, J1,2 9.0 Hz, H-1), 3.94 (dd, 1H, J6a,6b 12.4, J5,6a 2.3 Hz, H-6a), 3.91 (s, 3H, OCH3), 3.75

(dd, 1H, J6a,6b 12.4, J5,6b 5.8 Hz, H-6b), 3.58 (dd, 1H, J2,3 9.1, J3,4 9.0 Hz, H-3), 3.47 (ddd,

1H, J4,5 9.8, J5,6b 5.8, J5,6a 2.3 Hz, H-5), 3.41 (t, 1H, J4,5 9.8, J3,4 9.0 Hz, H-4), 3.40 (t, 1H,

J2,3 9.1, J1,2 9.0 Hz, H-2); 13C NMR (100 MHz, D2O): δ 170.2, 162.4, 129.5 (2), 124.5,

114.2 (2), 90.2, 77.1, 76.4, 70.9, 69.8, 61.0, 55.6; HRMS m/z calcd. for C14H20N2O7Na

(M+Na+) 351.1166, found 351.1162.

O

OH

HOHO

HOHN

NH

O

O

OH

HOHO

HOHN

NH

O

OCH3

84

 

N-(β-D-Glucopyranosyl)-p-chlorobenzoylhydrazide (3)

Using the same procedure as for compound 1, glucose

(150 mg, 0.8 mmol) and p-chlorobenzoylhydrazide (284

mg, 1.6 mmol) yielded 182 mg (66%) of 3. 1H NMR (400

MHz, D2O): δ 7.76 (d, 2H, J 8.5 Hz, Ar), 7.58 (d, 2H, J 8.5 Hz, Ar), 4.26 (d, 1H, J1,2 9.0

Hz, H-1), 3.94 (dd, 1H, J6a,6b 12.2, J5,6a 1.9 Hz, H-6a), 3.75 (dd, 1H, J6a,6b 12.2, J5,6b 5.8 Hz,

H-6b), 3.58 (t, 1H, J2,3 9.1, J3,4 9.0 Hz, H-3), 3.47 (ddd, 1H, J4,5 9.7, J5,6b 5.8, J5,6a 1.9 Hz,

H-5), 3.41 (t, J4,5 9.7, J3,4 9.0 Hz, 1H, H-4), 3.40 (t, 1H, J2,3 9.1, J1,2 9.0 Hz, H-2); 13C

NMR (100 MHz, D2O): δ 169.8, 138.1, 130.7, 129.0 (4), 90.0, 77.1, 76.4, 70.9, 69.7, 61.0;

HRMS m/z calcd. for C13H17N2O6ClNa (M+Na+) 355.0667, found 355.0667.

N-(β-D-Glucopyranosyl)-p-nitrobenzoylhydrazide (4)

Using the same procedure as for compound 1, glucose

(820 mg, 4.6 mmol) and p-nitrobenzoylhydrazide (1.5

g, 8.4 mmol gave 940 mg (60%) of 4. 1H NMR (400

MHz, D2O): δ 8.38 (d, 2H, J 8.7 Hz, Ar), 7.98 (d, 2H, J 8.7 Hz, Ar), 4.29 (d, 1H, J1,2 8.9

Hz, H-1), 3.93 (dd, 1H, J6a,6b 12.2, J5,6a 2.1 Hz, H-6a), 3.76 (dd, 1H, J6a,6b 12.2, J5,6b 5.8 Hz,

H-6b), 3.59 (t, 1H, J6a,6b 12.2, J3,4 9.0 Hz, H-3), 3.49 (ddd, 1H, J4,5 9.7, J5,6b 5.8, J5,6a 2.1

Hz, H-5), 3.42 (m, 2H, H-4, H-2); 13C NMR (100 MHz, D2O): δ 168.8, 149.8, 138.3, 128.8

(2), 124.0 (2), 90.0, 77.1, 76.5, 70.9, 69.7, 61.0; HRMS m/z calcd. for C13H17N3O8Na

(M+Na+) 366.0925, found 366.0907.

O-Benzyl-N-methylhydroxylamine (6)

Compound 8 (5.0 g, 21 mmol) was dissolved in 40 mL CH2Cl2-

TFA (1:1) and stirred at rt for 22 h. The solvent was then

removed under reduced pressure and the product purified by

column chromatography using pentane as eluent to afford 2.6 g of 6 as oil, yield 90%. 1H

NMR (400 MHz, CDCl3): δ 8.78 (s, 2H, NH2), 7.37 (s, 5H, Ar), 5.04 (s, 2H, OCH2), 2.91

(s, 3H, NCH3); 13C NMR (100 MHz, CDCl3): δ 133.0, 129.8, 129.5 (2), 129.1 (2), 76.4,

35.9; HRMS m/z calcd. for C8H12NO (M+H+) 138.0913, found 138.0915.

O

OH

HOHO

HOHN

NH

O

Cl

O

OH

HOHO

HOHN

NH

O

NO2

H2N

OO

F3C O-

85

 

N-Methyl-O-(N’-benzylacetamide)hydroxylamine (7)

Compound 12 (0.9 g, 3.1 mmol) was dissolved in 10 mL

CH2Cl2-TFA (1:1) and stirred at rt for 1 h. The solvent was

then removed under reduced pressure and the product was

recrystallized from Et2O-pentane to give 0.81 g of the TFA salt

7 as white plates, yield 87%. 1H NMR (400 MHz, CDCl3): δ 9.18 (s, 2H, NH2CH3), 7.36-

7.26 (m, 5H, Ar), 7.14 (s, 1H, CONH), 4.54 (s, 2H, OCH2), 4.46 (s, 2H, PhCH2), 2.88 (s,

3H, NCH3); 13C NMR (100 MHz, CDCl3): δ 169.4, 137.3, 129.0 (2), 128.0, 128.0 (2), 71.6,

43.5, 37.4; HRMS m/z calcd. for C10H15N2O2 (M+H+) 195.1128, found 195.1123.

tert-Butyl N-benzyloxy-N-methylcarbamate (8)

A 60% oil dispersion of NaH (1.6 g, 40 mmol) was added to t-butyl

N-methyl-N-hydroxycarbamate (5.3 g, 36 mmol) in 20 mL

anhydrous DMF and stirred at 0 °C for 30 min under nitrogen.

Benzyl bromide (4.7 mL, 40 mmol) was added and the reaction was stirred for 16 h at rt.

The reaction mixture was diluted with pentane (150 mL) and the organic layer was then

washed with water (3 x 50 mL) and brine (40 mL), dried over MgSO4, filtered and

concentrated. The residue was purified by column chromatography (CH2Cl2-pentane 2:3)

to afford 7.8 g of 8 as pale yellow oil, yield 85%. 1H NMR (400 MHz, CDCl3): δ 7.42-7.31

(m, 5H, Ar), 4.81 (s, 2H, OCH2), 3.05 (s, 3H, NCH3), 1.40 (s, 9H, C(CH3)3); 13C NMR (100

MHz, CDCl3): δ 157.2, 135.8, 129.6 (2), 128.6, 128.5 (2), 81.4, 76.6, 37.0, 28.4 (3);

HRMS m/z calcd. for C13H19NO3 (M+Na+) 260.1257, found 260.1260.

Ethyl (tert-butoxycarbonyl-N-methylaminooxy)acetate (9)

A 60% oil dispersion of NaH (136 mg, 3.4 mmol) was added to a

solution of t-butyl N-methyl-N-hydroxycarbamate (500 mg, 3.4 mmol)

in THF (10 mL) and stirred at 0 °C for 30 min under nitrogen. Ethyl

bromoacetate (452 μL, 4.1 mmol) was added and the reaction was stirred for 4 h at rt. The

reaction mixture was then diluted with EtOAc (150 mL) and the organic layer was washed

with water (3 x 50 mL) and brine (2 x 15 mL), dried over MgSO4, filtered and

concentrated. The residue was purified by column chromatography (EtOAc-pentane 1:9) to

NO

OO

NO

O

OBoc

H2NO

HN

O

O

F3C O-

86

 

afford 755 mg of 9 as pale yellow oil, yield 95%. 1H NMR (400 MHz, CDCl3): δ 4.45 (s,

2H, OCH2CO), 4.24 (q, 2H, J 7.1 Hz, OCH2CH3), 3.21 (s, 3H, NCH3), 1.49 (s, 9H,

C(CH3)3), 1.30 (t, 3H, J 7.1 Hz, OCH2CH3); 13C NMR (100 MHz, CDCl3): δ 169.5, 157.9,

82.1, 72.2, 61.2, 38.5, 28.4 (3), 14.3; HRMS m/z calcd. for C10H19NO5Na (M+Na+)

256.1155, found 256.1154.

(tert-Butoxycarbonyl-N-methylaminooxy)acetic acid (10)

Compound 9 (689 mg, 3.0 mmol) was dissolved in THF (6 mL) and a

solution of NaOH (240 mg, 6.0 mmol) in H2O (2 mL) was added, and the

solution was stirred for 4 h at rt. The reaction mixture was then diluted

with EtOAc (50 mL) and the organic layer was washed with 1 M HCl (2 x 50 mL), water (2

x 50 mL) and brine (1 x 20 mL), dried over MgSO4, filtered and concentrated. The residue

was purified by column chromatography (CH2Cl2) to afford 519 mg of 10 as pale yellow

oil, yield 86%. 1H NMR (400 MHz, CDCl3): δ 4.46 (s, 2H, OCH2), 3.14 (s, 3H, NCH3),

1.51 (s, 9H, C(CH3)3); 13C NMR (100 MHz, CDCl3): δ 170.7, 159.9, 85.2, 73.4, 37.9, 28.2

(3); HRMS m/z calcd. for C8H15NO5Na (M+Na+) 228.0842, found 228.0850.

(tert-Butoxycarbonyl-N’-methylaminooxyacetyl)-N-hydroxysuccinimide ester (11)

To a stirred solution of 10 (750 mg, 3.6 mmol) in EtOAc (25 mL)

was added N-hydroxysuccinimide (630 mg, 1.5 equiv) and DCC (900

mg, 1.2 equiv). The reaction mixture was then stirred at rt for 2 h.

The solid was removed by filtration and washed with EtOAc. The organics were then

washed with 1 M NaHCO3 (2 x 30 mL) and dried over MgSO4. After filtration, the solvent

was removed under vacuum and the resulting white solid was recrystallized from EtOAc-

hexanes giving 960 mg of 11, yield 87%. 1H NMR (400 MHz, CDCl3): δ 4.80 (s, 2H,

OCH2), 3.19 (s, 3H, NCH3), 2.86 (s, 4H, NCOCH2), 1.50 (s, 9H, C(CH3)3); 13C NMR (100

MHz, CDCl3): δ 168.8 (2), 165.2, 158.0, 82.6, 70.0, 38.9, 28.3 (3), 25.7 (2); HRMS m/z

calcd. for C12H18N2O7Na (M+Na+) 325.1013, found 325.1006.

NO

O

OHBoc

NO

O

ON

O

OBoc

87

 

(tert-Butoxycarbonyl-N-methyl-O-(N’-benzylacetamide))hydroxylamine (12)

To a solution of 11 (186 mg, 0.62 mmol) in CH3CN (15 mL) was

added benzyl amine (107 μL, 0.74 mmol). The reaction was

stirred under nitrogen for 30 min. The reaction mixture was then

diluted with EtOAc (100 mL) and the organic layer was washed with 1 M NaHCO3 (2 x 50

mL), 1 M HCl (2 x 50 mL), H2O (2 x 50 mL) and brine (50 mL), dried over MgSO4,

filtered and concentrated. This afforded 12 as a colorless oil (169 mg), yield 93%. 1H

NMR (400 MHz, CDCl3): δ 8.51 (s, 1H, CONH), 7.27-7.18 (m, 5H, Ar), 4.47 (d, 2H, J 6.0

Hz, ArCH2), 4.32 (s, 2H, OCH2), 3.05 (s, 3H, NCH3), 1.37 (s, 9H, C(CH3)3); 13C NMR (100

MHz, CDCl3): δ 168.9, 158.0, 138.2, 128.6 (2), 127.8 (2), 127.3, 83.0, 73.5, 42.9, 37.4,

28.1 (3); HRMS m/z calcd. for C15H22N2O4Na (M+Na+) 317.1471, found 317.1465.

N-(β-D-Xylopyranosyl)-p-toluenesulfonohydrazide (13)

Compound 13 (3.5 g, 11 mmol) was prepared by

making a solution containing 0.75 M D-xylose (2.0 g,

13.3 mmol) and p-toluenesulfonylhydrazide 5 (2.6 g,

13.7 mmol) in 2 M NH4OAc buffer pH 4.5, and incubating at 37 °C for 72 h. The solution

was then lyophilized and the product was recrystallized from isopropanol, yield 83%. 1H

NMR (400 MHz, CD3OD): δ 7.79 (d, 2H, J 8.3 Hz, Ar), 7.39 (d, 2H, J 8.3 Hz, Ar), 3.79

(dd, 1H, J5a,5b 11.3, J4,5a 5.4 Hz, H-5a), 3.70 (d, 1H, J1,2 8.7 Hz, H-1), 3.47-3.40 (m, 2H, H-

2, H-4), 3.28 (under CD3OD peak, 1H, H-3), 3.07 (dd, J5a,5b 11.3, J4,5b 10.7 Hz, H-5b), 2.43

(s, 3H, ArCH3); 13C NMR (100 MHz, CD3OD): δ 145.1, 137.1, 130.6 (2), 129.1 (2), 92.4,

78.2, 71.4, 71.2, 68.3, 21.5; HRMS m/z calcd. for C12H18N2O6NaS (M+Na+) 341.0777,

found 341.0788.

N-Methyl-O-benzyl-N-(β-D-xylopyranosyl)hydroxylamine (14)

Compound 14 (152 mg, 0.56 mmol) was prepared by

making a solution containing 0.75 M D-xylose (100 mg,

0.67 mmol) and compound 6 (110 mg, 0.8 mmol) in 2 M

NH4OAc buffer pH 4.5, and incubating at 37 °C for 72 h. The product was purified by

column chromatography (5% MeOH in CH2Cl2), yield 84%. 1H NMR (400 MHz,

NO

O

HN

Boc

O

OH

HOHO

HN

NH

SO

O

O

OH

HOHO N

O

88

 

CD3OD): δ 7.39-7.27 (m, 5H, Ar), 4.75 (s, 2H, ArCH2), 3.95 (d, 1H, J1,2 9.0 Hz, H-1), 3.88

(dd, 1H, J5a,5b 11.2, J4,5a 5.4 Hz, H-5a), 3.49-3.42 (m, 2H, H-2, H-4), 3.31 (under CD3OD

peak, 1H, H-3), 3.14 (dd, J5a,5b 11.2, J4,5b 10.9 Hz, H-5b), 2.69 (s, 3H, ArCH3); 13C NMR

(100 MHz, CD3OD): δ 138.5, 130.2 (2), 129.3 (2), 129.0, 96.4, 79.4, 76.4, 71.7, 71.1, 68.9,

39.3; HRMS m/z calcd. for C13H20NO5 (M+H+) 270.1335, found 270.1328.

N-Methyl-O-(N’-benzylacetamide)-N-(β-D-xylopyranosyl)hydroxylamine (15)

Using the same procedure as described for

compound 14, D-xylose (41 mg, 0.27 mmol) and 7

(100 mg, 0.32 mmol) gave 76 mg of 15 as white

amorphous solid (86%). 1H NMR (400 MHz, CD3OD): δ 7.33-7.21 (m, 5H, Ar), 4.45 (d,

1H, OCH2a, J 15.0 Hz), 4.41 (d, 1H, OCH2b, J 15.0 Hz), 4.34 (d, 1H, ArCH2a, J 15.1 Hz),

4.28 (d, 1H, ArCH2b, J 15.1 Hz), 3.98 (d, 1H, J1,2 9.0 Hz, H-1), 3.88 (dd, 1H, J5a,5b 11.2,

J4,5a 5.4 Hz, H-5a), 3.48-3.41 (m, 2H, H-2, H-4), 3.31 (under CD3OD peak, 1H, H-3), 3.16

(dd, J5a,5b 11.2, J4.5b 10.6 Hz, H-5b), 2.73 (s, 3H, Ac); 13C NMR (100 MHz, CD3OD): δ

172.1, 139.6, 129.6 (2), 128.7 (2), 128.3, 96.3, 79.1, 72.4, 71.6, 71.1, 68.9, 43.7, 38.8;

HRMS m/z calcd. for C15H23N2O6 (M+H+) 327.1550, found 327.1566.

N-(β-D-Glucopyranosyl)-p-toluenesulfonohydrazide (16)

Using the same procedure as described for compound

13, D-glucose (2.0 g, 11.1 mmol) and p-

toluenesulfonylhydrazide (2.13 g, 11.43 mmol) gave

3.43 g of compound 16 as white solid (89%). 1H NMR (400 MHz, D2O): δ 7.81 (d, 2H, J

8.3 Hz, Ar), 7.40 (d, 2H, J 8.3 Hz, Ar), 3.86 (dd, 1H, J6a,6b 11.7, J5,6a 1.9 Hz, H-6a), 3.67 (d,

1H, J1,2 8.5 Hz, H-1), 3.58 (dd, 1H, J6a,6b 11.7, J5,6b 6.2 Hz, H-6b), 3.34 (under CD3OD

peak, 1H, H-3), 3.29 (under CD3OD peak, 1H, H-4), 3.20-3.11 (m, 2H, H-2, H-5), 2.44 (s,

3H, ArCH3); 13C NMR (100 MHz, CD3OD): δ 145.1, 137.4, 130.6 (2), 129.1 (2), 91.5,

79.2, 78.2, 71.8 (2), 63.2, 21.5; HRMS m/z calcd. for C13H20N2O7NaS (M+Na+) 371.0883,

found 371.0901.

O

OH

HOHO

HOHN

NH

SO

O

O

OH

HOHO N

OO

HN

89

 

N-Methyl-O-benzyl-N-(β-D-glucopyranosyl)hydroxylamine (17)

Using the same procedure as described for compound 14, D-

glucose (103 mg, 0.57 mmol) and 6 (94 mg, 0.68 mmol)

gave 150 mg of 17 as white solid (88%). 1H NMR (400

MHz, D2O): δ 7.41-7.30 (m, 5H, Ar), 4.81 (d, 1H, J 10.4 Hz, OCH2a), 4.78 (d, 1H, J 10.4

Hz, OCH2b), 4.04 (d, 1H, J1,2 8.9 Hz, H-1), 3.84 (dd, 1H, J6a,6b 12.1, J5,6a 2.2 Hz, H-6a),

3.68 (dd, 1H, J6a,6b 12.1, J5,6b 5.1 Hz, H-6b), 3.49 (t, 1H, J3,4 8.9 Hz, H-3), 3.39 (t, 1H, J3,4

8.9 Hz, H-4), 3.28 (under CD3OD peak, 1H, H-2), 3.22 (ddd, 1H, J4,5 9.5, J5,6b 5.1, J5,6a 2.2

Hz, H-5), 2.76 (s, 3H, NCH3); 13C NMR (100 MHz, CD3OD): δ 138.1, 130.5 (2), 129.4 (2),

129.3, 95.4, 79.6, 79.3, 76.7, 71.8, 71.1, 62.7, 39.4; HRMS m/z calcd. for C14H22NO6

(M+H+) 300.1441, found 300.1454.

N-Methyl-O-(N’-benzylacetamide)-N-(β-D-glucopyranosyl)hydroxylamine (18)

Using the same procedure as described for

compound 14, D-glucose (23 mg, 0.13 mmol) and 7

(50 mg, 0.16 mmol) gave 38 mg of 18 as white

amorphous solid (82%). 1H NMR (400 MHz, CD3OD): δ 7.34-7.22 (m, 5H, Ar), 4.47 (d,

1H, J 14.9 Hz, OCH2a), 4.43 (d, 1H, J 14.9 Hz, OCH2b), 4.37 (d, 1H, J 15.1 Hz, ArCH2a),

4.31 (d, 1H, J 15.1 Hz, ArCH2b), 4.05 (d, 1H, J1,2 8.8 Hz, H-1), 3.87 (dd, 1H, J6a,6b 11.9,

J5,6a 2.1 Hz, H-6a), 3.68 (dd, 1H, J6a,6b 11.9, J5,6b 5.2 Hz, H-6b), 3.45 (t, 1H, J 8.8 Hz, H-3),

3.37 (t, 1H, J 8.7 Hz, H-4), 3.30-3.21 (m, 2H, H-2, H-5), 2.78 (s, 3H, NCH3); 13C NMR

(100 MHz, CD3OD): δ 172.1, 139.7, 129.6 (2), 128.6 (2), 128.3, 95.4, 79.7, 79.2, 72.5,

71.7, 71.4, 62.7, 43.7, 38.8; HRMS m/z calcd. for C16H24N2O7Na (M+Na+) 379.1475,

found 379.1462.

N-(2-Acetamido-2-deoxy-β-D-glucopyranosyl)-p-toluenesulfonohydrazide (19)

Using the same procedure as described for compound

13, D-N-acetylglucosamine (1.0 g, 9.0 mmol) and p-

toluenesulfonylhydrazide 5 (0.85 g, 9.1 mmol) gave

2.9 g of compound 19 as white solid (83%). 1H NMR (400 MHz, CD3OD): δ 7.74 (d, 2H, J

8.3 Hz, Ar), 7.38 (d, 2H, J 8.3 Hz, Ar), 3.94 (d, 1H, J1,2 9.2 Hz, H-1), 3.89 (dd, 1H, J6a,6b

O

NHAc

HOHO

HOHN

NH

SO

O

O

OH

HOHO

HO NO

O

OH

HOHO

HO NO

O

HN

90

 

11.7, J5,6a 1.7 Hz, H-6a), 3.60 (m, 1H, H-6b), 3.46 (t, 1H, J2,3 10.1, J1,2 9.2 Hz, H-2), 3.42-

3.37 (m, 1H, H-3), 3.20-3.18 (m, 2H, H-4, H-5), 2.43 (s, 3H, ArCH3), 2.01 (s, 3H, Ac); 13C

NMR (100 MHz, CD3OD): δ 173.9, 145.1, 137.2, 130.5 (2), 129.1 (2), 91.9, 78.9, 76.2,

72.4, 63.2, 55.0, 23.0, 21.5; HRMS m/z calcd. for C15H23N3O7NaS (M+Na+) 412.1148,

found 412.1156.

N-Methyl-O-benzyl-N-(2-acetamido-2-deoxy-β-D-glucopyranosyl)hydroxylamine (20)

Using the same procedure as described for compound 14, D-

N-acetylglucosamine (147 mg, 0.6 7 mmol) and 6 (110 mg,

0.8 mmol) gave 196 mg of 20 as white amorphous solid

(86%). 1H NMR (400 MHz, CD3OD): δ 7.42-7.40 (m, 2H, Ar), 7.36-7.30 (m, 3H, Ar),

4.68 (d, 1H, J 10.0 Hz, ArCH2a), 4.62 (d, 1H, J 10.0 Hz, ArCH2b), 4.19 (d, 1H, J1,2 9.8 Hz,

H-1), 3.95 (t, 1H, J1,2 9.8 Hz, H-2), 3.85 (dd, 1H, J6a,6b 12.1, J5,6a 2.2 Hz, H-6a), 3.69 (dd,

1H, J6a,6b 12.1, J5,6b 5.3 Hz, H-6b), 3.42 (t, 1H, J3,4 8.9, J2,3 9.8 Hz, H-3), 3.33 (t, 1H, J4,5

9.5, J3,4 8.9 Hz, H-4), 3.23 (ddd, 1H, J4,5 9.5, J5,6b 5.3, J5,6a 2.2 Hz, H-5), 2.72 (s, 3H,

NCH3), 2.01 (s, 3H, Ac); 13C NMR (100 MHz, CD3OD): δ 173.4, 138.1, 130.7 (2), 129.3

(2), 129.2, 93.7, 79.6, 77.6, 76.1, 71.6, 62.8, 54.1, 39.1, 23.1; HRMS m/z calcd. for

C16H24N2O6Na (M+Na+) 363.1526, found 363.1530.

N-Methyl-O-(N’-benzylacetamide)-N-(2-acetamido-2-deoxy-β-D-glucopyranosyl)

hydroxylamine (21)

Using the same procedure as described for

compound 14, D-N-acetylglucosamine (29 mg, 0.13

mmol) and 7 (31 mg, 0.16 mmol) gave 44 mg of 21

as white amorphous solid (85%). 1H NMR (400 MHz, CD3OD): δ 7.34-7.28 (m, 4H, Ar),

7.26-7.22 (m, 1H, Ar), 4.47 (d, 1H, J 14.8 Hz, OCH2a), 4.42 (d, 1H, J 14.8 Hz, OCH2b),

4.27 (d, 1H, J 14.7 Hz, ArCH2a), 4.25 (d, 1H, J1,2 9.7 Hz, H-1), 4.18 (d, 1H, J 14.7 Hz

ArCH2b), 3.89 (dd, 1H, J6a,6b 11.9, J5,6a 2.1 Hz, H-6a), 3.82 (t, 1H, J 9.7, Hz, H-2), 3.71 (dd,

1H, J6a,6b 11.9, J5,6b 5.4 Hz, H-6b), 3.42 (dd, 1H, J4,5 9.9, J3,4 8.5 Hz, H-3), 3.30 (under

CD3OD, H-4), 3.25 (ddd, 1H, J4,5 9.6, J5,6b 5.4, J5,6a 2.1 Hz, H-5), 2.75 (s, 3H, NCH3), 1.95

(s, 3H, Ac); 13C NMR (100 MHz, CD3OD): δ 173.5, 171.9, 139.8, 129.6 (2), 128.6 (2),

O

NHAc

HOHO

HO NO

O

OH

HOHO

HO NO

O

HN

91

 

128.3, 93.6, 79.7, 77.3, 72.9, 71.9, 62.7, 54.1, 43.6, 38.6, 23.0; HRMS m/z calcd. for

C18H27N3O7Na (M+Na+) 420.1741, found 420.1726.

Octylsulfonyl hydrazide (22)

A solution of hydrazide monohydrate (872 μL, 8.2 mmol) in 4 mL of THF

was cooled to -10 °C. Octylsulfonyl chloride (1.6 mL, 8.2 mmol) was

added slowly to the mixture over a period of 30 min and the reaction stirred was at 0 °C for

another 2 h. The solvent was then evaporated and the residue was then taken up in 30 mL

CH2Cl2 and poured into a separatory funnel. The organic layer was washed with H2O

(3x30 mL) and brine (1x30 mL), and then concentrated in vacuo. The product was then

recrystallized from EtOAc-pentane, (1.6 g, 7.4 mmol), yield 91%. 1H NMR (400 MHz,

CD3Cl): δ 4.27 (br s, 2H, NH2) 3.12-3.07 (m, 2H, SO2CH2), 1.86-1.76 (m, 2H,

SO2CH2CH2), 1.48-1.38 (m, 2H, SO2(CH2)2CH2), 1.34-1.25 (m, 8H, CH2), 0.88 (t, 3H, J

6.7 Hz, CH3); 13C NMR (100 MHz, CD3OD): δ 49.2, 31.7, 29.0, 28.9, 28.2, 23.1, 22.5,

14.0; HRMS m/z calcd. for C8H21N2O2S (M+H+) 209.1318, found 209.1338.

N’-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-octylsulfonohydrazide (23)

Comound 23 (860 mg, 2.1 mmol) was prepared by suspending

N-acetylglucosamine (500mg, 2.4 mmol) and

octylsulfonylhydrazide (542 mg, 2.6 mmol) in DMF (10 mL).

A catalytic amount of AcOH was added to the mixture which was then incubated at 37° C

for 48 h. The DMF was then evaporated and the product was purified via C-18 reverse

phase chromatography (15-50% CH3CN in H2O, 4-40 min gradient), yield 87%. 1H NMR

(400 MHz, CD3OD): δ 4.11 (d, 1H, J1,2 9.4 Hz, H-1), 3.93 (dd, 1H, J6a,6b 11.7, J5,6a 2.1 Hz,

H-6a), 3.63 (dd, 1H, J6a,6b 11.7, J5,6b 6.5 Hz, H-6b), 3.55 (dd, 1H, J2,3 10.1, J1,2 9.4 Hz, H-

2), 3.48 (dd, 1H, J2,3 10.1, J3,4 8.1 Hz, H-3), 3.28 (under CD3OH peak, H-5), 3.23 (dd, 1H,

J3,4 9.7, J3,4 8.1 Hz, H-4), 3.13-3.04 (m, 2H, SO2CH2), 1.99 (s, 3H, Ac), 1.78-1.70 (m, 2H,

SO2CH2CH2), 1.48-1.40 (m, 2H, SO2(CH2)2CH2), 1.37-1.29 (m, 8H, CH2), 0.91 (t, 3H, J

6.8 Hz, CH3); 13C NMR (100 MHz, CD3OD): δ 174.1, 92.1, 79.0, 76.3, 72.5, 63.3, 55.2,

50.0, 32.9, 30.3, 30.2, 29.4, 24.4, 23.7, 23.0, 14.4; HRMS m/z calcd. for C16H34N3O7S

(M+H+) 412.2111, found 412.2120.

H2N NH

SO

O 7

O

NHAc

HOHO

HOHN

NH

SO O

7

92

 

N’-(2-acetamido-4-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-2-deoxy-D-β-

glucopyranoside)-p-toluenesulfonohydrazide (24)

Compound 24 (100 mg, 0.17 mmol) was

prepared by suspending 2-acetamido-4-O-(2-

acetamido-2-deoxy-β-D-glucopyranosyl)-2-

deoxy-D-glucopyranoside (80 mg, 0.19 mmol) and p-toluenesulfonylhydrazide 5 (49 mg,

0.26 mmol) in DMF (1.5 mL). A catalytic amount of AcOH was added to the mixture

which was incubated at 37 °C for 48h. The solvent was then removed under reduced

pressure and the product purified with C-18 reverse phase HPLC (15-40% CH3CN in H2O,

4-40 min gradient), yield 90%. 1H NMR (400 MHz, D2O): δ 7.75 (d, 2H, J 8.2 Hz, Ar),

7.50 (d, 2H, J 8.5 Hz, Ar), 4.57 (d, 1H, J1,2 8.6 Hz, H-1’), 3.92 (dd, 1H, J6a,6b 12.4, J5,6a 1.7

Hz, H-6’a), 3.91 (d, 1H, J1,2 9.4 Hz, H-1), 3.83 (dd, 1H, J6a,6b 12.1, J5,6a 1.8 Hz, H-6a),

3.76-3.71 (m, 2H, H-2’, H-6’b), 3.63-3.46 (m, 7H, H-6b, H-4, H-3, H-2, H-5’, H-4’, H-3’),

3.37-3.34 (m, 1H, H-5), 2.47 (s, 3H, ArCH3), 2.07 (s, 3H, Ac), 2.03 (s, 3H, Ac); 13C NMR

(100 MHz, D2O): δ 174.8, 174.4, 145.8, 133.2, 130.0 (2), 128.0 (2), 101.6, 89.8, 79.5, 76.0,

75.4, 73.6, 72.9, 69.8, 60.7, 60.3, 55.7, 52.8, 22.3 (2), 20.9; HRMS m/z calcd. for

C23H37N4O12S (M+H+) 593.2123, found 593.2123.

N’-(2-acetamido-6-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-2-deoxy-D-β-

glucopyranoside)-p-toluenesulfonohydrazide (25)

Compound 25 (175 mg, 0.29 mmol) was prepared

by suspending 2-acetamido-6-O-(2-acetamido-2-

deoxy-β-D-glucopyranosyl)-2-deoxy-D-gluco-

pyranoside (142 mg, 0.33 mmol) and p-

toluenesulfonylhydrazide 5 (70 mg, 0.38 mmol) in DMF (0.5 mL). A catalytic amount of

AcOH was added to the mixture which was incubated at 37 °C for 48 h. The solvent was

then removed under reduced pressure and the product was purified by HPLC with a Prevail

carbohydrate column (gradient: 30-50% H2O in CH3CN, 6-30 min; 50-90% H2O in

CH3CN, 30-50 min), yield 88%. 1H NMR (500 MHz, D2O): δ 7.75 (d, 2H, J 8.3 Hz, Ar),

7.50 (d, 2H, J 8.3 Hz, Ar), 4.60 (d, 1H, J1,2 8.6 Hz, H-1’), 4.15 (dd, 1H, J6a,6b 11.7, J5,6a 1.8

Hz, H-6a), 4.00 (d, 1H, J1,2 9.3 Hz, H-1), 3.96 (dd, 1H, J6a,6b 12.3, J5,6a 1.6 Hz, H-6’a),

O

NHAc

HOO HO

O

NHAc

HOHO

HOHN

NH

S TolO O

O

NHAc

O

HOHO

O

NHAc

HOHO

HOHN

NH

S TolO O

93

 

3.79-3.73 (m, 3H, H-6b, H-6’b, H-2’), 3.59 (dd, 1H, J2,3 10.3, J3,4 8.7 Hz, H-3’), 3.52-3.46

(m, 4H, H-2, H-3, H-4’, H-5’), 3.45-3.41 (m, 1H, H-5), 3.33 (dd, 1H, J4,5 9.7, J3,4 9.0 Hz,

H-4), 2.47 (s, 3H, ArCH3), 2.09 (s, 3H, Ac), 2.04 (s, 3H, Ac); 13C NMR (100 MHz, D2O): δ

174.8, 174.4, 145.8, 133.1, 130.0 (2), 128.0 (2), 102.0, 90.1, 76.0, 75.8, 74.3, 73.9, 70.1,

69.9, 68.9, 60.9, 55.7, 53.3, 22.5, 22.4, 21.0; HRMS m/z calcd. for C23H37N4O12S (M+H+)

593.2123, found 593.2123.

Methyl 2-acetamido-2-deoxy-β-D-glucopyranoside (28)

Tosylhydrazine donor 19 (50 mg, 0.13 mmol) was dissolved in extra

dry DMF (1.0 mL), and pre-dried methanol (200 μL, 5.1 mmol) was

added to the solution. N-Bromosuccinimide (55 mg, 0.31 mmol) was

then added at rt. After 10 min of stirring, Amberlite resin (-OH) was added to quench the

reaction and the solution was stirred until the yellow color disappeared. The resin was

filtered, washed with MeOH and solvent was removed under reduced pressure. The residue

was then purified by flash chromatography (MeOH-CH2Cl2 1:11.5) to afford a white solid,

(29 mg, 0.11 mmol) yield 87% (β:α, 10:1). 1H NMR (400 MHz, CD3OD): δ 4.30 (d, 1H,

J1,2 8.4 Hz, H-1), 3.90 (dd, 1H, J6a,6b 11.9, J5,6a 2.2 Hz, H-6a), 3.70 (dd, 1H, J6a,6b 11.9, J5,6b

5.6 Hz, H-6b), 3.64 (dd, 1H, J1,2 8.4, J2,3 10.2 Hz, H-2), 3.46 (s, 3H, OCH3), 3.43 (dd, 1H,

J2,3 10.2, J3,4 8.4 Hz, H-3), 3.31 (under CD3OD peak, 1H, H-4), 3.26 (ddd, 1H, J4,5 9.5, J5,6b

5.6, J5,6a 2.2 Hz, H-5), 1.97 (s, 3H, Ac); 13C NMR (125 MHz, CD3OD): δ 173.8, 103.6,

78.0, 76.3, 72.2, 62.8, 57.3, 57.0, 22.9; HRMS m/z calcd. for C9H17NO6Na (M+Na+)

258.0948, found 258.0945.

3-Chloropropyl 2-acetamido-2-deoxy-β-D-glucopyranoside (29)

Compound 29 (28 mg, 0.094 mmol) was prepared as

compound 28 with 3-chloropropanol (159 μL, 1.9 mmol) and

purified by column chromatography (MeOH-CH2Cl2 1:11.5),

yield 72% (β:α, 7:1). 1H NMR (400 MHz, D2O): δ 4.55 (d, 1H, J1,2 8.4 Hz, H-1), 4.05 (q,

1H, J 10.4, J 5.2 Hz, OCHaHbCH2CH2Cl), 3.97 (dd, 1H, J6a,6b 12.3, J5,6a 1.8 Hz, H-6a),

3.80-3.75 (m, 2H, OCHaHbCH2CH2Cl, H-6b), 3.73-3.62 (m, 3H, H-2, H3,

OCH2CH2CHeHfCl), 3.57 (dd, 1H, J3,4 8.7, J4,5 10.3 Hz, H-4), 3.51-3.43 (m, 2H, H-5,

O

AcHN

HOHO

HO O

O

AcHN

HOHO

HO O Cl

94

 

OCH2CH2CHeHfCl), 2.12-1.93 (m, 5H, Ac, OCH2CH2CH2Cl); 13C NMR (100 MHz,

D2O): δ 174.8, 101.6, 76.0, 73.9, 70.1, 67.1, 60.9, 55.7, 41.8, 31.6, 22.3; HRMS m/z calcd.

for C11H20NO6ClNa (M+Na+) 320.0871, found 320.0886.

Octyl 2-acetamido-2-deoxy-β-D-glucopyranoside (30)

Comound 30 (31 mg, 0.093 mmol) was prepared as compound 28

with 1-octanol (292 μL, 1.9 mmol) and purified by column

chromatography (MeOH-CH2Cl2 1:11.5), yield 75% (β:α, 8:1). 1H

NMR (400 MHz, CD3OD): δ 4.40 (d, 1H, J1,2 8.4 Hz, H-1), 3.91-3.86 (m, 2H, H-6a,

OCHaHbCH2), 3.69 (dd, 1H, J6a,6b 11.9, J5,6b 5.6 Hz, H-6b), 3.62 (dd, 1H, J2,3 10.3, J1,2 8.4

Hz, H-2), 3.48- 3.42 (m, 2H, H-3, OCHaHbCH2), 3.30 (under CD3OD peak, 1H, H-4), 3.25

(ddd, 1H, J4,5 9.7, J5,6b 5.6, J5,6a 2.2 Hz, H-5), 1.97 (s, 3H, Ac), 1.58-1.51 (m, 2H,

OCH2CH2), 1.40-1.23 (m, 10H, octyl), 0.90 (t, 3H, J 6.8 Hz, CH2CH3); 13C NMR (100

MHz, CD3OD): δ 173.6, 102.7, 78.0, 76.1, 72.2, 70.6, 62.8, 57.5, 33.0, 30.7, 30.5, 27.2,

23.7, 23.0, 14.4; HRMS m/z calcd. for C16H31NO6Na (M+Na+) 356.2043, found 356.2050.

Allyl 2-acetamido-2-deoxy-β-D-glucopyranoside (31)

Compound 31 (25 mg, 0.096 mmol) was prepared as compound

28 with allyl alcohol (128 μL, 1.9 mmol) except that N-

iodosuccinimide (52 mg, 0.23 mmol) was used as an oxidizing

agent and the product was purified by column chromatography (MeOH-CH2Cl2 1:11.5),

yield 75% (β:α, 7:1). 1H NMR (400 MHz, D2O): δ 5.98-5.88 (m, 1H, allyl), 5.36-5.30 (m,

1H, allyl), 5.30-5.27 (m, 1H, allyl), 4.60 (d, 1H, J1,2 8.4 Hz, H-1), 4.39-4.34 (m, 1H, allyl),

4.21-4.16 (m, 1H, allyl), 3.96 (dd, 1H, J6a,6b 12.3, J5,6a 1.7 Hz, H-6a), 3.78 (m, 1H, H-6b),

3.79-3.74 (dd, 1H, J2,3 10.3, J1,2 8.4 Hz, H-2), 3.58-3.53 (m, 1H, H-3), 3.49-3.43 (m, 2H, H-

4, H-5), 2.06 (s, 3H, Ac); 13C NMR (100 MHz, D2O): δ 174.7, 133.5, 118.3, 100.2, 76.0,

74.0, 70.6, 70.1, 60.9, 55.7, 22.3; HRMS m/z calcd. for C11H19NO6Na (M+Na+) 284.1104,

found 284.1102.

O

NHAc

HOHO

HO7

O

O

AcHN

HOHO

HO O

95

 

Isopropyl 2-acetamido-2-deoxy-β-D-glucopyranoside (32)

Compound 32 (25 mg, 0.095 mmol) was prepared as compound 28

with isopropyl alcohol (145 μL, 1.9 mmol) and purified by column

chromatography (MeOH-CH2Cl2 1:11.5), yield 74% (β:α, 6:1). 1H

NMR (400 MHz, CD3OD): δ 4.51 (d, 1H, J1,2 8.1 Hz, H-1), 3.96 (sept, 1H, J 6.2 Hz,

OCH(CH3)2), 3.88 (dd, 1H, J6a,6b 11.8, J5,6a 2.2 Hz, H-6a), 3.68 (dd, 1H, J6a,6b 11.8, J5,6b 5.6

Hz, H-6b), 3.55 (dd, 1H, J1,2 8.1, J2,3 10.2 Hz, H-2), 3.48 (dd, 1H, J2,3 10.2, J3,4 8.0 Hz, H-

3), 3.30 (under CD3OD peak, 1H, H-4), 3.25 (ddd, 1H, J4,5 9.6, J5,6b 5.6, J5,6a 2.2 Hz, H-5),

1.97 (s, 3H, Ac), 1.19 (d, 3H, J 6.2 Hz, CHCH3), 1.12 (d, 3H, J 6.2 Hz, CHCH3); 13C NMR

(100 MHz, CD3OD): δ 173.6, 101.2, 77.9, 76.0, 73.0, 72.2, 62.9, 57.8, 23.7, 22.9, 22.2;

HRMS m/z calcd. for C11H21NO6Na (M+Na+) 286.1261, found 286.1269.

Benzyl 2-acetamido-2-deoxy-β-D-glucopyranoside (33)

Compound 33 (32 mg, 0.10 mmol) was prepared as compound

28 with benzyl alcohol (197 μL, 1.9 mmol) and purified by

column chromatography (MeOH-CH2Cl2 1:11.5), yield 80%

(β:α, 7:1). 1H NMR (400 MHz, CD3OD) δ 7.33-7.31 (m, 4H, Ar), 7.30-7.24 (m, 1H, Ar),

4.90 (d, 1H, J 12.2 Hz, CH2Ar), 4.62 (d, 1H, J 12.2 Hz, CH2Ar), 4.48 (d, 1H, J1,2 8.5 Hz,

H-1), 3.92 (dd, 1H, J6a,6b 12.1, J5,6a 2.1 Hz, H-6a), 3.74-3.69 (m, 2H, H-6b, H-2), 3.44 (dd,

1H, J3,4 10.4, J2,3 8.4 Hz, H-3), 3.33 (dd, 1H, J3,4 9.7, J4,5 8.4 Hz, H-4), 3.27 (ddd, 1H, J4,5

9.7, J5,6b 5.7, J5,6a 2.1 Hz, H-5), 1.95 (s, 3H, Ac); 13C NMR (100 MHz, CD3OD): δ 173.7,

139.2, 129.3 (2), 128.8 (2), 128.7, 101.8, 78.1, 76.0, 72.2, 71.5, 62.9, 57.4, 23.0; HRMS

m/z calcd. for C15H21NO6Na (M+Na+) 334.1261, found 334.1270.

Cyclohexyl 2-acetamido-2-deoxy-β-D-glucopyranoside (34)

The title compound (28 mg, 0.092 mmol) was prepared as

compound 28 with cyclohexanol (201 μL, 1.9 mmol) and

purified by column chromatography (MeOH-CH2Cl2 1:11.5),

yield 72% (β:α, 6:1). 1H NMR (400 MHz, CD3OD): δ 4.54 (d, 1H, J1,2 8.2 Hz, H-1), 3.88

(dd, 1H, J6a,6b 11.9, J5,6a 2.2 Hz, H-6a), 3.72-3.65 (m, 2H, H-6b, OCH), 3.57 (dd, 1H, J2,3

10.4, J1,2 8.2 Hz, H-2), 3.49 (dd, 1H, J2,3 10.4, J3,4 8.3 Hz, H-3), 3.31 (under CD3OD peak,

O

AcHN

HOHO

HO O

O

AcHN

HOHO

HO O

O

AcHN

HOHO

HO O

96

 

1H, H-4), 3.25 (m, 1H, J4,5 9.5, J5,6b 5.5, J5,6a 2.2 Hz, H-5), 1.97 (s, 3H, Ac), 1.85-1.78 (m,

2H, cyclohexyl), 1.74-1.64 (m, 2H, cyclohexyl), 1.50-1.27 (m, 6H, cyclohexyl); 13C NMR

(100 MHz, CD3OD): δ 173.6, 100.9, 77.9, 75.9, 72.2, 62.8, 57.8, 34.4, 32.5, 26.8 (2), 24.7,

24.5, 23.0; HRMS m/z calcd. for C14H25NO6Na (M+Na+) 326.1574, found 326.1586.

Methyl 2-acetamido-4-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-2-deoxy-D-β-

glucopyranoside (35)

T-GSH donor 24 (7.6 mg, 0.013 mmol) was dissolved

in extra dry DMF (90 μL), and anhydrous methanol

(10.4 μL, 0.26 mmol) was added to the solution. N-

Bromosuccinimide (5.5 mg, 0.031 mmol) was added at rt. After 10 min of stirring,

Amberlite resin (-OH) was added to quench the reaction and the solution was stirred until

the yellow color disappeared. The resin was filtered, washed with MeOH and solvent was

removed under reduced pressure. The residue was than purified by HPLC on a Prevail

carbohydrate column (gradient: 30-50% H2O in CH3CN, 6-30 min; 50-90% H2O in

CH3CN, 30-50 min), to afford a white solid (4 mg, 0.0091 mmol), yield 71% (β:α, 9:1). 1H

NMR (400 MHz, D2O): δ 4.61 (d, 1H, J1,2 8.4 Hz, H-1’), 4.46 (d, 1H, J1,2 8.0 Hz, H-1),

3.95 (dd, 1H, J6a,6b 12.3, J5,6a 1.6 Hz, H-6’a), 3.89 (dd, 1H, J6a,6b 12.1, J5,6a 1.8 Hz, H-6a),

3.79-3.70 (m, 4H, H-6’b, H-2’, H-2, H-4), 3.67 (dd, 1H, J6a,6b 12.1, J5,6b 6.4 Hz, H-6b),

3.62-3.56 (m, 2H, H-3, H-3’), 3.55-3.46 (m, 6H, H-5, H-5’, H-4’, OCH3), 2.09 (s, 3H, Ac),

2.04 (s, 3H, Ac); 13C NMR (100 MHz, D2O): δ 174.8, 174.7, 102.0, 101.6, 79.6, 76.0, 74.6,

73.6, 72.7, 69.8, 60.7, 60.3, 57.3, 55.7, 55.0 22.3, 22.2; HRMS m/z calcd. for

C17H30N2O11Na (M+Na+) 461.1741, found 461.1758.

3-Chloropropyl 2-acetamido-4-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-2-deoxy-

D-β-glucopyranoside (36)

T-GSH donor 24 (10 mg, 0.016 mmol) was

dissolved in extra dry DMF (200 μL), and 3-

chloropropanol (28 μL, 0.33 mmol) was added

to the solution. N-Bromosuccinimide (7 mg, 0.039 mmol) was then added at room

temperature. After 10 min of stirring, Amberlite resin (-OH) was added to quench the

O

NHAc

HOO HO

O

NHAc

HOHO

HO OCH3

O

NHAc

HOO HO

O

NHAc

HOHO

HO O Cl

97

 

reaction and the solution was stirred until the yellow color disappears. The resin was

filtered, washed with MeOH and solvent was removed under reduced pressure. The residue

was than purified by HPLC on a Prevail carbohydrate column (gradient: 30-50% H2O in

CH3CN, 6-30 min; 50-90% H2O in CH3CN, 30-50 min), to afford a white solid (6 mg,

0.012 mmol), yield 70% (β:α, 7:1). 1H NMR (400 MHz, D2O): 4.61 (d, 1H, J1,2 8.4 Hz, H-

1’), 4.53 (d, 1H, J1,2 8.3 Hz, H-1), 4.03 (q, 1H, J 10.4, J 5.2 Hz, OCHaHbCH2CH2Cl) 3.95

(dd, 1H, J6a,6b 12.3, J5,6a 1.8 Hz, H-6’a), 3.88 (dd, 1H, J6a,6b 12.1, J5,6a 1.9, H-6a), 3.78-3.70

(m, 5H, H-6’b, H-2’, H-2, H-4, OCHaHbCH2CH2Cl), 3.69-3.58 (m, 5H, H-6b, H-3, H-3’,

H-4, OCH2CH2CH2Cl), 3.55-3.46 (m, 3H, H-5, H-5’, H-4’), 2.11-1.94 (m, 8H, Ac,

OCH2CH2CH2Cl, Ac); 13C NMR (100 MHz, D2O): δ 174.8 (2), 101.6, 101.5, 79.6, 76.1,

74.6, 73.6, 72.6, 69.9, 67.2, 60.7, 60.3, 55.7, 55.1, 41.8, 31.6, 22.3 (2); HRMS m/z calcd.

for C19H34N2O11Cl (M+H+) 501.1845, found 501.1861.

Methyl 2-acetamido-6-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-2-deoxy-D-β-

glucopyranoside (37)

T-GSH donor 25 (30 mg, 0.051) was dissolved in extra dry

DMF (0.5 mL), anhydrous methanol (41 μL, 1.0 mmol) was

added to the solution. N-Bromosuccinimide (22 mg, 0.12

mmol) was then added at rt. After 10 min of stirring,

Amberlite resin (-OH) was added to quench the reaction and the solution was stirred until

the yellow color disappeared. The resin was filtered, washed with MeOH and solvent was

removed under reduced pressure. The residue was then purified by HPLC on a Prevail

carbohydrate column (gradient: 30-50% H2O in CH3CN, 6-30 min; 50-90% H2O in

CH3CN, 30-50 min), to afford a white solid (16 mg), yield 73% (β:α, 9:1). 1H NMR (500

MHz, D2O): δ 4.57 (d, 1H, J1,2 8.5 Hz, H-1’), 4.45 (d, 1H, J1,2 8.5 Hz, H-1), 4.25 (dd, 1H,

J6a,6b 11.2, J5,6a 2.0 Hz, H-6a), 3.96 (dd, 1H, J6a,6b 12.3, J5,6a 1.6 Hz, H-6’a), 3.79-3.73 (m,

3H, H-6b, H-2’, H-6’b), 3.68 (dd, 1H, J1,2 8.5, J2,3 10.3 Hz, H-2), 3.60-3.56 (m, 2H, H-5,

H-3’), 3.54 (dd, 1H, J2,3 10.3, J3,4 8.9 Hz, H-3), 3.50 (s, 3H, OCH3), 3.49-3.47 (m, 2H, H-

4’, H-5’), 3.41 (dd, 1H, J4,5 9.9, J3,4 8.9 Hz, H-4), 2.06 (s, 3H, Ac), 2.05 (s, 3H, Ac); 13C

NMR (100 MHz, D2O): δ 177.5, 177.3, 104.7, 104.3, 78.7, 77.4, 76.8, 76.5, 72.8, 72.7,

O

NHAc

O

HOHO

O

NHAc

HOHO

HO

O

98

 

71.4, 65.5, 59.9, 58.3, 58.2, 25.1, 25.0; HRMS m/z calcd. for C17H30N2O11Na (M+Na+)

461.1741, found 461.1720.

3-Chloropropyl 2-acetamido-6-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-2-deoxy-

D-β-glucopyranoside (38)

T-GSH donor 25 (15 mg, 0.025 mmol) was

dissolved in extra dry DMF (300 μL), and 3-

chloropropanol (42 μL, 0.050 mmol) was added to

the solution. N-Bromosuccinimide (11 mg, 0.062

mmol) was then added at rt. After 10 min of stirring, Amberlite resin (-OH) was added to

quench the reaction and the solution was stirred until the yellow color disappeared. The

resin was filtered, washed with MeOH and solvent was removed under reduced pressure.

The residue was than purified by HPLC on a Prevail carbohydrate column (gradient: 30-

50% H2O in CH3CN, 6-30 min; 50-90% H2O in CH3CN, 30-50 min), to afford a white solid

(9 mg), yield 71% (β:α, 9:1). 1H NMR (400 MHz, D2O): δ 4.56 (d, 1H, J1,2 8.5 Hz, H-1’),

4.51 (d, 1H, J1,2 8.4 Hz, H-1), 4.22 (dd, 1H, J6a,6b 11.2, J5,6a 1.6 Hz, H-6a), 3.99 (q, 1H, J

10.3, J 5.2 Hz, OCHaHbCH2CH2Cl), 3.95 (dd, 1H, J6a,6b 12.2, J5,6a 1.2 Hz, H-6’a), 3.78-

3.69 (m, 4H, OCHaHbCH2CH2Cl, H-6b, H-2’, H-6’b), 3.68-3.60 (m, 3H, H-2,

OCH2CH2CH2Cl), 3.59-3.52 (m, 3H, H-5, H-3’, H-3), 3.49-3.44 (m, 2H, H-4’, H-5’), 3.40

(dd, 1H, J4,5 9.8, J3,4 9.0 Hz, H-4), 2.08-1.92 (m, 8H, Ac, OCH2CH2CH2Cl, Ac); 13C NMR

(100 MHz, D2O): δ 174.8, 174.6, 101.6, 101.5, 76.0, 74.7, 73.9, 73.8, 70.1 (2), 68.7, 66.9,

60.9, 55.7, 55.6, 41.8, 31.6, 22.4, 22.3; HRMS m/z calcd. for C19H34N2O11Cl (M+H+)

501.1845, found 501.1865.

Azide 2-acetamido-2-deoxy-β-D-glucopyranoside (41)

T-GSH donor 19 (100 mg, 0.26 mmol), tetrabutylammonium chloride

(383 mg, 1.3 mmol) and 2,6-lutidine (149 μL, 1.3 mmol) were added to

extra dry DMF (2.0 mL). N-Bromosuccinimide (110 mg, 0.61 mmol)

was then added at rt. After 15 min of stirring, NaN3 (84 mg, 1.28 mmol) was added to the

reaction mixture which was stirred at rt overnight. Water (10 mL) was added to the

reaction and the aqueous layer was washed with CH2Cl2 (3 x 30 mL). The solvent was then

O

NHAc

O

HOHO

O

NHAc

HOHO

HO

O Cl

O

AcHN

HOHO

HO N3

99

 

removed under reduced pressure. Compound 41 (46 mg, 0.19 mmol) was obtained pure by

column chromatography (MeOH-CH2Cl2 1:11.5), yield 73%. 1H NMR (400 MHz, D2O): δ

4.76 (d, 1H, J1,2 9.4 Hz, H-1), 3.96 (dd, 1H, J6a,6b 12.4, J5,6a 2.1 Hz, H-6a), 3.80 (dd, 1H,

J6a,6b 12.4, J5,6b 5.4 Hz, H-6b), 3.73 (dd, 1H, J1,2 9.4, J2,3 9.8 Hz, H-2), 3.60 (dd, 1H, J2,3

9.8, J3,4 8.7 Hz, H-3), 3.56 (ddd, 1H, J4,5 9.7, J5,6b 5.4, J5,6a 2.2 Hz, H-5), 3.50 (dd, 1H, J3,4

8.7 , J4,5 9.7 Hz, H-4), 2.07 (s, 3H, Ac); 13C NMR (100 MHz, D2O): δ 175.0, 88.8, 78.1,

73.8, 69.7, 60.7, 55.2, 22.3; HRMS m/z calcd. for C8H14N4O5Na (M+Na+) 269.0856, found

269.0871.

2-acetamido-2-deoxy-3-O-(4-deoxy-α-L-threo-hex-4-enopyranosyluronicacid)-4-sulfate

-D-galactopyranoside (48)

Chondroitin sulfate polymer from bovine trachea (500 mg)

was cleaved using 30 mL of 30 mM NH4OAc buffer pH 7.0

and purified Chondroitin Lyase C in the presence of 0.05%

NaN3 overnight at rt. The reaction mixture was then purified using a MonoQ anion

exchange column (0-100% 20 mM NaHPO4 pH 6.5 1M NaCl in 20 mM NaHPO4 pH 6.5, 0

min – 60 min gradient). After lyophilization the chondroitin sulfate disaccharide was

desalted using size exclusion chromatography on a Bio-Gel P-2 (Bio-Rad) column or

dialysis (MWCO 100 da, Spectra/Por). The chondroitin sulfate 4-SO3- isomer was isolated

using a strong anion exchange column (SAX column) under isocratic conditions, with 20

mM NaOAc, 40 mM NaCl pH 3.5. Dialysis (MWCO 100 Da, Spectra/Por) then gave

compound 48 as a pure isomer. 1H NMR (400 MHz, D2O): δ 5.93-5.92 (m, 1H, H-4’), 5.27

(d, 0.5H, J1,2 4.2 Hz, H-1’α), 5.23 (d, 0.5H, J1,2 3.7 Hz, H-1α), 5.17 (d, 0.5H, J1,2 3.9 Hz, H-

1’β), 4.75 (d, 0.5H, J1,2 8.3 Hz, H-1β), 4.31 (dd, 0.5H, J2,3 11.2, J1,2 3.7 Hz, H-2α), 4.20 (d,

0.5H, J3,4 2.9 Hz, H-4α), 4.17 (dd, 0.5H, J6a,6b 7.6, J5,6a 4.7 Hz, H-6aα), 4.14-4.10 (m, 2H, H-

3’, H-3α, H-4β), 4.01 (dd, 0.5H, J2,3 10.9, J1,2 8.3 Hz, H-2β), 3.95 (dd, 0.5H, J2,3 10.9, J3,4 3.1

Hz, H-3β), 3.85-3.82 (m, 1H, H-2’), 3.79-3.71 (m, 2.5H, H-5α, H-5β, H-6bα, H-6aβ, H-6aβ),

2.08 (s, 3H, Ac); 13C NMR δ 174.7, 174.4, 169.2 (2), 144.2, 144.1, 107.2 (2), 101.0 (2),

95.0, 91.1, 79.8, 77.0, 75.0, 70.4, 69.4 (2), 68.3, 67.5, 65.6 (2), 61.2, 61.0, 52.3, 48.9, 22.1,

21.9; HRMS m/z calcd. for C14H20NO14S (M-H+) 458.0610, found 458.0612.

O

OH

-OOC

HOO

NHAc

OH-O3SO

O OH

100

 

2-acetamido-2-deoxy-3-O-(4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid)-6-

sulfate-D-galactopyranoside (49)

Chondroitin sulfate polymer from bovine trachea (500 mg)

was cleaved using 30 mL of 30 mM NH4OAc buffer pH 7.0

and purified Chondroitin Lyase C in the presence of 0.05%

NaN3 overnight at rt. The reaction mixture was then purified using a MonoQ anion

exchange column (0-100% 20 mM NaHPO4 pH 6.5 1M NaCl in 20 mM NaHPO4 pH 6.5, 0

min – 60 min gradient). After lyophilization the chondroitin sulfate disaccharide was

desalted using size exclusion chromatography on a Bio-Gel P-2 (Bio-Rad) column or

dialysis (MWCO 100 da, Spectra/Por). The chondroitin sulfate 6-SO3- isomer was isolated

using a strong anion exchange column (SAX column) under isocratic conditions, with 20

mM NaOAc, 40 mM NaCl pH 3.5. Dialysis (MWCO 100 da, Spectra/Por) then gave

compound 49 as a pure isomer. 1H NMR (400 MHz, D2O): δ 5.90-5.88 (m, 1H, H-4’), 5.22

(d, 0.5H, J1,2 5.0 Hz, H-1’α), 5.20 (d, 0.5H, J1,2 5.0 Hz, H-1’β), 5.19 (d, 0.5H, J1,2 3.2 Hz, H-

1α), 4.79 (under HOD, H-1β), 4.19-4.16 (m, 1H, H-3’), 4.09 (dd, 0.5H, J2,3 10.5, J1,2 3.3 Hz,

H-2α), 4.03 (dd, 0.5H, J2,3 10.5, J3,4 8.2 Hz, H-3α), 3.94-3.88 (m, 1H, H-5α, H-6aα), 3.86-

3.84 (m, 1.5H, H-6bα, H-6aβ, H-2β), 3.81-3.76 (m, 2H, H-2’, H-3β, H-5β), 3.57 (dd, 0.5H,

J6a,6b 10.1, J5,6b 8.4 Hz, H-6bβ), 3.54-3.52 (m, 1H, H-4α, H-4β), 2.08 (s, 3H, Ac); HRMS m/z

calcd. for C14H20NO14S (M-H+) 458.0610, found 458.0612.

1,2,3,4,6-Penta-O-acetyl-β-D-galactopyranose (50)

D-galactose (5.1 g, 0.0284 mol) and KOAc (2.7 g, 0.0284 mol) were

suspended in 45 mL acetic anhydride-acetic acid (8:1). The mixture

was stirred under reflux for 2h. The residue was then concentrated in

vacuo and the concentrate was poured into 1 L of ice water which was then stirred

overnight. The precipitate was filtered, washed with water and dried under vacuum which

gave 7.3 g of the product, yield 66%. 1H NMR (400 MHz, CDCl3): δ 5.70 (d, 1H, J1,2 8.3

Hz, H-1), 5.41 (d, 1H, J3,4 3.4, J4,5 0.9 Hz, H-4), 5.32 (dd, 1H, J2,3 10.4, J1,2 8.3 Hz, H-2),

5.08 (dd, 1H, J2,3 10.4, J3,4 3.4 Hz, H-3), 4.17-4.09 (m, 2H, H-6a, H-6b), 4.04 (m, 1H, H-5),

2.15 (s, 3H, OAc), 2.11 (s, 3H, OAc), 2.03 (s, 6H, OAc), 1.98 (s, 3H, OAc); 13C NMR (100

O

OAc

OAc

OAc

OAc

AcO

O

OH

-OOC

HOO

NHAc

OSO3-HO

O OH

101

 

MHz, CDCl3): δ 170.4, 170.2, 170.0, 169.5, 169.1, 92.3, 71.8, 71.0, 68.0, 66.9, 61.1, 20.9,

20.7 (3), 20.5; HRMS m/z calcd. for C16H22O11Na (M+Na+) 413.1054, found 413.1069.

Phenyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranose (51)

Phenol (9.4 g, 0.101 mol) and compound 50 (7.5 g, 0.0192 mol) were

dissolved in 2 mL CH2Cl2 and BF3·OEt2 (4.8 mL, 0.0385 mol) was

then added and the mixture stirred for 5 h at rt. The mixture was then

diluted with 700 mL of CH2Cl2 and the organic phase was washed with saturated NaHCO3

(3 x 100 mL), water (2 x 100 mL) and brine (100 mL), dried over MgSO4 and concentrated

in vacuo. Recrystallization with EtOAc-pentane afforded product 51 (6.9 g, 0.0159 mmol)

as white solid, yield 84%. 1H NMR (400 MHz, CDCl3): δ 7.32-7.28 (m, 2H, Ar), 7.09-7.05

(m, 1H, Ar), 7.01-6.99 (m, 1H, Ar), 5.49 (dd, 1H, J1,2 7.9, J2,3 10.4 Hz, H-2), 5.46 (d, 1H,

J3,4 3.4, J4,5 1.1 Hz, H-4), 5.11 (dd, 1H, J2,3 10.4, J3,4 3.4 Hz, H-3), 5.05 (d, 1H, J1,2 7.9 Hz,

H-1), 4.24 (dd, 1H, J6a,6b 11.2, J5,6a 7.0 Hz, H-6a), 4.16 (dd, 1H, J6a,6b 11.2, J5,6b 6.2 Hz, H-

6b), 4.06 (ddd, 1H, J5,6a 7.0, J5,6b 6.2, J4,5 1.1 Hz, H-5), 2.18 (s, 3H, OAc), 2.06 (s, 3H,

OAc), 2.05 (s, 3H, OAc), 2.01 (s, 3H, OAc); 13C NMR (100MHz, CDCl3): δ 170.5, 170.4,

170.3, 169.5, 157.1, 129.7 (2), 123.5, 117.1 (2), 99.8, 71.1, 71.0, 68.8, 67.0, 61.5, 20.9,

20.8 (2), 20.6; HRMS m/z calcd. for C20H24O10Na (M+Na+) 447.1261, found 447.1254.

Phenyl 4,6-benzylidine-β-D-galactopyranose (52)

A catalytic amount of sodium metal was dissolved in 30 mL of MeOH,

then compound 51 (2.5 g, 5.85 mmol) was added to the solution, and

the reaction mixture was stirred for 3 h at rt. The solvent was

evaporated in vacuo and the residue suspended in 35 mL CH3CN. The

suspension was made acidic with p-toluenesulfonic acid, then benzaldehyde dimethylacetal

(4.40 mL, 29.2 mmol) was added and the reaction mixture stirred for 5 h at rt. After

neutralizing the solution with triethylamine, solvent was removed in vacuo and the product

was recrystallized from MeOH giving compound 52 (1.8 g, 5.21 mmol) as white crystals,

yield 89%. 1H NMR (400 MHz, (CD3)2SO): δ 7.48-7.45 (m, 2H, Ar), 7.40-7.36 (m, 3H,

Ar), 7.32-7.29 (m, 2H, Ar), 7.07-7.05 (m, 2H, Ar), 7.02-6.98 (m, 1H, Ar), 5.59 (s, 1H,

ArCH), 5.28 (d, 1H, J2,OH 4.7 Hz, C2-OH), 5.06 (d, 1H, J3,OH 5.4 Hz, C3-OH), 4.99 (d, 1H,

O

OAc

OAc

OPh

OAc

AcO

O

OH

O

OPh

O

HO

Ph

102

 

J1,2 6.9 Hz, H-1), 4.15 (d, 1H, J3,4 2.9 Hz, H-4), 4.06 (m, 2H, H-6a, H-6b), 3.76 (d, 1H, J4,5

1.0 Hz, H-5), 3.66-3.57 (m, 2H, H-2, H-3); 13C NMR (100 MHz, (CD3)2SO): δ 157.3,

138.6, 129.4 (2), 128.6, 127.9 (2), 126.2 (2), 121.7, 116.2 (2), 100.3, 99.7, 75.8, 71.7, 69.7,

68.4, 66.0; HRMS m/z calcd. for C19H20O6Na (M+Na+) 345.1332, found 345.1322.

Phenyl 2,3-di-O-benzoyl-4,6-benzylidine-β-D-galactopyranose (53)

To a solution of 52 (400 mg, 1.2 mmol) in 20 mL pyridine, was added

benzoyl chloride (2.7 mL, 23 mmol) and the reaction was stirred at rt

for 2 h. The solvent was removed in vacuo and chromatography on a

silica gel column (toluene → toluene-EtOAc 2:3) and subsequent

recrystallization with EtOAc-pentane provided the desired product 53 (567 mg, 1.05 mmol)

as a white solid, yield 88%. 1H NMR (400 MHz, CDCl3): δ 8.02-7.97 (m, 4H, Ar), 7.56-

7.48 (m, 4H, Ar), 7.40-7.35 (m, 7H, Ar), 7.27-7.23 (m, 2H, Ar), 7.05-7.03 (m, 3H, Ar),

6.14 (dd, 1H, J2,3 10.4, J1,2 8.1 Hz H-2), 5.58 (s, 1H, ArCH), 5.45 (dd, 1H, J2,3 10.4, J3,4 3.4

Hz, H-3), 5.34 (d, 1H, J1,2 8.1 Hz, H-1), 4.67 (d, 1H, J3,4 3.4 Hz, H-4), 4.47 (d, 1H, J6a,6b

12.5 Hz, H-6a), 4.19 (d, 1H, J6a,6b 12.5 Hz, H-6b), 3.82 (s, 1H, H-5); 13C NMR (100MHz,

CDCl3): δ 166.4, 165.3, 157.4, 137.6, 133.6, 130.1 (2), 129.9 (2), 129.7, 129.6 (2), 129.2,

129.1, 128.6 (2), 128.5 (2), 128.3 (3), 126.5 (2), 123.3, 117.8 (2), 101.1, 100.4, 73.6, 72.9,

69.1, 69.0, 66.9; HRMS m/z calcd. for C33H28O8Na (M+Na+) 575.1676, found 575.1648.

Phenyl-2,3-di-O-benzoyl-β-D-galactopyranoside (54)

A suspension of 53 (1.02 g, 1.84 mmol) in 40 mL acetic acid was

stirred at 95°C for 15 min. The solvent was removed in vacuo and

recrystallization in EtOAc-pentane gave 54 (772 mg, 1.66 mmol) as

white crystals, yield 90%. 1H NMR (400 MHz, (CD3)2SO): δ 7.90-7.89 (m, 4H, Ar), 7.62-

7.57 (m, 2H, Ar), 7.49-7.43 (m, 4H, Ar), 7.29-7.26 (m, 2H, Ar), 7.02-6.98 (m, 3H, Ar),

5.78 (dd, 1H, J2,3 10.2, J1,2 8.0 Hz, H-2), 5.60 (d, 1H, J1,2 8.0 Hz, H-1), 5.50 (d, 1H, J4,OH

5.8 Hz, C4-OH), 5.39 (dd, 1H, J2,3 10.2, J3,4 3.0 Hz, H-3), 4.85 (t, 1H, J6,OH 5.5 Hz, C6-OH),

4.23 (dd, 1H, J4,OH 5.8, J3,4 3.0 Hz, H-4), 3.98 (t, 1H, J5,6 6.2 Hz, H-5), 3.68-3.56 (m, 2H,

H-6a, H-6b); 13C NMR (100 MHz, (CD3)2SO): δ 165.2, 165.0, 156.9, 133.6, 133.5, 129.6

(2), 129.3, 129.2 (2), 129.1, 129.0 (2), 128.8 (2), 128.6 (2), 122.5, 116.4 (2), 98.3, 75.3,

O

OBz

OH

OPh

OH

BzO

O

OBz

O

OPh

O

BzO

Ph

103

 

74.7, 69.9, 65.4, 59.7; HRMS m/z calcd. for C26H24O8Na (M+Na+) 487.1363, found

487.1357.

Phenyl-2,3-di-O-benzoyl-β-D-galactopyranuronic acid (55)

To a solution of 54 (1.4 g, 3.03 mmol) in CH2Cl2 (6 mL) containing

TEMPO (10 mg, 0.06 mmol) was added a solution of saturated

NaHCO3 (6 mL, with its pH adjusted to 9.5 with saturated Na2CO3)

containing KBr (73 mg, 0.61 mmol) and Bu4N+Br- (195 mg, 0.61 mmol). The mixture was

cooled to 0°C. Under vigorous stirring Ca(OCl)2 (868 mg, 6.07 mmol) was added slowly

in small portions. After 3 h at 0 °C the reaction was quenched with 600 mg sodium

metabisulfate. After addition of water (6 mL) and CH2Cl2 (6 mL), 1 M HCl was added to

adjust the final pH to pH 3. Then the organic phase was separated, and the remaining

aqueous phase was extracted with CH2Cl2. The combined organic phases were washed

with brine, dried over with MgSO4 and concentrated in vacuo. Chromatography on a silica

gel column (pentane-EtOAc 2:1 → pentane-EtOAc-AcOH 10:10:1) provided the product as

white solid in 1.37 g yield, 94%. 1H NMR (400 MHz, CDCl3): δ 7.95-7.90 (m, 4H, Ar),

7.49-7.41 (m, 2H, Ar), 7.32-7.25 (m, 4H, Ar), 7.18-7.14 (m, 2H, Ar), 6.98-6.96 (m, 3H,

Ar), 6.07 (dd, 1H, J1,2 8.0, J2,3 10.2 Hz, H-2), 5.51 (dd, 1H, J2,3 10.2, J3,4 2.8 Hz, H-3), 5.33

(d, 1H, J1,2 8.0 Hz, H-1), 4.77 (d, 1H, J3,4 2.8 Hz, H-4), 4.53 (s, 1H, H-5); 13C NMR (100

MHz, CDCl3): δ 170.2, 166.1, 165.6, 157,1, 133.5, 133.3, 130.1 (2), 129.9 (2), 129.6 (2),

129.3, 129.0, 128.5 (2), 128.4 (2), 123.4, 117.7 (2), 100.0, 74.1, 73.5, 69.4, 68.3; HRMS

m/z calcd. for C26H21O9 (M+H+) 477.1191, found 477.1189.

Phenyl-2,3-di-O-benzoyl-Δ4-β-D-galactopyranuronic acid (56)

Compound 55 (217 mg, 0.45 mmol) and Ac2O (480 μL, 5.18 mmol)

were added to 5 mL pyridine and stirred at 70°C. After 3 h, the

solvent was removed and a silica column chromatography (toluene →

toluene-EtOAc-AcOH 80:10:1) yielded 168 mg of compound 56 as a clear oil, yield 81%. 1H NMR (400 MHz, CDCl3): δ 8.14-8.12 (m, 2H, Ar), 8.06-8.03 (m, 2H, Ar), 7.63-7.59 (m,

2H, Ar), 7.50-7.44 (m, 4H, Ar), 7.35-7.31 (m, 2H, Ar), 7.16-7.14 (m, 2H, Ar), 7.111-7.07

(m, 1H, Ar), 6.63 (dd, 1H, J3,4 4.5 Hz, H-4), 6.09 (d, 1H, J1,2 2.4 Hz, H-1), 5.73-5.70 (m

O

OBz

COOHOPh

HO

BzO

O

OBz

-OOC

BzO OPh

104

 

2H, H-2, H-3); 13C NMR (100 MHz, CDCl3): δ 165.6, 165.2, 156.2, 142.0, 134.0, 133.7,

130.2 (2), 130.1 (2), 129.9 (2), 129.5, 128.8, 128.7 (5), 123.6, 117.1 (2), 109.6, 94.8, 68.5,

64.2; HRMS m/z calcd. for C26H21O9 (M-H+), 459.1085, found 459.1081.

Phenyl -Δ4-β-D-galactopyranuronic acid (57)

A catalytic amount of sodium was added to 5 mL MeOH, to which

compound 56 (578 mg, 1.26 mmol) was added, and stirred at rt for 9

h. The reaction was then diluted with water (80 mL), washed with

CH2Cl2 (2 x 20 mL) and lyophilized. Purification with reverse phase HPLC

chromatography (2-30% CH3CN in H2O, 4-40 min gradient) produced compound 57 (281

mg, 1.12 mmol) as white solid, yield 89%. 1H NMR (400 MHz, D2O): δ 7.45-7.42 (m, 2H,

Ar), 7.23-7.18 (m, 3H, Ar), 5.97 (d, 1H, J3,4 3.9 Hz, H-4), 5.74 (d, 1H, J1,2 5.3 Hz, H-1),

4.32 (t, 1H, J2,3 4.6 Hz, H-3), 4.07 (t, J2,3 4.6 Hz, 1H, H-2); 13C NMR (100 MHz, D2O): δ

168.9, 156.1, 144.6, 129.9 (2), 123.6, 117.6 (2), 107.6, 98.5, 69.8, 66.4; HRMS m/z calcd.

for C12H11O6 (M-H+) 251.0561, found 251.0568.

Phenyl 4-(2-N-acetylethanethio)-β-D-galactopyranuronic acid (58)

The Δ4-uronic acid 57 (5 mg, 0.020 mmol), 4,4’-azobis(4-

cyanovaleric acid (22 mg, 0.080 mmol) and N-

acetylcysteamine (21.3 μL, 0.200 mmol) were dissolved in

1.0 mL H2O-MeOH (1:1). The solution was spurged with

N2(g) for 20 min and the reaction was stirred at 80 °C for 4 h. The product was then purified

using reverse phase HPLC (2-30% CH3CN in H2O, 4-40 min gradient) giving 4.4 mg of

compound 58 in 60% yield. 1H NMR (400 MHz, CD3OD): δ 7.20-7.17 (m, 2H, Ar), 7.07-

7.05 (m, 2H, Ar), 6.93-6.90 (m, 1H, Ar), 4.74 (d, 1H, J1,2 7.6 Hz, H-1), 4.20 (d, 1H, J4,5 1.8

Hz, H-5), 3.86 (dd, 1H, J2,3 9.6, J3,4 4.5 Hz, H-3), 3.58 (dd, 1H, J2,3 9.6, J1,2 7.6 Hz, H-2),

3.44 (dd, 1H ,J3,4 4.5, J4,5 1.8 Hz, H-4), 3.41-3.36 (m, 1H, SCHaHb), 3.33-3.28 (m, 1H,

SCHaHb), 2.70 (t, J 6.1 Hz, 2H, SCH2CH2N) 1.90 (s, Ac); 13C NMR (100 MHz, CD3OD):

δ 175.2, 173.3, 159.5, 130.3 (2), 123.3, 118.2 (2), 103.3, 76.9, 75.0, 73.3, 53.9, 40.2, 34.7,

22.7; HRMS m/z calcd. for C16H20NO7S (M-H+) 370.0965, found 370.0977.

O

OH

COOH

OPhHO

SNH

O

O

OH

-OOC

HO OPh

105

 

Phenyl 4-(2-aminoethylthio)-β-D-galactopyranuronic acid (59)

The Δ4-uronic acid 58 (12 mg, 0.0476 mmol) and cysteamine

hydrochloride (100 mg, 0.885 mmol) were dissolved in 1 mL

H2O and added to 1 mL of DMF containing benzophenone (50

mg, 0.274 mmol). The solution was purged with N2(g) for 20 min and then the reaction was

stirred at rt under UV light (365 nm) for 2 h. The solution was diluted with 10 mL H2O and

passed through a C18 plug, which was washed with H2O and eluted with 30% CH3CN.

After lyophilization the residue was purified using reverse phase HPLC (2-30% CH3CN in

H2O, 4-40 min gradient) affording compound 34 (11 mg, 0.0320 mmol) as white solid,

yield 60%. 1H NMR (400 MHz, D2O): δ 7.45-7.41 (m, 2H, Ar), 7.20-7.16 (m, 3H, Ar),

5.06 (d, 1H, J1,2 7.8 Hz, H-1), 4.48 (d, 1H ,J4,5 1.9 Hz, H-5), 4.16 (dd, 1H, J3,4 4.5 Hz, H-

3), 3.74 (dd, 1H, J2,3 9.7 Hz, H-2), 3.51 (dd, 1H, J4,5 1.9 Hz, H-4), 3.28 (t, 1H, J 6.4 Hz,

CH2S), 2.99 (t, 1H, J 6.4 Hz, CH2NH2) ppm; 13C NMR (100 MHz, D2O): δ 174.8, 156.8,

130.1 (2), 123.6, 117.1 (2), 100.9, 75.2, 72.5, 71.5, 52.2, 38.7, 30.8; HRMS m/z calcd. for

C14H20NO6S (M+H+) 330.1005, found 330.1015.

N-(2-acetamido-2-deoxy-3-O-(4-deoxy-α-L-threo-hex-4-enopyranosyl uronic acid)-4/6-

sulfate-β-D-galactopyranoside)octylsulfonohydrazide (60)

Chondroitin sulfate 48/49 (30 mg, 0.065 mmol) and

tetraethylammonium chloride (11 mg, 0.065 mmol)

were dissolved in 20 μL H2O and added to it was a

solution of octylhydrazide 22 (20 mg, 0.098 mmol)

in DMF (200 μL), which was then made acidic with AcOH and incubated at 37 °C for 24 h.

The reaction mixture was then diluted with water (5 mL) and purified with reverse phase

HPLC (15-50% CH3CN in H2O, 4-40 min gradient) and lyophilized to afford 53 mg of 60

as the tetraethylammonium salt, and predominantly the 4-SO3- isomer, yield 82%. 1H

NMR (400 MHz, D2O): δ 6.04 (d, 1H, J3,4 4.7 Hz, H-4’), 5.32 (d, 1H, J1,2 2.1 Hz, H-1’),

4.64 (d, 1H, J3,4 2.8 Hz, H-4), 4.34 (d, 1H, J1,2 9.7 Hz, H-1), 4.23 (d, 1H, J2,3 10.8, J3,4 2.7

Hz, H-3), 4.08 (dd, 1H, J2,3 10.8, J1,2 9.7 Hz, H-2), 3.98-3.96 (m, 1H, H-3’), 3.88-3.86 (m,

1H, H-2’), 3.85-3.75 (m, 3H, H-5, H-6a, H-6b), 3.27-3.19 (m, 18H, SO2CH2, NCH2CH3),

2.10 (s, 3H, Ac), 1.81-1.74 (m, 2H, SO2CH2CH2), 1.46-1.43 (m, 2H, SO2(CH2)2CH2), 1.36-

O

OH

COOH

OPhHO

SH2N

7

O

OH

OOC

HOO

NHAc

OROR

OHN N

HSO

OR = SO3

- or H

106

 

1.28 (m, 32H, octyl, NCH2CH3), 0.88 (t, 3H, J 6.9 Hz, CH3); 13C NMR (125 MHz, D2O): δ

174.4, 168.7, 143.4, 107.0, 99.9, 90.0, 76.3, 76.1, 75.3, 68.4, 64.3, 61.3, 49.5, 48.8, 46.6

(8), 31.0, 28.0 (2), 27.3, 22.3, 22.2, 22.0, 13.4, 8.2 (8); HRMS m/z calcd. for

C22H38N3O15S2 (M+H+) 648.1749, found 648.1751.

tert-Butoxycarbonyl-N-methyl-O-octylhydroxylamine (61)

A 60% oil dispersion of NaH (230 mg, 5.71 mmol) was added to t-

butyl N-methyl-N- hydroxycarbamate (600 mg, 4.1 mmol) in 10 mL

anhydrous DMF and stirred at 0 °C for 45 min under nitrogen. Octyl

iodide (1.0 mL, 5.71 mmol) was added and the reaction was stirred for 15 h. The reaction

mixture was diluted with pentane (200 mL) and the organic layer was then washed with

water (3 x 100 mL) and brine (100 mL), dried over MgSO4, filtered and concentrated. The

residue was purified by column chromatography (pentane → pentane-EtOAc 4:1) to afford

1.02 g of 61 as pale oil, yield 96%. 1H NMR (400 MHz, CDCl3): δ 3.82 (t, 2H, OCH2) 3.09

(s, 3H, NCH3), 1.64-1.54 (m, 2H, OCH2CH2), 1.49 (s, 9H, O(CH3)3), 1.41-1.28 (m, 8H,

CH2), 0.88 (t, 3H, J 6.8 Hz, CH3); 13C NMR (100 MHz, CDCl3): δ 157.0, 81.2, 74.4, 36.5,

31.9, 29.6, 29.3, 28.5 (3), 28.4, 26.2, 22.8, 14.2; HRMS m/z calcd. for C9H22NO (M+H+)

160.1695, found 160.1703.

N-Methyl-O-octylhydroxylamine (62)

Compound 61 (1.0 g, 3.86 mmol) was dissolved in 8.4 mL TFA-H2O-

Triisopropylsilane (95:2.5:2.5) and stirred at rt for 1 h. The TFA was then

removed using N2(g) purging and the product was taken up in pentane (100 mL) and washed

with saturated NaHCO3 (3 x 50 mL), H2O (50 mL) and finally brine (50 mL). The

resulting layer was dried over MgSO4, filtered and concentrated. The residue was purified

by column chromatography (pentane → pentane-EtOAc 3:1) to afford 508 mg of 62 as pale

oil, yield 83%. 1H NMR (400 MHz, CDCl3): δ 3.70 (t, 2H, J 6.7 Hz, OCH2), 2.73 (s, 3H,

NCH3), 1.62-1.53 (m, 2H, OCH2CH2), 1.36-1.24 (m, 8H, CH2), 0.88 (t, 3H, J 6.8 Hz, CH3); 13C NMR (100 MHz, CDCl3): δ 73.8, 39.1, 32.0, 29.6, 29.4, 28.9, 26.2, 22.8, 14.2; HRMS

m/z calcd. for C14H29NO3Na (M+Na+) 282.2039, found 282.2027.

HN O 7

N O 7O

O

107

 

N-Methyl-O-octyl-N-(2-acetamido-2-deoxy-3-O-(4-deoxy-α-L-threo-hex-4-

enopyranosyl uronic acid)-4/6-sulfate-β-D-galactopyranoside)hydroxylamine (63)

Chondroitin sulfate 48/49 DIPEA salt (40 mg, 0.053

mmol) was dissolved in 100 μL of DMF, to which was

added a solution of N-methyl-O-octylhydroxylamine

62 (17 mg, 0.107 mmol) in DMF (100 μL), made

acidic with AcOH, and incubated at 37 °C for 48 h. The reaction mixture was then diluted

with water (5 mL) and pre-purified using a C-18 column plug. The product was purified

using a MonoQ anion exchange column (0-100% 1 M DIPEA⋅HCl pH 7.0 in 50 mM

DIPEA⋅HCl pH 7.0; 0–60 min gradient). Excess DIPEA salt was removed using a C-18

plug where the product was eluted with CH3CN to give compound 63 (18 mg, 0.0214

mmol), predominantly the 4-SO3- isomer as the DIPEA salt, yield 40%. 1H NMR (400

MHz, D2O): δ 6.05 (d, 1H, J3,4 4.8 Hz, H-4’), 5.30 (d, 1H, J1,2 2.6 Hz, H-1’), 4.61 (d, 1H,

J3,4 2.1 Hz, H-4), 4.34 (dd, 1H, J2,3 10.1, J1,2 9.6 Hz, H-2), 4.30-4.21 (d, 1H, J1,2 9.6 Hz, H-

1), 4.15 (dd, 1H, J2,3 10.1, J3,4 2.1 Hz, H-3), 3.97-3.95 (m, 1H, H-3’), 3.90-3.86 (m, 1H, H-

2’), 3.84-3.70 (m, 8H, H-5, H-6a, H-6b, OCHa, NCH(CH3)2), 3.63-3.57 (m, 1H, OCHb),

3.23 (q, 4H, J 7.4 Hz, NCH2CH3), 2.76 (s, 3H, NCH3), 2.10 (s, 3H, Ac), 1.59-1.51 (m, 2H,

OCH2CH2), 1.41-1.34 (m, 30H, NCH2CH3, (CH3)2HCNCH(CH3)2), 1.34-1.26 (m, 10H,

octyl CH2), 0.88 (t, 3H, J 6.8 Hz, CH3); 13C NMR (100 MHz, D2O): δ 174.0, 169.2, 144.0,

107.0, 100.2, 92.0, 77.2, 76.6, 76.0, 73.1, 68.7, 64.5, 61.4, 54.5 (4), 48.8, 42.7 (2), 38.9,

31.3, 28.9, 28.6, 27.9, 25.5, 22.5, 22.2, 17.9 (4), 16.4 (4), 13.6, 12.3 (2); HRMS m/z calcd.

for C23H39N2O14S (M-H+) 599.2127, found 599.2127.

N-Methyl-O-octyl-N-(2-acetamido-2-deoxy-3-O-(methyl-4-deoxy-α-L-threo-hex-4-eno

pyranosyluronate)-4/6-sulfate-β-D-galactopyranoside)hydroxylamine (64)

Compound 63 (7 mg, 0.0081 mmol) was dissolved in

400 μL DMF and K2CO3 (0.6 mg, 0.004 mmol) was

added to the solution. Subsequently, MeI (0.5 μL,

0.0086 mmol) was added and the reaction mixture was

stirred at rt for 2 h. The product was purified using reverse phase HPLC (2-50% CH3CN in

H2O, 4-60 min gradient) and lyophilized, affording compound 64 (5 mg, 0.0067 mmol),

7O

OH

OOC

HOO

NHAc

OROR

O N O

R = SO3- or H

7O

OHHO

O

NHAc

OROR

O N O

O O

108

 

predominantly the 4-SO3- isomer as the DIPEA salt, yield 83%. 1H NMR (400 MHz, D2O):

δ 6.35 (d, 1H, J3,4 4.9 Hz, H-4’), 5.35 (d, 1H, J1,2 1.5 Hz, H-1’), 4.64 (d, 1H, J3,4 2.9 Hz, H-

4), 4.36 (dd, 1H, J2,3 10.2, J1,2 9.6 Hz, H-2), 4.08 (d, 1H, J1,2 9.6 Hz, H-1), 4.08 (dd, 1H, J2,3

10.2, J3,4 2.8 Hz, H-3), 4.00-3.98 (m, 1H, H-3’), 3.95-3.94 (m, 1H, H-2’), 3.87 (s, 3H,

COOCH3), 3.83-3.78 (m, 2H, H-5, H-6a,), 3.77-3.71 (m, 4H, H-6b, NOCHaHb,

N(CH(CH3)2)2), 3.63-3.57 (m, 1H, NOCHaHb), 3.23 (q, 2H, J3,4 7.4 Hz, NCH2CH3), 2.76

(s, 3H, NCH3), 2.10 (s, 3H, Ac), 1.58-1.51 (m, 2H, NOCH2H2), 1.37-1.26 (m, 25H,

NCH2CH3, octyl CH2, (CH3)2HCNCH(CH3)2), 0.88 (t, 3H, J 6.8 Hz, CH3); 13C NMR (100

MHz, D2O): δ 174.0, 164.5, 139.7, 111.7, 100.9, 91.8, 79.2, 76.7, 75.7, 73.1, 68.3, 61.1,

54.5 (2), 53.1, 48.5, 42.7, 38.9, 31.2, 28.7, 28.5, 27.8, 25.4, 22.4, 22.1, 17.8 (4), 16.3, 13.5,

12.3; HRMS m/z calcd. for C24H41N2O14S (M-H+) 613.2273, found 613.2290.

N-Methyl-O-octyl-N-(2-acetamido-2-deoxy-3-O-(4-iodo-5-dehydro-4-deoxy-5-methoxy

-β-L-iduronic acid)-4/6-sulfate-β-D-galactopyranoside)hydroxylamine (65)

Compound 63 (4 mg, 0.0047 mmol) was dissolved in

300 μL MeOH to which NIS (1.3 mg, 0.0056 mmol)

was added, and the reaction mixture was stirred at rt

for 1.5 h. The product was purified using reverse phase HPLC and lyophilized to afford 3.7

mg of 65, predominantly the 4SO3- isomer as the DIPEA salt, yield 77%. 1H NMR (400

MHz, D2O): δ 5.08 (d, 1H, J1,2 7.6 Hz, H-1’), 4.84 (d, 1H, J3,4 2.8 Hz, H-4), 4.36-4.31 (m,

2H, H-2, H-4’), 4.24 (d, 1H, J1,2 9.9 Hz, H-1) 4.16 (dd, 1H, J3,4 10.0 Hz, H-4’), , 3.86-3.71

(m, 8H, H-3, H-6a, H-6b, NOCHaHb, N(CH(CH3)2)2), 3.65-3.58 (m, 1H, NOCHaHb),

3.45-3.40 (m, 4H, H-2’, OCH3), 3.23 (q, 4H, J3,4 7.4 Hz, NCH2CH3), 2.75 (s, 3H, NCH3),

2.04 (s, 3H, Ac), 1.57-1.51 (m, 2H, NOCH2H2), 1.38-1.28 (m, 38H, NCH2CH3, octyl CH2,

(CH3)2HCNCH(CH3)2), 0.88 (t, 3H, J 6.8 Hz, CH3); HRMS m/z calcd. for C24H42N2O15SI

(M-H+) 757.1356, found 757.1341.

O

NHAc

OROR

OO

OH-OOC

IHO

OCH3

7N O

109

 

References

1. Watson, J. D.; Crick, F. H. C. Nature 1953, 171, 737-738.

2. Watson, J. D.; Crick, F. H. C. Nature 1953, 171, 964-967.

3. Crick, F. H. C. Symp. Soc. Exp. Biol. 1958, 171, 138-163.

4. Walsh, C. T. Posttranslational Modification of Proteins: Expanding Nature’s inventory; Roberts & Co. Publishers: Greenwood Village, 2005.

5. Varki, A. Glycobiology 1993, 3, 97-130.

6. Dwek, R. A. Chem. Rev. 1996, 96, 683-720.

7. Sears, P.; Wong, C. –H. Cell. Mol. Life Sci. 1998, 54, 223-252.

8. Marshal, R. D.; Annu. Rev. Biochem. 1972, 41, 673-702.

9. Imberty, A.; Pérez, S. Protein Eng. 1995, 8, 699-709.

10. Hunt, L. T.; Dayhoff, M. O. Biochem. Biophys. Res. Commun. 1970, 39, 757-765.

11. Yamashita, K.; Kamerling, J. P.; Kobata, A. J. Biol. Chem. 1982, 257, 12809-12814.

12. Fukuda, M.; Spooncer, E.; Oates, J. E.; Dell, A.; Klock, J. C. J. Biol. Chem. 1984, 259, 10925-10935.

13. Molecular and Cellular Glycobiology, Fukuda, M., Hindsgaul, O., Eds.; Oxford University Press: New York, 2000.

14. Strous, G. J.; Dekker, J. Crit. Rev. Biochem. Mol. Biol. 1992, 27, 57-92.

15. Brocke, C.; Kunz, H. Bioorg. Med. Chem. 2002, 10, 3085-3112.

16. Essentials of Glycobiology, Varki, A., Cummings, R., Esko, J.; Freeze, H.; Hart, G.; Marth, J. Eds.; Oxford Cold Spring Harbor Laboratory Press: Plainview, NY, 1999.

17. Livingston, P. O; Immunol. Rev. 1995, 145, 147-166.

18. Joe, M.; Bai, Y.; Nacario, R. C.; Lowary, T. L. J. Am. Chem. Soc. 2007, 129, 9885–9901.

19. Sharon, H. L. N. J. Biol. Chem. 1978, 253 3468-3476.

110

 

20. Kajihara, Y.; Suzuki, Y.; Yamamoto, N.; Sasaki, K.; Sakakibara, T.; Juneja, L. R.

Chem. Eur. J. 2004, 10, 971-985.

21. Dorland, van Halbeek, H.; Vliegenthart, J. F. G. J. Biol. Chem. 1978, 253, 3468-3476.

22. Liu, L.; Bennett, C. S.; Wong, C. –H. Chem. Comm. 2006, 21-33.

23. Hardingham, T. E.; Fosang, A. J. FASEB J. 1992, 6, 861-870.

24. Hanessian, S.; Lou, B. L. Chem. Rev. 2000, 100, 4443–4463.

25. Fischer, E. Chem. Ber. 1893, 26, 2400-2412.

26. Capon, B. Chem. Rev. 1969, 69, 407-498.

27. Lubineau, A.; Fischer, J. –C. Synth. Commun. 1991, 21, 815-818.

28. Ferriéres, V.; Bertho, J. –N.; Plusquellec, D. Tetrahedron Lett. 1995, 36, 2749-2752.

29. Bertho, J. –N.; Ferriéres, V.; Plusquellec, D. J. Chem. Soc. Chem. Commun. 1995, 1391-1393.

30. Park, T. –J.; Weiwer, M.; Yuan, X.; Baytas, S. N.; Munoz, E. M.; Murugesan, S.; Linhardt, R. J. Carbohydr. Res. 2007, 342, 614-620.

31. Hanessian, S.; Bacquet, C.; LeHong, N. Carbohydr. Res. 1980, 80, C17-C22.

32. Lou, B.; Reddy, G. V.; Wang, H.; Hanessian, S. In Preparative Carbohydrate Chemistry, Hanessian, S., Ed.; Dekker: New York, 1997; p389.

33. Hanessian, S.; Lu, P. –P. Ishida, H. J. Am. Chem. Soc. 1998, 120, 13296-13300.

34. Noyori, R.; Kurimuto, I. J. Org. Chem. 1986, 51, 4320-4322.

35. Balavoine, G.; Gref, A.; Fischer, J. –C.; Lubineau, A. Tetrahedron Lett. 1990, 31, 5761-5764.

36. Li, Z. –J.; Cai, L. –N.; Cai, M. –S. Synth. Commun. 1992, 22, 2121-2124.

37. Deng, S.; Gangadharmath, U.; Chang, C. –W. T. J. Org. Chem. 2006, 71, 5179-5185.

38. Marks, G. S.; Neuberger, A. J. Chem. Soc. 1961, 4872-4879.

111

 

39. Likhosherstov, L. M.; Novikova, O. S.; Derevitskaja, V. A.; Kochetkov, N. K.

Carbohydr. Res. 1986, 146, C1-C5.

40. Collins, P.; Ferrier, R. Monosaccharides: Their Chemistry and Their Roles in Natural Products; Wiley & Sons: Chichester, U.K., 1995.

41. Bejugam, M.; Flitsch, S. L. Org. Lett. 2004, 6, 4001-4004.

42. Wen, S.; Guo, Z. Org. Lett. 2001, 3, 3773-3776.

43. Larabi, M. –L.; Fréchou, C.; Demailly, G. Tetrahedron Lett. 1994, 35, 2175-2178.

44. Meslouti, A. E.; Beaupère, D.; Demailly, G.; Uzan, R. Tetrahedron Lett. 1994, 35, 3913-3916.

45. Inazu, T.; Kobayashi, K. Synlett 1993, 869-870.

46. He, Y.; Hinklin, R. J.; Chang, J.; Kiessling, L. L. Org. Lett. 2004, 6, 4479-4482.

47. Sjölin, P.; Eloffson, M.; Kihlberg, J. J. Org. Chem. 1996, 61, 560-565.

48. Kunz, H. Angew. Chem. Int. Ed. Engl. 1987, 26, 294-308.

49. Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007-2010.

50. Doores, K. J.; Mimura, Y.; Dwek, R. A.; Rudd, P. M. Elliott, T.; Davis, B. G. Chem. Comm. 2006, 1401-1403.

51. Gray, G. R. Arch. Biochem. Biophys. 1974, 163, 426-428.

52. Fischer, E. Chem. Ber. 1884, 17, 579-584.

53. Rischbieth, P. Chem. Ber. 1887, 20, 2673-2674.

54. Cervigni, S. E.; Dumy, P.; Mutter, M. Angew. Chem. Int. Ed. Engl. 1996, 35, 1230-1232.

55. Peri, F.; Dumy, P.; Mutter, M. Tetrahedron 1998, 54, 12269-12278.

56. Fox, H. H. J. Org. Chem. 1953, 18, 990-993.

57. Helferich, B.; Schirp, H. Chem. Ber. 1953, 86, 547-556.

58. Bendiak, B. Carbohydr. Res. 1997, 304, 85-90.

59. Wolfrom, M. L.; Soltzberg, S. J. Am. Chem. Soc. 1936, 58, 1783-1785.

60. Sah, P. P. T.; Daniels, T. C. Recl. Trav. Chim. Pays-Bas 1950, 69, 1545-1556.

112

 

61. Ojala, C. R.; Ostman, J. M.; Ojala, W. H. Carbohydr. Res. 2002, 337, 21-29.

62. Takeda, Y. Carbohydr. Res. 1979, 77, 9-23.

63. Ojala, W. H.; Ojala, C. R.; Gleason, W. B. J. Chem. Crystallogr. 1999, 29, 19-26.

64. Leteux, C.; Childs, R. A.; Chai, W.; Stoll, M. S.; Kogelberg, H.; Feizi, T. Glycobiology 1998, 8, 227-236.

65. Ridley, B. L.; Spiro, M. D.; Glushka, J.; Albersheim, P.; Darvill, A.; Mohnen, D. Anal. Biochem. 1997, 249, 10-19.

66. Naven, T. J. P.; Harvey, D. J. Rapid Commun. Mass Spectrom. 1996, 10, 829-834.

67. Kameyama, A.; Kaneda, Y.; Yamanaka, H.; Yoshimine, H.; Narimatsu, H.; Shinohara, Y. Anal. Biochem. 2004, 76, 4537-4542.

68. Zhang, Z.; Zhang, R.; Liu, G. J. Chromatogr. A. 1996, 728, 343-350.

69. Lin, J. -K.; Wu, S. S. Anal. Biochem. 1987, 59, 1320-1326.

70. Lee, M.-r.; Shin, I. Org. Lett. 2005, 7, 4269–4272.

71. Zhi, Z. -l.; Powell, A. K.; Turnbull, J. E. Anal. Biochem. 2006, 78, 4786-4793.

72. Bohorov, O.; Andersson-Sand, H.; Hoffmann, J.; Blixt, O. Glycobiology 2006, 16, 21C-27C.

73. Peluso, S.; Imperiali, B. Tetrahedron Lett. 2001, 42, 2085–2087.

74. Peluso, S.; Ufret, M. de L.; O'Reilly, M. K.; Imperiali, B. Chem. Biol. 2002, 9, 1323-1328.

75. Carrasco, M. R.; Nguyen, M. J.; Burnell, D. R.; MacLaren, M. D.; Hengel, S. M. Tetrahedron Lett. 2002, 43, 5727-5729.

76. Carrasco, M. R.; Brown, R. T. J. Org. Chem. 2003, 68, 8853-8858.

77. Filira, F.; Biondi, B.; Biondi, L.; Giannini, E.; Gobbo, M.; Negri, L.; Rocchi, R. Org. Biomol. Chem. 2003, 1, 3059-3063.

78. Flinn, N. S.; Quibell, M.; Monk, T. P.; Ramjee, M. K.; Urch, C. J., Bioconjugate Chem. 2005, 16, 722-728.

79. Langenhan, J. M.; Peters, N. R.; Guzei, I. A.; Hoffmann, M.; Thorson, J. S. Proc. Nat. Acad. Sci. U.S.A. 2005, 102, 12305-12310.

113

 

80. Ahmed, A.; Peters, N. R.; Fitzgerald, M. K.; Watson, J. A., Jr.; Hoffmann, F. M.;

Thorson, J. S. J. Am. Chem. Soc. 2006, 128, 14224-14225.

81. Griffith, B. R.; Krepel, C.; Fu, X.; Blanchard, S.; Ahmed, A.; Edmiston, C. E.; Thorson, J. S. J. Am. Chem. Soc. 2007, 129, 8150-8155.

82. Jandik, K. A.; Gu, K.; Linhardt, R. J. Glycobiology 1994, 4, 289-296.

83. Gemma, E.; Meyer, O.; Uhrín, D.; Hulme, A. N. Mol. Biosys. 2008, 4, 481-495.

84. Viehe, H. G. Janousek, Z.; Merényi, R. Acc. Chem. Res. 1985, 18, 148-154.

85. Ludwigs, U.; Elgavish, A.; Esko, J. D.; Meezan, E.; Rodén, L. Biochem. J. 1987, 245, 795-804.

86. Skidmore, M. A.; Patey, S. J.; Thanh, N. T. K.; Fernig, D. G.; Turnbull, J. E.; Yates, E. A. Chem. Comm. 2004, 2700-2701.

87. Selkala, S. A.; Alakurtti, S.; Koskinen, A. M. P. Tetrahedron Lett. 2001, 42, 3215-3217.

88. Bazin, H. G.; Wolff, M. W.; Linhardt, R. J. J. Org. Chem. 1999, 64, 144-152.

89. Toole, B. P. Nat. Rev. Cancer 2004, 4, 528-539.

90. Carrasco, M. R.; Brown, R. T.; Serafimova, I. M.; Silva, O. J. Org. Chem. 2003, 68, 195-197.

91. Niikura, K.; Kamitani, R.; Kurogochi, M.; Uematsu, R.; Shinohara, Y.; Nakagawa, H.; Deguchi, K.; Monde, K.; Kondo, H.; Nishimura, S. -I. Chem. Eur. J. 2005, 11, 3825-3834.

92. Peri, F.; Jiménez-Barbero, J.; García-Aparicio, V.; Tvaroška, I.; Nicotra, F. Chem. Eur. J. 2004, 10, 1433-1444.

93. Kice, J. L.; Guaraldi, G.; Venier, C. G. J. Org. Chem. 1966, 31, 3561-3567.

94. Lerch, U.; Moffatt, J. G. J. Org. Chem. 1971, 36, 3861-3869.

95. Toma, T.; Shimokawa, J.; Fukuyama, T. Org. Lett. 2007, 9, 3195-3197.

96. Timell, T. E.; Enterman, E.; Spencer F.; Soltes, E. J. Can. J. Chem. 1965, 43, 2296-2305.

97. Saunders, M. E.; Timell, T. E. Carbohydr. Res. 1968, 6, 12-17.

114

 

98. Namchuk, M. N.; McCarter, J. D.; Becalski, A.; Andrews, T.; Withers, S. G. J. Am.

Chem. Soc. 2000, 122, 1270-1277.

99. Piszkiew, D.; Bruice, T. C. J. Am. Chem. Soc. 1968, 90, 5844-5848.

100. Titov, E. V.; Korzhenevskaya, N. G.; Rybachenko, V. I. Ukr. Khim. Zh. (Russ. Ed.) 1968, 34, 1253-1256.

101. Jencks, W. P. Prog. Phys. Org. Chem. 1964, 2, 63-128.

102. Rosenberg, S.; Silver, S. M.; Sayer, J. M.; Jencks, W. P. J. Am. Chem. Soc. 1974, 96, 7986-7998.

103. Egberink, H.; Van Heerden, C. Anal. Chim. Acta 1980, 118, 359-368.

104. Thorson, J. S.; Vogt, T.; In Carbohydrate-Based Drug Discovery, Wong, C –H., Ed.; Wiley-VCH: Weinheim, Germany, 2003; p685.

105. Geyer, H.; Holschback, C.; Hunsmann, G.; Schneider, J. J. Biol. Chem. 1988, 263 11760-11767.

106. Mascola, J. R.; Montefiori, D. C. Nature 2003, 9, 393-394.

107. Sanders, R. W.; Venturi, M.; Schiffner, L.; Kalyanaraman, R.; Katinger, H.; Lloyd, K. O.; Kwong, P. D.; Moore, J. P. J. Virol. 2002, 76, 7293-7305.

108. Scanlan, C. N.; Pantophlet, R.; Wormald, M. R.; Saphire, E. O.; Stanfield, R.; Wilson, I. A.; Katinger, H.; Dwek, R. A.; Rudd, P. M.; Burton, D. R. J. Virol. 2002, 76, 7306-7321.

109. Danishefsky, S. J.; Allen, J. R. Angew. Chem. Int. Ed. Engl. 2000, 39, 836-863.

110. Mangholz, S. E.; Vasella, A. Helv. Chim. Acta 1991, 74, 2100– 2111.

111. Lubineau, A.; Gallic, J. L.; Malleron, A. Tetrahedron Lett. 1987, 28, 5041–5044.

112 Helferich, B.; Schirp, H. Chem. Ber. 1953, 86, 547–556.

113. Zinner, H.; Brenken, H.; Braun, W.; Falk, I.; Fechtner, E.; Hahner, E. Liebigs Ann. Chem. 1959, 622, 133–149.

114. Lin, J. K.; Wu, S. S. Anal. Chem. 1987, 59, 1320–1326.

115. Muramoto, K.; Yamauchi, F.; Kamiya, H. Biosci. Biotechnol. Biochem. 1994, 58, 1013–1017.

116. Ojala, W. H.; Ojala, C. R.; Gleason, W. B. J. Chem. Crystallogr. 1999, 29, 19–26.

115

 

117. See equilibrium constants in Table 2.1, Chapter 2.

118. Cusack, N. J.; Reese, C. B.; Risius, A. C. Roozpeikar, B. Tetrahedron 1976, 32, 2157-2162.

119. Falk, M.; Smith, D. G.; McLachlan, J.; McInnes, A. G. Can. J. Chem. 1966, 44, 2269-2281.

120. The biofilm specialists Carmen Leung, Anthony Chibba and Heather Griffiths are gratefully acknowledged for synthesizing the PNAG oligosaccharides used in subsequent experiments.

121. Lee, K. P.; Trochimowicz, H. J. J. Am. J. Pathol. 1982, 106, 8-19.

122. Kundu, T. Synlett 2006, 498-499.

123. Palmieri, G. Tetrahedron 1983, 39, 4097–4101.

124. Yang, D. Y.; Han, O. S.; Liu, H. W. J. Org. Chem. 1989, 54, 5402–5406.

125. Hale, K. J.; Cai, J. Chem. Commun. 1997, 2319–2320.

126. Carsten, P.; Waldmann, H. J. Org. Chem. 2003, 68, 6053–6055.

127. Camarero, J. A.; Hackel, B. J.; De Yoreo, J. J.; Mitchell, A. R. J. Org. Chem. 2004, 69, 4145–4151.

128. Kwon, Y.; Welsh, K.; Mitchell, A. R.; Camarero, J. A. Org. Lett. 2004, 6, 3801–3804.

129. Nishida, Y; Shingu, Y.; Dohi, H.; Kobayashi, K Org. Lett. 2003, 5, 2377–2380.

130. Cai, Y.; Ling, C. C.; Bundle, D. R. Org. Lett. 2005, 7, 4021–4024.

131. Lemieux, R. U.; Driguez, H. J. Am. Chem. Soc. 1975, 97, 4063–4069.

132. Wang, X.-L. Carbohydr. Res. 2008, 343, 1509–1522.

133. Kobayashi, S.; Kiyosada, T.; Shoda, S.-i. Tetrahedron Lett. 1997, 328, 2111-2112.

134. Zurabyan, S. E.; Volosyuk, T. P.; Khorlin, A. Y. Carbohydr. Res. 1969, 9, 215–220.

135. Urabyan, S. E.; Antonenko, T. S.; Khorlin, A. Y. Carbohydr. Res. 1970, 15, 21–27.

136. Doores, K. J.; Mimura, Y.; Dwek, R. A.; Rudd, P. M.; Elliott, T.; Davis, B. G. Chem. Commun. 2006, 1401–1403.

116

 

137. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. 2001, 40, 2005–

2021.

138. Li, B.; Zeng, Y.; Hauser, S.; Song, H.; Wang, L. –X. J. Am. Chem. Soc. 2005, 127, 9692-9693.

139. Itoh, Y.; Wang, X.; Hinnebusch, B. J.; Preston, J. F., III; Romeo, T. J. Bacteriol. 2005, 187, 382-387.

140. Leung, C.; Chibba, A.; Gómez-Biagi, R. F.; Nitz, M. Carbohydr. Res. 2009, 344, 570-575.

141. Previous work done by Lin Ding a member of the Nitz lab 2004-2005.

142. Pojasek, K.; Shriver, Z.; Kiley, P.; Venkataraman, G.; Sasiekharan, R. Biochem. Biophys. Res. Comm. 2001, 286, 343-351.

143. Ernst, S.; Venkataraman, G.; Winkler, S.; Godavarti, R.; Langer, R.; Cooney, C. L.; Sasisekharan, R. Biochem. J. 1996, 315, 589-597.

144. D’arcy, S. M. T.; Carney, S. L.; Howe, T. J. Carbohydr. Res. 1994, 255, 41-59.

145. Rees, M. D.; Hawkins, C. L.; Davies, M. J. J. Am. Chem. Soc. 2003, 125, 13719-13733.

146. Bazin, H. G.; Wolff, M. W.; Linhardt, R. J. J. Org. Chem. 1999, 64, 144–152.

147. Rye, C. S.; Withers, S. G. Carbohydr. Res. 2004, 339, 699-703.

148. Hancock, S. M.; Vaughan, M. D.; Withers, S. G. Curr. Opin. Chem. Biol. 2006, 10, 509-519.

149. Huang, W.; Boju, L.; Tkalec, L.; Su, H.; Yang, H. –O.; Gunay, N. S.; Linhardt, R. J.; Kim, Y. S.; Matte, A.; Cygler, M. Biochemistry, 2001, 40, 2359-2372.

150. Zhang, L. –S.; Mummert, M. E. Anal. Biochem. 2008, 379, 80-85.

117

 

Appendix A

Hydrolysis data for glycoconjugates in Chapter 2

118

 

0 20 40 60 80 100 120 140 160 1800

20

40

60

80

100

% G

lyco

conj

ugat

e 1

rem

aini

ng

Time (min)

Figure A.1 Hydrolysis of N-(β-D-glucopyranosyl)benzoylhydrazide 1 at 50 °C ( ) 50 mM NaOAc pH 4.0;

(●) 50 mM NaOAc pH 5.0; ( ) 50 mM Na2HPO4 pH 6.0. Each value represents the average of two

experiments; standard deviation was between 3-8%, 5% MeOH was needed to maintain solubility. Lines

indicate best fit of data to a first-order rate law.

0 20 40 60 80 100 120 140 160 180

0

20

40

60

80

100

% G

lyco

conj

ugat

e 2

rem

aini

ng

Time (min)

Figure A.2 Hydrolysis of N-(β-D-glucopyranosyl)-p-methoxybenzoylhydrazide 2 at 50 °C ( ) 50 mM

NaOAc pH 4.0; (●) 50 mM NaOAc pH 5.0; ( ) 50 mM Na2HPO4 pH 6.0. Each value represents the average

of two experiments; standard deviation was between 3-8%, 5% MeOH was needed to maintain solubility.

Lines indicate best fit of data to a first-order rate law.

119

 

0 20 40 60 80 100 120 140 160 1800

20

40

60

80

100

% G

lyco

conj

ugat

e 3

rem

aini

ng

Time (min)

Figure A.3 Hydrolysis of N-(β-D-glucopyranosyl)-p-chlorobenzoylhydrazide 3 at 50 °C ( ) 50 mM NaOAc

pH 4.0; (●) 50 mM NaOAc pH 5.0; ( ) 50 mM Na2HPO4 pH 6.0. Each value represents the average of two

experiments; standard deviation was between 3-8%, 5% MeOH was needed to maintain solubility. Lines

indicate best fit of data to a first-order rate law.

0 20 40 60 80 100 120 140 160 180

20

40

60

80

100

% G

lyco

conj

ugat

e 4

rem

aini

ng

Time (min)

Figure A.4 Hydrolysis of N-(β-D-glucopyranosyl)-p-nitrobenzoylhydrazide 4 at 50 °C ( ) 50 mM NaOAc pH

4.0; (●) 50 mM NaOAc pH 5.0; ( ) 50 mM Na2HPO4 pH 6.0. Each value represents the average of two

experiments; standard deviation was between 3-8%, 5% MeOH was needed to maintain solubility. Lines

indicate best fit of data to a first-order rate law.

120

 

0 5 10 15 20 25 30 35 40

0

20

40

60

80

100

% G

lyco

conj

ugat

e 13

rem

aini

ng

Time (h)

Figure A.5 Hydrolysis of N-(β-D-xylopyranosyl)-p-toluenesulfonohydrazide 13 at 37 °C ( ) 20 mM NaOAc

pH 4.0; (●) 20 mM NaOAc pH 5.0; ( ) 20 mM Na2HPO4 pH 6.0. Each value represents the average of two

experiments; standard deviation was between 3-5%. 0.5% DMSO was needed to maintain solubility. Lines

indicate best fit of data to a first-order rate law.

0 20 40 60 80 100 120 140

0

20

40

60

80

100

% G

lyco

conj

ugat

e 14

rem

aini

ng

Time (h)

Figure A.6 Hydrolysis of N-methyl-O-benzyl-N-(β-D-xylopyranosyl)hydroxylamine 14 at 37 °C ( ) 20 mM

NaOAc pH 4.0; (●) 20 mM NaOAc pH 5.0; ( ) 20 mM Na2HPO4 pH 6.0. Each value represents the average

of two experiments; standard deviation was between 3-5%. Lines indicate best fit of data to a first-order rate

law.

121

 

0 50 100 150 200 250 300 350

0

20

40

60

80

100

% G

lyco

conj

ugat

e 15

rem

aini

ng

Time (h)

Figure A.7 Hydrolysis of N-methyl-O-(N’-benzylacetamide)-N-(β-D-xylopyranosyl)hydroxylamine 15 at 37

°C ( ) 20 mM NaOAc pH 4.0; (●) 20 mM NaOAc pH 5.0; ( ) 20 mM Na2HPO4 pH 6.0. Each value

represents the average of two experiments; standard deviation was between 3-5%. Lines indicate best fit of

data to a first-order rate law.

0 20 40 60 80 100

0

20

40

60

80

100

% G

lyco

conj

ugat

e 16

rem

aini

ng

Time (h)

Figure A.8 Hydrolysis of N-(β-D-glucopyranosyl)-p-toluenesulfonohydrazide 16 at 37 °C ( ) 20 mM NaOAc

pH 4.0; (●) 20 mM NaOAc pH 5.0; ( ) 20 mM Na2HPO4 pH 6.0. Each value represents the average of two

experiments; standard deviation was between 3-5%. 0.5% DMSO was needed to maintain solubility. Lines

indicate best fit of data to a first-order rate law.

122

 

0 50 100 150 200 250

0

20

40

60

80

100

% G

lyco

conj

ugat

e 17

rem

aini

ng

Time (h)

Figure A.9 Hydrolysis of N-methyl-O-benzyl-N-(β-D-glucopyranosyl)hydroxylamine 17 at 37 °C ( ) 20 mM

NaOAc pH 4.0; (●) 20 mM NaOAc pH 5.0; ( ) 20 mM Na2HPO4 pH 6.0. Each value represents the average

of two experiments; standard deviation was between 3-5%. Lines indicate best fit of data to a first-order rate

law.

0 150 300 450 600 750 900

0

20

40

60

80

100

% G

lyco

conj

ugat

e 18

rem

aini

ng

Time (h)

Figure A.10 Hydrolysis of N-methyl-O-(N’-benzylacetamide)-N-(β-D-glucopyranosyl)hydroxylamine 18 at

37 °C ( ) 20 mM NaOAc pH 4.0; (●) 20 mM NaOAc pH 5.0; ( ) 20 mM Na2HPO4 pH 6.0. Each value

represents the average of two experiments; standard deviation was between 3-5%. Lines indicate best fit of

data to a first-order rate law.

123

 

0 200 400 600 800 1000 1200 1400 1600

0

20

40

60

80

100

% G

lyco

conj

ugat

e 19

rem

aini

ng

Time (h)

Figure A.11 Hydrolysis of N-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-p-toluenesulfonohydrazide 19 at 37

°C ( ) 20 mM NaOAc pH 4.0; (●) 20 mM NaOAc pH 5.0; ( ) 20 mM Na2HPO4 pH 6.0. Each value

represents the average of two experiments; standard deviation was between 3-5%. 0.5% DMSO was needed to

maintain solubility. Lines indicate best fit of data to a first-order rate law.

0 500 1000 1500 2000

0

20

40

60

80

100

% G

lyco

conj

ugat

e 20

rem

aini

ng

Time (h)

Figure A.12 Hydrolysis of N-methyl-O-benzyl-N-(2-acetamido-2-deoxy-β-D-glucopyranosyl)hydroxylamine

20 at 37 °C ( ) 20 mM NaOAc pH 4.0; (●) 20 mM NaOAc pH 5.0; ( ) 20 mM Na2HPO4 pH 6.0. Each value

represents the average of two experiments; standard deviation was between 3-5%. Lines indicate best fit of

data to a first-order rate law.

124

 

0 750 1500 2250 3000 3750 4500

0

20

40

60

80

100

% G

lyco

conj

ugat

e 21

rem

aini

ng

Time (h)

Figure A.13 Hydrolysis of N-methyl-O-(N’-benzylacetamide)-N-(2-acetamido-2-deoxy-β-D-glucopyranosyl)

hydroxylamine 21 at 37 °C ( ) 20 mM NaOAc pH 4.0; (●) 20 mM NaOAc pH 5.0; ( ) 20 mM Na2HPO4 pH

6.0. Each value represents the average of two experiments; standard deviation was between 3-5%. Lines

indicate best fit of data to a first-order rate law.

125

 

Appendix B

Selected NMR spectra

126

 

23

23

O

NHAc

HOHO

HOHN

NH

SO O

7

127

 

O

NHAc

HOO HO

O

NHAc

HOHO

HO

24

HN

NH

S TolO O

128

 

O

NHAc

O

HO HO

O

NHAc

HOHO

HOHN

NH

S TolO O

25

129

 

O

NHAc

HOO HO

O

NHAc

HOHO

HO

35

OCH3

130

 

O

NHAc

HOO HO

O

NHAc

HOHO

HO

36

O Cl

131

 

O

NHAc

O

HO HO

O

NHAc

HOHO

HO

OCH3

37

132

 

O

NHAc

O

HO HO

O

NHAc

HOHO

HO

O

38

Cl

133

 

O

OAc

OAc

OAc

OAc

AcO

50

134

 

O

OAc

OAc

OPh

OAc

AcO51

135

 

O

OH

O

OPh

O

HO

Ph

52

136

 

O

OBz

O

OPh

O

BzO

Ph

53

137

 

O

OBz

OH

OPh

OH

BzO

54

138

 

O

OBz

COOHOPh

HO

BzO

55

139

 

56

O

OBz

-OOC

BzO OPh

140

 

57

O

OH

-OOC

HO OPh

141

 

O

OH

COOH

OPhHO

58

SNH

O

142

 

O

OH

COOH

OPhHO

59

SH2N

143

 

7

60

O

OH

OOC

HOO

NHAc

OROR

OHN N

HSO

ODIPEA salt

144

 

63

7O

OH

OOC

HOO

NHAc

OROR

O N O

R = SO3- or H DIPEA salt

145

 

64

7O

OHHO

O

NHAc

OROR

O N O

O O

DIPEA salt