Studies on the Hydrolysis Mechanisms of Sialosides and Synthesis and Evaluation of a Bicycl0[4.1.0]Heptyl Analogue of Glucose-1-Phosphate

STUDIES ON THE HYDROLYSIS MECHANISMS OF SIALOSIDES AND SYNTHESIS AND EVALUATION OF A BICYCL0[4.1.0]HEPTYL ANALOGUE OF GLUCOSE-1-PHOSPHATE

Veedeeta Dookhun B .Sc. (Hons) University of Mauritius, 1998

THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

In the Department of Chemistry

O Veedeeta Dookhun 2004

SIMON FRASER UNIVERSITY

November 2004

Name: Veedeeta Dookhun

Degree: Doctor of Philosophy

Title of Thesis: Studies on the hydrolysis mechanisms of aryl sialosides and synthesis & evaluation of a bicyclo[4.1 .O]heptyl analogue of glucose- 1 -phosphate

Examining Committee: Chair: Dr. D.J. Vocadlo (Assistant Professor)

Dr. A.J. Bennet (Professor) Senior Supervisor

Dr. E. Plettner (Assistant Professor) Committee Member

Dr. S. Holdcroft (Professor) Committee Member

Dr. P.D. Wilson (Assistant Professor) Internal Examiner

Dr. M.E. Tanner (Professor) External Examiner Department of Chemistry University of British Columbia

Date Approved: 224 .. 11 SIMON FRASER UNIVERSITY

PARTIAL COPYRIGHT LICENCE

The author, whose copyright is declared on the title page of this work, has granted to Simon Fraser University the right to lend this thesis, project or extended essay to users of the Simon Fraser University Library, and to make partial or single copies only for such users or in response to a request from the library of any other university, or other educational institution, on its own behalf or for one of its users. The author has further granted permission to Simon Fraser University to keep or make a digital copy for use in its circulating collection. The author has further agreed that permission for multiple copying of this work for scholarly purposes may be granted by either the author or the Dean of Graduate Studies. It is understood that copying or publication of this work for financial gain shall not be allowed without the author's written permission. \ Permission for public performance, or limited permission for private scholarly use, of any multimedia materials forming part of this work, may have been granted by the author. This information may be found on the separately catalogued multimedia material and in the signed Partial Copyright Licence. The original Partial Copyright Licence attesting to these terms, and signed by this author, may be found in the original bound copy of this work, retained in the Simon Fraser University Archive. W. A. C. Bennett Library Simon Fraser University Burnaby, BC, Canada ABSTRACT

Sialic acids, which are nine-carbon acidic keto sugar residues, are very often terminally linked to complex oligosaccharide chains in living organisms. Their highly exposed positions allow them to be actively involved in cell-cell and cell-protein recognition processes. Sialidases are enzymes that catalyze the hydrolysis of natural a linked sialosides of these glycoconjugates with retention of configuration going via a tyrosinyl P-sialoside intermediate.

A series of aryl a-D-sialosides were synthesized in order to characterize the wild type M. Viridifaciens enzyme and its mutants Y370G and D92G. The tyrosine residue, the nucleophile, formed the P-linked enzyme-substrate intermediate in the active site while the aspartate residue acted as the general acidhase catalyst.

A panel of seven aryl P-D-sialosides, were synthesized and characterized in order to study their hydrolysis mechanisms in aqueous medium. 4-nitrophenyl P-D-N-sialoside was chosen for a comparative mechanistic study with its corresponding a-anomer. It was found that the (3 anomer was more than hundred times more stable than the a-anomer.

Detailed kinetic and product studies pointed towards a dissociative mechanism (SN1)for both anomers with solvation of the carboxylate moiety being the driving force in the hydrolysis reaction. Our result disproves the a lactone intermediate proposed for the a anomer by Ashwell et al. The biological significance of these results will be discussed.

The aryl P-D-sialosides were screened against the Y370G mutant of M. viridifaciens and it was found that 3-chloropheny! P-D-sialoside was the best inhibitor with a Ki 1.7 pM. iii Finally, the synthesis of a bicyclo[4.l.O]heptyl analogue of glucose-l-phosphate, (lR, 2R,

3,4S, 5S, 6S)-5-phosphate-2,3,4-trihydroxy-1-hydroxymethyl)-bicyclo[4.1 .O]heptan-2-

yl dihydrogen phosphate (4.5) in eleven steps is reported. Methyl a-D-glucopyranoside

was used as starting material. Compound 4.5 was tested as a potential substrate of UTP:

a-D-glucose-1-phosphate uridylyltransferase, the enzyme that converts a-D-glucose 1- phosphate into UDP-glucose. However, compound 4.5 was found to be a weakly binding inhibitor (12% inhibition at a concentration of 0.1 mM). DEDICATION

To my parents Vidyotma and Ubhuychand Dookhun

who believed in me all the way. ACKNOWLEDGEMENTS

I would like to express my deepest gratitude to my senior supervisor Dr. Andrew J.

Bennet for all his help and guidance during the past 5 years. It has been an honour working for him. A special thank you to members of my supervisory committee Dr. E.

Pletter and Dr. S. Holdcroft, my external and internal examiners Dr. M. Tanner and Dr. P.

Wilson for their valuable suggestions concerning this thesis. Credit goes to Dr J. N.

Watson for providing the enzymes for the kinetic experiments and helping with the enzyme kinetics. I would like to acknowledge Mrs. M. Tracy and Dr A. Tracy for all the

NMR work; Dr D. McGillivray from University of Victoria for all the LR and HR MS;

Mr P. Ferreira for MS and Mr. M. Yang for elemental analysis. Members of the Bennet lab-past and present and my close friends whose good humour made this journey enjoyable. My heartfelt gratitude to my family (Dookhun and De Camprieu) whose love and support is priceless. TABLE OF CONTENTS

APPROVAL i1

ABSTRACT i11

DEDICATION v

ACKNOWLEDGEMENTS VI

TABLE OF CONTENTS VII

LIST OF FIGURES XI1

LIST OF TABLES XVI

LIST OF ABBREVIATIONS AND ACRONYMS XVII

1 CHAPTER ONE: GENERAL INTRODUCTION TO GLYCOSYLTRANSFER REA CTZONS

1.1 GENERALINTRODUCTION

1.2.1 Retaining Glycosidases

1.2.1.1 Brcansted Plots

1.2.1.2 Oxacarbenium ion-like transition state

1.2.2 Retaining Glycosidases

1.2.2.1 Brcansted Plots

1.2.2.2 Oxacarbenium ion-like transition state

1.2.2.3 Substrate distortion during catalysis

1.2.3 Retaining PN-a~et~lhexosaminidases

1.2.4 Inverting Glycosidases

1.2.5 Similarities and Differences between a- and flGlycosidases 1.3 GLYCOSYLTRANSFERASES

1.3.1 Glycosyltransferases: Mechanism

1.3.2 Retaining glycosyltransferases

1.3.3 Inverting Glycosyltransferases

1.3.4 Common Structural Features of Glycosyltransferases

1.3.4.1 DXD Domain

1.3.4.2 Acceptor and Donor Binding Sites

1.3.4.3 Reaction Mechanism

1.3.5 Sialyltransferases

1.4 SIALICACIDS: GENERALBACKGROUND

1.5 BIOLOGICAL ROLES OF SIALICACIDS

1.6 SIALIDASEAND INFLUENZA

1.7 INHIBITORS OF THE SIALIDASE

1.8 CHEMICAL0-SIALYLATION METHODS

1.8.1 2-Halo derivatives

1.8.2 2-Thio Derivatives

1.8.3 2-Phosphite Derivatives

1.8.4 Synthesis of sialosides using neighbouring group participation at C-3

1.9 SYNTHESISOF CHROMOGENIC ACTIVATED SIALIDASE SUBSTRATES

2 CHAPTER TWO: STUDIES ON THE MECHANISM OF SIALIDASES

2.1 INTRODUCTION

2.2 ROLE OF THE CONSERVED TYROSINE RESIDUE IN CATALYSIS

2.3 ROLE OF THE CONSERVED ASPARTIC RESIDUE IN CATALYSIS

2.4 PROPOSEDMECHANISM FOR A RETAINING SIALIDASE

viii 2.5 SN1OR SN2MECHANISM? 45

2.5.1 Introduction 45

2.5.2 '3~Kinetic Isotope Effects 46

2.5.3 Kinetic Isotope Effect studies in Enzymatic Reactions 48

2.5.4 Materials and Methods 50

2.5.5 Results and Discussion 53

2.5.6 Conclusion 56

2.6 SYNTHESISOF ARYL CI-D-SIALOSIDES 56

2.7 EXPERIMENTAL 58

3 CHAPTER THREE: UNEXPECTED STABILITY OF ARYL P-D-N- ACETYLNEURAMINIDES IN NEUTRAL SOLUTION: BIOLOGICAL IMPLICATIONS FOR SIALYL TRANSFER REACTIONS 67

3.1 INTRODUCTION 67

3.2 SYNTHESIS OF ARYL b-D-N-ACETYLNEURAMINIDES 69

3.2.1 Spectral Assignment of the 4-nitrophenyl-PD-N-acetylneuraminide 70

3.3 MATERIALAND METHODS 73

3.3.1 Kinetic Experiments 73

3.3.2 Buffer systems 73

3.3.3 Hydrolysis Kinetics 74

3.3.3.1 Acid-Catalyzed Hydrolysis of 3.7a-g 75

3.3.3.2 Spontaneous Hydrolysis of 3.7b-d 76

3.4 RESULTSAND DISCUSSION 77

3.4.1 pH-Rate profile for the hydrolysis of 4-nitrophenyl PD-N-acetylneuramide (3.7b) and 4-nitrophenyl a-D-N-acetylneuramide (2.4~)at 50 *C 78

3.4.1.1 Kinetic Parameters 80

3.5 PH INDEPENDENT REGION 83

ix 3.5.1 Activation Parameters

3.5.2 Brgnsted Plots

3.5.3 Ethanolysis Studies

3.5.4 Product studies to investigate participation of carboxylate group

3.5.5 Analysis of the products from ethanolysis reaction using NMR spectroscopy

3.5.5.1 Ethanolysis product studies for 4-nitrophenyl-P- D-N-acetylneuraminide

3.5.5.2 Product distribution

3.5.6 Analysis of products from ethanolysis reaction using GC-MS

3.5.7 Proposed transition states at pH independent region

3.6 INVESTIGATION OF THE BASE-CATALYZED PROCESS

3.6.1 Exchange Experiment with ~2~~0

3.6.2 Investigation into the carboxylate group participation

3.6.3 Primary Kinetic Isotope Effect Study

3.6.4 Possible mechanism for the base catalyzed hydrolysis of aryl PD-sialoside

3.7 LOWPH DOMAIN

3.8 BIOLOGICALIMPLICATIONS FOR SIALYL TRANSFER REACTION

3.9 ARYLp-D-N-ACETYLNEURAMINIDES AS SUBSTRATES OF SIALIDASES.

3.9.1 Materials and Method

3.9.2 Enzyme Kinetics

3.9.3 Results and Discussion

3.9.4 Conclusion

3.10 EXPERIMENTAL

4 CHAPTER FOUR SYNTHESIS AND EVALUATION OF A BICYCL0[4.1.0]HEPTYL ANALOGUE OF GLUCOSE-1-PHOSPHATE

4.1 INTRODUCTION SYNTHETICTARGET

RESULTSAND DISCUSSION

ENZYMATICSTUDIES

ATTEMPTEDCHEMICAL SYNTHESIS OF UDP ANALOGUE

CONCLUSIONAND FUTUREWORK

EXPERIMENTAL

REFERENCES LIST OF FIGURES

Figure 1.1-1 Glycosidic Bond 1

Figure 1.2-1 Classification of glycosidases 3

Figure 1.2-2 Double displacement mechanism for a retaining a-galactosidase 5

Figure 1.2-3 Double displacement mechanism for a retaining a-galactosidase 7

Figure 1.2-4 Chemical structure of the thiooligosaccharide ([methyl S-P-D-glucopyranosyl- (l+4)-S-4-thio-~-~-glucopyranosyl-(1+~)-~-~-thio-~-~-glucopyranosyl-(l+4)-S-4- thio-P-D-glucopyranosyl-(1+4)4-thio-a-~+glucopyranoside

Figure 1.2-5 Distorted substrate at the enzymatic transition state of Endoglucanase I

Figure 1.2-6 Retaining j3-N-acetylhexosaminidases

Figure 1.2-7 General mechanism for an inverting a-galactosidase

Figure 1.3-1 General mechanism for a retaining glycosyltransferase

Figure 1.3-2 General mechanism for an inverting glycosyltransferase

Figure 1.3-3 Important binding characteristic of the donor

Figure 1.3-4 Oxacarbenium-ion like character in the transition state of GnT 1

Figure 1.3-5 CMP-NeuAc

Figure 1.4-1 Conformation of neuraminic acid

Figure 1.4-2 N-acetylated forms of sialic acid

Figure 1.7-1 Some sialidase inhibitors

Figure 1.8-1 Glycal formation by 2,3 elimination

Figure 1.8-2 Coupling method using 2-halo derivatives

Figure 1.8-3 Coupling strategy using 2-thio derivatives

Figure 1.8-4 Coupling strategy using 2-phosphite derivative

Figure 1.8-5 General scheme for C-3 participating auxiliaries Figure 1.8-6 General scheme for C-3 participating auxiliaries 32

Figure 1.8-7 An example of the use of auxiliaries in coupling reactions 33

Figure 1.9-1 Unnatural activated sialidase substrates 34

Figure 2.1-1 a-Ketosidically linked sialic acid 35

Figure 2.2-1 Bransted plots. Effect of leaving group ability on a) kcatfor the wild-type sialidase (a) and mutant Y370D (0). Reprinted with permission from The American Chemical Society 37

Figure 2.2-2 Bransted plots. Effect of leaving group ability on kCatlK,,,forthe wild-type sialidase (a) and mutant Y370D (0). Reprinted with permission from The American Chemical Society 38

Figure 2.2-3 Time course of M. viridifaciens Y370D mutant sialidase reaction monitored by 600 MHz 'H NMR spectroscopy. Reprinted with permission from The American Chemical Society 39

Figure 2.2-4 Proposed catalytic mechanism for the inverting Y370D mutant sialidase 40

Figure 2.4-1 Proposed mechanism for a retaining a-sialidase 44

Figure 2.5-1 Potential energy diagram 46

Figure 2.5-2 SN1mechanism 47

Figure 2.5-3 Possible mechanisms for the glycosylation step in sialidases 48

Figure 2.5-4 13C NMR spectra of a 1:2 mixture of C-3 and C-3, C-2 labelled 3-chlorophenyl (5-acetamido-3,5-dideoxy-~-g~ycero-a-~-gaacto-non--uopyranosyonicacid) substrates with ~42-l~~)alanine at pH 5.24 (100 mM acetate) in a 15% D20in water as solvent. 51

Figure 2.5-5 Plot of R/% ratio of the '3~-labelledsubstrates versus fraction of reaction for the Y370G catalyzed hydrolysis reation (R = '3~/12~at time t, % = '3~/12~at time = 0 for the substrate) 54

Figure 2.5-6 Plot of R/Ro ratio of 13C labelled substrate versus fraction of reaction for the D92G catalyzed hydrolysis reaction (R = '3~/12~at time t, % = '3~/'2~at time = 0 for the substrate) 54

Figure 2.5-7 13C NMR spectrum of 1:2 mixture of C-3 and C-3, C-2 labelled 3-chlorophenyl (5-acetamido-3,5-dideoxy-~-g~ycero-a-~-ga~acto-non-2-ulopyranosylonicacid) substrates with a-sialic acid product pH 5.24 (100 mM acetate) in a 15% D20in water as solvent. 55

Figure 2.6-1 Synthesis of aryl a-D-sialosides(2.4a-e) 57

xiii Figure 3.1-1 CMP-$-D-N-acetylneuraminide 68

Figure 3.1-2 Sialosyl oxacarbenium ion 69

Figure 3.2-1 Synthesis of aryl $-D-N-acetylneuraminides 70

Figure 3.2-2 'H NMR spectrum of 4-nitrophenyl-P-D-N-acetylneuraminidein D20 7 1

Figure 3.2-3 13cNMR spectrum of 4-nitrophenyl-P-D-N-acetylneuraminidein D20 72

Figure 3.3-1 The two anomers of 4-nitrophenyl-D-N-acetylneuraminidechosen for this study 74

Figure 3.4-1 Plot of log(kob,)versus pH for the hydrolyses of 4-nitrophenyl a- and P-D-N acetylneuraminides, T= 50 OC. The included solid lines are the best non-linear fits to equation 1 and 2. The dashed line is generated from kinetic rate constants reported for the hydrolysis of 4-nitrophenyl ~-D-Nacetylneuraminide, T= 50 OC 83 78

Figure 3.5-1 Pyridinium a-D-N-acetylneuraminides 83

Figure 3.5-2 Erying plot for the hydrolysis of 4-nitrophenyl a-D-N-acetylneuraminide. The displayed line is the calculated fit to the Eyring equation 85

Figure 3.5-3 Erying plot for the hydrolysis of 4-nitrophenyl $-D-N-acetylneuraminide.The displayed line is the calculated fit to the Eyring equation 86

Figure 3.5-4 Bransted plot, log (kobs) versus the pKa of the conjugate acid of the leaving group for the spontaneous reactions of compounds 3.7a-g measured at pH 1.00 at 50 OC88

Figure 3.5-5 Bransted plot at pH 8.08 at 100 OC 90

Figure 3.5-6 Plot of log kobsversus Y for the hydrolysis of 4-nitrophenyl ~-D-N- acetylneuraminide (2.4a) at 75 OC. 92

Figure 3.5-7 Plot of log kobsversus YPicfor the hydrolysis of 4-nitrophenyl $-D-N- acetylneuraminide (3.7b) at 75 OC. 94

Figure 3.5-8 Intramolecular participation of the carboxylate in hydrolysis 96

Figure 3.5-9 'H NMR of the ethanolysis products for the hydrolysis of 4-nitrophenyl $-D-N- acetylneuraminide 97

Figure 3.5-10 'H NMR of the ethanolysis product for the hydrolysis of 4-nitrophenyl a-D-N- acetylneuraminide 98

Figure 3.5-11 "0 labelled carboxylate group in 2.4a and 3.7b 100

xiv Figure 3.5-12 Possible transition states for the hydrolysis of compounds 3.4b and 2.4a in the pH independent region. 100

Figure 3.61 Attack of a hydroxide ion at the ipso carbon 103

Figure 3.6-2 Attack of the carboxylate oxygen on the ipso carbon 104

Figure 3.6-3 [~R-~H4 nitrophenyl 1-P-D-sialoside 105

Figure 3.6-4 Trans-diaxial elimination mechanism 105

Figure 3.6-5 Possible mechanism for the base-catalyzed pathway for the hydrolysis of aryl sialosides 106

Figure 4.1-1 Biosynthesis of UDP-Glc 127

Figure 4.1-2 Analogues of glucose-1-phosphate 128

Figure 4.2-1 Carbocyclic cyclopropanated motif for inhibitors 129

Figure 4.3-1 Synthesis of key intermediate enone 4.14 131

Figure 4.3-2 Observed nOe contact for compound 4.16 132

Figure 4.3-3 Synthesis of Glc-1-P analogue 133

Figure 4.4-1 Phosphate Assay 134

Figure 4.5-1 Uridine-5'-monomorpholidophosphate 135

Figure 4.5-2 Migration of the phosphate group 136

Figure 4.5-3 Proposed pathway for formation of 4.19 137

Figure 4.6-1 Alternate coupling method to avoid migration of phosphate group 137 LIST OF TAB LES

Table 2.3-1 Relative catalytic activity of the D92G mutant and the wild-type sialidase. 42

Table 3.4-1 Calculated pKas and rate constants for the hydrolyses of 4-nitrophenyl a- and P-D-N-acetylneuraminide at 50 "C (p = 0.3, NaC104) 80

Table 3.4-2 Observed Rate Constants for the Hydrolysis of 4-Nitrophenyl P-D-N- acetylneuraminic acid (3.7b) at 50 "C (p = 0.3, NaC104) 81

Table 3.4-3 Observed rate constants for the hydrolysis of 4-Nitrophenyl a-D-N- acetylneurarninide (2.4a) at 50 "C (p = 0.3, NaC104)a'b'" 82

Table 3.5-1 Observed rate constants for the hydrolyses of 4-nitrophenyl a-D-N- acetylneuraminide as a function of temperature at pH 8.08, (p = 0.3, NaC104) " 85

Table 3.5-2 Observed rate constants for the hydrolyses of 4-nitrophenyl P-D-N- acetylneuraminide as a function of temperature at pH 8.08, (p = 0.3, NaC104)" 86

Table 3.5-3 Activation parameters derived for the spontaneous hydrolysis of 4- nitrophenyl a- and P-D-sialosides at pH 8.08, (p = 0.3 M, NaC104) 87

Table 3.5-4 Observed rate constants for the hydrolyses of aryl P-D-N-acetylneuraminic acids at at 50 "C and a pH of 1.00 (p = 0.3, NaC104) " 89

Table 3.5-5 Observed rate constants for the hydrolyses of aryl P-D-N-acetylneuraminide at 100 "C and a pH of 8.08 (p = 0.3, NaC104) 90

Table 3.5-6 Observed rate constants (kobs)for the hydrolyses of Cnitrophenyl a-D-N- acetylneuraminide (2.4a) as a function of %EtOH in phosphate buffer, 15 mM at 75 "C, pH 7.25. 93

Table 3.5-7 Observed rate constants (kobs)for the spontaneous hydrolysis of 4-nitrophenyl P-D-N-acetylneuraminide (3.7b) as a function of %EtOH in phosphate buffer, 15 mM at 75 "C, pH 7.25. 94

Table 3.5-8 Observed product percentages formed during solvolysis of 2.4a and 3.7b in aqueous ethanol (vlv) solvent mixtures at 75 "c~'~ 99

Table 3.9-1 Kinetic Parameters of 3-chloropheny! P-D-sialoside with Y370G enzyme. 110

xvi LIST OF ABBRE 4ND ACRO

A Angstrom

Ac Acetyl

Ac20 Acetic Anhydride

AcOH Acetic Acid

AIBN 2,2'-Azobisisobutyronitrile bs Broad singlet

BnCl Benzyl chloride

BnOH Benzyl alcohol n-BuLi n-Butyl Lithium d Doublet dd Doublet of doublet

DANA 2-Deoxy-2,3-didehydro-D-N-acetylneuraminicacid

DMF Dimethyl formamide

DMSO Dimethyl sulfoxide

DTBP 2,6-ditert-butyl pyridine

EtOAc Ethyl acetate

h Hour

HPLC High performance liquid chromatography

J Coupling constant

m Multiplet

MeCN Acetonitrile

xvii MHz Megahertz

Min Minute mP Melting point

MeOH Methanol

NeuAc N-Acetylneuraminic acid

NBS N-Bromosuccinimide

NIS N-Iodosuccinimide nOe Nuclear Overhauser effect

NMR Nuclear Magnetic Resonance

PPm Parts per million

9 Quartet

S Singlet

TEA Triethylamine

THF Tetrahydrofuran

TfOH Trifluoromethanesulfonic acid

TFAA Trifluoroacetic anhydride

TMSOTf Tetramethyl silyl triflate

UDP Uridine diphosphate

UMP Uridine monophosphate

uv Ultraviolet

vis Visible

xviii CHAPTER ONE: GENERAL INTRODUCTION TO GLYCOSYLTRANSFER REACTIONS

1 .I General introduction

Carbohydrates are involved in many important roles in nature. Their functions range from acting as an important source of energy in living organisms to more complex roles when they are linked to lipids, proteins etc. They are actively involved in energy storage and release, cell-cell recognition and other crucial biological functions in the b~d~.',~,~

Monosaccharides are highly functionalized and complex molecules. For example glucose

(R=H) in Figure 1.1-1 has five different hydroxyl groups sites where glycosylation or other chemical modifications can occur. Furthermore, there are two possible configurations at the anomeric centre: that is alpha linkages when the aglycon is axial and beta linkages when the aglycon is equatorial.

Sugar

Glycosidic bond

Anomeric Centre

Figure 1.1-1 Glycosidic Bond In order to handle such complex molecules, nature has devised numerous ways to catalyze the formation and cleavage of glycosidic linkages. There are four different types of carbohydrate processing enzymes, namely:

1. Glycosidases which cleave longer oligosaccharides into smaller chains or

units and which replace the aglycon moiety of the oligosaccharide by a

hydroxyl group.

2. Glycosyltranferases which use activated carbohydrate donor molecules in

order to extend oligosaccharide chains via glycosidic linkages.

3. Polysaccharide lyases cleave polysaccharide chains through a beta

elimination pathway. This results in the formation of a double bond at the

newly formed non-reducing terminus.

4. Carbohydrate esterases catalyze the de-0 or de-N acetylation of

substituted sugar molecules.

A listing of members from these four enzyme families can be found at

http://afmb.cnrs-mrs.fr/-cazy/CAZY/index.html. This thesis details research on members from the glycosidase and glycosyltranferase families.

1.2 Glycosidases

Glycosidases can be sub-classified into two families: retaining and inverting enzymes, and this classification is based on the stereochemical outcome of the enzyme-catalyzed reaction (Figure 1.2-1)

Retaining fl glycosidase

HO HO

OH fl-linkedsubstrate a-linked substrate OR

Inverting fl glycosidase Retaining a glycosidase

a product 6~

Figure 1.2-1 Classification of glycosidases

That is, inverting glycosidases operate with inversion of configuration at the anomeric centre, whereas retaining glycosidases hydrolyze the glycosidic bond with retention of configuration. 1.2.1 Retaining Glycosidases

In 1953, Koshland proposed that retaining glycosidases operate through a two-step double displacement mechanism that involves formation of a glycosyl-enzyme

intem~ediate.~There are two catalytic acidic amino acid residues in the enzyme active

site, one of which acts as the nu~leo~hile.~The other residue has a dual catalytic role

acting as a general base in the glycosylation stage and a general base in the

deglycosylation stage.

The first step, glycosylation, involves attack of the nucleophile at the anomeric centre of the glycoside, the other residue, the general acid catalyst, assists in the departure of the leaving group (OR) by protonating it. This leads to the formation of a glycosyl-enzyme intermediate. In the second step, deglycosylation, the covalent glycosyl-enzyme intermediate is cleaved by a water molecule acting as the nucleophile with assistance by the general base residue to form the retained hemiacetal product. Both steps proceed through an oxacarbenium ion-like transition state. Shown below is the general mechanism for a retaining glycosidase (Figure 1.2-2) Nucleophile

\OH + ROH + Y-H General acid

Figure 1.2-2 Double displacement mechanism for a retaining a-galactosidase

1.2.1.1 Bronsted Plots

In order to characterize fully the rates of various steps in the hydrolytic mechanism it is necessary to identify substrates for which glycosylation and deglycosylation steps are rate limiting. Hence a series of aryl glycosides have been used to construct Brmsted plots for various glycosidases.6~7One example is that of aryl P-glycosides with Agrobacteriurn sp.

~-~lucosidase(~b~).*The plot of log kc,, for the enzymatic hydrolysis of a series of aryl p glucoside substrates versus the pK, of the conjugate acid of the various leaving groups was biphasic, concave downwards. From this observation it was concluded that there was a change in rate determining step as the leaving group ability decreased. For substrates with pKa > 8, it was concluded that glycosylation was rate-limiting because of a large negative slope (81, = -0.7).~ On the other hand, the value of log kc,, was independent of leaving group for substrates with a pKa < 8. Hence on the basis of this observation and the results from nucleophilic competition studies, it was concluded that deglycosylation was rate-determining for these substrate^.^'^

1.2.1.2 Oxacarbenium ion-like transition state a-Secondary deuterium kinetic isotope effect (aSDKE) measurements on the aryl glucosides provided insight into the transition state structure of the Abg catalyzed hydrolysis rea~tion.~A higher average value of kHlkDwas observed for substrates in which deglycosylation (kHlkD= 1.11) was rate limiting than when glycosylation was rate- determining (kHlkD = 1.06). These observations were taken to indicate that the deglycosylation transition state has more oxacarbenium ion like character than the corresponding glycosylation transition state.

1.2.2 Retaining Glycosidases

In 1953, Koshland proposed that retaining glycosidases operate through a two-step double displacement mechanism that involves formation of a glycosyl-enzyme intermedi~ite.~There are two catalytic acidic amino acid residues in the enzyme active site, one of which acts as the nu~leo~hile.~The other residue has a dual catalytic role acting as a general base in the glycosylation stage and a general base in the deglycosylation stage.

The first step, glycosylation, involves attack of the nucleophile at the anomeric centre of the glycoside, the other residue, the general acid catalyst, assists in the departure of the

6 leaving group (OR) by protonating it. This leads to the formation of a glycosyl-enzyme intermediate. In the second step, deglycosylation, the covalent glycosyl-enzyme intermediate is cleaved by a water molecule acting as the nucleophile with assistance by the general base residue to form the retained hemiacetal product. Both steps proceed through an oxacarbenium ion-like transition state. Shown below is the general mechanism for a retaining glycosidase (Figure 1.2-3).

Nucleophile

+ ROH

& General acid 1

+ H20 * I Nucleofuge

y:eral Base

Figure 1.2-3 Double displacement mechanism for a retaining a-galactosidase 1.2.2.1 Brwsted Plots

In order to characterize fully the rates of various steps in the hydrolytic mechanism it is necessary to identify substrates for which glycosylation and deglycosylation steps are rate limiting. Hence a series of aryl glycosides have been used to construct Bronsted plots for various glycosidases.6~7One example is that of aryl P-glycosides with Agrobacteriurn sp.

~-~lucosidase(~b~).~The plot of log kcatfor the enzymatic hydrolysis of a series of aryl p glucoside substrates versus the pKa of the conjugate acid of the various leaving groups was biphasic, concave downwards. From this observation it was concluded that there was a change in rate determining step as the leaving group ability decreased. For substrates with pKa > 8, it was concluded that glycosylation was rate-limiting because of a large negative slope (PI, = -0.7).~ On the other hand, the value of log kc,, was independent of leaving group for substrates with a pK, < 8. Hence on the basis of this observation and the results from nucleophilic competition studies, it was concluded that deglycosylation was rate-determining for these substrates.839

1.2.2.2 Oxacarbenium ion-like transition state a-Secondary deuterium kinetic isotope effect (aSDKIE) measurements on the aryl glucosides provided insight into the transition state structure of the Abg catalyzed hydrolysis reaction.* A higher average value of kH/kDwas observed for substrates in which deglycosylation (kH/kD= 1.11) was rate limiting than when glycosylation was rate- determining (kH/kD = 1.06). These observations were taken to indicate that the deglycosylation transition state has more oxacarbenium ion like character than the corresponding glycosylation transition state. 1.2.2.3 Substrate distortion during catalysis

Crystal structures of P-glycosidases with non-hydrolyzable substrates bound in the active

site have revealed that binding induces significant distortion of the pyranoside ring.''-l2

One such example is that of the F. oxyporum Endoglucanase I (EG I), a cellulase, which

hydrolyzes the 0-1,4 linkages of cellulose.'' The solved crystal structure of this glycosyl

hydrolase contained the hydrolytically stable thiooligosaccharide substrate analog shown

in Figure 1.2-4

Figure 1.2-4 Chemical structure of the thiooligosaccharide ([methyl S-P-~-glucopyranosyl-(l+4)-S- 4-thio-~-~-glucopyranosyl-(1+4)-S-4-thio-~-~-glucopyranosyl-(1+4)-S-4-thio-~-~-glucopyranosyl- (1+4) 4-thio-a-D-glucopyranoside

The glucopyranose unit in the -1 subsite was found to be distorted from the 4~1to a

distorted boat conformation where the C-1 atom is slightly above the plane formed by C-

2, C-3 and 0-5. This placed the anomeric linkage into a quasi-axial orientation such that

the leaving group occupies a more accessible position for protonation by the general acid

catalyst and hence facilitates scission of the glycosidic bond. Furthermore, this permits

an unhindered line of attack for the incoming nucleophile at C-1 of the sugar (Figure 1.2-

5). Figure 1.2-5 Distorted substrate at the enzymatic transition state of Endoglucanase I

1.2.3 Retaining P-N-acetylhexosaminidases

A different mechanism has been proposed for some retaining P-N-acetylhexosaminidases where the N-acetyl moiety acts as an intramolecular nucleophile. The reaction goes through an oxazolidinium inte~mediate.'~"~

Figure 1.26 Retaining P-N-acetylhexosaminidases 1.2.4 Inverting Glycosidases

Inverting glycosidases operate by a single displacement mechanism. The active site general base deprotonates a water molecule as it nucleophilically attacks the anomeric carbon of the glycoside. The departure of the aglycon is assisted by the general acid catalyst thus resulting in formation of the product having the opposite configuration at the anomeric centre. The reaction goes through an oxacarbenium ion-like transition state that is similar to those of the retaining glycosidases (Figure 1.2-7).

General base --T--J

& General acid

Figure 1.2-7 General mechanism for an inverting a-galactosidase

1.2.5 Similarities and Differences between a- and P-Glycosidases

The retaining and inverting enzymes are quite similar. Both enzymes use acidic amino acid residues (aspartate or glutamate) as their catalytic residues.15 Hydrolytic transition states in both cases have oxacarbenium ion-like character and there is significant distortion of the sugar ring. 1,16,17,18 The major difference between the two enzyme families is the distance between the two catalytic amino acid residues in the active site. The distance is about 10.5A in inverting enzymes but only about 4.5A in retaining enzymes.'" In the case of inverting enzymes, the 10.5 A separation between catalytic residues provides enough space for both the substrate and nucleophilic water molecule to bind between the two carboxylate residues. 5,'7,'5 However, retaining enzymes can only fit the substrate and this compact arrangement gives rise to a double displacement mechanism that results in a retained configuration in the product.

1.3 Glycosyltransferases

The formation of glycosidic bonds is one of the most important reactions in the biological kingdom. This type of chemistry is involved in the biosynthesis of simple carbohydrates, such as sucrose and lactose, and complex glycoconjugates that include polysaccharides, glycolipids and glycoproteins. The sugar component in these structures is critical to many crucial biological processes like fertilization, immune defense, viral replication, parasitic infection, cell growth, cell-cell adhesion, and inflammati~n.',~

Oligosaccharides are synthesized in eukaryotic cells by different enzymes that are present in an assembly line situated in the endoplasmic reticulum and the Golgi apparatus.19 The glycosyltransferases have their own donor, acceptor and linkage Even though these enzymes are so profoundly important, not much was known about their structure and mechanism until the mid 2990's. The molecular basis of donor and acceptor specificity is also unclear. However, these enzymes remain as potential drug targets in the fight against many diseases including cancer, and viral, bacterial and fungal-based infections.

1.3.1 Glycosyltransferases: Mechanism

There are two ways by which glycosidic linkages can be made. The non-Leloir pathway which uses glycosyl phosphates as donors and the nucleotide dependent pathway (Leloir pathway) which uses monosaccharides donors activated as glycoesters of nucleoside mono or diphosphates.22-24 These glycosylation reactions can proceed either with retention or inversion of configuration at the anomeric centre of the donor sugar.

1.3.2 Retaining glycosyltransferases

Glycosyltransferases are believed to share a similar mechanism to that proposed for glycosidases.25 The reactions of retaining glycosyltransferases are thought to proceed via two oxacarbenium ion-like transition states and a glycosyl-enzyme intermediate.26 The enzyme nucleophile attacks the anomeric centre of the donor whilst the leaving group departure is assisted by metal ion catalysis, this generally involves either Mn2+or Mg2+, rather than by general-acid catalysis as occurs in the glycosidases. In a subsequent step, the hydroxyl group of an acceptor molecule reacts at C-1 of the donor-enzyme intemediate with general-base catalysis to form the glycoside with retained configuration

(Figure 1.3-1). Nucleophile

M"' Donor Sugar Nucleotide "\ww 1 Acceptor

Enzyme General Base

Newly formed glycosidic bond

Figure 1.3-1 General mechanism for a retaining glycosyltransferase

Another mechanism that has been proposed for a retaining glycosyltransferase involves an internal return mechanism. Specifically, Persson et al. postulated that the galactosyltransferase LgtC from Neisseria meningitidis proceeds through an unusual SNi mechanism27 whereby the departure of the leaving group generates an ion-pair that collapses by the nucleophilic attack of the acceptor hydroxyl group on the same face of the sugar. Evidence for this mechanism includes the failure to trap the covalent glycosyl- enzyme intermediate using 5-fluorogalactosyl fluoride.28 1.3.3 Inverting Glycosyltransferases

Inverting glycosyltransferases are believed to operate through a single SN2-like displacement reaction. An acceptor hydroxyl group acts as a nucleophile, with assistance by a general-base residue in the enzyme's active site, to attack the C-1 of the donor to give an inversion of stereochemistry at the anomeric carbon (Figure 1.3-2).

Enzyme Genaral Base --f""-

UDP t

,"I Donor Sugar Nucleotide Newly formed glycosidic bond

Figure 1.3-2 General mechanism for an inverting glycosyltransferase

1.3.4 Common Structural Features of Glycosyltransferases

Glycosyltransferases being mostly membrane bound enzymes are difficult to express and purify. Hence, there has been a considerable delay in solving their structures through crystallographic studies. However, tremendous advances have been made during the past five years in structure-function relationships for this family of enzymes. This section will provide a general overview of the active site structure and the mechanism of glycosyltranferases using specific examples from the literature.

The important common features in the glycosyltransferases are a conserved DXD motif responsible for metal binding, the donor and acceptor binding sites, the catalytic

15 residues and a flexible loop that senses the presence of the donor sugar anomeric carbon, and the phosphate linkage responsible for glycosylated product release.

1.3.4.1 DXD Domain

The DXD amino acid motif has been observed in many glycosyltransferase families.29 The motif contains two aspartic acid residues (D) and 'X' can be any amino acid residue. This entity is responsible for the binding of the donor sugar in the enzyme active site. From the crystal structure of the rabbit N-acetylglucosaminyltransferase I 25 it was observed that the ~n~'ion was coordinated with the two oxygen atoms of the diphosphate linkage and the second acidic residue of the DXD domain. This restriction imposed by the metal ion forces the first acidic residue in the DXD domain to interact with the UDP-GlcNAc residue in the active site. The ~n~+ionis believed to assist in the departure of the UDP moiety during glycosylation by stabilizing the development of an additional negative charge.

1.3.4.2 Acceptor and Donor Binding Sites

As enzyme-catalyzed coupling reactions between two carbohydrates are highly specific with respect to both C-1 configuration of the donor and the site of attachment of the sugar residue on the acceptor, each enzyme has a unique domain structure for substrate recognition and binding. For example, in the b-1,4-galactosyltransferase 30 it has been found that the UDP-Gal is embedded in the catalytic pocket with all its hydroxyl groups interacting with amino acid residues. A weak H-bond between the 0-4 oxygen of the galactose moiety and an aspartate residue, is only possible with galactose because of its axial hydroxyl group at the C-4 position (Figure 1.3-3).

Asp 318

H OUDP

Figure 1.3-3 Important binding characteristic of the donor

Removal or modification of this axial OH group on the C-4 position results in loss of activity of the enzyme. The nucleotide recognising domain (NRD) is responsible for the recognition of a pyrimidine n~cleotide.~' The rabbit N-acetylglucosaminyl transferase crystal structure revealed that both the uracil ring and the hydroxyl groups of the ribose makes significant contact to amino acid residues in the donor binding sites.25

Another important feature to point out is the presence of a structured loop made up of about thirteen residues that is formed upon binding of the donor. Only the tip of this loop makes contact with the diphosphate moiety of the donor. This flexible loop becomes disordered when UDP is formed upon glycosylation, and this leads to the release of the glycosylated product

Less is known about acceptor binding, but it has been observed that in the case of

Gal-T1 transferase binding of the donor induces a conformational change to create an acceptor binding site that can accommodate an extended sugar acceptor.30 Following binding, an acceptor OH group is pointing towards the C-1 of the bound donor ready for the glycosylation reaction. 1.3.4.3 Reaction Mechanism

The reaction mechanism for inverting glycosyltransferases involves a general base that assists the deprotonation of a hydroxyl group of the acceptor.

For example in the GnT 1, the general base is D291 which is located 4.7A from the anomeric centre of donor UDP GICNAC.~'This position allows proper alignment of the acceptor for nucleophilic attack at C-1 of GlcNAc. These reactions proceed through an oxacarbenium ion-like transition state as shown in Figure 1.3-4, similar to that proposed for glycosidase reactions.32 ow ever, it has been suggested that a distorted nucleotide-sugar would be more prone to undesirable hydrolysis.25 Also, acceptor binding is required for driving the reaction toward the transition state.

Figure 1.3-4 Oxacarbenium-ion like character in the transition state of GnT 1 1.3.5 Sialyltransferases

Sialyltransferases transfer the sialic acid moiety from cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to the terminii of growing oligosaccharide chains.

Figure 1.3-5 CMP-NeuAc

Recently the crystal structure of the sialyltransferase from Campylobacter jejuni was published.33 The enzyme was co-crystallized with an inert analogue of the donor sugar, namely CMP-3-fluoro-N-acetylneuraminic acid. It was found that the substrate analogue was bound in a distorted skew boat conformation such that C-1, C-2, C-3 and the ring oxygen are coplanar. This conformation also places the leaving group above the plane of the ring such that there is an electrostatic interaction between the non-bridging oxygen of the phosphate moiety with the ring oxygen of the sialyl molecule. This arrangement optimizes the stabilization of the developing oxacarbenium ion character on the pyranosyl ring as the glycosidic linkage is being broken. Sialyltransferases differ from other glycosyltransferases in that a monophosphate donor is utilized and this difference in leaving groups does not require a metal ion for catalysis. Thus, sialyltransferases do not contain a DXD motif.33 1.4 Sialic Acids: General background

Sialic acids are members of a family of compounds that possess a nine carbon amino acid sugar skeleton known as neuraminic acid. The IUPAC name for neuraminic acid is 5- amino-3,5-dideoxy-D-glycero-~-galacto-2-nonulopyranosonicacid (Figure 1.4-1).34,35

1 C02H 2 co1 3 I 0 H

a-neuraminic acid

D- galacto = 1

D- glycero C02H

p-neuraminic acid

Figure 1.4-1 Conformation of neurarninic acid

There are more than 70 derivatives of the core N-acetylated neuraminic acid (e.g.,

NeuSAc and NeuSGc are shown in Figure 1.4-2) have been discovered. Other derivatives include the possible presence of functional group modifications such as acetyl, lactyl, methyl, sulphate and phosphate groups to the C-4, C-7, C-8 and C-9 hydroxyl groups. Molecules having a double bond between C-2 and C-3 have also been isolated. 34,35,36 N-acetylneuraminic acid (NeuSAc) N-glycoloylneurarninic acid (NeuSGc)

Figure 1.4-2 N-acetylated forms of sialic acid

1.5 Biological Roles of Sialic acids

Sialic acid residues are usually found on the terminii of the carbohydrate moieties in glycoconjugates. The highly exposed location of sialic acid residues on the cell surfaces is ideal for many carbohydrate-protein interactions results in sialic acid being an important determinant in molecular and cellular recognition events and in many disease states.

Listed below are some structure function relationships of sialic acid.

'. Sialic acids are large, hydrophilic and acidic, they can influence and

stabilize the conformation of glycoconjugates.37,38,39

2. The negatively charged carboxylate group in the sialic acid residues

allows them to play an important role in transport and binding of Ca 2+ or

other positively charged ions.36

3. Salic acid derivatives act as receptors for bacteria and viruses, enzymes,

hormones, lectins et~.~' 4. Another important role is that sialyl moieties can act as biological masks

to prevent recognition of penultimate galactose residues. Hence the

glycoconjugate is protected against unwanted degradation. For example,

serum glycoproteins are invisible to the Ashwell receptors in the liver

since the galactose residue is shielded by the sialic acids present on the

terminal site^.^' Once the sialic acids are cleaved by sialidases, the

asialoglycoproteins are recognized by the Ashwell receptors and removed

from circulation by the hepatocytes.42,43

5. SLex (Sialyl lewis x) mediate cell-cell adhesion through lectins. For

example selectins interact with sialyl lewis x during recruitment of

leukocytes in the inflammation process. 44

1.6 Sialidase and Influenza

Influenza is a highly infectious illness affecting the respiratory system. The influenza virus can change in two ways. Antigenic Drift occurs when small changes in the virus happen continuously over time. The new virus strains are not recognised by the body's immune system. As the strain of virus is new, few people have a built-in immunity from past exposures. It is not unusual that reinfection occurs more than one time in a given flu season. However, a new strain of influenza virus that is foreign to human beings suddenly becomes highly contagious, passing from person to person.45 This change is called antigenic shift which results in a an abrupt and major change in the surface antigens on the influenza A viruses resulting in a new influenza A subtype. When this shift happens (usually every 10 to 40 years), the risk of a pandemic is very high. Even the people who have been vaccinated have no defense against the disease; since flu shots protect against influenza variants that health experts have anticipated will be active in a given flu season, not against other unexpected viral strains.46 vaccines prime the immune system to prevent viruses from gaining a foothold in the body, whereas standard home remedies ease symptoms but have no effect on the infection itself.

Another way of treating the disease is by targeting the active site of one of the viruses' surface proteins which is a sialidase. This is an ideal target since there is little variation in the enzymes' active site between virus strains. It is this protein that is responsible for the infectivity of the virus in the body.

Sialidases (EC 3.2.1.18, N-acetylneuraminosyl glycohydrolases, neuraminidases) play an important role in the infectivity of the influenza virus. They are present on the viral surface and assist in the release and propagation of progeny viruses from infected cells.47

For a single copy of an influenza virus to enter a human cell, the surface protein hemagglutinin on the virus must bind to sialic acid, on the surface of the cell. This binding event induces the cell to engulf the virus. The viral genes and internal proteins are freed, and they work their way into the cell nucleus. Once there, some of the viral proteins set about replicating the viral RNA and also constructing messenger RNA that can be read out and translated into proteins by the cell's protein-making machinery.

Eventually the newly made viral RNA and proteins assemble and bud from the cell as new viral particles. The emerging particles are coated with sialic acid, the very substance that binds influenza

viruses to the cells they attempt to invade. If the sialic acid were to remain on the virus

and on the surface of a virus-making cell, hemagglutinin on the newly minted viral

particles would bind to the sialic acid, causing the particles to clump together on the cell

and thus render the virus inactive. However, the new viral particles contain the

neuraminidase enzyme on their surfaces, and it is this enzyme that cleaves the surface

coating of sialic acid. The neuraminidase releases the virus particles, thus enabling them

to travel and infect new ~ells.~~,~~

1.7 Inhibitors of the sialidase

In view of rapidly mutating influenza viruses, the search for more potent inhibitors is

ongoing. The rational design of sialidase inhibitors really started off with the elucidation

of the crystal structure of enzyme's active site with and without 2-deoxy-2,3-didehydro-

D-N-acetylneuraminic acid (DANA).49,50 It was reported earlier that DANA was an

inhibitor of influenza neuraminidase with an IC50 value of 5-10 pM. 51

The key features of how DANA binds to the active site were used in the design of new

.inhibitors. For example, the double bond forces DANA to adopt a half-chair

conformation thus, mimicking the ring distortion present at the oxacarbenium ion-like

transition state.

The carboxylate group has very important electrostatic interactions with the enzyme's

arginine triad. The glycerol side chain fits in a hydrophobic pocket in the active site.

The C-4 hydroxyl is positioned into another pocket whereas the acetamido group on C-5

seems to to fit into a hydrophobic hole.j2 HO

AcH N C02H AcHN HO HO

Sialic Acid DANA

H00c02HAcHN '4 AcHN acOZH

H2N

Zanamivir Oseltamivir

Figure 1.7-1 Some sialidase inhibitors Shown in Figure 1.7-1 on the previous page are some of the current generation of sialidase inhibitors. The structural features of the inhibitors that contain a six membered ring are similar to those of DANA. For example, Zamamivir, which is marketed as

Relenza, has a double bond between C-2 and C-3 but it contains a guanidino group at C-4 that has favourable interactions with a negatively charged pocket in the influenza sialidase active site.52,53,54-56 However, Oseltamivir also known as Tamiflu has an O- isopentyl group instead of the glycerol side chain and this group makes contact with a hydrophobic pocket in the active site.57-59

BCX-1812 (Peramivir) is based on a saturated cyclopentane system but it still retains all the functionalities that are essential for binding.60 Specifically, BCX-1812 incorporates the guanidino group from Zanamivir, the isopentyl group from Oseltamivir, the acetamido and carboxylate from DANA. This is a strong indication that the 3D orientation of the groups is more important than the ring system used to orient them.61

Due to the development of resistance to the current drugs available on the market, the search for new drugs for influenza is ongoing. 1.8 Chemical 0-sialylation methods

In order to determine the biological roles of complex sialylated oligosaccharides, they have to be synthesized. However, the synthesis of sialosides is challenging because of the presence of the carboxylate group at the anomeric centre and the absence of functionality at the C-3 position. The carboxylate group makes the anomeric carbon C-2 more sterically hindered and this results in a decreased efficiency for synthesis of the glycosidic bond. Furthermore, the leaving group at C-2 makes many of the various sialic acid derivatives more prone to 2,3-elimination during the coupling reaction. This poses a serious problem since the glycal formed is a protected form of 2-deoxy-2,3-didehydro-D-

N-acetylneuraminic acid (DANA) which is an inhibitor of sialidases (Figure 1.8-1). The absence of any functional groups on C-3 excludes the use of neighbouring group participation to direct the stereochemical outcome of the glycosidation reaction.

Furthermore the P-anomer is thermodynamically more stable than the required a-anomer.

Thus, coupling sialic acid to other residues is difficult.

AcOo

____)

AcHN / AcO

Figure 1.8-1 Glycal formation by 2,3 elimination

Over the years, various synthetic methods have been developed to enhance coupling efficiencies. This section will highlight some of the more efficient coupling strategies.62

27 1.8.1 2-Halo derivatives

The fully protected sialosyl chloride (1) can be synthesized from sialic acid in two steps:

1) esterification with MeOH in the presence H+; and 2) protection and derivatization via treatment with HClIAcCl 63, Tic14 64 or AcClIMeOH. 65

OAc

C02Me - AcHN OR AcHN

Figure 1.8-2 Coupling method using 2-halo derivatives

The coupling method involving sialosyl chloride (1) and the acceptor is highly effective for simple alcohols like phenol and methanol but yields mainly the glycal with less reactive or sterically hindered alcohols. Promoters that have been used include Hg(CN)2/

~~~r2~~;mild lewis acids such as ZnBr2, SnC12 67 and polymer based silver salts.68 In addition, coupling reactions using phase transfer catalysts work quite well for the synthesis of aryl sialosides." However, the phase transfer reaction has a drawback in that the aglycon must be water soluble. 1.8.2 2-Thio Derivatives

These derivatives (3) where R' = alkyl, aryl, require thiophilic activators like

(dimethyl(methylthio)sulfonium trifluoromethanesulfonate (DMTST) (Figure 1.8-

3).70,71,72

OAc OAc

Ac0Oe

SR' - OR AcHN AcH N AcO 4

Figure 1.8-3 Coupling strategy using 2-thio derivatives

NISI TfOH is a particularly effective activator as higher anomeric stereoselectivity for the a-anomer is obtained in the glycosylation of sterically crowded hydroxyl groups. Also the use of acetonitrile as solvent significantly improves the stereoselectivity of the coupling reaction.73

1.8.3 2-Phosphite Derivatives

OAc - AcHN AcHN

Figure 1.8-4 Coupling strategy using 2-phosphite derivative Schmidt and Wong simultaneously developed sialyl phosphites (5) as glycosyl donors in

0-sialylation reactions (Figure These donors require a very small amount of

TMSOTf (10-20 mol %) for activation. The dimethyl phosphite when activated with

ZnC12/AgC104in CH2C12gave a higher yield (80%) and a high a-stereosele~tivit~.~~

1.8.4 Synthesis of sialosides using neighbouring group participation at C-3

Sialic acid derivatives do not have a C-3 substituent that can act as a neighbouring group to direct glycosylation reactions. One way to circumvent this problem is to introduce an easily removable functionality at C-3 position (Figure 1.8-5). Hence, the double bond functionality in a protected 2,3-dehydrosialic acid is used to incorporate the auxiliary at

C-3. For example, epoxidation of, or addition to the double bond are the most commonly used reactions for this purpose. Thus a P-glycoside will be obtained with equatorial auxiliaries and a-glycoside with axial auxiliaries at C-3 position. The advantage with this strategy is that 2,3 elimination, which is a major side reaction in coupling reactions, is avoided. R

Introduction of Aulrilliary AB C02Me AcHN / C02Me - RO AcHN A

Promoter ROH

.\17---3\ RemovalSubstituent of C-3 OR + AcHN A AcHN RO RO 10 9

A = 0-,S-, Br- or Se- auxilliary B = Halide, phosphite

Figure 1.8-5 General scheme for C-3 participating auxiliaries

This methodology was developed by Okamoto et a1 in the 1980's.~~-~'

For example, glycal (11) is converted to a 2-thiomethyl-3-thiophenyl compound (12) upon reaction with ArSCl in high yield, a derivative which is highly crystalline and therefore easily isolated (Figure 1.8-6). 82 1) ArSCV CH2C12

b SMe AcHN AcHN SAr 2) MeSNa/CH3CN AcO 12

PhSC I/ AgOTf / DTBP 1 MeCN I ROH

C02Me Ph,SnH/AIBN AcO in toluene OR OR AcHN AcHN SAr AcO AcO

Figure 1.8-6 General scheme for C-3 participating auxiliaries

This methodology was developed by Okamoto et a1 in the 1980's.~~-~'

w SMe AcHN AcHN SAr 2) MeSNa/CH3CN

Snin tolueneWAIBN

4 ~~~s OR OR AcHN AcHN SAr AcO AcO

Figure 1.8-7 An example of the use of auxiliaries in coupling reactions

Coupling of this glycosyl donor (12) with the acceptor in the presence of a promoter yields compound 13. Removal of the auxiliary at the C-3 position affords the desired product 14. The disadvantage with using auxiliaries is that additional steps are introduced into the synthetic route.

1.9 Synthesis of chromogenic activated sialidase substrates

The mechanism and activity of sialidases can be studied in detail by making use of a variety of substrates. These substrates can be of natural origin (e.g., sialyl lactose) or they can be chemically synthesized (e.g., 4-nitrophenyl a-D-sialoside). A good substrate for enzymatic assay should have the following attributes: 1) it should be reactive, i.e. bear good leaving groups; and 2) the by-product(s) of catalysis should be easily detected with high sensitivity. Examples of unnatural chromogenic substrates are shown below 4- nitrophenyl a-D-sialoside (PNP-aNeuAc) and 4-methylumbelliferyl a-D-sialoside (MU- aNeuAc)

HO- HO-

AcHN HO

NO2 PNP-aNeu5Ac

Figure 1.9-1 Unnatural activated sialidase substrates

Upon hydrolysis, 4-nitrophenyl a-D-sialoside releases 4-nitrophenol which has a high extinction coefficient. The solution and enzyme catalyzed hydrolyses of a series of aryl a-~-sialosides~~~~~and pyridinium a-~-sialosides~~have been studied. CHAPTER TWO: STUDIES ON THE MECHANISM OF SIALIDASES

2.1 Introduction

Sialidases (EC 3.2.1.18: N-acetylneuraminosyl glycohydrolases) are a family enzymes that catalyze the hydrolysis of terminal sialic acid residues that are bound to glycoproteins, glycolipids and polysaccharides through an a-ketosidic linkage (Figure

0-R AcHN HO R = glycoprotein etc I Sialic Acid a-ketosidic linkage

Figure 2.1-1 a-Ketosidically linked sialic acid

These enzymes hydrolyze sialic acid derivatives with retention of configuration at the anomeric carbon. 87,88,89 The active site within the sialidase superfamily has been shown to contain a conserved tyrosine residue, a pair of acidic residues (glutamic or aspartic acid) and an arginine triad which binds to the carboxylate group of the substrate via electrostatic interaction^.^' Results from X-ray crystallography studies have shown that sialic acid is bound in the active site of influenza type A sialidase (Tokyo 3/67) in a boat conformation such that there is a strong electrostatic interaction between the carboxylate of the sialoside and the arginine (Arg371) of the enzyme.91392

2.2 Role of the conserved tyrosine residue in catalysis

Prior to May 2003, it was widely believed that the glutamate was key in stabilizing the sialosyl-enzyme intermediate either electrostatically or nucleophilically. However, Watts et al. demonstrated that the tyrosine acts as the nucleophile of the trans-sialidase from

Trypanosoma cruzi. The activated fluoro sugar analogue, 2,3-difluoro sialic acid covalently labels the active site tyrosine

As part of a collaborative project within our group, the role of the conserved tyrosine residue in catalysis in sialidases was investigated. Dr. Watson expressed and purified three tyrosine mutants Y370G, Y370A and Y370D of the Micromonospora viridifaciens

(M. viridifaciens) sialidase enzyme. I synthesized the activated substrates, namely 4- cyano, 3-chloro, 4-nitro, 3-nitrophenyl a-D-sialosides, that were needed to construct the

Brgnsted plots. The synthesis and experimental details are described in section 2.6 at the end of this chapter.

Dr. Watson performed detailed kinetic experiments on three tyrosine mutants Y370G,

Y370A and Y370D of the Micromonospora viridifaciens (M. viridifaciens) sialidase enzyme to demonstrate the importance of the tyrosine residue to catalysis. It was shown that all three mutants could hydrolyse the activated substrate 4-methylumbelliferyl a-D sialoside with near wild-type efficiency.95 In order to probe the mechanism further, rate constant data was measured for a series of aryl a-D-sialosides and two isomeric sialyl- lactose substrates.

The Bronsted plots were analyzed and it was found that cleavage of the glycosidic C-0 bond was independent of kc,, for the wild type enzyme (Dlg = 0.02 + 0.03). A non- chemical step, conformational change, was likely the rate-determining step. On the other hand, the kc,, parameter of Y370D mutant showed a strong dependence (Pig = -0.55 +

0.03) on substrates at pK, value greater than 9.09. This was indicative of glycosidic bond cleavage being rate limiting.g5

Figure 2.2-1 Brmsted plots. Effect of leaving group ability on a) kc,, for the wild-type sialidase (0) and mutant Y370D (0). Reprinted with permission from The American Chemical Society

All experiments were performed at 37OC and pH 5.25. Leaving group ability represented as pK, (BH') as follows: 4-nitrophenol (7.18); 4-methylumbelliferone (7.80); 4-cyanophenol (7.96); 3- nitrophenol (8.27); 3-chlorophenol (9.09); 3-methoxyphenol (9.60); phenol (9.92), and lactose (3'-OH (-13.6) and 6'-OH (-13.8)). It was found that the Brgnsted plot of Y370D mutant indicated a bigger dependence of k,,JK, parameter on leaving group ability (Pig = -0.74 + 0.04) than the wild type enzyme

(Pig = -0.30 + 0.04)

Figure 2.2-2 Br~nstedplots. Effect of leaving group ability on k,,,lK,,,for the wild-type sialidase (0) and mutant Y370D (0). Reprinted with permission from The American Chemical Society

When comparing mutant enzymes with the wild-type enzyme, it is important to include natural substrates. For example, when looking at the activated 4-nitrophenyl substrate, only an eighteen fold decrease is observed for the Y370D mutant. However, for the natural substrate, a lo4 fold reduction in loss of activity is noted which is consistent with the loss of activity associated with removal of the catalytic nucleophile. Such valuable information can easily be overseen if conclusions are based on activated substrates only.

However, the groundbreaking observation was that mutagenesis of the conserved tyrosine residue changed the mechanism from that of retention of configuration to an inverting rne~hanism.~~

220 rnin

166 rnin

66 rnin

35 rnin

20 rnin

8 rnin

0 min H MU-deu5Ac 3e!- 7 .-,* T- , T -,---T *------I-Tf --, , ----,. ppm :8 2.6 2.4 2.2 2.0 3.8 : E

Figure 2.2-3 Time course of M. viridifaciens Y370D mutant sialidase reaction monitored by 600 MHz 'H NMR spectroscopy. Reprinted with permission from The American Chemical Society

Conditions: 20 mM MU-aNeu5Ac in 10 mM tartrate buffer, pD 5.2 at 25 "C; 1 U enzyme in 0.6 mL. Time zero spectrum was recorded before the addition of enzyme.

This was demonstrated by a time course NMR experiment where the first formed product was p-sialic acid (Figure 2.2-3).95 This was explained by the fact that a new hole was created in the active site of the mutants which could accommodate a water molecule that acted as a nucleophile (Figure 2.2-4).

Asp 370

Glu

AcHN HO

Asp92 / Enz

Figure 2.2-4 Proposed catalytic mechanism for the inverting Y370D mutant sialidase

Another interesting observation is that at low pH, the Y370D mutant has a higher activity than the wild type enzyme. At this pH, the glutamate residue, the presumed general base in the active site is very likely to be protonated. This suggests that the Y370D mutant could be reacting through an SNl mechanism involving a short-lived sialyl cation which is trapped by a water molecule. 2.3 Role of the conserved aspartic residue in catalysis

As part of a collaborative project within our group and in partnership with the Centre of

Biomolecular Science (Professor Garry Taylor's Group) at University of St Andrews, the role of the conserved aspartate residue in catalysis in sialidases was investigated.96 The conserved aspartic acid residue in the active site is presumed to act as a general-acid to catalyze the departure of the aglycon and as a general-base to deprotonate a water molecule to attack the enzyme-substrate intermediate (Figure2.4-1). We studied the role of this residue by performing a series of experiments that are similar to those utilized for the tyrosine mutant studies.95.

The D92G mutant of the M. viridifaciens sialidase was cloned, expressed, purified and

~haracterized.~~The purified D92G mutant possessed similar catalytic activity to that of the wild-type enzyme when measured using activated substrate, e.g. 4-nitrophenyl a-D- sialoside. Through 'H NMR studies it was found that the D92G mutant was a retaining enzyme as the first formed product was a-sialic acid, similar to the wild-type enzyme.

This result was consistent with the fact that the tyrosine acts as the nucleophile in the

D92G mutant. The difference between the wild-type and the D92G mutant lies in their reactivity towards a-sialosides having poor leaving group, e.g. 3'-lactose (Table 2.3-1). Table 2.3-1 Relative catalytic activity of the D92G mutant and the wild-type sialidase. 95

Leaving Group Relative kcata Relative kcatlKm

4-ni trophenol 7.8 1.5

" equals kcat(wild-type)/kcat(D92G) beq~al~kcadKm(wild-type)/ kcadKm(D92G)

The PI, parameters derived from the Brgnsted plots for the D92G mutant enzyme- catalyzed hydrolysis are -0.37 f 0.02 and -0.72 f 0.03 for the kinetic terms kcat and kcadK, respectively. Substitution of the conserved aspartic acid by a glycine residue causes glycosidic bond cleavage to be partially rate-limiting for kcat. However, in the wild-type catalyzed reaction, kcatis limited by a step (most likely conformational change) that occurs after the scission of the glycosidic bond.95 A greater catalytic effect is displayed when the substrate possesses a poor leaving, such as lactose which is one of the natural aglycons.

The crystal structure of the D92G mutant complexed with DANA which is a transition state mimic was studied using X-ray crystallography. It was found that replacement of the conserved aspartic acid by the more flexible glycine did not cause any significant perturbation of the active site as compared to the wild type structure. A water molecule is positioned at a point between the oxygens of the carboxylate group of D92 in the wild- type structure and is H-bonded to the 04of DANA. 2.4 Proposed mechanism for a retaining sialidase

It was proposed that in the wild type enzyme from M. viridajaciens the glutamate residue acts as a general base deprotonating the tyrosine which attacks at the C-2 of the substrate forming the enzyme-sialyl inter~nediate.~~The aspartate residue then assists in the deprotonation of a water molecule which breaks down the enzyme-sialyl intermediate into sialic acid (Figure 2.4-1).

2.5 SNl or SN2 mechanism?

2.5.1 Introduction

There are two possible variants in the mechanism shown in Figure 2.4-1:

1) where the reaction goes through a positively charged intermediate (oxacarbenium

ion) or

2) where the departure of the leaving group coincides with the attachment of the

tyrosine residue with little build up of positive charge on the anomeric carbon.

The two possibilities can be distinguished by making use of the lunetic isotope effect.

The origin of kinetic isotope effect is founded on the quantum mechanical prediction that the zero point energy of an isotopically substituted molecule is different from that of its unlabelled counterpart.97 Isotopic substitution has its main effects on the vibration frequencies of the normal modes in which the bonds to which the isotope is attached have a large motion. To a just approximation the vibration in question is to be from a diatomic molecule, the vibrational frequency is given by Equation 1.

= --Jn ( :=)I Equation 1 (Hooks7sLaw)

where k is the force constant of the bond and ~HXis the reduced mass of the diatomic molecule. The reduced mass is calculated using Equation 2

1 1 1 -- - -+- Equation 2 kX M~ M~ For example, the zero point energy of isotopically substituted molecule (C-D) lies lower in the energy potential well compared to the unlabelled one (C-H) (Figure 2.5-1). Hence, isotopic labelling at or near a reaction centre results in a change in the reaction rate when compared to the unlabelled material. The magnitude of this effect depends on the force constant changes between ground state and transition state.

Bond Length (C-L where L = H or D)

Figure 2.5-1 Potential energy diagram

2.5.2 13cKinetic Isotope Effects

SN1reactions are commonly associated with values of around 1.00-1.01 for the reaction centre carbon isotope effect (k12ClkI3c). This situation arises because the loss of zero- point energy caused by breaking of the bond to the leaving group (C-X) is greatly compensated by the increase in zero point energy associated with double bond character in the bonds connecting the reaction centre to the groups which stabilize positive charge

(Figure 2.5-2). Reduction in force constants

\ I - - products

Strengthening of force constants

Figure 2.5-2 SN1mechanism

SN2 reactions are associated with higher values for the reaction centre I3c-IC1I3 (1.03-

1.09) because the loss in zero point energy of the carbon nucleofuge bond is counteracted to a lesser degree by tightening of the bonds surrounding the reaction centre carbon atom.

In an SN1mechanism, the ketosidic linkage between the leaving group and the anomeric carbon would be completely cleaved prior to bond formation between the incoming nucleophile and C-2. The reaction would proceed through a short-lived oxacarbenium ion intermediate. In the SN2 pathway, there would be assistance to leaving group departure from the active-site nucleophile. In other words, at the SN2transition state for glycosylation there is both partial bond formation between the nucleophile and C-2, and partial bond cleavage between the nucleofuge and C-2 whereas at the SN1transition state the leaving group departs without the assistance of the nucleophile (Figure 2.5-3). TY~ Nucleophile Glu

A General Acid Asp

SN~ SN~

Figure 2.5-3 Possible mechanisms for the glycosylation step in sialidases

2.5.3 Kinetic Isotope Effect studies in Enzymatic Reactions

There is one important requirement when determining the kinetic isotope effect by competitive method: the step showing an isotope effect is the actual chemical conversion of the enzyme-substrate (ES) complex into the enzyme-product (EP) complex. Shown below is an example of an enzyme-catalyzed reaction.97 Michaelis-Menten Equation

kl k2 k3 E+S - - ES - EP- E+P k-1

u = V[S] where K = k3(km1+ k2)

Where u is the initial reaction rate; [El is the total enzyme concentration; [S] is the substrate; P is the product; V is the maximum reaction rate.

The rate expression for two isotopic species S and S* in a mixture can be derived in the same manner as for ordinary competitive inhihibitors. The major difference is that a true competitive inhibitor prevents any reaction at the active site it occupies whereas an isotope just prevents reaction by the other isotopic species at the same site as the active sites occupied by S are unavailable to S* and vice-versa.

Hence, the determination of isotope effects by competitive methods gives an isotope effect on VIK. In order to probe whether the transition state through which the wild type sialidase and its mutants proceed is associative (SN2)or dissociative (SN~),it was decided to measure the

13C-kinetic isotope effect for the hydrolysis of "c-labelled 3-chlorophenyl (5-acetamido-

3,5-dideoxy-~-glycero-a-D-galacto-non-2-ulopyranosylonicacid). This experiment will give us an insight into the nature of the transition state of the sialidase.

The reasons for choosing this particular substrate are: (1) the measured PI, values for these reactions are known and these values allow a determination of when bond cleavage is rate limiting for the enzyme catalyzed hydrolysis reaction; and (2) the required substrate can be easily synthesized from "C labelled sialic acid in 4 steps. The enzymes chosen for this study were the D92G and Y370G mutants of M. viridifaciens and the wild-type Vibrio cholerae enzyme.95,98

2.5.4 Materials and Methods

D-(3-13C)sialic acid and ~-(2,3-'~~2)sialic acid were purchased from Omicron and were used to synthesize "c-labelled 3-chlorophenyl (5-acetamido-3,5-dideoxy-D-glycero-a-D- galacto-non-2-ulopyranosylonic acid) substrates according to the procedure described on pages 61 and 65 in section 2.6 at the end of this chapter. The internal standard L-(~-'~c) alanine (99 atom% 13C) was purchased from Sigma-Aldrich. Figure 2.5-4 13cNMR spectra of a 1:2 mixture of C-3 and C-3, C-2 labelled 3-chlorophenyl(5- acetamido-3,5-dideoxy-~-g~ycero-a-~-ga~-non-2-1opyranosyonicacid) substrates with L-(~-'~c) alanine at pH 5.24 (100 mM acetate) in a 15% DzO in water as solvent.

In the proton decoupled 13cNMR spectrum of (3-13c) 3-chlorophenyl (5-acetamido-3,5- dideoxy-~-g~ycero-a-~-galacto-non-2-ulopyranosyonicacid) the C-3 signal appears as a singlet with a chemical shift of 43.54 ppm. The corresponding C-3 signal for the (2,3-

13c2)labelled substrate is a doublet with the two peaks appearing at 43.70 and 43.37 ppm respectively. Therefore it is expected that enhancement of the intensity for the (2,3-13c2) labelled substrate relative to the (3-13c) labelled substrate will occur if the value of the

13canomeric KIE is greater than 1. Thus, the integration of the peaks associated with each isotopomer would give a value of the 12~/13~in the reaction mixture.

The reaction was performed in a 5 mm NMR tube. The 600 pL reaction volume contained 100 mM acetate buffer at pH 5.25 (for the D92G and Y370G mutants) or 50 mM acetate buffer, 150 mM NaCl and 9 mM CaC12 at pH 5.50 for the Vibrio cholerae enzyme, 10 mM substrate (I:1 mixture of singly and doubly labelled substrates), D20 (90 pL) containing 0.4 mg of "C labelled alanine. The reaction was initiated by the addition of enzyme (provided by Dr. J. N.Watson). The amount of enzyme was adjusted such that the reaction would reach at least 60% conversion during the 16 hour time course of the experiment.

Standard "C NMR spectra were collected on a Varian 500 MHz spectrometer at 125 Hz thermostated at constant temperature of 25 "C with a spectral window between 80 and 11 ppm. Each spectrum was made up of 640 scans with a delay of 20 minutes in between acquisitions. The 65536 point FID was zero filled to 64K points with an apodization of

0.1 Hz. The individual spectra were integrated using line fitting on the MestRe-C version

Beta 3.7.9.0 software and using the L-(2-"c) alanine signal set to 1000 as the internal reference. 2.5.5 Results and Discussion

The background rate of hydrolysis for the unlabelled 3-chlorophenyl (5-acetamido-3,5- dideoxy-~-glycero-a-D-galacto-non-2-ulopyranosylonicacid) in 100 mM acetate buffer pH 5.25 at 25 OC measured was (3.94 k 0.42) x s-' which is negligible compared to the enzyme-catalyzed reaction. Hence no corrections were made for background hydrolysis.

The data was fitted to Equation 3 shown below using the computer program

KIE = [In ( 1-F)]/ ln [(1 -F) FUR,)] Equation 3

F = Fraction of reaction

R = "c/'~c in remaining starting material at time t

Ro = l3C/l2cin starting material at time = 0 0.95 0- 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 Fraction of reaction

Figure 2.5-5 Plot of R/% ratio of the 13c-labelled substrates versus fraction of reaction for the Y370G catalyzed hydrolysis reation (R = '3~/12~at time t, & = 13~/12~at time = 0 for the substrate)

+ +

I I I I 0.20 0.30 0.40 0.50 0.60 Fraction of reaction

Figure 2.5-6 Plot of R/Ro ratio of 13clabelled substrate versus fraction of reaction for the D92G catalyzed hydrolysis reaction (R = 13~/12~at time t, R, = 13C/ 12C at time = 0 for the substrate) Unfortunately, we noticed that the C-3 signals of the a-sialic acid product appeared at similar chemical shifts to the unreacted substrate peaks. A good baseline separation of the peaks could not be achieved. This resulted in inaccurate integration values for the C-

3 signals of the unreacted substrate peaks and hence the scatter in the calculated "C/'~C ratios.

* Umacted substrate

Figure 2.5-7 13cNMR spectrum of 1:2 mixture of C-3 and C-3, C-2 labelled 3-chlorophenyl(5- acetamido-3,5-dideoxy-~-glycero-a-~-galacto-non-2-ulopyranosylonicacid) substrates with a-sialic acid product pH 5.24 (100 mM acetate) in a 15 % DzO in water as solvent.

In the case of the vibrio cholerae enzyme, the a-sialic acid peaks could not be resolved

from the substrate peaks at all, presumably due to either the pH of the reaction or the

presence of ca2+ions in the reaction mixture, resulting in accidental shift equivalence. 2.5.6 Conclusion

We could not draw any conclusions from this study. However, it should be ascertained

that there is good baseline separation of the unreacted substrate peak and the a-sialic acid

formed during the course of the reaction which is the major obstacle that prevented

success of these experiments.

2.6 Synthesis of aryl a-D-sialosides

Different methods for synthesizing aryl a-sialosides have been reported in literature. The

first method involved the use of P-sialosyl chloride with the sodium salt of the phenol in

DMF as solvent. 99,100,101,102,103 Another method made use of phase transfer catalysts.69.

However, in our hands these methods proved to be problematic since we obtained poor

yields.

So the a-aryl sialosides were synthesized according to another literature procedure.'04

This reaction involves coupling the P-sialosyl chloride with the desired phenol in the presence of Hunig's base using acetonitrile as solvent. The fully protected aryl sialosides were purified by column chromatography and crystallized from diethyl ether to afford the coupled product free of elimination by-product. Deprotection in two steps followed by precipitation gave the required aryl a-D-sial~sides.~~The 'H NMR spectra of these

sialosides were identical to those reported in literature. HO% C02H a C02Me

AcHN - AcHN HO

Sialic acid 2.1

~~0% b - C02Me - AcHN AcHN AcO

2.2 2.3 a-e

d, e

AcHN

2.4 a-e

a) MeOH, Amberlite IR-120 (H'), b) Acetyl chloride, acetic acid; c) phenol, Hunig's base, CH3CN, d) NaOMe, MeOH e) LiOH. HzO, 3:l vlv THF/H20

Figure 2.6-1 Synthesis of aryl a-D-sialosides (2.4a-e) 2.7 Experimental

General Acetonitrile and methanol were distilled from CaH2 and Mg respectively prior to use. NMR spectra were recorded on Varian 500 MHz spectrometer. The powdered molecular sieves were flame dried before use. Sodium methoxide solution (10 mg/mL in dry methanol) were made fresh for each reaction.

Methyl (5-acetamido-3,5-dideoxy-~-glycero-~-~-galacto-non-2-ulopyranosyl)onate

(2.1)

Sialic acid (6.0g1 19.4 mmol) and Amberlite IR-120 (H+) were dried under vacuum for about 2 hours. Dry methanol (100 mL) was added and the mixture was stirred for 24 hours at room temperature. The clear solution was filtered and evaporated to give a colorless syrup. The product was crystallised from MeOH/Et20 ether mixture as a white powder (4.8g, 76%); mp 191-192 OC (lit. 63,69,'05 mp 193.5-194.7): 'H NMR (400 MHz,

D20)6: 1.90 (t, 1 H, J3a, 3e+ J3a.4 = 24 HZ,H-3a), 2.02 (s, 3 H, CH3), 2.27 (dd, 1 H, J3a,3e=

12 HZ, H-3a, J3e,4= 5 HZ, H-3e), 3.50 (dd, 1 H, J7,6 = 1.5 HZ,J7,8 = 10 HZ, H-7), 3.57 (dd,

1 H, J9a,9b= 12 HZ, Jga,8= 6 HZ, H-9a), 3.68 (td, 1 H Js,9a= 6 HZ, J8,9b=3.0 HZ,J8,7= 10.0

Hz, H-8), 3.76-3.83 (m, 4 H, H-9b, 0CH3), 3.88 (t, I H, J5,4+ J5,6= 22.0 Hz, H-5), 3.97-

4.06 (m, 2 H, H-4, H-7) Methyl (5-acetamido-4,7,8,9-tetra-0-acetyl-3,5-dideoxy-~-glycero-~-~-galacto-non-

2-ulopyranosy1)onate chloride (2.2)

Compound 2.1 (4.0g, 12.3 mmol) was added to freshly distilled acetyl chloride (64 mL) at 0 OC. A mixture of MeOH (12 mL) and glacial acetic acid (24 rnL) was added dropwise to the above mixture. The flask was sealed and the reaction mixture was stirred for 48 hrs at room temperature. The solution was evaporated to dryness and coevaporated several times with dry toluene. The sialosyl chloride was obtained as an off- white foam. The crude product was purified by column chromatography (silica gel: eluant hexane: acetone (4:3 (vlv)) to afford a white foamy solid (4.3g, 68%). 'H NMR

(400 MHz, CDC13) 6: 2.01-2.13 (m, 15 H, CH3), 2.28 (t, 1 H, J3a,3e + J3e,4=24.0 HZ, H-

3a), 2.78 (dd, 1 H, J3e,3a = 12.0 HZ, J3e,4 = 5.0 HZ, H-3e), 3.88 (s, 3 H, 0CH3), 4.05 (dd, I

H J9a,9b=12.0 Hz,J~~,~=~.OHZ,H-9a), 4.19 (dd, 1 H, J5,4~J~,~zJ*,N~= 30.0H~, H-5),

4.34 (dd, 1 H, J6,5 = 10.0 HZ, J6,7 = 2.5 HZ, H-6), 4.41 (dd, 1 H, J9a,9b= 12.0 HZ, J9b,8=2.5

HZ), 5.18 (td,IH,J8,9a=6.0H~,Jg,9b=2.5H~,J8,7=10.0Hz,H-8), 5.33-5.45 (m, 2H,H-

4, H-7).

Methyl [(4-nitrophenyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-~-glycero-a-

~-ga~acto-non-2-ulopyranosyl)]onate(2.3a)

Compound 2.2 (lg, 1.95 mmol) and 4-nitrophenol (2.7g, 19.5 mmol)) were dissolved in dry CH3CN (200 mL) in an ice bath at 0 OC. Hunig's base (2.7 rnL, 28.2 mmol) was added to the solution. The reaction mixture was stirred for 18 hrs, the temperature was allowed to warm up gradually to room temperature. The solvent was evaporated under reduced pressure to afford a yellow syrup which was dissolved in CH2C12(300 mL). The organic phase was washed with a saturated solution of NaHC03 (2 x 150 mL), 10%

H2SO4 (1 x lOOrnL), brine (150 rnL). The organic layer was dried (Na2S04) and concentrated to dryness. The crude product was purified by flash column chromatography (silica gel: eluant hexane: acetone (4:3 (vlv)) to afford a pale yellow syrup which was crystallized from diethyl ether-petroleum ether twice to give white powder (500 mg, 42% yield); mp 98-100 OC (lit 4' mp 104-108 "C); 'H NMR (400 MHz,

CDC13) 6: 1.93, 2.05, 2.06, 2.11, 2.19 (5 x S, 15 H, CH3), 2.30 (t, 1 H, J3a,3e + J3e,4 = 25.0

HZ, H-3a), 2.74 (dd, 1 H, J3e,3a= 13.0 HZ, J3e,4= 5.0 HZ, H-3e), 3.66 (s, 3 H, 0CH3),

4.07-4.15 (m, 2 H, H-5 H-9a), 4.22 (dd, 1 H, J9~,9b= 12.4 HZ, J9b,* = 2.3 HZ, H-9b), 4.60

(dd, 1 H, J6,5= 11.0 HZ, J6,,= 2.0 HZ, H-6), 4.98 (td, 1 H, J4,3a= 12.5 HZ, J4,3e= 5.0 HZ,

J4,5=13.0Hz, H-4), 5.23 (d, 1 H J5,NH=lO.OHz,NH), 5.32-5.40 (m, 2H, H-7,H-8),

7.11-7.18 (m, 2 H, Ar-H), 8.16-8.22 (m, 2 H, Ar-H).

Methyl [(3-nitrophenyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-~-glycero-a-

D-galacto-non-2-ulopyranosyl)]onate(2.3b)

Compound 2.2 (lg, 1.95 mmol) and 3-nitrophenol (2.728, 19.5 mmol) were dissolved in dry CH3CN (200 mL) in an ice bath at O•‹C. Hunig's base (2.8 mL, 29.2 mmol) was added to the solution. The reaction mixture was stirred for 18 hrs, the temperature was allowed to warm up gradually to room temperature. Same work up and purification procedure as for compound 2.3a. A white solid (460 mg, 39% yield) was obtained as

60 product; mp 78-82 OC (lit "mp 101- 102 "C); 'H NMR (400 MHz, CDC13) 6: 1.92, 2.03,

2.05, 2.13, 2.18 (5 x S, 15 H, CH3), 2.27 (t, 1 H, J3a,3e= J3a,4= 13.0 HZ, H-3a), 2.75 (dd, 1

H, J3e,3a = 13.0 HZ,J3e,4 = 5.0 HZ, H-3e), 3.63 (s, 3 H, 0CH3), 4.03-4.17 (m, 2 H, H-5, H-

9a), 4.21 (dd, 1 H, Jga,9b= 12.6 HZ, Jgb,g= 2.6 HZ, H-9b), 4.38 (dd, 1 H, 10.8 HZ, J6,~

= 1.9Hz,H-6),4.95 (td, 1 H, J4,3a= 12.5Hz, J4,3e=4.6Hz, J4,5= 10.3 Hz,H-4),5.27 (d,

1 H J5,~"=10.0 HZ, NH), 5.33-5.38 (m, 2 H, H-7, H-8), 7.34-7.51 (m, 2 H, Ar-H), 7.88-

7.96 (m, 2 H, Ar-H).

Compound 2.2 (250 mg, 0.98 mmol) and 3-chlorophenol (1.25g, 9.80 mmol) were dissolved in dry CH3CN (200 mL) in an ice bath at 0 OC. Hunig's base (1.4 mL, 14.6 mmol) was added to the solution. The reaction mixture was stirred for 18 hours, the temperature was allowed to warm up gradually to room temperature. Same work up and purification procedure as for compound 2.3a. A white solid (353 mg, 60% yield) was obtained as product; mp 77-80 OC (litg3mp 102-103 OC); 'H NMR (400 MHz, CDC13) 6:

1.92, 2.04, 2.05, 2.14 (5 x S, 15 H, CH3), 2.19 (t, 1 H, J3a,3e+ J3e,4 = 25.0 HZ, H-3a), 2.67

(dd, 1 H, J3e,3a = 12.0 HZ, J3q4 = 4.8 HZ, H-3e), 3.70 (s, 3 H, 0CH3), 4.07-4.17 (m, 2 H,

H-5,H-9a),4.31 (dd, I H, J9a,9b= 13.0H~,J9b,8=2.0H~,H-9b),4.38 (dd, 1 H,J6,5= 11.0

HZ, J6,7= 1.5 HZ,H-6), 4.97 (td, 1 H, J4,3a= 12.5 HZ, J4,3e= 5.0 HZ, J4,5 = 13.0 HZ, H-4),

5.18 (d, 1 H, JS,NH=lO.OHz, NH), 5.32-5.38 (m, 2H, H-7, H-8), 6.08-7.02 (m, 1 H,Ar-

H), 7.06-7.11 (m, 2 H, Ar-H), 7.19-7.25 (m, 1 H, Ar-H).

6 1 Methyl (4-cyanophenyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-~-glycero-a-

D-galacto-non-2-ulopyran0syl)onate(2.3d)

Compound 2.2 (500 mg, 0.98 mmol) and 4-cyanophenol (1.16g, 9.80 mmol) were dissolved in dry CH3CN (200 mL) in an ice bath at 0 "C. Hunig's base (1.3 mL, 13.6 mmol) was added to the solution. The reaction mixture was stirred for 18 hrs, the temperature was allowed to warm up gradually to room temperature. Same work up and purification procedure as for compound 2.3a. A white solid (300 mg, 53% yield) was obtained as product; mp 85-89 "C (lit 4' mp 104-10S•‹C);'H NMR (400 MHz, CDC13) 6:

1.92, 2.03, 2.05, 2.13, 2.18 (5 x S, 15 H, CH3), 2.27 (t, 1 H, J3a,3e+J3e,4= 25.0 HZ,H-3a),

2.72 (dd, 1 H, J3e,3a= 13.0 HZ, J3q4 = 5.0 HZ,H-3e), 3.63 (s, 3 H, 0CH3), 4.06-4.14 (m, 2

H, H-5, H-9a), 4.23 (dd, 1 H, J9a,9b= 13.0 HZ, Jgb,*= 2.0 HZ, H-9b), 4.57 (dd, 1 H, J6,5=

11.0 HZ, J6,7=1.5 HZ, H-6), 4.95 (td, 1 H, J4,3a= 12.5 HZ, J4,3e= 5.0 HZ,J4,5 = 13.0 HZ,H-

4), 5.27 (d, 1 H, JS,NH= 10.0 HZ, NH), 5.32-5.38 (m, 2 H, H-7, H-8),7.08-7.14 (m, 2 H,

Ar-H), 7.56-7.62 (m, 2 H, Ar-H).

Compound 2.2 (250 mg, 0.49 mmol) and phenol (920 mg, 4.9 mmol) were dissolved in dry CH3CN (100 mL) in an ice bath at 0 OC. Hunig's base (0.7 mL, 7.3 mmol) was added to the solution. The reaction mixture was stirred for 18 hrs, the temperature gradually

62 warming to room temperature. Same work up and purification procedure as for compound 2.3a. A white solid (103 mg, 37% yield) was obtained as product; mp 79-82

"C (lit 4'383369mp 102-106•‹C);'H NMR (400 MHz, CDC13) 6: 1.90, 2.01, 2.03, 2.15 (5 x s,

15 H, CH3), 2.22 (t, 1 H, J3a,3e + J3e,4 = 25.0 HZ, H-3a), 2.7 1 (dd, 1 H, J3e,3a = 13.0 HZ,J3e,4

= 5.0 Hz, H-3e), 3.65 (s, 3 H, 0CH3), 4.05-4.21 (m, 2 H, H-5, H-9a), 4.33 (dd, 1 H, J9a,9b

= 13.0Hz, J9b,8=2.0H~,H-9b), 4.39 (dd, 1 H,J6,5= ll.0Hz,J6,~=1.5 HZ, H-6),4.97

(td, 1 H, J4,3a = 12.5 HZ, J4,3e= 5.0 HZ,J4,5 = 13.0 HZ, H-4), 5.16 (d, 1 H, J5,NH= 10.0 HZ,

NH), 5.34-5.41 (m, 2 H, H-7, H-8), 7.04-7.11 (m, 3 H, Ar-H), 7.25-7.31 (m, 2 H, Ar-H).

4-Nitrophenyl(5-acetamido-3,5-dideoxy-~-glycero-a-~-galacto-non-2- ulopyranosylonic acid) (2.4a)

Compound 2.3a (100 mg, 0.16 mmol) was dissolved in anhydrous methanol (5 rnL) and a methanolic sodium methoxide solution (2.0 mL, 0.87 mmol) was added. The mixture was stirred at 0•‹C for 15 min. Dowex 50W HCR-W2 (H+)(prewashed with methanol) cation exchange resin was added to neutralize the solution. The resin was filtered off and washed several times with methanol. The solvent was evaporated under reduced pressure. The methyl ester was precipitated out using a mixture of MeOH/Et20 and cooling to -25 "C as a white powder which was dissolved in a 3: 1 v/v THFJwater mixture

(2 rnL ) at 0 OC. LiOH.H20 (32 mg, 0.76 mmol) was added to the mixture. After 15 min of stirring at 0 OC, the mixture was neutralized with Dowex 50W HCR-W2 (H') resin.

The resin was filtered off and washed with MeOH. The filtrate was evaporated to dryness under reduced pressure. The product was precipitated out using a mixture of MeOWEtzO to give a white solid (42.7 mg, 60%): mp 110-113 "C (lit99mp 113-1 15 OC)

.1 H NMR (400 MHz, D20) 6: 1.%-LO4 (m, 4 H, H-3a, CH3), 2.80 (dd, 1 H, J3e,3a= 13.0

Hz, J3e.4 = 5.0 HZ, H-3e), 3.52-3.61 (m, 2 H, H-7, H-9a), 3.76-3.82 (m, 3 H, H-4, H-8, H-

9b), 3.94(t, 1 H, J5,6=J5,4= lO.OH~,H-5),4.17(dd,1 H,J6,5=10.5Hz,J6,,= 1.5HzlH-

6), 7.21-7.30 (m, 2 H, Ar-H), 8.15-8.23(m, 2 H, Ar-H).

3-Nitrophenyl(5-acetamido-3,5-dideoxy-~-glycero-a-~-galacto-non-2- ulopyranosylonic acid) (2.4b)

Compound 2.3b (100 mg, 0.16 mmol) was dissolved in anhydrous methanol (5 mL) and a methanolic sodium methoxide solution (2.0 mL, 0.87 mmol) was added. The mixture was stirred at 0 OC for 15 min. Then Dowex 50W HCR-W2 (H+) (prewashed with methanol) cation exchange resin was added to neutralize the solution. The resin was filtered off and washed several times with methanol. The solvent was evaporated under reduced pressure. The residue was dissolved in a 3: 1 vlv THFlwater mixture (2 mL) at 0

"C. LiOH.H20 (0.32 mg, 0.76 mmol) was added to the mixture. After 15 min of stirring at O•‹C, the mixture was neutralized with Dowex 50W HCR-W2 (H+) resin. The resin was filtered off and washed with MeOH. The filtrate was evaporated to dryness under reduced pressure. The product was precipitated out using a mixture of MeOWEt20 to give a white solid (25 mg, 34 %). mp 110-1 13 "C (litg3mp 116-118 "C). 'H NMR (400

MHz, D20) 6: 1.93-2.03 (m, 4 H, H-3a, CH3), 2.88 (dd, 1 H, J3e,3a = 12.6 HZ, J3e,4= 4.8

Hz, H-3e), 3.5 1-3.61 (m, 2 H, H-7, H-9a), 3.75-3.82 (m, 3 H, H-4, H-8, H-9b), 3.91 (t, 1 H,J5,6+ J5,4=21.4 HZ, H-5),4.17 (dd, 1 H,J6,5= 10.5 Hz,J6,7= 1.5 HZ, H-6), 7.19-7.23

(m, 2 H, Ar-H), 7.64-7.71 (m, 2 H, Ar-H).

3-Chlorophenyl(5-acetamido-3,5-dideoxy-D-glycero-a-~-galacto-non-2- ulopyranosylonic acid) (2.4~)

Compound 2.3~(100 mg, 0.16 mmol) was dissolved in anhydrous methanol (5 mL) and a methanolic sodium methoxide solution (1.9 mL, 0.83 mmol) was added. The mixture was stirred at 0 OC for 15 min. Then Dowex 50W HCR-W2 (Hf) (prewashed with methanol) cation exchange resin was added to neutralize the solution. The resin was filtered off and washed several times with methanol. The solvent was evaporated under reduced pressure. The methyl ester was precipitated out using a mixture of MeOH/Et20 and cooling to -25 "C as a white powder (47 mg, 66%) of which 17 mg was dissolved in a 3: 1 V/VTHFIwater mixture (2 mL) at 0 OC. LiOH.H20 (7 mg, 0.17 mmol) was added to the mixture. After 15 min of stirring at 0 OC, the mixture was neutralized with Dowex

50W HCR-W2 (H+) resin. The resin was filtered off and washed with MeOH. The filtrate was evaporated to dryness under reduced pressure. The product was precipitated out using a mixture of MeOWEt20 to give a white solid (12 mg, 47%) mp 116-119 OC

(litg3mp 114-115 "C). 'H NMR (400 MHz, D20) 6: 1.97-2.03 (m, 4 H, H-3a, CH3), 2.81

(dd, 1 H, J3e,3a=12.6Hz, J3e,4=4.8H~,H-3e),3.51-3.61(m,2H,H-7,H-9a),3.75-3.82

(m, 3H,H-4,H-8,H-9b),3.91 (t, 1 H, J5,6+J5,4=21.4Hz,H-5),4.17 (dd, 1 H, J6,5=

10.5 Hz, J6,7 = 1.5 HZ,H-6), 7.19-7.23 (m, 2 H, Ar-H), 7.64-7.7 1 (m, 2 H, Ar-H). 4-cyanophenyl (5-acetamido-3,5-dideoxy-~-glycero-a-~-galacto-non-2- ulopyranosylonic acid) (2.4d)

Compound 2.3d (30 mg, 0.05 mmol) was dissolved in anhydrous methanol (5 mL) and a methanolic sodium methoxide solution (0.6 mL, 0.26 mmol) was added. The mixture was stirred at 0 OC for 15 min. Then Dowex 50W HCR-W2 (H+) (prewashed with methanol) cation exchange resin was added to neutralize the solution. The resin was filtered off and washed several times with methanol. The solvent was evaporated under reduced pressure and the residue was dissolved in a 3:l vlv THFIwater mixture (2 mL) at

0 "C. LiOH.H20 (9 mg, 0.21 mmol) was added to the mixture. After 15 min of stirring at

0 OC, the mixture was neutralized with Dowex 50W HCR-W2 (H+) resin. The resin was filtered off and washed with MeOH. The filtrate was evaporated to dryness under reduced pressure. The product was then precipitated out using a mixture of MeOH/EtzO to give a white solid (12 mg, 57%). mp 118-122 "C (lit83mp 126-128 OC). 'H NMR (400

MHz, D20) 6: 1.97-2.03 (m, 4 H, H-3a, CH3), 2.8 1 (dd, 1 H, J3e,3a = 12.6 HZ, J3e,4= 4.8

Hz, H-3e), 3.51-3.61 (m, 2 H, H-7, H-9a), 3.75-3.82 (m, 3 H, H-4, H-8, H-9b), 3.91 (t, 1

H, J5,6+ J5.4 = 21.4 HZ, H-5), 4.17 (dd, 1 H, J6,5 = 10.5 HZ, J6,7 = 1.5 HZ,H-6), 7.19-7.23

(m, 2 H, Ar-H), 7.64-7.7 1 (m, 2 H, Ar-H). CHAPTER THREE: UNEXPECTED STABILITY OF ARYL B-D- N-ACETYLNEURAMINIDES IN NEUTRAL SOLUTION: BIOLOGICAL IMPLICATIONS FOR SIALYL TRANSFER REACTIONS

3.1 Introduction

Many specific biological processes are mediated by carbohydratelprotein interactions,

such as receptorlligand recognition and glycoconjugate intracellular trafficking. 106,107 In many of these biologically important interactions the sugar component is N-

acetylneuraminic acid (sialic acid), a common constituent of the glycolipids and

glycoproteins produced by animal cells.lo8 Indeed, N-acetylneuraminic acid is frequently found at the non-reducing termini of cell surface oligosaccharide moieties 86 and this prominence is in keeping with its importance in cellular and molecular recognition events.'09

Three distinct families of sialyl-transferring enzymes are found in nature: (a) sialidases

(N-acetylneuraminyl glycohydrolases) are retaining glycosidases that remove sialic acid from glycoconjugates;110-112 87,113-115 (b) trans-sialidases are retaining enzymes expressed by trypanosomes that enable the parasite to transfer sialic acid from an external source onto its own surface carb~h~drates;"~and (c) sialyltransferases are inverting enzymes that utilize CMP-b-D-N-acetylneuraminide (3.1) as the sialyl donor to make a-sialoside linkages. 'I7 Figure 3.1-1 CMP-P-D-N-acetylneuraminide

The results detailed in Chapter 2 point to the conserved tyrosine residue being the nucleophile for both classes of retaining sialyl transferring enzymes, that is, exo- sia1ida~es;~hndtrans-~ialidases.~~~~~. The proposed enzymatic mechanism for sialidases is shown in Figure 2.4-1 on page 44. A central feature of this mechanism is that formation of an N-acetylneuraminosylium ion (sialyl oxacarbenium ion; 3.2) intermediate is avoided, a species that has an estimated lifetime in aqueous solution of 2 3 x lo-"

S. 118,119

This chapter details a series of kinetic and product studies performed on analogues of the sialosyl-enzyme intermediate, i.e., aryl P-D-N-acetylneuraminides (3.7a-g) in aqueous media. In addition, a specific comparison is made between 3.7b and 4-nitrophenyl a-D-

N-acetylneuraminide (2.4a), the anomer of 3.7b. The results from these studies address critical questions concerning the intrinsic reactivity of sialosides and the solution lifetime of the sialosyl oxacarbenium ion (3.2). Figure 3.1-2 Sialosyl oxacarbenium ion

3.2 Synthesis of aryl PD-N-acetylneurarninides

The synthesis of aryl 0-D-N-acetylneuraminidesinvolved the use of P-D-sialosyl fluoride

(3.2), which was made from the peracetylated methyl ester of sialic acid and a 70% solution of ~-~~ridine.'~~The sialosyl fluoride was reacted with the different phenols in the presence of BF3.0Et2and dried ground 4A molecular sieves. The reaction yielded the desired product along with an elimination product, the a$-unsaturated ester. Since these two compounds have identical Rf, they could not be separated by column chromatography. A solution of the crude product in diethyl ether (5 rnL) was sonicated with a glass rod in the flask until a fine powder was obtained, which was filtered and washed with a small amount of ether. The product of the coupling reaction hence obtained was free of elimination product.

Deacetylation of the protected aryl P-D (3.6a-g) sialosides was achieved by use of a solution of sodium methoxide in methanol. The methyl ester was then hydrolysed with lithium hydroxide in a solvent of 3:l v/v THF/H20. Neutralization followed by lyophilization afforded the final compounds (3.7a-g). C02Me d AcHN - AcHN

a) MeOH, Amberlite IR-120 (H+) b) Acetic anhydride, Pyridine, DMAP c) 70% Pyridine- HF d) Phenols, BFJ.0Et2,molecular sieves, CHzClze) NaOMe, MeOH f) LiOH HzO, 3:l vlv THFJH20

Figure 3.2-1 Synthesis of aryl P-D-N-acetylneuraminides

3.2.1 Spectral Assignment of the 4-nitrophenyl-P-D-N-acetylneuraminide

The stereochemistry of the aryl sialosides was assigned using 2-D homonuclear ('H-'H), heteronuclear ('H-I3c correlated), conventional 'H and 13c-NMR experiments. Typical

'H and 13c-NMR spectra for one of the aryl P-D-sialosides (3.7b) is shown in Figures

3.2-2 and 3.2-3.

3.3 Material and methods 3.3.1 Kinetic Experiments

The buffers 2-(N-morpho1ino)ethanesulfonic acid (MES), N-tris[hydroxymethyl]methyl-

3-amino-propanesulfonic acid (TAPS) and (cyclohexylamino)-1-propanesulfonic acid

(CAPS) were purchased from Sigma and used without further purification. N-

Acetylneuraminic acid was purchased from Rose Scientific and used without further purification, Milli-Q water (18.2 Mi2 cm-') was used for kinetic and product studies experiments. Perchloric acid and sodium hydroxide solutions were made by dilutions of a standard 1.00 M solution. All other salts used in the hydrolysis runs were of analytical grade and were used without purification. 99% ethanol was distilled from the corresponding magnesium alkoxide prior to use. CH2C12 was distilled over CaH2. NMR spectra were acquired on a Bruker AMX-400 spectrometer.

3.3.2 Buffer systems

The neutral acid-sodium hydroxide buffer systems were used between the following pH values indicated: 2.10-3.19, malonic acid; 3.76-4.02, succinic acid; 4.46-5.36, acetic acid;

5.50-6.30, 2-(N-morpholino)ethanesulfonic acid (MES); 7.00-8.20, N- tris[hydroxymethyl]methyl-3-amino-propanesulfonic acid (TAPS); 10.00-11.13, 3-

(cyclohexylamino)-1-propanesulfonic acid (CAPS). Buffer pH values were measured at room temperature. No temperature correction was done for the acid based buffers as they were used for the pH-independent portion of the curve. For solution where [w]> 0.01

M perchloric acid-sodium perchlorate was used, and when [OH-] > 0.01 M sodium hydroxide-sodium perchlorate was used. Ionic strength was maintained at 0.3 M with sodium perchlorate except when [H'] > 0.3 M or [OH-] > 0.3 M.

3.3.3 Hydrolysis Kinetics

All absorbance measurements were made using 1-cm path length cuvettes that were housed in a Cary 3E UV-vis spectrophotometer, fitted with a Cary six-cell Peltier constant-temperature accessory. The respective a- and 0- anomer of 4-nitrophenyl sialoside will be referred as 2.4a and 3.7b respectively throughout this chapter (Figure

3.3-1).

HO,J0& HO,J0& 0

AcH N HO

Figure 3.3-1 The two anomers of 4-nitrophenyl-D-N-acetylneuraminidechosen for this study

The hydrolysis reactions of 3.7b were monitored using four different protocols. For pH values < 4.50 and > 11.20, a methanol stock solution of 3.7b (50 pL, 2.3 pM) was injected into a cuvette containing 3 mL of the required buffer that had been pre- equilibrated for 10 minutes at 50 OC. The absorbance versus time data was monitored for three half lives at 340 nm and 408 nm for the low and high pH reactions, respectively.

Rate constants were calculated by fitting the absorbance versus time data to a standard first order rate equation using the nonlinear least-squares routine computer program

Grafit. For pH values between 4.50 and 6.33, a discontinuous assay method was used. A solution of 3.7b (4 mg) in 20 mL of the required buffer was aliquoted into 1 mL samples in 2 mL glass ampoules which were sealed. The ampoules were then placed in a water bath held at 50 OC. The ampoules were taken out of the water bath at regular intervals and the reaction was quenched by cooling to 0 "C. The reactions were monitored in this fashion until they were about 5% complete. In order to determine the reaction end point two ampoules were heated at 100 OC for two days. A portion of the solution from each ampoule (200 pL) was added to pH 10.0 CAPS buffer (0.3 M, 400 1L) and the absorbance of the resultant mixture was measured at 408 nm.

Between pH 6.40 and 11.20 the changes in absorbance at 408 nm were monitored at 50

"C until about 34% of the hydrolytic reaction had occurred and then the cell-block temperature was raised to 75 "C until the reaction was complete.

3.3.3.1 Acid-Catalyzed Hydrolysis of 3.7a-g

For the acid catalyzed region of the pH-rate profile and the Bronsted plot at pH 1.00, the change in absorbance was measured in the cuvette itself. The data was fitted to a standard first order rate equation using grafit. The wavelengths used were 340 nm for the

4-nitrophenyl, 3-nitrophenyl, 270 nm for the phenyl, and 280 for all the other runs. 3.3.3.2 Spontaneous Hydrolysis of 3.7b-d

The hydrolyses of 3.7b-e were monitored at a pH of 8.08 using ampoules as outlined above. The ampoules were maintained at a temperature of 100 "C by placing them in a

"boiler" where they were immersed in the vapor generated from a boiling water bath.

Ampoules were taken out at regular intervals for about two to three half-lives of hydrolysis and the reactions were quenched by cooling the ampoules in an icelwater bath.

The rate constant for hydrolysis of 3.7b under these conditions (pH 8.08, T = 100 "C) was estimated by extrapolation of rate constant data measured at 50, 65, 75 and 85 "C

(Table 3.5-2 on page 86 ). 3.4 Results and Discussion

Figure 3.4-1 shows the logarithms of the observed rate constants (kobs) as a function of pH for the hydrolysis of 4-nitrophenyl B-D-N-acetylneuramide (3.7b) and 4-nitrophenyl a-D-N-acetylneuramide (2.4a). The observed rate constants for the hydrolysis of 3.7b were fitted to Equation 1, where K, is the ionization constant of 3.7b, while kH+,ko, k,,, and koH-K, are the respective rate constants for:

the acid-catalyzed reaction of the neutral molecule;

the acid-catalyzed reaction of the carboxylate form or the spontaneous

reaction of the neutral molecule;

the spontaneous reaction of the anion; and

a base promoted reaction.

Equation 1

log kobs= log{k H+ [H'] 1 (1 + Ka 1 [H']) + ko I( 1 + Kal[H']) +

kspI (1 + [Hf ] I Ka)+ koH-Kw1 [H'] (1 + [H'] 1Ka) } 3.4.1 pH-Rate profile for the hydrolysis of 4-nitrophenyl P-D-N-acetylneuramide

(3.7b) and 4-nitrophenyl a-D-N-acetylneuramide(2.4a) at 50 OC

Figure 3.4-1 Plot of log(kob,)versus pH for the hydrolyses of 4-nitrophenyl a-and P-D-N acetylneuraminides, T= 50 OC. The included solid lines are the best non-linear fits to equation 1 and 2. The dashed line is generated from kinetic rate constants reported for the hydrolysis of 4- nitrophenyl a-D-N acetylneuraminide, T= 50 OC 83

First, a comment is warranted on the difference between the rate constant for spontaneous hydrolysis of 2.4a reported by Ashwell et aL8)and that measured in the current study

(Table 3.4-3 on page 82). Although, in the current study no temperature corrections were applied to the buffer pH values (measured at 25 OC), all of the measured rate constants for hydrolysis of 2.4a (pH 10.50 up to pH 14.00 {[NaOH] = 1.00 M)) were identical within experimental error. Also, the rate constants measured in TAPS (0.3 M) buffer, with and without ionic strength adjustment, are identical within experimental error. Therefore, neither solution acidity nor ionic strength differences can account for the variation in rate constant noted above. The most likely explanation is a difference in temperature between the two data sets. Indeed, given the activation parameters reported on page 87 (Table

3.5-3), an imbalance of 4 "C, between the two studies, is sufficient to rationalize a rate difference of two-three-fold. Nevertheless, it is logical to compare the newly acquired rate data for hydrolysis of 2.4a with that evaluated for 3.7b because these data was measured using the same instrumental setup.

Remarkably, the spontaneous hydrolysis of 2.4a, the thermodynamically less stable diastereomer, occurs at a rate that is over hundred times faster than corresponding reaction of 3.7b. This large difference in rate constants can be contrasted to those for the base-promoted reaction (koH-Kw) and those for pH values less than three where 2.4a only reacts about three-four times faster than does 3.7b.

Also shown in Table 3.4-3 on page 82 are the recently measured rate constants for pH values between 7.3 and 14.0 and the best fit line for the previously reported kinetic data

(pH 0.8-10.00) for the hydrolysis of 4-nitrophenyl-a-D-N-acetylneuraminide 2.4a. The newly measured rate constants for a-anomer were fitted to Equation 2 where kspand koH.

Kw represent the same rate constants detailed above.

Equation 2 3.4.1.1 Kinetic Parameters

Table 3.4-1 Calculated pKas and rate constants for the hydrolyses of 4-nitrophenyl a- and P-D-N-acetylneuraminide at 50 OC (p = 0.3, NaC104)

Parameter

a This work unless stated b Values taken from reference 83 Table 3.4-2 Observed Rate Constants for the Hydrolysis of 4-Nitrophenyl P-D-N- acetylneuraminic acid (3.7b) at 50 OC (p = 0.3, NaC104)

"Mean value of three kinetic runs; quoted error = 0,-1

1onic strength = 1.0 at pH =1.00 and 14.00 Table 3.4-3 Observed rate constants for the hydrolysis of 4-Nitrophenyl a-D-N- acetylneuraminide (2.4a) at 50 OC (p = 0.3, NaC104)

"Mean value of three lunetic runs; quoted error = on.,

Ionic strength = 1.0 at pH 14.00

" The rate constants measured in TAPS buffer (0.3 M, pH 7.9 were identical within experimental error. 3.5 pH independent region

The spontaneous hydrolysis of 2.4a (a-anomer), the thermodynamically less stable diastereomer, occurs at a rate that is over 100-times faster than corresponding reaction of

3.7b (B-anomer). This remarkably large difference in rate constants can be contrasted to those for the base-promoted reaction (koH-Kw) where 2.4a reacts about 2.5 times faster than does 3.7b.

With regard to previous mechanistic studies on sialosides, conflicting interpretations on the role of the carboxylate group during non-enzymatic hydrolysis reactions have been published. Thus, Ashwell et al. proposed that the C-1 carboxylate group assists nucleophilically in both the acid-catalyzed departure of a phenol (ko term) and the spontaneous departure of a phenolate anion (ksp term) during the hydrolysis reactions of the carboxylate form of aryl a-D-N-a~et~lneuraminides.~'Whereas, Horenstein and co- workers proposed that the carboxylate group is coplanar to the oxacarbenium ion at the transition state for hydrolysis of CMP P-D-N-acetylneuraminide (i.e., nucleophilic participation cannot occur).'" In addition, based on similar Pt values for the hydrolysis of the carboxylate and carboxylic acid forms of pyridinium a-D-N-acetylneuraminide

(3.8) Chou et a1 also argued that the anomeric carboxylate group does not assist in the departure of neutral pyridine leaving groups (Figure 3.5-1).~'

Figure 3.5-1 Pyridinium a-D-N-acetylneuraminides 3.5.1 Activation Parameters

The hydrolyses of both the a- and P-D-4-nitrophenyl sialoside were performed at four different temperatures in order to determine the enthalpy and entropy of activation in the pH independent region of the pH rate profile. The activation parameters were determined by fitting the kinetic data to the Erying equation (Equation 3). The observed rate constants are listed in Tables 3.5-1 & 3.5-2.

ln(kh1 kB T)= - AH' / RT + AS'/ R Equation 3

Where kB is Boltzmann's constant, h is Planck's constant, and R is the molar gas constant

$ Mechanistic significance of AH : This is a measure of the enthalpic barrier the reactants need to overcome to be converted into products. It is also an indication of the extent of bond-breaking and bond-making in the transition state.

t Mechanistic significance of AS : This value provides some insight into the structure of the transition state. A negative value signifies that the entropy of the transition state is smaller than that of the reactants, i.e. the transition state is more ordered than the reactant ground state. Figure 3.5-2 Erying plot for the hydrolysis of 4-nitrophenyl a-D-N-acetylneuraminide.The displayed line is the calculated fit to the Eyring equation

Table 3.5-1 Observed rate constants for the hydrolyses of 4-nitrophenyl a-D-N- acetylneuraminide as a function of temperature at pH 8.08, (j.~= 0.3, NaC104) a

Temperature (OC)I 10 x kobs

"Mean value of three kinetic runs; quoted error = o .., Figure 3.5-3 Erying plot for the hydrolysis of 4-nitrophenyl P-D-N-acetylneuraminide.The displayed line is the calculated fit to the Eyring equation

Table 3.5-2 Observed rate constants for the hydrolyses of 4-nitrophenyl f3-D-N- acetylneuraminide as a function of temperature at pH 8.08, (p = 0.3, NaC104)"

Temperature (OC) 10 x kObs( sml)

- --

"Mean value of three kinetic runs; quoted error = o ..I Table 3.5-3 Activation parameters derived for the spontaneous hydrolysis of 4- nitrophenyl a- and P-D-sialosidesat pH 8.08, (p = 0.3 M, NaC104)

27 f 4

59 + 8

% The AS values suggest that both anomers could be operating via a dissociative mechanism.

3.5.2 Brcinsted Plots

Linear free energy relationships (LFER) are correlations between the effect of a structural change on a thermodynamic parameter and the corresponding effect on a lunetic parameter. For instance, a Brgnsted plot is a graph of the logarithm of the rate parameter versus a pKa value. The slope of the plot (PI,) indicates sensitivity of the reaction rate to changing pKa of the conjugate acid of the leaving group and hence it is possible to infer the degree of protonation of the leaving group at the reaction transition state. Figure 3.5-4 Brmsted plot, log (kobs) versus the pKa of the conjugate acid of the leaving group for the spontaneous reactions of compounds 3.7a-g measured at pH 1-00 at 50 OC Table 3.5-4 Observed rate constants for the hydrolyses of aryl P-D-N- acetylneuraminic acids at at 50 "C and a pH of 1.00 (p = 0.3, NaC104) a

Phenol pK, (ArOH) 10 x kobs(s-I)

4-Methylphenol

3-Methylphenol

Phenol

4-Chlorophenol

3-Chlorophenol

3-Nitrophenol

4-Ni trophenol

"Mean value of three kinetic runs; quoted error = o .-I

The calculated Pig value at pH 1.00 = 0.14 f 0.08.

For the rates of hydrolysis at pH 8.08 and 100 OC, the discontinuous assay method was used. That is, sealed ampoules containing the reaction solutions were placed in the vapours of a boiling water bath. Ampoules were removed from the heat source at regular intervals and the reaction was quenched by cooling the ampoules in an ice bath. The absorbance of each reaction solution (500 pL) was read at 408 nm in a 1 cm path length cuvette. The reaction was followed in this manner until no further change in absorbance was noted. For 4-nitrophenyl P-D-sialoside (3.7b), the rate at 100 OC was estimated using the activation parameters given in Table 3.5-3 on page 87. Figure 3.5-5 Bransted plot at pH 8.08 at 100 OC

Table 3.5-5 Observed rate constants for the hydrolyses of aryl P-D-N- acetylneuraminide at 100 OC and a pH of 8.08 (p = 0.3, NaC104)

Phenol

" Rate of hydrolysis of 4-nitrophenyl P-D-N-acetylneuraminide was estimated using the activation parameters listed in Table 3.5-3. A PI, value of -1.24 f 0.16 at pH 8.08 indicates that rate is highly dependent on the pK, of the conjugate acid of the leaving group. Thus, it can be concluded that there is insignificant protonation of the leaving group oxygen at the reaction transition state. On the other hand, a PI, value of 0.14 f 0.08 at pH 1.00 indicates that the leaving group is completely protonated at the reaction transition state, the leaving group is departing as a neutral molecule.

3.5.3 Ethanolysis Studies

Ethanolysis studies provide an insight into the effects of changing solvent polarity has on the reaction rate. The standard Grunwald-Winstein equation (Equation 4) is commonly used to analyze solvent effects on DN + AN (SN1) substitution reactions. Equation 4 shown below is a modification of the Grunwald-Winstein equation which correlates k, the solvolytic rate in a particular solvent and that measured in 80% ethanol-water (k,) for solvolysis of a series of substrates to a scale (Y,) of the solvent ionizing power based on the reactions of adamantyl-based substrates possessing an identical, if practical, leaving

group x.'~~

Equation 4

solvated-) (R+)solvated + (X-) solvated

log (klko)~~= my, + c

The slope m, a sensitivity parameter, of such a plot will provide an indication about the charge distribution at the transition state. For our study, the data was fit to Equation 4 using Ypi, values calculated from the solvolysis of 1-adamantyl picrate at 25 OC. 123,124

Figure 3.5-6 Plot of log kobs versus Y ,i, for the hydrolysis of 4-nitrophenyl a-D-N-acetylneuraminide (2.4a) at 75 OC. Table 3.5-6 Observed rate constants (kobs)for the hydrolyses of 4-nitrophenyl a-D- N-acetylneuraminide (2.4a) as a function of %EtOH in phosphate buffer, 15 mM at 75 OC, pH 7.25.

% EtOH

A small positive of m = 0.23 f 0.02 for 2.4a indicates some minor decrease in reaction rate as the polarity of the solvent is decreased. Figure 3.5-7 Plot of log kobsversus YPi, for the hydrolysis of 4-nitrophenyl P-D-N-acetylneuraminide (3.7b) at 75 OC.

Table 3.5-7 Observed rate constants (kobs)for the spontaneous hydrolysis of 4- nitrophenyl P-D-N-acetylneuraminide(3.7b) as a function of %EtOH in phosphate buffer, 15 mM at 75 OC, pH 7.25.

% EtOH

60 In this case of the b-anomer, the calculated m value is -0.04 f 0.01, which is close to zero. This value is consistent with the transition state having very similar solvation requirements to the ground state and thus the rate of hydrolysis is not affected by a change in polarity of the reaction solvent. On the other hand, it appears that for the a- anomer the transition state is more highly solvated than the ground state since m = 0.23 f

0.02.

3.5.4 Product studies to investigate participation of carboxylate group

In order to clarify the mechanistic picture of the hydrolysis of 3.7b1we decided to investigate the intramolecular participation of the carboxylate group during hydrolysis.

This type of participation results in the retention of configuration in the product. There are two possible sites of attack on the molecule (Figure 3.5-8): (1) the anomeric centre forming the shortlived a-lactone intermediate, mechanism proposed for 2.4a by Ashwell et al., (Pathway 1) and (2) the ips0 carbon of the aromatic ring (Pathway 2) Attack of the carboxylate oxygen on the ipso-carbon of the aryl group \Pathway 2

Figure 3.5-8 Intramolecular participation of the carboxylate in hydrolysis

3.5.5 Analysis of the products from ethanolysis reaction using NMR spectroscopy

Hydrolysis of 3.7b and 2.4a (3 mg) in ethanollwater mixtures (2 rnL) was performed in sealed glass ampoules at 75 OC and each ampoule contained 3 equiv of N- methylmorpholine so as to ensure that the ethyl sialoside product is stable to the solvolytic conditions. The reactions were allowed to proceed for about nine half-lives. The resultant solution was lyophilized and the solid residue was analyzed using 'H NMR spectroscopy.

3.5.5.1 Ethanolysis product studies for 4-nitrophenyl-p D-N-acetylneuraminide

50% EtOH

100% EtOH

Figure 3.5-9 'H NMR of the ethanolysis products for the hydrolysis of 4-nitrophenyl P-D-N- acetylneuraminide

Hydrolysis of 3.7b in 100% ethanol gives the ethyl P-sialoside as the major product. No detectable elimination product (glycal) was observed. However, one should note that the ethyl ester of sialic acid was not formed during hydrolysis as the expected peaks for H-3e at 2.13 ppm for the p-anomer and at 2.55 ppm for the a-anomer were absent. 50% EtOH

Ii, 100% EtOH

Figure 3.5-10 'H NMR of the ethanolysis product for the hydrolysis of 4-nitrophenyl a-D-N- acety lneuraminide

Hydrolysis of 2.4a in 100% ethanol gave the ethyl P-sialoside as the major product.

However, hydrolysis in 50% EtOH for nine half lives resulted in extensive degradation of the sialic acid skeleton to give several unidentifiable products.

The similarity noted above and the observed products formed during the ethanolysis of 4- nitrophenyl a- and P-sialosides strongly indicates that a strained a-lactone intermediate is not a viable intermediate during these reactions. That is, reactions that involve intramolecular nucleophilic participation should give retained ethyl sialosides andlor ethyl ester products. 3.5.5.2 Product distribution

Table 3.5-8 Observed product percentages formed during solvolysis of 2.4a and 3.7b in aqueous ethanol (vlv) solvent mixtures at 75 "c",~

% EtOH Glycal NeuAc a-GlyOEt P-GlyOEt Unknown

" all solvents contained 3 mole equivalent of N-methylmorpholine

Estimated errors f 5%

tr = trace

No values are reported for 50 % EtOH for 3.7b because under these conditions extensive decomposition was noted.

3.5.6 Analysis of products from ethanolysis reaction using GC-MS

"0 label was incorporated in the carboxylate group of the aryl sialosides by performing the final hydrolysis of the methyl ester in a 3: 1 vlv THF/''OH~ (95 atom % "0;Marshall

Isotopes Ltd., batch number 02041nw). The compounds obtained after lyophilization (3 mg) were subjected to similar ethanolysis conditions (100 % and 50 % ethanol vlv with three equivalents of N-methyl morpholine) as described in Section 3.5.5 on page 106.

After lyophilization, the product was dissolved in 0.1 M AcOH (2 mL) and extracted with distilled dichloromethane (2 x 2 rnL). The organic extracts were then dried over anhydrous MgS04 and subjected to GC-MS

99 AcHN

'NO,

3.7b-"0 acid 2.4a-"0 acid

Figure 3.5-11 ''0 labelled carboxylate group in 2.4a and 3.7b

The mass spectra of the products showed no incorporation of ''0 in the 4-nitrophenol obtained upon hydrolysis. This result rules out the possibility of the carboxylate attacking the ipso-carbon of the aryl ring (Pathway 2). Solvolysis studies rule out

Pathway 1.

3.5.7 Proposed transition states at pH independent region

The only plausible explanation that is consistent with our results for both 3.7b and 2.4a is that hydrolysis is occurring through a dissociative mechanism as indicated primarily by the large positive AS values observed for both anomers. 3.4b and 2.4a (Figure 3.6-12).

Figure 3.5-12 Possible transition states for the hydrolysis of compounds 3.4b and 2.4a in the pH independent region. The spontaneous hydrolysis of 2.4b, the thermodynamically less stable diastereomer, occurs at rate that is over 100-times faster than the corresponding reaction of 3.4a. The enhanced reactivity of the a-anomer is not caused by intramolecular nucleophilic catalysis by the C-1 carboxylate group during its spontaneous hydrolysis for the following reasons:

1. The respective Pc values on kspfor the a- and p-anomers of -1.3 (60 and -

1.24 f 0.16 (100 "C) are indistinguishable, thus suggesting similar late transition states for both anomers.

2. Both retained and inverted substitution products are formed during aqueous ethanolyses of 2.4a and 3.7b, an observation that is inconsistent with intramolecular nucleophilic participation because such reactions yield only retained ethyl sialoside products. At this time there is no evidence for the occurence of intramolecular assistance by the anomeric carboxylate during the hydrolysis reactions of sialosides, a conclusion that was reached by other workers. 85,121

The faster spontaneous hydrolysis of the a-anomer (2.4a) relative to the p-anomer

(3.7b) is likely caused by the relief of steric crowding at the anomeric ketal center on approaching the hydrolytic transition state. This additional steric effect is significantly smaller in the spontaneous hydrolysis reactions of typical carbohydrate acetals, such as methyl a- and p-glucopyranosides (3.8 and 3.9).12' In this case, the thermodynamically less stable anomer (3.8) only reacts two to three times faster than does 3.9.125

t The AS for 3.7b and 2.4a are 59 and 27 ~mol-IK-' respectively. The smaller value for the entropy of activation for the a-anomer (2.4a) is consistent with a greater loss in entropy. This originates from the fact that the axial carboxylate in the a-anomer at ground state is less solvated compared to the transition state where the carboxylates adopts a pseudo-equatorial position which is sterically less hindered. The difference in the solvation of the carboxylate in the p-anomer is not significant from the ground state to the transition state.

The 18~-exchangeexperiments in which the carboxylate groups in both 3.7b and 2.4a were labelled rules out the possibility that intramolecular attack on the ipso-carbon since no isotopic enrichment was observed in the 4-nitrophenol product.

3.6 Investigation of the base-catalyzed process

3.6.1 Exchange Experiment with ~2~~0

For the base-promoted reaction 18~-incoporationinto the 4-nitrophenol product was monitored at [OH-] = 1.0 M. Specifically, an approximate 1:l ~2~~01~2~~0mixture was made by adding equal volumes of ~~''0(95 atom % "0; Marshall Isotopes Ltd., batch number 02041nw) and freshly made-up aqueous NaOH (2 M). To the aqueous media (0.4 mL) either 3.7b or 2.4a (2 mg, 4.6 pmol) was added and the resulting solutions were maintained at 50 OC for approximately five half-lives for hydrolysis. Following neutralization with malonate buffer (0.3 M, 5 rnL, pH 2.69) the resultant aqueous media was extracted with distilled dichloromethane (2 x 2 mL). The combined organic extracts were then dried over Na2S04 and following removal of the solvent under reduced pressure the resulting solid residues were analyzed by standard EI mass spectrometry.

A 2.1% incorporation of "0 in phenol was observed for both the 4 nitrophenyl a- and the p- sialosides consistent with "0~2exchange with free phenolate in solution.

Figure 3.6-1 Attack of a hydroxide ion at the ipso carbon

The results from the '80-incorporation experiments (in H~"o) rule out the possibility that the base-promoted reactions of 3.7b and 2.4a occur by nucleophilic attack of hydroxide ion on the @so-carbon of the nitrophenyl ring. The observation that no "0 exchanged into the leaving group rules out a nucleophilic aromatic substitution of the p- nitrophenoxide ion (Figure 3.6-1).'~~ 3.6.2 Investigation into the carboxylate group participation

Compounds 3.7b-"0 and 2.4a-"0 (3 mg) were subjected to similar hydrolysis conditions (pH 14.00, 1.00 M NaOH) in Section 3.6.4 on page 106. After neutralization

(2 M AcOH), extraction with distilled CH2C12 (2 x 2 mL) and drying (Na2S04),the samples were subjected to EI-MS.

No incorporation of '$0into the liberated 4-nitrophenol was observed in either anomers.

Therefore it can be concluded that the carboxylate is not participating in the base- catalyzed hydrolysis reaction (Figure 3.6-2).

Figure 3.6-2 Attack of the carboxylate oxygen on the ipso carbon

3.6.3 Primary Kinetic Isotope Effect Study

Deuterium was incorporated at C-3 position of sialic acid according to published procedure and this material was used to synthesize compound below according to synthetic scheme on 68 (Figure 3.2-1). '27'128 4-Nitrophenyl (3R)-5-acetamido-3-deuterio-

3,5-dideoxy-~-~lycero-~-~-galacto-non-2-ulopyranosyonicacid) was made using the same procedure as for the synthesis of 3.7b. The NMR spectrum of [~R-~H4- nitrophenyll-0-D-sialosidewas identical to the unlabelled compound (3.7b) except for the absence of the triplet at 1.85 ppm and the presence of a doublet at 2.60 ppm instead of a pair of doublets. The reaction rates were measured at 50 OC at pH 14.00 (1 M NaOH)

Figure 3.6-3 [~R-~H4 nitrophenyl 1-P-D-sialoside

The possibility that the reaction involves an E2 elimination mechanism is precluded, at least in the case of 3.7b, because the observed deuterium KIE (kHlkD = 1.096 f 0.025) is too small to be a primary effect that should be associated with a trans-diaxial elimination reaction (Figure 3.6-4)

pJN02

AcHN AcHN ~6, HO

Figure 3.6-4 Trans-diaxial elimination mechanism 3.6.4 Possible mechanism for the base catalyzed hydrolysis of aryl P-D-sialoside

At this point we have been able to rule out the three mechanisms shown in Figures 3.6-1,

3.6-2, 3.6-4. As the carbohydrate product(s) of this reaction cannot be identified because extensive decomposition occurs in these strongly basic solutions a positive assignment of the mechanism is not possible. However, it is possible that the mechanism is that the hydroxide ion directly attacks the anomeric center ejecting 4-nitrophenoxide ion. The sialic acid formed is then rapidly degraded under the strongly basic conditions.

Degradation under co; OH - - - strongly basic OH conditions

Figure 3.6-5 Possible mechanism for the base-catalyzed pathway for the hydrolysis of aryl sialosides

Our mechanism is different from that proposed by Ashwell et a1 as we could not see a titration curve for the ionization of a hydroxide in our pH-rate profile for both an~mers.~~

3.7 Low pH domain

The rate constants for hydrolysis of 3.7b display a point of inflection, which is presumably associated with protonation of its carboxylate group. The calculated pK, value of 3.7b (axial carboxylate) is 1.58 a value that is 1.27 units more acidic than the corresponding ionization associated with 2.4a (equatorial carb~x~late).~~A similar trend in acidity constants has been reported for the cis- and trans-isomers of 4-tert- butylcyclohexanecarboxylic acid, where in 50% aqueous ethanol the equatorial carboxylic acid (trans) is more acidic than the axial isomer by 0.46 pK, units.'29

3.8 Biological implications for sialyl transfer reaction

Exo-sialidases have evolved to use a tyrosine residue as the nucleophile instead of the usual carboxylate residues found in most retaining glycosidases. I,IO,I~,I~OIt has been

suggested that there would be unfavourable electrostatic interaction with the carboxylate group of the sialoside if the nucleophile carried a negative charge.93,94 However, the positively-charged arginine triad in the enzyme's active site counteracts the charge on the substrate upon binding.91'92Our study on the aryl P-D-sialosides reveals that the anomers possess a smaller intrinsic reactivity (one hundred-fold) in comparison to the a-anomers.

This may be a possible reason why CMP P-D-sialoside is the donor in sialyltransferases: the a-anomer would be extremely labile. Specifically, the spontaneous rate of hydrolysis of CMP P-D-sialoside is 1.0 f 0.1 x s-' at 37 "c."~Hence one would expect the spontaneous rate of hydrolysis of CMP a-D-sialoside to be about 1.0 x s-', which corresponds into a half-life of about 10 minutes. Such a high reactivity is counterproductive as the donor should have a lifetime long enough to be transported from the site where it is biosynthesized to the sialyltransferase active site. A similar logic applies to sialidases and trans-sialidases: nature has chosen to stabilize the sialyl-enzyme intermediate by malung the leaving group worse. 3.9 Aryl D- N-acetylneuraminides as substrates of sialidases.

We decided to test the aryl P-D-N-acetylneurarninidesas potential substrates of the Y370G mutant neuraminidase from M. viridifaciens. The purpose of this study was to gain insight into the binding and hydrolysis of these compounds by the mutant enzyme, especially whether these unnatural sialosides would fit into the hole created when the tyrosine residue is replaced by a much smaller glycine residue.

3.9.1 Materials and Method

The wild type and the Y370G mutant enzyme from M. viridafaciens were provided by

Dr. J. N. Watson. MUNANA and the sialic acid quantitation kit were purchased from

SigmaIAldrich.

3.9.2 Enzyme Kinetics

Kinetic measurements were performed at 37 "C in 100 mM acetate buffer at pH 5.25 in a

1 cm path length cuvette. The enzyme catalyzed hydrolysis of MUNANA (1 pM) was

monitored by following the absorbance change over time at 347 nm using a Cary-3E

spectrophotometer equipped with a Peltier constant temperature accessory. The reaction

mixture was incubated at 37 "C for 3 min and the reaction was initiated by the addition of

thermally equilibrated enzyme (50 pL) ICso values for 3-chlorophenyl. P-D-sialoside

with the wild type and mutant enzymes were estimated from the rates of hydrolyses at

four different inhibitor concentrations. The binding constant (Ki) for 3-chlorophenyl, P-D-sialoside with mutant enzyme was calculated from the concentration of substrate used (MUNANA) and its K, with the enzyme under similar conditions (Equation 5). This calculation was based on the assumption that 3-chlorophenyl. P-D-sialoside is competing with MUNANA for the enzyme's active sites.

Equation 5

Values for kcat were estimated in the following manner: 3-chlorophenyl. P-D-sialoside (2 mM) was incubated with the enzyme (180 yL) in 20 mM acetate buffer at pH 5.25 at 37

OC for a total of 23 h (200 yL reaction). An aliquot of the reaction mixture (90 yL) was then quenched by addition of 100 mM Tris buffer (35 pL) at pH 7.60. The sample was then assayed for sialic acid (Sialic Acid Quantitation Kit). The kcat value was obtained by dividing the maximum rate of hydrolysis by the enzyme concentration.

3.9.3 Results and Discussion

An initial screen of all aryl. P-D-sialosides (Compounds 3.4a-g) with the Y370G mutant showed that the phenyl- and 3-chlorophenyl. P-D-sialosides were bound the tightest. 3- chlorophenyl P-D-sialoside was chosen for an indepth kinetic study to determine whether the mutant enzyme could catalyze its hydrolysis. Table 3.9-1 Kinetic Parameters of 3-chloropheny! P-D-sialoside with Y370G enzyme.

Y370G Ki (M) kcat (s-')

MUNANA 3.5 10-5 44.3

3-Chlorophenyl P-D-sialoside 1.7x10-~ 6x10-~"~~

Note " This is a lower limit for the k,,value since the detection limit of the assay is 10 uM sialic acid.

"his value is based on a single measurement

These results show that indeed the 3-chlorophenyl. p-D-sialoside is tightly bound in the active site of the Y370G mutant compared to the MUNANA substrate (K, = 3.5 x

M). Even though the rest of the catalytic machinery is intact, the enzyme is only able to break down the unnatural 3-chloropheny! P-D-sialoside very slowly.

3.9.4 Conclusion

This is the first example of an unnatural P-linked substrate of the Y370G mutant enzyme from M. viridifaciens. The novelty with this compound is the fact that the enzyme-bound intermediate in the wild type enzyme was mimicked in this Y370G system. These unnatural sialosides (p-anomer) are tightly bound into the hole that is created when the tyrosine nucleophile is substituted by a smaller glycine residue. 3.10 Experimental

General Dichloromethane and methanol were distilled from CaH2 and Mg respectively prior to use. NMR spectra were recorded on Varian 500 MHz spectrometer. The powdered molecular sieves were flame dried under vacuum before use. Sodium methoxide solution (10 mg/mL in dry methanol) were made fresh for each reaction.

Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-~-glycero-~-~-galacto-non-

2-ulopyranosy1)onate fluoride (3.5)

Compound 3.4 (6.0g. 18.5 mmol) was dissolved in dry pyridine (72 mL) and the resulting solution was cooled to 0 "C in an ice bath. Acetic anhydride (84 mL, 890 mmol) and a catalytic amount of diaminopyridine was added then to this mixture which was stirred for about 4 hours at 0 OC and then for 48 hours at room temperature. The resulting solution was evaporated at low temperature and repeatedly coevaporated with dry toluene (4 x 50 mL) to remove traces of pyridine. The foamy solid was then stirred with 70% hydrogen fluoride-pyridine solution (45 mL) in a Teflon vessel at 0 "C for 5 hours. The mixture was carefully poured on ice (200 mL) and the resulting solution was neutralised with a saturated solution of NaHC03 until effervescence ceased. The mixture was then extracted with dichloromethane (5 x 200 mL). The combine organic phase was then washed with

10% H2SO4 solution (300 mL), water (300 mL), brine (300 mL) and dried over anhydrous Na2S04. Evaporation of the solvent afforded a pale yellow syrup which triturated with petroleum ether to afford a pale white solid. The product was recrystallised using ethyl acetateldiethy1 ether mixture. The product (5.6g, 65%) was obtained as a white powder. mp 139-141 "C 'H NMR (400 MHz, CDC13) 6: 1.66, 1.90, 2.04, 2.13 (15

111 H, 5-CH3), 2.22 (m, 1 H, J3a,~=35.8 HZ, J3a,3e= 13.0 HZ,J3a,4= 11.5 HZ, H-3a), 2.52 (dt,

1 H, J3e,3a = 13.8 HZ,J3e,4 = J3e,~= 4.7 HZ, H-3e), 3.84 (s, 3 H, 0CH3), 4.04 (dd, 1 H, J9a,9b

= 12.5 HZ, Jga,8= 6.0 HZ, H-9a), 4.18-4.30 (m, 2 H, H-5, H-6), 4.40 (dd, 1 H, J9b,9a=12.5

Hz, J91,,8= 2.7 Hz, H-9b), 5.18 (m, 1 H, JX,7= 12.0Hz1H-8),5.24(m1 1 H, H-4), 5.35 (d,

1 H, JNH,S = 9.4 HZ, NH), 5.41 (dd, 1 H, J7,6= 2.0 HZ H-7); 13cNMR (100 MHz, CDC13)

6: 20.9, 20.9, 21.0, 21.0, 23.3 (CH3), 35.6 (d, JC-3, F = 28 HZ, C-3), 48.7 (C-5), 53.6

(OCH3), 62.5 (C-9), 67.2 (C-4), 68.5 (C-7), 70.3 (C-8), 73.2 (C-6), 106.9 (d, Jc.2, F =

230.4 HZ C-2), 164.7 (dl Jc-,,F= 29.5 HZ,C-1),170.0, 170.2, 170.6, 170.8, 171.2 (C=O)

To dried powdered 4A molecular sieves (5 g), phenol (404 mg, 4.30 mmol) and sialosyl

fluoride 3.5 (400 mg, 0.83 mmol) were added the resulting solid mixture was maintained under vacuum (14 mmHg/torr) with stirring for 30 min. To this mixture, dry CH2C12(30 mL) was added and the resultant mixture was stirred for an additional 1 h. Then a

solution of BF3.0Et2 (0.72 mL, 0.83 mmol) in CH2C12 (4 mL) was added and the mixture

was stirred overnight at room temperature. The resultant mixture was filtered and the

solid residue was washed thoroughly with CH2C12. The combined filtrates were washed with a saturated solution of NaHC03 (150 mL), water (150 mL), brine (150 mL) and the resulting solution was dried with Na2S04. A pale yellow syrup was obtained after evaporation of the solvent and this material was crystallized from Et20 to give a white powder (307 mg, 83% yield). mp 210-213 OC ; [a120D=-19.0 (c = 1.05, CH,C12); 'H NMR (400 MHz, CDC13) 6: 1.69, 1.88, 2.05, 2.16 (4 x s, 12 H, CH3), 1.99 (m, 1 H, H-

3a), 2.64 (dd, 1 H, J3e,3a = 12.9 HZ, J3e,4 = 5.0 HZ, H-3e), 3.70 (s, 3 H, 0CH3), 4.08 (dd, 1

H, J6,5 = 10.4 HZ,J6J = 2.4 HZ, H-6), 4.14 (dd, 1 H, J9a,9b= 12.5 HZ, Jsa3= 6.8 HZ, H-9a),

4.20 (q, 1H, J5,4 + J5,6 + J~,NH= 31.2 HZ, H-5), 4.64 (ddd, 1 H, J9b,8= 2.7 HZ, H-9b), 4.95

(td, 1 H, J8,7= 8.7 HZ, H-8), 5.24 (d, 1 H, JNH,~= 10.3 HZ, NH), 5.37 (dd, 1 H, H-7), 5.48

(td, 1 H, J4,3a = 10.9 HZ, H-4), 6.83-7.08 (m, 2 H, Ar-H), 7.18-7.29 (m, 2 H, Ar-H); 13c

NMR (100 MHz, CDC13) 6: 20.5, 20.6, 20.7, 20.9, 23.1 (CH3), 38.5 (C-3), 49.2 (C-5),

53.0 (OCH3), 61.9 (C-9), 67.8 (C-4), 68.6 (C-7), 71.6 (C-8), 72.0 (C-6), 99.0 (C-2),

116.5, 122.9, 129.8 (Ar-C), 167.4, 170.0, 170.2, 170.4, 170.5, 171.0 (C=O, C-1) Anal

calcd: C, 55.02; H, 5.86; N, 2.47. Found: C, 54.76; H, 5.97; N, 2.60.

Methyl [4-nitrophenyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-~-glycero-~-

D-galacto-non-2-ulopyranosyl)]onate(3.6b)

To dried powdered 4A molecular sieves a mixture of 4-nitrophenol (580 mg, 4.17 mmol) and sialosyl fluoride 3.5 (400 mg, 0.83 mmol) was added and the resultant solid mixture was maintained under vacuum (6 mmHg) for 30 min. The solid mixture was then placed under an N2 atmosphere and dry CH2C12 (30 mL) was added via a syringe and this mixture was stirred for 1 h. Then a solution of BF3.0Et2 (0.72 mL, 0.83 mmol) in

CH2C12 (4 mL) was added and the mixture was stirred overnight at room temperature.

Same work up and purification procedure as for compound 3.6a. A pale yellow syrup was obtained after evaporation of the solvent and this material was crystallized from Et20 to give a white powder (196 mg, 39% yield), mp 187-189 OC. [a]20D = -77.8 (c = 1.26, CH2C12). 'H NMR (400 MHz, CDC13) 6: 1.75, 1.89, 2.06, 2.08, 2.17 (5 x s, 15 H, CH3),

2.03 (m, 1 H, H-3a), 2.68 (dd, 1 H, J3e,3a= 12.9 HZ, J3e,4= 5.0 HZ, H-3e), 3.76 (s, 3 H,

0CH3), 4.04 (dd, 1 H, J6,5 = 10.4 HZ, J6,7= 2.4 HZ, H-6), 4.12 (dd, 1 H, J9a,9b= 12.5 HZ,

J9,,g = 6.8 Hz, H-9a), 4.20 (q, 1 H, J5,4 + J5,6 + JS,~~= 31.2 Hz, H-5), 4.64 (ddd, 1 H, J9b,8

=2.7 Hz,H-9b),4.90(ddd, 1 H, J8,~=8.7,H-8),5.26(d, 1 H, JNH,5= 10.3 Hz,NH), 5.35

(dd, 1 H, H-7), 5.48 (td, 1 H, J4,3a + J4,5 = 21.8 HZ, J4,3e = 5.0 HZ, H-4), 7.08-7.15 (m, 2

H, Ar-H), 8.12-8.22 (m, 2 H, Ar-H); 13cNMR (100 MHz, CDC13) 6: 20.6. 20.7, 20.8,

23.1 (CH3), 38.5 (C-3), 49.1 (C-5), 53.5 (OCH3), 61.8 (C-9), 67.8 (C-4, C-7), 71.5 (C-8),

72.9 (C-6), 99.7 (C-2), 116.8, 125.9, 143.1, 158.8 (Ar-C), 170.0, 170.2, 170.3, 170.5,

170.9 (C=O, C-1) Anal calcd for C26H32N2015:C, 50.98; H, 5.27; N, 4.57. Found: C,

50.75; H, 5.44; N, 4.34.

Methyl [3-nitrophenyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-~-glycero~~-

D-galacto-non-2-ulopyranosyl)]onate(3.6~)

To dried powdered 4A molecular sieves (5 g), 3-nitrophenol (580 mg, 4.17 mmol) and sialosyl fluoride 3.5 (400 mg, 0.83 mmol) were added. The resulting solid mixture was maintained under vacuum (14 mmHg1torr) with stirring for 30 min. To this mixture, dry

CH2C12(30 mL) was added and the resultant mixture was stirred for an additional 1 h.

Then a solution of BF3.0Et2 (0.72 mL, 0.83 mmol) in CH2C12 (4 mL) was added and the mixture was stirred overnight at room temperature. Same work up and purification procedure as for compound 3.6a A pale yellow syrup was obtained after evaporation of the solvent and this material was crystallized from Et20 to give a white powder (130 mg, 14% yield), mp 108-1 10 "C. [a]20D = -34.3 (c = 1.O5, CH2C12). 'H NMR (400 MHz,

CDC13)6: 1.77, 1.89,2.04,2.06,2.17 (5 xs, 15H,CH3),2.05 (m, 1 H,H-3a),2.69 (dd, 1

H, J3e,3a = 12.9 HZ, J3e,4 = 5.0 HZ, H-3e), 3.78 (s, 3 H, 0CH3), 4.05 (dd, 1 H, J6,5 = 10.4

Hz,J6,7=2.4Hz, H-6), 4.11 (dd, lH, J9a,9b=12.5 Hz,J~~,~=~.~Hz,H-9a),4.21 (q, 1 H,

J5,4 + J5,6 + J~,NH= 10.4 HZ, H-5), 4.64 (dd, 1 H, J9b,g = 2.7 HZ, H-9b), 4.93 (ddd, 1 H, J8,~

= 8.7 Hz, H-8), 5.27 (d, 1 H, JNH,5 = 10.3 HZ, NH), 5.48 (m, 1 H, H-7), 5.49 (td, 1 H, J4,3a

= 10.9 Hz, H-4), 7.35-7.55 (m, 2 H, Ar-H), 7.83-7.95 (m, 2 H, Ar-H); 13cNMR (100

MHz, CDC13) 6: 20.5, 20.6, 23.0 (CH3), 38.4 (C-3), 49.6 (C-5), 53.3 (OCH3), 61.9 (C-9),

68.1 (C-7), 68.4 (C-4), 71.4 (C-8), 72.9 (C-6), 99.9 (C-2), 123.2, 130.4, 149.3, 154.4 (Ar-

c), 166.6, 169.9, 170.1, 170.4, 170.7 (C=O, C-1) Anal calcd: C, 50.98; H, 5.26; N, 4.57.

Found: C, 51.20; H, 5.21; N, 4.72.

Methyl [4-chlorophenyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-~-glycero-

. P-D-galacto-non-2-ulopyranosyl)]onate(3.6d)

To dried powdered 4A molecular sieves (5 g), 4-chlorophenol (521 mg, 4.10 mmol) and

sialosyl fluoride 3.5 (500 mg, 1.03 mmol) were added and the resulting solid mixture was

maintained under vacuum (14 mmHg1to1-r) with stirring for 30 min. To this mixture, dry

CH2C12(30 mL) was added and the resultant mixture was stirred for an additional 1 h.

Then a solution of BF3.0Et2(0.90 mL, 1.03 mmol) in CH2C12(4 mL) was added and the

mixture was stirred overnight at room temperature. Same work up and purification

procedure as for compound 3.6a. A pale green syrup was obtained after evaporation of

the solvent and this material was crystallized from Et20 to give a white powder (310 mg,

115 62 % yield). mp 187-189 "C; [a]20~ = -23.6 (c = 1.10, CH2C12);'H NMR (400 MHz,

CDC13) 6: 1.74, 1.89, 2.05, 2.06, 2.16 (5 x s, 15 H, CH3), 1.98 (m, 1 H, H-3a), 2.63 (dd, 1

H, J3e,3a= 12.9 HZ, J3e,4=5.0H~1H-3e), 3.71 (s, 3 H, 0CH3),4.06 (dd, 1 H, J6,5 = 10.4

Hz,J6,7=2.4Hz,H-6),4.13 (dd, 1 H, J9a,gb= 12.5Hz,J9a,8=6.8 Hz,H-9a),4.19(q, 1 H,

J5,4 + J5,6 + J~,NH= 31.2 HZ, H-5), 4.61 (ddd, 1 H, J9b,8= 2.7 HZ,H-9b), 4.94 (td, 1 H, J8,7

= 8.7 HZ,H-8), 5.27 (dl 1 Hl JNH,~= 10.3 HZ,NH), 5.36 (dd, 1 H, H-7), 5.47 (td, 1 H, J4,3a

= 10.9 Hz, H-4), 6.88-6.94 (m, 2 H, Ar-H), 7.16-7.22 (m, 2 H, Ar-H); 13cNMR (100

MHz, CDC13) 6: 20.6, 20.7, 20.8, 23.2 (CH3), 38.5 (C-3), 49.2 (C-5), 53.2 (OCH3), 61.8

(C-9), 67.7 (C-4), 68.3 (C-7), 71.4 (C-8), 72.2 (C-6), 99.2 (C-2), 117.9, 129.7, (Ar-C),

167.0, 170.0, 170.2, 170.4 (C=O, C-1) Anal calcd: C, 51.87; H, 5.36; N, 2.32. Found: C,

51.75; H, 5.43; N, 2.56.

Methyl [3-chlorophenyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-~-glycero-

. P-D-galacto-non-2-ulopyranosyl)]onate(3.6e)

To dried powdered 4A molecular sieves (5 g), 3-chlorophenol (521 mg, 4.10 mmol) and

sialosyl fluoride 3.5 (500 mg, 1.03 mmol) were added. The resulting solid mixture was

maintained under vacuum (14 mmHg/tom) with stirring for 30 min. To this mixture, dry

CH2C12(30 mL) was added and the resultant mixture was stirred for an additional 1 h.

Then a solution of BF3.0Et2(0.90 mL, 0.83 mmol) in CH2C12(4 mL) was added and the

mixture was stirred overnight at room temperature. Same work up and purification

procedure as for compound 3.6a. A pale green syrup was obtained after evaporation of

the solvent and this material was crystallized from Et20 to give a white powder (235 mg,

116 38% yield). mp 170-172 "C; [a]20D = -35.0 (c = 1.00, CH2C12);'H NMR (400 MHz,

CDC13)6: 1.57, 1.79, 1.89,2.05,2.16(5 xs, 15H,CH3),2.05(m, 1 H,H-3a),2.64(dd, 1

H, J3e,3a = 12.9 HZ, J3e,4 = 5.0 HZ, H-3e), 3.73 (s, 3 H, 0CH3), 4.05 (dd, 1 H, J(j5= 10.4

HZ, J6,7=2.4Hz,H-6),4.12(dd, 1 H, J9a,9b=12.5Hz, J9a,8=6.8H~,H-9a),4.21(q, 1 H,

J5,4 + J5,6 + JS,NH= 31.2. HZ, H-5), 4.61 (ddd, 1 H, J9b,8 = 2.7 HZ,H-9b), 4.96 (td, 1 H, J8,7

= 8.7 HZ, H-8), 5.24-5.41 (m, 2 H, NH, H-7), 5.45 (td, 1 H, J4,3a = 10.9 HZ, H-4), 7.11-

7.18 (m, 2 H, Ar-H), 8.16-8.22 (m, 2 H, Ar-H); 13cNMR (100 MHz, CDC13) 6: 20.6,

23.0 (CH3), 38.5 (C-3), 49.5 (C-5), 53.2 (OCH3), 62.0 (C-9), 68.4 (C-7, C-4), 71.7 (C-8),

72.6 (C-6), 99.5 (C-2), 115.0, 117.9, 123.4, 130.6, (Ar-C), 169.9, 170.1, 170.3, 170.8

(C=O, C-1) Anal calcd: C, 5 1.87; H, 5.36; N, 2.32. Found: C, 51.97; H, 5.40; N, 2.55.

Methyl [4-methylphenyl-(5-acetamido-4,7,8,9-tetra-0-acetyl-3,5-dideoxy-~-glycero-

. P-D-galacto-non-2-ulopyranosyl)]onate(3.60

To dried powdered 4A molecular sieves (5 g), 4-methylphenol (521 mg, 4.82 mmol) and

sialosyl fluoride 3.5 (400 mg, 0.83 mmol) were added the resulting solid mixture was

maintained under vacuum (14 mmHg/torr) with stirring for 30 min. To this mixture, dry

CH2C12 (30 mL) was added and the resultant mixture was stirred for an additional 1 h.

Then a solution of BF3.0Et2(0.72 mL, 0.83 mmol) in CH2C12 (4 mL) was added and the

mixture was stirred overnight at room temperature. Same work up and purification

procedure as for compound 3.6a. A pale yellow syrup was obtained after evaporation of the solvent and this material was crystallized from Et20 to give a white powder (301 mg,

62% yield). mp 140-144 OC; [a]20D = -62.9 (c = 1.05, CH2C12);'H NMR (400 MHz,

CDCI3) 6: 1.57, 1.71, 1.88, 2.04, 2.05, 2.10 (6 x s, 18 H, CH3), 1.97 (m, 1 H, H-3a), 2.63

(dd, 1 H, J3q3a = 12.9 HZ, J3e,4= 5.0 HZ, H-3e), 3.69 (s, 3 H, 0CH3), 4.08 (dd, 1 H, J6,5 =

10.4 HZ, J6,7 = 2.4 HZ, H-6), 4.14 (dd, 1 H, J9a,9b=12.5 HZ,J9a,8 = 6.8 HZ, H-9a), 4.20 (q,

1 H, J5,4 + J5,6 + JSJH= 31.2 HZ, H-3, 4.64 (ddd, 1 H, J9b,8= 2.7 HZ, H-9b), 4.95 (td, 1

H, Jg,7 = 8.7 HZ, H-S), 5.24 (d, 1 H, JNH,5 = 10.3 HZ, NH), 5.37 (dd, 1 H, H-7), 5.48 (td, 1

H, J4,3a = 10.9 HZ, H-4), 6.80-6.87 (m, 2 H, Ar-H), 6.98-7.04 (m, 2 H, Ar-H); I3c NMR

(100 MHz, CDC13) 6: 20.5, 20.6, 20.7, 20.9, 23.1 (CH3), 38.5 (C-3), 49.2 (C-5), 53.0

(OCH3), 61.9 (C-9), 67.8 (C-4), 68.6 (C-7), 71.6 (C-8), 72.0 (C-6), 99.0 (C-2), 116.4,

130.1, (Ar-C), 170.0, 170.3, 170.4 (C=O, C-1) Anal calcd: C, 55.76; H, 6.06; N, 2.41.

Found: C, 56.0; H, 6.18; N, 2.30.

Methyl [3-methylphenyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-~-glycero-

, f!-D-galacto-non-2-ulopyranosyl)]onate(3.6g)

To dried powdered 4A molecular sieves (5 g) m-cresol (548 mg, 4.10 mmol) and sialosyl

fluoride 3.5 (500 mg, 1.03 mmol) were added and the resulting solid mixture was

maintained under vacuum (14 mmHg1torr) with stirring for 30 min. Dry CH2C12(30 mL)

was added via a syringe and the mixture was stirred for 1 hour. BF3.0Et2(0.90 mL, 1.03

mmol) was added and the mixture was stirred overnight at room temperature. Same work

up and purification procedure as for compound 3.6a. A pale yellow syrup was obtained

after evaporation of the solvent and this material was crystallized from diethyl ether to

1 I8 give a white powder (385 mg, 65% yield). mp 165-167 "C ; [a]20D = -11.4 (c = 1.05,

CH2C12);'H NMR (400 MHz, CDC13) 6: 1.71, 1.88, 2.04, 2.05, 2.16 (5 x s, 15 H, CH3),

1.97 (m, 1 H, H-3a), 2.63 (dd, 1 H, J3e,3a = 12.9 HZ, J3e,4 = 5.0 HZ, H-3e), 3.71 (s, 3 H,

OCH3), 4.07 (dd, 1 H, J6,5 = 10.4 HZ, J6,7 = 2.4 HZ, H-6), 4.16 (dd, 1 H, J9a,9b = 12.5 HZ,

J9,,8 = 6.8 Hz, H-9a), 4.21 (q, 1 H, J5,4 + J5,6 + JS,NH= 31.2 Hz, H-5), 4.63 (ddd, 1 H, J9b,8

= 2.7 HZ, H-9b), 4.94 (td, 1 H, J8,7= 8.7 HZ, H-8), 5.27 (d, 1 H, JNH,5= 10.3 HZ, NH),

5.37 (d, 1 H, H-7), 5.48 (td, 1 H, J4,3a = 10.9 HZ, H-4), 6.70-6.86 (m, 3 H, Ar-H), 7.09

(m, 1 H, Ar-H); 13cNMR (100 MHz, CDC13) 6: 20.6, 20.7, 20.9, 21.4, 23.2 (CH3), 38.5

(C-3), 49.2 (C-5), 53.0 (OCH3), 61.9 (C-9), 67.8 (C-7), 68.6 (C-4), 71.6 (C-8), 72.2 (C-

6), 98.9 (C-2), 113.3, 117.4, 123.8, 129.5 (Ar-C), 167.4, 170.0, 170.2, 170.3, 170.5, 171.0

(C=O, C-1) Anal calcd: C, 55.76; H, 6.07; N, 2.41. Found: C, 55.47; H, 6.20; N, 2.61.

Phenyl(5-acetamido-3,5-dideoxy-~-glycero~~-~-galacto-non-2-ulopyranosylonic acid) (3.7a)

Compound 3.6a (100 mg, 0.18 mmol) was dissolved in anhydrous methanol (5 mL) and a methanolic sodium methoxide solution (2.1 rnL, 0.90 mmol) was added. The mixture was stirred at 0 OC for 15 min. Then Dowex 50W HCR-W2 (p)(prewashed with methanol) cation exchange resin was added to neutralize the solution. The resin was filtered off and washed several times with methanol. The filtrate was evaporated under reduced pressure and the residue was dissolved in a 3: 1 vlv THFIwater mixture (2 mL) at

0 "C. LiOH.H20 (36 mg, 0.85 mmol) was added to the mixture. After 15 min of stirring at 0 OC, the mixture was neutralized with Dowex 50W HCR-W2 (H+) resin. The resin

119 was filtered off and washed with MeOH. The filtrate was evaporated under reduced pressure to remove the MeOH and the remaining aqueous solution was lyophilized to give a white solid (63 mg, 92%). [a]20D = -26.7 (c = 1.16, H20). 'H NMR (400 MHz,

D20) 6: 1.83 (t, 1 H, J3a,3e+ J744 = 24.4 HZ, H-3a), 2.00 (s, 3H, CH3), 2.55 (dd, 1 H, J3e,3a

= 13.0 Hz, J3e,4 = 4.9 Hz, H-3e), 3.44 (d, 1 H, J7,8= 9.5 Hz, H-7), 3.61 (dd, 1 H, J9a,9b=

11.8 HZ, J9a,8 = 5.2 HZ, H-9a ), 3.65- 3.79 (m, 4 H, NH, H-9b, H-6, H-8), 3.97 (t, 1 H, J5

+J5,6=20.8HZ, H-5 ), 4.25 (td, 1 H, J4,3a=11.0H~, J4,5= ll.OHz,H-4), 7.02-7.10 (m, 3

H, Ar-H), 7.27-7.37 (m, 2 H, Ar-H); "C NMR (100 MHz, D20) 6: 19.0 (CH3), 35.4 (C-

3), 48.9 (C-5), 60.1 (C-9), 63.7 (C-4), 65.2 (C-7), 67.1 (C-8), 67.7 (C-6), 92.2 (C-2),

112.0, 117.6, 127.1, 152.3 (Ar-C), 170.2, 171.9(C=O, C-1); HRMS (FAB) m/z (M-W)

C17H22N09requires 384.1295 found 384.1300.

4-Nitrophenyl(5-acetamido-3,5-dideoxy-~-glycero-~-~-galacto-non-2- ulopyranosylonic acid) (3.7b)

To a solution of 3.6b (50 mg, 0.08 mmol) in anhydrous methanol (5 mL) was added a methanolic sodium methoxide solution (1 mL, 0.40 mmol) and this mixture solution was stirred at 0 OC for 15 min. Dowex 50W HCR-W2 (H+) cation exchange resin (prewashed with methanol) was added to neutralize the solution. After removal of the resin by filtration it was washed several times with methanol. The combined solvent was evaporated under reduced pressure and the resultant residue was dissolved in a 3:l v/v

THFIwater mixture (2 mL) at 0 OC. To this solution LiOH.H20 (17 mg, 0.40 mmol) was added. After stirring at 0 OC for 25 min, the mixture was neutralized with Dowex 50W

120 HCR-W2 (H+) resin. Following removal of the resin, the filtrate was concentrated under reduced pressure. The remaining aqueous solution was then lyophilized to give a white solid (33 mg, 92%); [a]20~ = -86.9 (c = 1.22, H20) 'H NMR (400 MHz, DzO) 6: 1.85 (t,

1 H, J3e,3a+ J3a,4 = 24.5 HZ, H-3a), 2.04 (s, 3 H, CH3), 2.60 (dd, 1 H, J3e,3a= 12.9 HZ, J3e,4

= 5.0 HZ, H-3e), 3.45 (d, 1 H, J7,8 = 9.0 HZ, H-7), 3.60 (dd, 1 H, J9a,9b= 11.8 HZ, J9a,8=

5.2 Hz, H-9a), 3.64-3.77 (m, 4 H, NH, H-6, H-8, H-9b), 4.00 (t, 1 H, J5,4 + J5$= 20.8 HZ,

H-5 ), 4.25 (td, 1 H, J4,3a + J4,5 = 21.8 HZ, J4,3e= 5.0 HZ, H-4), 6.85-7.21 (m, 2 H, Ar-H),

8.20-8.38 (m, 2 H, Ar-H). I3cNMR (100 MHz, D20)6: 19.0 (CH3), 37.5 (C-3), 48.6 (C-

5), 60.3 (C-9), 63.4 (C-4), 65.2 (C-7), 66.8 (C-8), 68.5 (C-6), 98.1 (C-2), 113.8, 122.7,

138.9, 156.8 (Ar-C), 170.3, 171.7 (C=O, C-1); HRMS (FAB) dz(M-H', C17H21N2011 requires 419.1 145 found 429.1 156.

3-Nitrophenyl(5-acetamido-3,5-dideoxy-~-glycero~~-~-galacto-non-2- ulopyranosylonic acid) (3.7~)

Compound 3.6~(100 mg, 0.16mmol) was dissolved in anhydrous methanol (5 mL) and a methanolic sodium methoxide solution (1.9 mL, 0.82 mmol) was added and this mixture was stirred at 0 "C for 15 min. Then Dowex 50W HCR-W2 (H') (prewashed with methanol) cation exchange resin was added to neutralize the solution. The resin was filtered off and washed several times with methanol. The filtrate was evaporated under reduced pressure and the residue was dissolved in a 3: 1 vlv THFIwater mixture (2 mL) at

0 "C. LiOH.H20 (32 mg, 0.76 mmol) was added to the mixture. After 15 min of stirring at 0 "C, the mixture was neutralized with Dowex 50W HCR-W2 (Hf) resin. The resin

121 was filtered off and washed with MeOH. The filtrate was evaporated under reduced pressure to remove the MeOH and the remaining aqueous solution was lyophilized to give a white solid (66 mg, 93%). [a]20D = -24.3 (c = 1.03, H20) 'H NMR (400 MHz,

D20) 6: 1.85 (t, 1 H, J3a,3e+J3a4 = 24.4 HZ, H-3a), 2.00 (s, 3 H, CH3), 2.58 (dd, 1 H, J3e,3a

= 13.0 HZ, J3e,4 = 4.6 HZ, H-3e), 3.45 (d, 1 H J7,*= 9.0 HZ, H-7), 3.57 (dd, lH, J9a,9b=

11.8 Hz, J9,,8 = 5.2 Hz, H-9a), 3.65-3.77 (m, 4 H, NH, H-9b, H-8, H-6), 4.00( t, 1 H, J5,4+

J5,6=20.8 HZ, H-5 ), 4.25 (td, 1 H, J4,3a= 10.9 HZ, J4,5 = 10.9 HZ, H-4), 7.38-7.53 (m, 2

H, Ar-H), 7.84-7.96 (m, 2 H, Ar-H); 13cNMR (100 MHz, D20) 6: 22.2 (CH3), 43.5 (C-

3), 52.0 (C-5), 63.3 (C-9), 66.4 (C-4), 68.0 (C-7), 70.1 (C-8), 71.4 (C-6), 100.8 (C-2),

111.8, 117.4, 124.3, 130.6, 148.4, 154.7 (Ar-C), 173.2, 175.4 (C=O, C-1); HRMS (FAB) dz(M-H') requires 429.1 151 found 429.1 145

4-Chlorophenyl(5-acetamido-3,5-dideoxy-~-glycero~~-~-galacto-non-2- ulopyranosylonic acid)(3.7d)

Compound 3.6d (100 mg, 0.17mmol) was dissolved in anhydrous methanol (5 mL) and a methanolic sodium methoxide solution (1.9 mL, 0.82 mmol) was added. The mixture was stirred at 0 OC for 15 min. Then Dowex 50W HCR-W2 (H+)(prewashed with methanol) cation exchange resin was added to neutralize the solution. The resin was filtered off and washed several times with methanol. The filtrate was evaporated under reduced pressure and the residue was dissolved in a 3: 1 v/v THFIwater mixture (2 mL) at 0 OC. LiOH.H20

(34 mg, 0.81 mmol) was added to the mixture. After 15 min of stirring at 0 OC, the mixture was neutralized with Dowex 50W HCR-W2 (H') resin. The resin was filtered

122 off and washed with MeOH. The filtrate was evaporated under reduced pressure to remove the MeOH and the remaining aqueous solution was lyophilized to give a white solid (67 mg, 95%). [a]20~= -36.5 (c = 1.15, H20). 'H NMR (400 MHz, D20) 6: 1.81

(t, 1 H, J3a,3e + J3a,4 = 24.4 HZ, H-3a), 2.00 (s, 3 H, CH3), 2.52 (dd, 1 H, J3e,3a= 13.0 HZ,

J3e,4=4.9H~,H-3e),3.43(d, 1 H,J~,~=~.OHZ,H-~),3.57 (dd, 1 H, J9a,9b=11.8H~, J9a,8

= 5.2 Hz, H-9a), 3.61- 3.77 (m, 4 H, NH, H-9b, H-8, H-6), 3.96 (t, 1 H, J5,4+ J5,6= 20.8

Hz, H-5),4.22 (td, 1 H, J4,3a= 10.9Hz, J4,5 = 10.9Hz, H-4), 6.96-7.04 (m, 2H,Ar-H),

7.22-7.32 (m, 2 H, Ar-H); 13cNMR (100 MHz, D20) 6 :24.7 (CH3), 43.0 (C-3), 54.5 (C-

5), 65.8 (C-9), 69.2 (C-4), 69.2 (C-7), 70.8 (C-8), 72.9 (C-6), 98.0 (C-2), 119.6, 120.9,

131.8.3, 155.4 (Ar-C), 176.1, 177.4 (C=O, C-1); HRMS (FAB) m/z (M-H')

C17H21C1N09requires 41 8.0905 found 41 8.0905.

3-Chlorophenyl(S-acetamido-3,5-dideoxy-~-glycero~~-~-galacto-non-2- ulopyranosylonic acid) (3.7e)

Compound 3.6e (100 mg, 0.17mmol) was dissolved in anhydrous methanol (5mL) and a methanolic sodium methoxide solution (1.9 mL, 0.82 mmol) was added. The mixture was stirred at 0 OC for 15 min. Then Dowex 50W HCR-W2 (H+) (prewashed with methanol) cation exchange resin was added to neutralize the solution. The resin was filtered off and washed several times with methanol. The filtrate was evaporated under reduced pressure and the residue was dissolved in a 3: 1 vlv THFIwater mixture (2 rnL) at

0 "C. LiOH.H20 (34 mg, 0.81 mmol) was added to the mixture. After 15 min of stirring at 0 "C, the mixture was neutralized with Dowex 50W HCR-W2 (H+) resin. The resin

123 was filtered off and washed with MeOH. The filtrate was evaporated under reduced pressure to removed the MeOH and the remaining aqueous solution was lyophilized to give a white solid (61 mg, 86%); [a]20D = -48.0 (c = 1.00, H20) 'H NMR (400 MHz,

DzO) 6: 1.80 (t, 1 H, J3a,3e+J3a,4 = 24.4 HZ, H-3a), 1.99 (s, 3 H, CH3), 2.53 (dd, 1 H, J3e,3a

= 13.0 HZ, J3e,4 = 4.9 HZ, H-3e), 3.43 (d, 1 H, J7,8 = 9.0 HZ, H-7), 3.56 (dd, 1 H, J9a,9b=

11.8 Hz, J9a,8 = 5.2 Hz, H-9a), 3.61- 3.76 (m, 4 H, NH, H-9b, H-8, H-6), 3.96 (t, 1 H, J5,4

+ J5,6=20.8 HZ, H-5 ), 4.21 (td, 1 H, J4,3a= 10.9 HZ,J4,5 = 10.9 HZ,H-4), 6.91-7.09 (m, 3

H, Ar-H), 7.19-7.26 (m, 1 H, Ar-H); 13cNMR (100 MHz, D20) 6: 24.7(CH3),42.9 (C-3),

54.5 (C-5), 65.8 (C-9), 69.1 (C-4), 70.7 (C-7), 73.0 (C-8), 73.9 (C-6), 97.9 (C-2), 116.6,

118.3, 123.0, 133.2, 136.7, 157.2 (Ar-C), 175.8, 177.4 (C=O, C-1); HRMS (FAB) dz

(M-H') C17H21C1N09requires 4 18.0905 found 418.0909.

4-Methylphenyl(5-acetamido-3,5-dideoxy-~-glycero~~-~-galacto-non-2- ulopyranosylonic acid) (3.7f)

Compound 3.6f (100 mg, 0.17 mmol) was dissolved in anhydrous methanol (5 mL) and a methanolic sodium methoxide solution (2.0 rnL, 0.87 mmol) was added. The mixture was stirred at 0 "C for 15 min. Then Dowex 50W HCR-W2 (H') (prewashed with methanol) cation exchange resin was added to neutralize the solution. The resin was filtered off and washed several times with methanol. The filtrate was evaporated under reduced pressure and the residue was dissolved in a 3: 1 v/v THFJwater mixture (2 mL) at

0 "C. LiOH.H20 (34 mg, 0.81 mmol) was added to the mixture. After 15 min of stirring at 0 "C, the mixture was neutralized with Dowex 50W HCR-W2 (H') resin. The resin

124 was filtered off and washed with MeOH. The filtrate was evaporated under reduced pressure to removed the MeOH and the remaining aqueous solution was lyophilized to

give a white solid (66 mg, 94%); [a]20~ = -18.3 (c = 1.04, H20) 'H NMR (400 MHz,

D20) 6: 1.80 (t, 1 H, J3a,3e+J3a,4 = 24.4 HZ, H-3a), 1.98 (s, 3 H, CH3), 2.21 (s, 3 H, Ar

CH3), 2.52 (dd, 1 H, J3e,3a = 13.0 HZ,J3e,4 = 4.9 HZ,H-3e), 3.41 (d, 1 H, J7,8 = 9.1 HZ, H-

7), 3.52 (dd, IH, J9a,9b= 11.8 HZ, Jga,8 = 5.2 HZ, H-9a), 3.60- 3.79 (m, 4 H, NH, H-9b, H-

8, H-6), 3.95 (t, 1 H, J5,4 + J5,6 = 20.8 HZ,H-5 ), 4.21 (td, 1 H, J4,3a = 10.9 HZ,J4,5 = 10.9

Hz, H-4), 6.90-6.97 (m, 2 H, Ar-H), 7.10-7.15 (m, 2 H, Ar-H); 13cNMR (100 MHz,

D20) 6: 19.6, 22.1 (CH3), 38.9 (C-3), 52.1 (C-5), 63.3 (C-9), 66.8 (C-4), 68.3 (C-7), 70.2

(C-8), 70.5 (C-6), 95.4 (C-2), 115.5, 130.3, 153.0 (Ar-C), 173.5, 175.0 (C=O, C-1);

HRMS (FAB) dz(M-H+) ClgH24N09requires 398.145 1 found 398.1459.

3-Methylphenyl(5-acetamido-3,5-dideoxy-~-glycero-~-~-galacto-non-2- ulopyranosylonic acid) (3.7g)

Compound 3.6g (100 mg, 0.17 mmol) was dissolved in anhydrous methanol (5 mL) and a methanolic sodium methoxide solution (2.0 mL, 0.87 mmol) was added. The mixture was stirred at 0 OC for 15 min. Then Dowex 50W HCR-W2 (H+) (prewashed with methanol) cation exchange resin was added to neutralize the solution. The resin was filtered off and washed several times with methanol. The filtrate was evaporated under reduced pressure and the residue was dissolved in a 3: 1 v/v THFIwater mixture (2 mL) at 0 OC. LiOH.H20

(34 mg, 0.81 mmol) was added to the mixture. After 15 min of stirring at 0 OC, the mixture was neutralized with Dowex 50W HCR-W2 (H') resin. The resin was filtered

125 off and washed with MeOH. The filtrate was evaporated under reduced pressure to

removed the MeOH and the remaining aqueous solution was lyophilized to give a white

solid (56 mg, 85%); [a]20~ = -40.4 (r = 1.09, H2O) 'H NMR (400 MHz, D20) 6: 1.81 (t,

1 H, J3a,3e + J3a,4 = 24.4 HZ, H-3a), 2.00 (s, 3 H, CH3), 2.26 (s, 3 H, Ar-CH3), 2.53 (dd, 1

H, J3e,3a= 13.0H~,J3e,4=4.9H~,H-3e), 3.42 (d, 1 H,J7,8=9.0H~,H-7),3.53 (dd, 1 H,

J9a,9b= 1 1.8 HZ, J9a,8 = 5.2 HZ, H-9a), 3.69- 3.76 (m, 4 H, NH, H-9b, H-8, H-6), 3.96 (t, 1

H, J5,4+ J5,6= 20.8 HZ,H-5 ), 4.24 (td, 1 H, J4,3a = 10.9 HZ, J4,5 = 10.9 HZ, H-4), 6.80-

6.93 (m, 3 H, Ar-H), 7.16-7.23 (m, 1 H, Ar-H); "C NMR (100 MHz, D20)6:19.1, 17.4

(CH3), 37.5 (C-3), 49.0 (C-5), 60.1 (C-9), 63.6 (C-4), 65.1 (C-7), 67.4 (C-8), 68.1 (C-6),

96.6 (C-2), 109.3, 110.7, 138.2, 114.2, 118.0, 120.3, 152.4 (Ar-C), 170.3, 171.9 (C=O, C-

1); HRMS (FAB) dz(M-H') C18H24N09requires 398.145 1 found 398.1463. CHAPTER FOUR SYNTHESIS AND EVALUATION OF A BICYCL0[4.1.0]HEPTYL ANALOGUE OF GLUCOSE-1-PHOSPHATE

4.1 Introduction

Complex oligosaccharides are involved in numerous biological recognition processes, examples include fertillization, immune defense, viral and parasitic infections, cell growth, cell adhesion and inflammati~n.~.' The assembly of these oligosaccharides requires sugar nucleotide donors, compounds that are biosynthesized from glycosyl phosphates.13' For example, uridine 5' (a-D-glucopyranosyl diphosphate) (UDP-Glc), a key intermediate in many biochemical pathways including that of glycogen formation, is made enzymatically from a-D-glucopyranosyl phosphate (Glc- 1-P) and uridine 5'- triphosphate (UTP) (Figure 4.1- I).''~

Figure 4.1-1 Biosynthesis of UDP-Glc

The biological importance of glycosyl phosphates has resulted in many researchers investing considerable effort to synthesize analogues of this class of compounds. In the specific case of a-D-glucopyranosyl phosphate (4.1), analogues that have been made include: (i) 2-deoxy-2-fluoro-a-D-glucopyranosyl phosphate (4.2) a compound in which the C-2 hydroxyl group has been replaced by a fluorine atom;"3 (ii) C-(a-D- glucopyranosyl)methane phosphonate (4.3), an analogue that is hydrolytically

and (iii) 4.4 a mimic where the normal carbohydrate ring oxygen atom (0-5) has been substituted by a phosphorus atom. 136,137

'OH

4.3 4.4

Figure 4.1-2 Analogues of glucose-1-phosphate 4.2 Synthetic Target

The synthesis and characterization of a novel, conformationally rigid, carbocyclic

analogue of glucose-1-phosphate is described. The target compound 4.5 was chosen on

the basis of the reported potent inhibition of the yeast a-glucosidase by the carbocyclic

analogue 4.6, a structure that mimics a-D-glucopyranosyl amine.138. In compound 4.6

the cyclopropane ring forces the sin membered ring into half-chair c~nformation.'~~In

addition, 4.5 was evaluated as a potential substrate for yeast UTP:a-D-glucose-1-

phosphate uridylyltransferase in order to test whether the UDP-analogue 4.7 could be

made by a chemoenzymatic approach.

Figure 4.2-1 Carbocyclic cyclopropanated motif for inhibitors 4.3 Results and Discussion

Enone 4.14 was synthesised from methyl a-D-glucosylpyranoside (4.8) in seven steps according to a literature procedure (Figure 4.3-1).'~~The first step involved protection of the free hydroxyl groups in compound (4.8) with benzyl chloride in DMF using NaH as a base. After work up, the resulting crude perbenzylated product was hydrolysed to form hemiacetal (4.9) using boiling AcOH that contained 10% aqueous H2S04. The crude compound (4.9) was then precipitated from water, and after filtration, it was recrystallized twice from methanol to give a white crystalline solid in 50 % overall yield from methyl a-D-glucopyranoside. The lactone (4.10) was formed by oxidation of (4.9) with DMSO/ acetic anhydride and this material was then reacted with the anion of dimethy methylphosphonate to form hemiacetal (4.11). Reduction of (4.11) with NaBK in THF gave a mixture of diastereomeric diols (4.12) and these compounds were oxidized to give the diketone compound (4.13). OBn I C

BnO

OBn OBn I I e

BnO

OBn OBn

BnOkBnO OBn ,OMe BnO ,OMe

HO P10Me

a) BnCI, NaH, DMF b) HzS04,AcOH c) DMSO, AczO d) BuLi, THF, dimethylmethylphosphonate e)

NaBH4, THF f) DMSO, TFAA, CH2C12, triethyl amine g) TEA, LiCl, CHzClz

Figure 4.3-1 Synthesis of key intermediate enone 4.14

Triethylamine and LiCl were used for the cyclization step to synthesize enone (4.14).

This modification to the literature procedure "9 was made because cyclization proceeded faster with these reagents than it did when 18-crown-6 and K2C03was used. As a result, it was observed that the enone product was less prone to aromatize. Attempted reduction

131 of enone with several reagents such as LiAlK, L~AI(OBU')~H,Zn(BH& resulted in the formation of mixtures, of varying ratio, of the two diastereomeric alcohols. However, the use of L-Selectride in THF at 0 OC effected a stereoselective reduction that produced only the pseudo-axial alcohol (4.15) in 78% yield (Figure 4.3-3). Alcohol (4.15) was then reacted using Furukawa's cyclopropanation protocol to afford compound (4.16) as the only observable diastereomer in a 62% yield.

The stereochemistry of the bicyclo[4.1.O]heptane was confirmed by an nOe experiment to be equivalent to that of D-glucose rather than L-idose. Specifically, a strong nOe contact was observed between H-7s and H-4 (Figure 4.3-2).

Figure 4.3-2 Observed nOe contact for compound 4.16 (a) L-selectride, THF, 0 OC, (b) (MehZn, CH21z,toluene, -10 OC, (c) dibenzyldiisopropylphosphoramidate, tetrazol, CHzClz; bleach, THF (d) 10% Pd-C, MeOH.

Figure 4.3-3 Synthesis of Glc-1-P analogue

Reaction of compound (4.16) with reagents such as diphenylphosphochloridate 141,142 and tetraphenylpyrophosphate in the presence of various bases failed to yield any phosphorylated products. However, the use of dibenzyldiisopropylphosphoramidate in the presence of tetrazol gave the expected phosphite product. 144,145,146 The crude phosphite was immediately oxidized using bleach in THF. Standard work up procedures followed by column chromatography afforded analytically pure product (4.17) in a yield of 52%. Subsequent removal of the benzyl protecting groups was realized, in a quantitative yield, by hydrogenation in the presence of 10% Pd-C

4.4 Enzymatic Studies

Compound 4.5 was tested as a potential substrate for Baker's yeast UTP:a-D-glucose-1- phosphate uridylyltransferase (EC 2.7.7.9). Any formation of the UDP-glucose analogue

4.7 would be accompanied by the simultaneous production of pyrophosphate. Production of pyrophosphate was monitored by the use of a commercial assay (Figure 4.4-1).'~~

Unfortunately, compound 4.5 was shown not to be a substrate for the Baker's yeast uridylyl transferase as no pyrophosphate was detected. Moreover, addition of 4.5 (0.1 mM) to a UTP:a-D-glucose-1-phosphate uridylyltransferase-catalyzed reaction containing glucose-1-phosphate (0.1 mM) and UTP (0.1 mM) resulted in a 12% reduction in the rate of pyrophosphate production.

UDP-glucose- 1-P uridylyl transferase

Glucose 1-P - UDP-Glucose

UTP PPi 1 Pyrophosphorylase 2Pi + MESG(2-amino-6mercapto-7- methyl-purine riboside Purine Nucleoside Phosphorylase 1 PNP (2-amino-6-mercapto-7-methylpurine) monitored at 360 nm

Figure 4.4-1 Phosphate Assay Therefore, it can be concluded that compound 4.5 is weak inhibitor of UTP:a-D-glucose-

1-phosphate uridylyltransferase. A distorted sugar ring is not a transition state mimic in the case of UDP-glucose-1-P uridylyl transferase enzyme. This is different from glycosidases The hydroxyl group on the phosphate moiety acts as a nucleophile displacing pyrophosphate from UTP forming the UDP derivative.

However, if compound 4.5 was chemically transformed into the UDP-Glc analogue 4.7 which is the donor in glycosyltransferases then we could confirm our rationale that the bicyclo[4.l.O]heptyl motif does indeed mimic the transition state conformation in glucosyltransferase-catalyzed reactions.

4.5 Attempted Chemical Synthesis of UDP analogue

An attempted synthesis of compound (4.7) involved the use of uridine-5'- monomorpholidophosphate with anhydrous pyridine as s01vent.l~~.Phosphate (4.5) was converted to the tri-n-octyl ammonium salt first before coupling it to uridine-5'- monomorpholidophosphate 23 in the presence of tetrazole. The reaction mixture was stirred for three days under an N2 atmosphere.

4.18

Figure 4.5-1 Uridine-5'-monomorpholidophosphate The 'H NMR spectrum of the crude product (after removal of pyridine) showed no starting material was present in the mixture. This mixture was loaded onto a HPLC C-18 reversed phase column and eluted with 50 mM ammonium acetate (pH 5.50) at a flow rate of 3 mL min-'. A compound that eluted out of the column (retention time 13 min) after lyophilisation was shown by 'H NMR spectroscopy to contain no signals from a uridyl moiety.

Furthermore, based on the change in 'H -31 P coupling patterns this material was assigned to be compound 4.19

Figure 4.5-2 Migration of the phosphate group

It is speculated that the coupling reaction took place, but since the uridine monophosphate moiety is a good leaving group, an intramolecular rearrangement occurs in the presence of base, to give the isomeric phosphate 4.19 (Figure 4.5-2). Figure 4.5-3 Proposed pathway for formation of 4.19

4.6 Conclusion and Future Work

To prevent migration of the phosphate group, all of the hydroxyl groups will have to be protected by acetate groups during the coupling reaction (Figure 4.6-1). Alternatively, a different coupling strategy will need to be employed in the future.

Figure 4.6-1 Alternate coupling method to avoid migration of phosphate group Although we were unsuccessful in synthesizing the UDP-Glc derivative, much of the ground work has been done in order to optimize the installation and purification of the bicyclo[4.l.O]heptyl phosphates 4.5 and 4.20. We were able to demonstrate that the cyclopropane ring system is stable under various coupling reaction conditions studied.

Also HPLC appears to be the most appropriate method for purification of deprotected glycosyl phosphate analogues. 4.7 Experimental

General Toluene and dichloromethane were distilled from CaH2 prior to use. THF was

dried over sodium in the presence of benzophenone. NMR spectra were recorded on

Varian 500 MHz spectrometer. All glassware was flame dried before use.

Dibenzyldiisopropyl phosphoramite was stored as a 50 mg/mL solution in anhydrous

dichloromethane under an atmosphere of nitrogen.

60 % NaH (112.3g) was washed repeatedly with hexanes (1.5 L) and transferred as a

slurry in DMF (1.5 L) into a 3 neck 5 L flask fitted with a dropping funnel and a

mechanical stirrer at 0 OC under N2. A solution of methyl a-D-glucopyranoside 4.8 (75 g,

0.39 mol) in DMF (200 mL) was added dropwise until effervescence ceased. Benzyl chloride (100 mL) was added dropwise over a period of 15 minutes and the resulting

solution was heated to about 75 "C. The oil bath was then removed and the rest of the

benzyl chloride (332 mL) was added dropwise. The mixture was stirred overnight.

MeOH (500 mL) was added dropwise till effervescence stopped. The mixture was then

heated under a gentle reflux for about 1 hour. After cooling to room temperature, the

reaction mixture was poured into water (1.5 L). The product was extracted from the

aqueous phase with ether (4 x 500 mL). The combined organic extract was washed with

water (2 x 400 mL), brine (2 x 400 mL), dried (Na2S04), filtered and concentrated at

reduced pressure. The crude product was obtained as a pale yellow syrup. To a solution of crude product in AcOH (2 L) at reflux was added aqueous H2S04(10%

(vlv), 750 mL) in portions over 1.5 hour period. The solution was then allowed to cool down slightly and it was then poured into water (9 L). The resulting mixture was allowed to crystallize for 2 days. The solid was filtered and recrystallized from MeOH (3 L) to give product as white crystals (104 g, 50% for two steps): mp 157-158 "C {lit 151-152

"c"~Rf 0.15, EtOAcltoluene 1:6; IR (KBr) 3420 (OH) 3030 (Ar C-H) cm-'; 'H NMR

(CDC13) 6 3.15 (brs), 1 H, OH), 3.55-3.66 (m, 3 H, H-2, H-4, H-6'), 3.71 (dd, 1 H, J5,6 =

3.8 HZ,J6,6' = 10.6 HZ, H-6), 3.97 (t, 1 H, J2,3 + J3,4 = 18.6 HZ, H-3), 4.02 (ddd, 1 H, J5,6,

= 2.2 HZ, J4,5 = 10.1 HZ, H-5), 4.48 (d, 1 H, JA,B=12.2 HZ, CHAHBC6H5),4.59 (d, 1 H,

CH,A.H&~HS), 4.64 (d, 1 H, JE,F= 11.8 HZ, CHEHK~H~),4.78 (d, 1 H, CHfiK6H5), 4.82

(d, 1 H, CHCHDC~HS),4.82 (d, 1 H, JG,H= 10.9 HZ, CHG&C6H5), 4.96 (d, 1 H,

CHGHHC6HS),5.23 (d, 1 H, Jl,2 = 3.6 Hz, H-1), 7.11-7.20 ( m, 2 H, H-Ar), 7.20-7.39 (m,

18 H, H-Ar); I3cNMR (CDC13) 6 68.6, 70.4, 73.3,73.5, 75.0, 75.6,77.7, 80.1, 81.7, 91.3

(C-1), 127.5 (Ar), 128.8 (Ar), 127.9 (Ar), 128.0 (Ar), 128.1 (2C, Ar), 128.1 (Ar), 128.3 (2

C, Ar), 137.8 (Ar), 137.9 (Ar), 138.2 ( Ar).

To a solution of 4.9 (21 g, 39 mmol) in DMSO (1 10 mL) was added acetic anhydride (75 mL). The resulting solution was stirred at room temperature for 14 hrs. A water1 toluene mixture (1: 1 vlv, 50 mL) was then added and evaporated. This process was done three times. The product obtained was dried under high vacuum to afford a colourless syrup

(20.4 g, 97%): Rf 0.55 (toluene1 EtOAc 6:l); IR (nujol) 1753 (C=O) cm-'; 'H NMR

140 (toluene, d-8) 6 3.39-3.42 (m, 2 H, H-6), 3.72 (t, J2,3 + J3,4 = 13.7 Hz, 1 H, H-3), 3.85 (dd,

1 H, J3,4=7.0HzlJ~,~=S.SHZ,H-4), 3.97 (dl 1 H, J2,3=6.7Hz,H-2),4.14(dt11 H,H-

5), 4.18 (d, 1 H, JA,B= 12.0 HZ,CHAHBC~HS), 4.36 (dl 1 H, JC,D= 1 1.6 HZ, CHCHDC6HS),

4.38 (d, 1 H, JE,F = 11.8 HZ, CHECHK6H5),4.48 (d, 1 H, JG,~= 11.5 HZ, CHGHHC6H5),

4.60 (d, 1 H, CHCHDC~H~),4.57 (dl 1 H, CHEHK~HS),4.94 (d, 1 H, CHGHHC6H5),7.00-

7.19 ( m, 20 H, H-Ar); I3c NMR (toluene, d-8) 6 68.8 (C-6), 73.6 (CHAHBC6H5),73.7

(~cHDC~H~),73.8 (CHGHHC~H~), 73.9 (~EHK~H~),76.7 (C-4), 78.2 ((2-2, C-5), 81.8

(C-3), 138.0, 138.3,138.5, 138.6, 168.4 (GO).

3,4,5,7-Tetra-0-benzyl-l-deoxy-l-(dimethoxyphosphoryl)-a-~-gluco-2- heptulopyranose (4.1l)lj9

Under a nitrogen atmosphere, a stirring solution of dimethyl methylphosphonate (8.0 mL,

74 mmol) in dry THF (120 mL) was cooled to -70 to -75 "C (dry ice I acetone). n-

Butyllithium (30 rnL, 1 equiv, 2.5 M in hexane) was added and stirring was continued for a further 30 min. The above cooled solution was then transferred by cannula into a cold solution of the lactone 4.10 (20 g, 37 mmol) in THF (100 rnL) maintained at -70 to -75

"C (dry icelacetone) and the resulting solution was stirred at this temperature for 2 hours and gradually allowed to warm up to room temperature overnight. Aqueous ammonium chloride (250 mL, 30% wlv) was added. The product was extracted from the aqueous solution with EtOAc (3 x 250 mL) and the combined organic extracts were washed with aqueous H2SO4 (400 mL, 10% vlv), saturated aqueous NaHC03 (400 mL), brine (400 mL), dried (Na2S04),filtered, and concentrated at aspirator pressure to give a pale yellow

141 syrup. The product was crystallized from etherlpetroleum ether and refrigerated for two days. After filtration and drying at high vacuum a white powder (16.6 g, 67 %) was obtained. mp 85-86 "C [lit. mp 112-113"CI; Rf 0.43 (EtOAcIhexanes 1:l); 'H NMR

(CDC13) 6 1.65 (dd, 1 H, Jl,,,= 15.2 HZ, Jl,p= 18.4 HZ, H-1), 2.27 (dd, 1 H, Jl',p=17.2

Hz H-l'), 3.24 (d, 1 H, J3,4 = 9.2 Hz, H-3), 3.58-3.65 (m, 2 H, H-5, H-7), 3.61 (d, 3 H,

JH,P=ll.lH~,PoCH3),3.66(d,3H,J~~,p= 11.2H~,POCH~),3.71 (dd, 1H,J6,7,=11.1

Hz, J7,7'= 10.6 HZ, H-77, 4.06 (m, 1 H, H-6), 4.12 (t, 1 H, J3,4+ J4,5= 18.8 HZ, H-4),

4.47 (s, 2 H, CH~C~HS),4.57 (d, 1 H, JA,B~10.9 HZ, CHAH~C~H~),4.64 (d, 1 H, Jc,~=

11.7 Hz, CHcH~C6H5),5.63 (brs, 1 H, OH), 7.15-7.32 (m, 20 H, H-Ar); 13c NMR

(CDC13) 6 32.8 (d, Jc,p = 134.8 HZ, C-1), 51.8 (d, JC,p = 6.0 HZ, POCH3), 53.3 (d, JC,p =

6.0 HZ, Porn3), 68.8 (C-7), 71.2 (C-6), 73.4 (m2C6H5), 74.8 (mAHBC6H5),75.2

(~cHDC~HS),75.6 (~ZCGH~),78.6 (C-5), 82.9 (d, Jc,p = 13.1 HZ, C-3), 83.2 (d, JC,p =

4.0 Hz, C-4), 96.8 (d, JC,p= 8.0 HZ, C-2), 138.0 (Ar), 138.1 (Ar), 138.5 (Ar), 138.7 (Ar).

3,4,5,7-Tetra-0-benzyl-l-deoxy-l-(dimethoxyphosphoryl)-a-~-glycero-~-gulo- heptitol and 3,4,5,7-Tetra-0-benzyl-l-deoxy-l-(dimethoxyphosphoryl)-~-~-glycero-

D-ido-heptitol (4.12)'~~

To a stirred solution of 4.11 (lO.Og, 15.1 mmol) in THF (100 mL) NaBh (0.74g, 1.3 equiv) was added in portions and the resulting mixture was stirred at room temperature overnight. The mixture was filtered and the solvent evaporated at aspirator pressure. The resulting residue was partitioned between H20 (200 rnL) and EtOAc (200 mL) and the aqueous phase was washed with EtOAc (2 x 200 mL). The combined organic extracts

142 were washed with H20 (200 mL), brine (200 mL), dried (Na2S04), filtered, and concentrated at aspirator pressure to give a colourless syrup (9.94 g, 99%). Rf 0.22

(EtOAcIhexane 4:l); 'H NMR (CDC13) (3 1.66 (td, 1 H, J1,2= 3.5 Hz, J1,l,= 15.4 Hz, Jl,p=

19.0 Hz, H-1), 2.27 (dt, 1 H, J13,2= 9.7 Hz, JI,l,+ JI,,~=31.5 Hz, H-1'), 3.62-3.67 (m, 7

H, 2(OCH3), CHAHBOBn),3.71 (dd, 1 H, J = 2.4 Hz, J = 5.0 Hz,), 3.75 (dd, 1 H, J = 3.1

Hz, J = 7.3 Hz), 4.01-4.11 (m, 3 H), 4.48-4.62 (m, 5 H, 5 x CH2C6Hs),4.67 (d, 1 H, Jc,~

= 11.2 HZ, CHCHDC6H5),4.75 (d, 1 H, CHcHDC6H5),4.85 (d, 1 H, CHHC6H5),7.22-7.36

(m, 20 H, H-Ar).

To a solution of dry DMSO (16.5 rnL, 231 mmol) in CH2C12 (200 mL) at -70 OC (dry icelacetone), TFAA (21.5 mL, 69 mmol) was added in a dropwise manner. The mixture was stirred for about 45 minutes during which time a white precipitate was formed. To the above reaction mixture a solution of 4.12 (1 lg, 17 mmol) in CH2C12 (150 rnL) cooled to -70 OC (dry icelacetone) was transferred by cannula in a dropwise fashion. After the

addition was complete the solution was stirred for 1.5 hours at the same temperature. To this mixture Et3N (5 mL, 68 mmol) was added and the mixture was allowed to warm to

room temperature overnight. After the addition of cold aqueous H2SO4(2N, 200 mL) the organic layer was separated and washed with saturated aqueous NaHC03 (200 mL) and concentrated at aspirator pressure to afford a pale yellow syrup. The crude product was partially purified by flash column chromatography (2:l vlv tolueneEtOAc) to give

product (9.4 g, 85%) as a colourless syrup which was used immediately for the next step.

To a stirred solution of the diketone 4.13 (9.4 g, 14 mmol) in dry CH2C12(300 mL) was

added LiCl.H20 (0.30 g, 0.5 equiv) and Et3N (2.18 mL, 1.1 equiv). The reaction mixture

was stirred for about 6 hours. The organic phase was washed with aqueous H2SO4(100

mL, 5% v/v), saturated aqueous NaHC03 (100 mL), brine (100 mL) and concentrated at

the aspirator pressure to afford a yellow syrup. The enone was purified by flash column

chromatography (hexanesEtOAc 2:l) to give product (4.17 g, 55% for 2 steps) as a

colourless syrup: Rf 0.34 (EtOAcIhexane 1:4); IR 1697 (C=O) cm-'; 'H NMR (CDC13) 6

. 3.99-4.10 (m, 3 H, H-5, H-6, C&HBOBn), 4.27 (d, 1 H, JA,~= 16.1 HZ, CHAHBOBn),

4.37 (brd, 1 H, J4,5 = 7.8 Hz, H-4), 4.50 (d, 1 H, Jc,~= 13.0 Hz, CHCHDC6H5),4.52 (d, 1

H, CHCHDC6H5),4.69 (d, 1 H, JE,F= 11 .O HZ, CHEHK~H~),4.74 (d, 1 H, JG,~= 11.4 HZ,

CHGH~C6H5),4.75 (d, 1 H, J~,J= 10.9 HZ, CHIHJC~H~),4.90 (d, 1 H, CHaK6H5),5.00

(d, 1 H, CH1HJC6H5),5.1 1 (d, 1 H, CHGH~C6H5),6.2 1 (dd, 1 H, J2,6 = 1.7 HZ, J2,4= 3.7

Hz, H-2), 7.20-7.45 (m, 20 H, H-Ar); "C NMR (CDCll) 6 69.0 (CH20Bn), 73.2

(mCH~C6H5),74.4 (~GHHC~H~),75.6 (~EHK~HS), 75.7 (m1HjC6H5), 79.2 (C-4),

83.9 (C-6), 84.9 (C-5), 123.9 (C-2), 127.7, 127.8, 127.9, 128.0 (2 C), 128.1 (2 C), 128.2,

128.4 (2C), 128.5, 137.4 (Ar), 137.6 (Ar), 137.8 (Ar), 159.0, 196.7 (C-1). (IS, 4R, 5S, 6S)-4,5,6-Tribenzyloxy-3-(benzyloxymethyl)-2-cyclohexen-l-ol(4.15)

A 1 M solution of L-selectride in THF (1 1.7 mL, 11.7 mmol) was added in a dropwise manner to a stirred and cooled (0 "C) solution of enone 4.14 (4.17g, 7.80 mmol) in THF

(200 mL) under a nitrogen atmosphere. The mixture was stirred for 18 h as it warmed to room temperature gradually. A saturated solution of N&Cl (50 mL) was added and the resulting mixture was stirred for 15 min after which water (100 mL) was added. Then the aqueous solution was extracted with EtOAc (2 x 150 mL), the organic extracts were washed with brine and dried over Na2S04. A pale yellow syrup was obtained upon removal of the volatiles under reduce pressure. The resultant crude product was purified by column chromatography (2:l v/v hexanes1EtOAc) to afford allylic alcohol 4.15 as a colourless syrup (3.22 g, 77%) [a120D= -39.0 (C = 1.00, CH2C12)'H NMR (CDC13) 6

2.57 (s, 1 H, OH), 3.59 (dd, 1 H, J6,5= 9.3 HZ, J6,i= 4.1 HZ, H-6), 3.94 (d, 1 H, J7a,7b=

12.3 HZ, CH7aH7bOBn),4.05 (dd, 1 H, J5,6 = 9.3 HZ, J5,4 = 6.8 HZ, H-5), 4.16 (dd, 1 H,

J4,2 = 1.0 HZ, H-4), 4.23 (dd, 1 H, J7b,2 = 1.0 HZ, CH7aH7bOBn),4.30 (t, I H, J1,2 + J1,6 =

8.9 HZ, H- I), 4.46 (d, 1 H, JA,~= 11.9 HZ, CHACHeC6H5),4.48 (d, 1 H, CHACHBC6H5),

4.66 (d, 1 H, Jcp = 10.3 HZ, CHCHDC6H5),4.68 (d, 1 H, JE,F= 11.5 HZ, CHEHK6H5),

4.74 (d, 1 H, JG,"= 11.2 HZ, CHGH&jH5),4.78 (d, 1 H, CHc&C6H5), 4.79 (d, 1 H,

CHG6H5),4.87 (d, 1 H, CHGH~C6H5),5.91 (dd, 1 H, J2,1 = 4.9 HZ, J2,6 = 1.4 HZ, H-2)

7.10-7.52 (m, 20 H, H-Ar); 13c NMR (CDCl3) 6 65.1 (C-1), 70.2 (C-7), 72.6

(~AHBC~HS),72.9 (~HK~HS), 74.0 (~cHDC~HS), 74.7 (~GHHC~H~),78.7 (C-4),

78.8 (C-6), 79.1 (C-5), 124.6 (C-2), 127.7, 127.6, 127.7, 127.9, 127.9, 128.0, 128.3,

128.3, 128.4, 128.5, 137.9, 138.1, 138.5, 138.6 (Ar-C) Anal calcd. (C35H3705):C, 78.33;

H, 6.76 Found. C, 78.17; H, 6.65.

145 To rigorously dried allylic alcohol 4.15 (500 mg, 1.0 mmol) in dry toluene (100 mL)

under nitrogen at -10 OC was added a 2 M solution of Me2Zn in toluene (4.0 mL, 8.0

mmol) mmol) dropwise. The mixture was stirred for 15 min, following which CH212(1.4

mL, 18 mmol) was added dropwise and the resulting mixture was stirred overnight as the

solution was allowed to warm up to room temperature. A saturated solution of NH&l

(50 mL) was added and the mixture was stirred for another 30 min. The resulting

solution was then diluted with water (100 mL) and extracted with EtOAc (3 x 100 mL).

The combined organic extracts were washed with 10% H2SO4(150 mL), NaHC03 (150

mL) and brine (150 mL) and then dried over Na2S04 to afford after removal of the

volatiles a yellow oil. This residue was purified by column chromatography (2: 1 v/v

hexanes/EtOAc) to afford the cyclopropanated allylic alcohol 4.16 as a colourless syrup

(317 mg, 62%) [a]20~= +24.5 (c = 1.10, CH2C12)'H NMR (CDC13) 6 0.52 (dd, 1 H, J7R,1

= 9.5 Hz, J7R,7S= 5.8 HZ, H-7R), 1.14 (t, 1 H, J7S,7R+ J7S,1 = 11.4 Hz, H-7S), 1.34 (m, 1 H,

H-I), 2.55 (bs, 1 H, OH), 2.67 (d, 1 H, J8a,gb = 10.0 HZ CHsaH8~OBn),3.42 (dd, 1 H, J3,4

= 9.6 HZ, J3.2 = 4.6 HZ, H-3), 3.60 (dd, 1 H, J4,5 ~7.8HZ , H-4), 3.81 (d, 1 H, H-8b), 4.18

(dl 1 H, H-3, 4.31 (dl 1 H, JA,B = 12.1 HZ, CHAHBC~H~),4.38 (d, 1 H, CHAHBC6H5),

4.43 (dd, 1 H, J1.2 = 7.9 Hz, H-2), 4.6 1 (d, 1 H, Jc,~= 1 1.4 HZ, CHCHDC6H5),4.63 (dl 1

H, JE,F=11.5 HZ, CHEHFC~HS),4.70 (dl 1 H, JG,H= 10.8 HZ, CHGHHC6H5),4.78 (d, 1 H,

CHcH~C6H5),4.82 (dl 1 H, CHGHHC~HS),4.84 (d, 1 H, CHm6H5),7.18-7.42 (m, 20

H, H-Ar); 13C NMR (CDC13) 6: 8.8 (C-7), 22.3 (C-1), 28.3 (C-6), 64.6 (C-2), 72.7

(~AHBC~H~),73.1 (CHCHDC~H~), 74.5 (~EHFC~H~),74.9 (C-8, ~GHHC~H~),78.6

146 (C-4), 79.2 (C-5), 80.9 (C-3), 127.4, 127.6, 127.6, 127.7, 127.8, 128.2, 128.3, 128.4,

128.4 (Ar-C), 138.0, 138.2, 138.6, 139.0 (Ar-C). Anal calcd. (C36H3905):C, 78.52; H,

6.95 Found: C, 78.40; H, 6.95.

(IS,2S, 3R,4S, 5R, 6R)-3,4,5-Tris(benzy1oxy)-6-((benzyloxy)methyl)- bicyclo[4.1.0]heptan-2-yl dibenzyl phosphate (4.17)

Dibenzyl diisopropylphosphoramidite (358 mg, 1.03 mmol) and tetrazole (105 mg, 1.50 mmol) were mixed vigorously in dry CH2C12 (30 mL) under nitrogen for about 30 min after which time the tetrazole had partially dissolved. A solution of alcohol 4.16 (275 mg, 0.50 mmol), which had previously been coevaporated with dry toluene (3 x 50 mL), in dry CH2C12 (20 mL) was added to the above reaction mixture via a syringe. The resulting reaction mixture was stirred until the starting material had disappeared

(monitored by TLC 2: 1 vlv hexaneslethyl acetate). Then the solution was concentrated under reduced pressure and the resulting residue was purified by column chromatography

(2:l vlv hexanes/EtOAc) to give the expected phosphite. After this material had been dissolved in THF (75 mL) a bleach solution (2 rnL, 5 % vlv) was added and this mixture was stirred for about 1 h. The solvent was removed under reduced pressure, and then water (100 mL) was added and the aqueous solution was extracted with CH2C12 (3 x 50 mL). The combined organic extracts was washed with water (100 mL) and dried over

MgS04. After removal of the solvent under reduced pressure, the residue was purified by column chromatography (8: 1 vlv toluenelacetone) to afford a colourless oil (213 mg,

52%) [a]20n = +58.0 (C = 1.00, CH2C12) 'H NMR (CDC13) 6:. 0.53 (dd, 1 H, J7~,,= 9.5 Hz,J7~,7s=5.8HzH-7R), 1.12(t, 1 H,J~s,~R+J~s,~= 11.4Hz,H-7S), 1.41 (m, 1 H,H-1),

2.60 (d, 1 H, J8a,8b= 10.0 HZ, H-8a), 3.44 (m, 1 H, H-3), 3.56 (dd, 1 H, J4,5 = 8.1 HZ,J4,~

= 10.1 HZ, H-4), 3.72 (d, 1 H, H-8b), 4.16 (d, 1 H, H-5), 4.30 (d, 1 H, JA,~= 12.3 HZ,

CHACHBC~HS),4.31 (d, 1 H, CHACHsC6H5), 4.57-4.64 (m, 3 H, CHcHDC6H5,

CHEHFC~HS,CHGHHC~HS) 4.75-4.88 (m, 3 H, CHCHDC~H~,CaK6H5, CHGHHC6H5),

4.94-5.08 (m, 4 H, 2P(0)OCH20Ph), 5.33 (dt, 1 H, J2.3= 4.5 Hz, JI,~+ J2,P = 15.4 Hz, H-

2) 7.18-7.40 (m, 30 H, H-Ar); 13cNMR (CDC13) 6 10.0 (C-7), 22.1 (C-1), 29.1 (C-6),

69.3 (2C, P(0)OCH20Ph), 72.7 (C-2), 72.6 (mAHBC6H5),72.64 (mcHDC6H5),74.2

(C-8), 74.7 (~EHFC~H~),74.9(mGH&H5 ), 78.2 (C-4), 78.4 (C-3) 127.4, 127.5,

127.6, 127.7, 128.0, 128.1, 128.2, 128.3, 128.4, 128.5, 128.7, 135.5, 135.6, 136.1, 138.1,

138.5, 138.9 (Ar-C); 31~NMR 6 = - 0.53. Anal calcd (C50H5208P):C, 74.06; H, 6.34

Found: C, 73.76; H, 6.49.

(lR, 2R, 3S, 4S, 5S, 6s)-3,4,5-Trihydroxy-6-(hydroxymethy1)-bicyclo[4.1.O]heptan-2- yl dihydrogen phosphate (4.5)

To a solution of 4.17 (200 mg, 0.25 mmol) in dry methanol (20 mL) was added 10% Pd on carbon (84 mg). The mixture was stirred under an atmosphere of hydrogen for 24 h.

The solution was filtered and the residue was washed thoroughly with MeOH. The combined filtrate was evaporated to dryness and dried under vacuum to afford a colourless solid (66.4 mg, 99% yield). [a]20~ = + 23 (c = 1.00, H20) 'H NMR (D20) 6:

0.58 (dd, 1 H, J7~,1= 9.5 Hz, J~R,~s= 5.9 Hz H-7R), 0.91 (t, 1 H, J7S,7R+ J7~,i = 11.3 HZ,

H-7S), 1SO (m, 1 H, H-I), 2.84 (d, 1 H, Jga,gb = 11.9 HZ, H-8a), 3.29-3.39 (m, 2 H, H-3,

148 H-4), 3.94 (dl 1 H, H-8b) 4.00 (d, 1 H, JjYs= 7.6 HZ,H-5), 4.71 (td, 1 H, J2,3= 4.5 HZ,J2,1

13 + J2,p = 15.4 Hz, H-2); C NMR (D20)6 12.0 (C-7), 24.7 (C-1), 33.4 (C-7), 68.3 (C-8),

72.3 (C-3), 73.9 (C-4), 74.7 (C-5), 74.9 (C-2); "P NMR 6 = - 0.59.

(lR,2R, 3S, 4S, SS, 6S)-3,4,5-Trihydroxy-6-(hydroxymethyl)-bicyclo[4.l.O]heptan-2-

2-yl dihydrogen phosphate

To a solution of 4.5 (40.0 mg, 150 pmol) in water (3 mL) pyridine (5 mL) and tri-n- octylamine (64.0 pL, 150 pmol) were added. The resulting mixture was evaporated and coevaporated several times with anhydrous pyridine (8 x 1.5 mL). 4-morpholine-N,N- dicyclohexylcarboxamidinium uridine 5'-monophosphomorpholidate (172 mg, 236pmol) and 1H-tetrazole (33 mg, 472 pmol) were coevaporated with anhydrous pyridine (8 x 1.5 mL) and transferred as a solution in anhydrous pyridine (2 mL) to the reaction flask containing the dry phosphate salt. The resulting mixture was stirred under N2 at room temperature for 3 days. After 3 days, the pyridine was evaporated to dryness under reduced pressure and mixture was diluted with water (1 mL). The crude compound was purified by reversed phase HPLC using 50 mM ammonium acetate at pH 5.50 at a flow rate of 3 mL min-'. After lyophilisation, a colourless liquid was obtained (2.5 mg, 2%).

The 'H NMR spectra of the product 4.19 showed the presence of an impurity (-20%) that could not be identified.'~NMR (D20) 6 0.59 (q, J7~,6 = 9.5 Hz, J7R,7s = 5.9 Hz H-7R),

0.86 (t, 1 H, J~s,~R+J7~,6= 11.3 HZ, H-79, 1.42 (m, 1 H, H-6), 2.80 (d, 1 H, JA,~=11.9

HZ, CHAHBOH),3.37 (t, 1 H, J3.2 + J3,4 = 19.7 HZ, H-3), 3.85 (d, 1 H, J2,3 = 9.5 HZ, H-2), = 6.5 Hz, H-4), 4.96 (t, 1 H, J5,4 + Js,6 = 14.8 Hz, H-5). "P NMR 6 = + 16 ppm (H3PO4 reference)

Enzyme Kinetics Protocol

Each 1 mL run containing of 50 mM Tris HCl buffer pH 7.5; MESG substrate (2-amino-

6-mercapto-7-methylpurine riboside) (0.2 mM); pyrophosphorylase (1 x 10.' units), of purine nucleoside phosphorylase (PNP) (1 x units); glucose-l-phosphate (0.1 mM); compound 4.5 (0.1 mM) and UTP (0.1 rnM) was pre-incubated for 10 min at 22 "C. The reaction was initiated by the addition of UTP-glucose-l-phosphate uridylyl transferase (1 x units) and monitored at 360 nm for the production of 2-amino-6-mercapto-7- methylpurine.

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