1. Synthesis of C-glycoside sulfones via oxirane- thirane exchange 2. Preparation of sialic acid derivatives amenable to solid-phase synthesis 3. Conformational analysis of complex polysaccharides

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1

1. SYNTHESIS OF C-GLYCOSIDE SULFONES VIA OXIRANE-THIIRANE

EXCHANGE. 2. PREPARATION OF SL\LIC ACID DERIVATIVES AMENABLE TO

SOLID PHASE SYNTHESIS. 3. CONFORMATIONAL ANALYSIS OF COMPLEX

POLYSACCHARIDES.

by

Terrence Michael Flaherty

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF CHEMISTRY

in Partial Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

in the Graduate College

THE UNIVERSITY OF ARIZONA

1997 UMI Nuzober: 9729494

UMI Microform 9729494 Copyright 1997, by UMI Company. All rights reserved.

This microform edition is protected against unauthorized copying under Title 17, United States Code.

UMI 300 North Zeeb Road Ann Arbor, MI 48103 THE UNIVERSITY OF ARIZONA ® GRADUATE COLLEGE

As members of the Final Examination Committee, we certify that we have read the dissertation prepared by Terrence M. Flaherty entitled Synthesis of C-Glycoside Sulfones via Oxirane-Thiirane

Exchange. 2. Preparation of Sialic Acid Derivatives Amenable

to Solid Phase Synthesis. 3. Conformational Analysis of Complex Polysaccharides.

and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor Of Philosophy

Date Inhy Richard^ Glass Date

Kobin Holt Date

—M li, Fex> ?7 F. Ai>n Walker Date i3P^n David Wigley Date

Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. 3

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under the rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNED: 4

ACKOWLEDGMENTS

I would like to thank my advisor. Professor Jacquelyn Gervay, for giving me the opportunity to work on some very interesting chemistry and for challenging me to excellence both in the laboratory and in the classroom.

1 would also like to thank my parents, Lawrence and Margaret Flaherty, for their love, support, and encouragement throughout the years which was so important in making this possible. 5

TABLE OF CONTENTS

LIST OF FIGURES 6

LIST OF TABLES 11

ABSTRACT 12

CHAPTER 1. SYNTHESIS OF C-GLYCOSIDE SULFONES VIA OXIRANE- THIIRANE EXCHANGE 13

INTRODUCTION 14

RESULTS AND DISCUSSION 24

CONCLUSION 50

EXPERIMENTAL SECTION 50

CHAPTER 2. PREPARATION OF SIALIC ACID DERIVATIVES AMENABLE TO SOLID-PHASE SYNTHESIS 67

INTRODUCTION 68

RESULTS AND DISCUSSION 87

CONCLUSION 99

EXPERIMENTAL SECTION 99

CHAPTER 3. CONFORMATIONAL ANALYSIS OF COMPLEX POLY­ SACCHARIDES 106

INTRODUCTION 107

RESULTS AND DISCUSSION 118

CONCLUSION 130

EXPERIMENTAL SECTION 131

APPENDIX A. NMR AND IR SPECTRA 133

REFERENCES 236 6

LIST OF FIGURES

Figure 1.1. Inflammatory Response 15

Figure 1.2. Sialyl Lewis* determinant 16

Figure 1.3. Tunicamycin 18

Figure 1.4. Uridine sulfonamide 18

Figure 1.5. Methylene diphosphonate nucleotide 19

Figure 1.6. Proposed synthetic route to GDP fucose 20

Figure 1.7. Known synthetic routes to thiiranyl acetals 21

Figure 1.8. 1,2,3-Thiadiazoline decomposition products 22

Figure 1.9. Oxirane-thiirane exchange mechanism with alkali thiocyanates 22

Figure 1.10. Oxirane-thiirane exchange mechanism with triphenylphosphine sulfide 23

Figure 1.11. Oxirane-thiirane exchange mechanism with dimethylthioformamide 24

Figure 1.12. Synthetic scheme for initial model study of oxirane-thiirane exchange 25

Figure 1.13. Synthetic strategy to C-glycosyl sulfones via oxirane-thiirane exchange of exocyclic 1,2-anhydrosugars 27

Figure 1.14. Second generation approach to the preparation of C-fucosyl sulfones 29

Figure 1.15. Stereospecific epoxidation of glucal (top) and allal (bottom) 30

Figure 1.16. Hydride (deuteride) addition to 1,2-anhydrosugars 31

Figure 1.17. Hydride addition to exocyclic 1,2-anhydrosugars 33

Figure 1.18. Strategy for C-glycoside sulfoxides or sulfones via nucleophilic addition to endocyclic epoxides 35

Figure 1.19. Sttategy for the preparation of C-glycoside sulfones using methylmethanesulfonylacetate 37

Figure 1.20. Approach to C-glycoside sulfones via reaction of exocyclic 1,2-anhydrosugars with TMSI 39

Figure 1.21. Proposed mechanism of oxazoline formation from an epoxide 40 7

Figure 1.22. Oxazoline synthesis from an epoxide 41

Figure 1.23. Free radical addition of thiolacetic acid to tri-O-acetyl-D-glucal 42

Figure 1.24. Free radical addition of thiolacetic acid to perbenzyl exocyclic glycal 42

Figure 1.25. Proposed mechanism for radical addition of thiolacetic acid to an exocyclic glycal 43

Figure 1.26. Stereoselectivity for glycosyl radicals 44

Figure 1.27. Synthesis of p-C-glycosides by hydrogen atom transfer 44

Figure 1.28. Stereoselectivity of cyclohexyl radicals 45

Figure 1.29. Deprotection of the C-glycoside thiolacetate and methylation 45

Figure 1.30. Oxidation of C-glycoside sulfide to the sulfone with dimethyldioxirane 46

Figure 1.31. Oxidation of the C-glycoside sulfide to the sulfoxide 47

Figure 1.32. Synthetic scheme to C-fucosyl sulfone 48

Figure 1.33. Preparation of C-fucosyl sulfoxides by oxidation with m-CPBA... 49

Figure 1.34. Model study examining the coupling of C-fucosyl sulfone with a tosyl halide (X = F or CI) 50

Figure 2.1. Krepinsky's solid-phase method for the preparation of oligosaccharides 69

Figure 2.2. Kahne methodology for the solid-phase preparation of (3-linked oligosaccharides 70

Figure 2.3 Danishefsky's protocol for the preparation of oligosaccharides (P = protecting group, S = solid support) 71

Figure 2.4. Lewis^ blood group determinant 72

Figure 2.5. Kopper method for die solid-phase preparation of oligosaccharides 74

Figure 2.6. Wong's chemical-enzymatic approach to the synthesis of sialyl Lewis* glycopeptide 76

Figure 2.7. Synthetic Strategy to inhibitors of H. pylori 77

Figure 2.8. Prototypical carbohydrate amino acid 78 8

Figure 2.9. Carbohydrate amino acid prepared by Paulsen 79

Figure 2.10. Carbohydrate amino acids prepared by Lehmann 79

Figure 2.11. Yoshimura's disaccharide with an amide linkage 80

Figure 2.12. Monomeric unit for Wessel's tetrasaccharide synthesis 80

Figure 2.13. Wessel's block strategy to a carbohydrate-peptide tetramer 81

Figure 2.14. Kessler's synthesis of a carbohydrate amino acid 83

Figure 2.15. Merrer's methodology for the preparation of carbohydrate amino acids 84

Figure 2.16. General structure of Ichikawa's carbohydrate amino acid tetramer.. 85

Figure 2.17. Ichikawa strategy for the coupling of carbohydrate amino acids 85

Figure 2.18. Persulfated P( 1 -»6)-linked oligosaccharide synthesized by Ichikawa 86

Figure 2.19. Methyl 2,6-anhydro-D-^/ycero-D-^w/o-hepturonate 86

Figure 2.20. A^-acetyl neuraminic acid 87

Figure 2.21. Strategy to sialic acid free amine using Hanessian's methodology.. 88

Figure 2.22. Partially sffategy for the removal of tlie //-acetyl group on sialic acid via rerr-butoxycarbonylation and subsequent hydrazinolysis 89

Figure 2.23. Unsuccessful strategy to iV-deacylated sialic acid via a 1,4-lactone. 90

Figure 2.24. Proposed hydrazinolysis mechanism and subsequent addition of acetyl hydrazine to per-(9-benzoyl 1,4-lactone of sialic acid 91

Figure 2.25. Grieco's methodology for the methanolysis of lactams 92

Figure 2.26. Strategy to A/-BOC sialic acid derivative via imidate methanolysis.. 92

Figure 2.27. Lactonization of N-BOC sialic acid derivative upon acetylation 93

Figure 2.28. Fuch's methodology for N-deacylation 94

Figure 2.29. A'-deacylation of the methyl glycoside methyl ester of sialic acid... 94

Figure 2.30. Methyl ester protection of sialic acid free amine 95 9

Figure 2.31. Preparation of amide-linked sialic acid dimers 95

Figure 2.32. Attempted coupling of A/-BOC sialic acid derivative with the methyl glycoside methyl ester free amine of sialic acid 96

Figure 2.33. Preparation of free acid monomer unit 97

Figure 2.34. Revised strategy for the preparation of amide-linked sialic acid oligomers 98

Figure 3.1. Group A polysaccharide of Neisseria meningitidis 108

Figure 3.2. Group B polysaccharide of Neisseria meningitidis 108

Figure 3.3. Group C polysaccharide of Neisseria meningitidis 108

Figure 3.4. Preparation of iV-propionylated group B meningococcal polysaccharide Ill

Figure 3.5. Colominic acid 112

Figure 3.6. Internal esterification of die group C meningococcal polysaccharide 115

Figure 3.7. Internal esterification of the group B meningococcal polysaccharide 116

Figure 3.8. Aurintricarboxylic acid (left) and dexu-an sulfate (right) 117

Figure 3.9. 300 MHz NMR spectrum of colominic acid polylactone at 970c in DMSO-^/6 118

Figure 3.10. 75 MHz NMR spectrum of colominic acid polylactone 120

Figure 3.11. 75 MHz HET2DJ specuiim of colominic acid polylactone 121

Figure 3.12. 75 MHz HETCOR spectrum of colominic acid polylactone 123

Figure 3.13. NOE buildup curves of the colominic acid polylactone 124

Figure 3.14. Sialyllactone dimer used in conformational search 125

Figure 3.15. Boat conformation of the disialyllactone 126

Figure 3.16. Chair conformation of the disialyllactone with no hydrogen bonding 126

Figure 3.17. Chair conformation of the disialyllactone exhibiting intramolecular hydrogen bonding 126 10

Figure 3.18. Linear regression for the three low energy pyranose conformations of the sialyllactone dimer 127

Figure 3.19. Final calibration plot relating NOE to distance for the sialyllactone dimer 128

Figure 3.20. Two helical conformations of the colominic acid polylactone consistent with the NOE data 129

Figure 3.21. Gauche conformation of l-*4-linked C-disaccharide 130 11

LIST OF TABLES

Table 1.1. Summary of reaction conditions employed to effect oxirane- thiirane exchange in endocyclic 1,2-anhydrosugars 26

Table 1.2. Hydride addition of l,2-anhydro-3,4,6-tri-0-benzyl-D-glucose.. 32

Table 1.3 Summary of reagents used for hydride addition to exocyclic 1,2-anhydrosugars 34

Table 1.4. Reagents and conditions employed for addition of dimsyl carbanions to endocyclic 1,2-anhydrosugars 35

Table 1.5. Reagents and conditions employed for the attempted addition of dimethyl sulfone carbanion to an endocylic 1,2-anhydrosugar. 36

Table 1.6. Reagents and conditions employed for the addition of methyl methane-sulfonyl acetate carbanion to an endocyclic 1,2- anhydrosugar 38

Table 3.1. Complete chemical shift assignment of the colominic acid polylactone 119

Table 3.2. Carbon multiplicity and ^yc.H coupling constant data derived from the HET2DJ spectrum 122

Table 3.3. Complete *^C chemical shift assignment of colominic acid polylactone 123 12

ABSTRACT

As part of a program directed toward the synthesis of novel glycosyl transferase inhibitors possessing a sugar-CH2-S02-CH2-S02-CH2-nucleoside structure, P-C- glycoside sulfones have been prepared with high stereoselectivity. Both glucose and fucose derivatives were prepared. Sulfur incorporation was accomplished using free radical addition of thiolacetic acid to exocyclic glycals.

As part of a program directed toward the preparation of amide-linked sialic acid oligomers, a strategy was developed for the synthesis of sialic acid derivatives possessing either a free amine or a free acid functionality. Solution phase coupling of these monomers using standard peptide coupling techniques resulted in the synthesis of (l-»5)-amide linked sialic acid dimers.

As part of a program directed toward the identification of novel helical structures, the solution phase conformation of the polylactone of colominic acid was examined by

NMR and molecular modeling. The two structures generated from molecular modeling that were consistent with the NOE data were both helical. CHAPTER 1

PREPARATION OF C-GLYCOSIDE SULFONES VIA OXIRANE- THIIRANE EXCHANGE 14

INTRODUCTION

Carbohydrate interactions with proteins have been discovered to play an important role in inflammatory response. A family of these carbohydrate-binding proteins are known as selectins. The selectins are involved in the regulation of both lymphocyte recirculation and early events in blood cell interaction with endothelial cells during an inflammatory response. The three known types of these carbohydrate binding proteins (the L-, E-, and

P-selectins) have been indentified in lymphocytes, endothelial cells, and platelets. L- selectin is expressed on lymphocytes, E-selectin is expressed on activated endothelial cells, and P-selectin is expressed on platelets and endothelial cells.

L-selectin plays an important role in the adhesion of lymphocytes to high endothelial venules (HEV) and the recirculation of lymphocytes between blood and the lymph.^ L- selectin is expressed on all leukocytes, including neuU'ophils,'^ and it appears to play a role in the binding and extravasation of these neutrophils into sites of inflammation.^

E-selectin plays a crucial role in the infammatory response system of the body by sending neutrophils and monocytes to sites of inflammation (Figure 1.1).^ Upon inflammation, E-selectin is expressed on endothelial cells within 2-8 hours after the endothelial cells have been activated by interleukin-I (IL-1) and other cytokines. Within

24 hours, the level of E-selectin has been greatly reduced.^ E-selectin is believed to promote arrest of neutrophils during stress in the venous flow whereby the neutrophils are delivered to integrin receptors such as ICAMl or ICAM2 and transendothelial migration is achieved.®-^ 15

o cytokines endothelial cells

white blood cells

E-selectin

Figure 1.1. Inflammatory' reponse. Upon injury to tissue (A), cvtokines are released which promote the production of E-selectins (B). Adhesion occurs through the interaction of E-selectins and sialyl Lewis* on white blood cells (C, D). White blood cells reach the site of injury through spaces between endothelial cells (E).

P-selectin exists in the a-granules of platelets and the Weibel-Ralade bodies of vascular endothelial cells. ^ ^ ^ P-selectin is expressed on the surface of endothelial cells or platelets within several minutes after exposure to thrombin or oxygen radicals.'- P- 16 selectin is also believed to be involved in the early events of cell adhesion to inflamed sites and is necessary for transendothelial migration.

A great deal of research has focused on the carbohydrate binding specifities of the selectins. L-selectin has been found to interact with activated endothelium and high endothelial venules (HEV) if sialylated, fucosylated, or sulfated carbohydrates are expressed on leukocyte surfaces.'^ The E- and P-selectins, however, recognize specific carbohydrate determinants on the surfaces of neutrophils and monocytes. In particular, these selectins recognize determinants on the cell surfaces related to sialyl Lewis* (Figure

L2).

OH OH

OH OH OH NHAc

OR OH OH OH

AcHN HO

Figure 1.2. Sialyl Lewis* determinant.

Research by Lowe'^ and Paulson'^ have clearly demonstrated that only cells that expressed sialyl Lewis* on their surfaces were able to avidly bind E-selectin. Zhou'^ has shown that sialyl Lewis* on a cell surface is necessary but not sufficient for binding to P- selectin. Modification of the sialyl Lewis* ligand or other determinants is necessary for high avidity binding of P-selectin. 17

Studies on the interaction of sialyl Lewis* with the selectins are designed to more fully

understand the role of cell adhesion in inflammation and tumor metastasis. Sialyl Lewis*

is overexpressed on the cell suface of tumor cells. Because all selectins recognize

carbohydrate moieties on the surfaces of tumor cells, it is believed that the selectins may

play a significant role in the spread of malignancies.^® Magnani'^ has suggested that the

expression of the sialyl Lewis* antigen results in attachment of cells to E-selectin thereby

being a causative agent in the early events of cell adhesion leading to tumor cell

extravasation.

The goal of our research directed in this area is to develop potential glycosyl

transferase inhibitors which will prevent the glycosylation of either fucose or sialic acid

on the lactosaminoglycan core structure. Since sialic acid and fucose are the essential

carbohydrates necessary for the binding of cell surface carbohydrate determinants with

selectins, inhibition of either sialic acid or fucose incorporation to the glycan core

structure may result in a loss of selectin binding. It is hoped that this new class of

glycosyl transferase inhibitors can provide potential therapies against diseases where cell

adhesion plays an important role such as cancer,^®"^^ arthritis^^-^^-^'^ and Parkinson's

disease.^^

Several glycosoyl transferase inhibitors are known. Tunicamycin (Figure 1.3), an

isostere of nucleoside diphosphates, is a naturally occurring antitumor antibiotic that inhibits dolichol-mediated glycoprotein synthesis^^. However, due to its high toxicity,

tunicamycin is not a useful therapeutic agent. 18

0

H( O NHAc

OH OH

Figure 1.3. Tunicamycin.

Alternative analogs to tunicamycin have been developed which maintain the 5-bond distance between the sugar and the 5' nucleoside moieties and which attempt to provide a more stable linkage between the sugar and the nucleoside than a diphosphate linkage.

Gil-Fernandez has prepared a uridine sulfonamide glucose analog (Figure 1.4) which possesses antiviral activity.^'

,OBz

BzO- O BzO

OBz

OH OH

Figure 1.4. Uridine sulfonamide. 19

Vaghefi^® has prepared a methylene diphosphonate nucleotide (Figure 1.5) which was

shown to inhibit galactosyltransferase activity. This compound did not, however,

possess any antitumor or antiviral activity.

OH

HO

OH OH

Figure 1.5. Methylene diphosphonate nucleotide.

In our case, we were interested in preparing sugar nucleotide isosteres that have a sugar-CH2-S02-CH2-S02-CH2-nucleoside general structure. It is believed that these compounds will serve as strong inhibitors of fucosyl and sialyl transferase. Inhibition of either fucose or sialic acid on lactosaminoglycans will prevent the formation of the sialyl

Lewis* determinant, the component recognized by the selectins and thus believed to be involved in inflammation and tumor metastasis. Substitution of an (9-glycosidic linkage with a C-glycosidic linkage should render the target resistant to glycosidases. These isosteres have dimethylene sulfone linkages that are stable to hydrolysis rather than the phosphodiester linkages that occur in vivo. Sulfones are nonionic, achiral isosteres of phosphodiesters that are stable to both chemical and biochemical hydrolysis.^' Sulfones also increase membrane permeability thereby assisting penetration into cells.Methyl phosphonate oligonucleotide analogs have been prepared but diastereomers are created by 20

the chiral phosphorus.^' These diastereomers create binding affinity problems to natural oligonucleotides. This problem is avoided by using achiral sulfone analogs.

1.dimethyldioxirane 1. LAH, THF O'^^OBn CH2CI2 reflux

2. Ph3PS, TFA, PhH 2. CS2CO3, Mel Oxone OBn

1.1 VJ

> KHMDS, -78°C i -

I OBn OBn

ClOgS' N" "NRg

OBnOBn

3. H2. Pd(C)

OH OH

Figure 1.6. Proposed synthetic route to GDP-fucose. 21

The synthetic targets were the sulfone isosteres of GDP-fucose and CMP-sialic acid.

Our work focused exclusively on the synthesis of the GDP-fucose sulfone isostere. The

first generation synthetic strategy is given in Figure 1.6. The exocyclic methylene fucose

can be prepared in five steps from a-L-fucose. Epoxidation^^'^'^ with dimethyldioxirane^^

provided a 2:1 mixture of epoxides based on integration of the methyl singlets. The

unique feature of our methodology involved oxirane-thiirane exchange to produce a

thiiranyl acetal. The thiirane ring was then to be opened by hydride addition to the

anomeric center yielding a C-glycoside thiol. The thiol was to subsequently be

methylated and oxidized to a sulfone. The sulfone was then to be coupled to the guanine

sulfonyl chloride which upon global deprotection was to provide the desired sulfone

isostere of GDP-fucose.

The chemistry of thiiranyl acetals has not been well studied. Only two known

methods for the preparation of thiiranyl acetals have been reported (Figure 1.7).

Singh'^-^^ has prepared them by photochemical addition of alcohols to thioketenes, and

Beiner^®-'^ has prepared 2-alkoxy thiiranes via reaction of thioesters with diazocompounds.

s s

S R ROH

R

Figure 1.7. Known synthetic routes to thiiranyl acetals. 22

Beiner's methodology, however, suffers from poor yields and side product formation.

Addition of diazomethane across the thiocarbonyl produces a 1,2,3-thiadiazoline which

can decompose to give enol ethers or thiolesters (Figure 1.8).

OR' S Thermal 1 CH2N2 A Elimination R OR" R OR'

Alkyl insertion

R R OR' OR'

Figure 1.8. 1,2,3-Thiadiazoline decomposition products.

M M M S— SCN" Y N-

O— / + GCN" S Me' S"

Figure 1.9. Oxirane-thiirane exchange mechanism with alkali thiocyanates. 23

Our methodology for the preparation of thiiranyl acetals involves the use of oxirane- thiirane exchange. There are four reported procedures for oxirane-thiirane exchange.

Kirk^® reported the first preparation of thiiranes from epoxides by reaction with alkali thiocyanates. The oxirane-thiirane exchange proceeds by trans opening of the epoxide by the thiocyanate. Then, a second Walden inversion takes place whereby trans closure of the thiirane ring occurs (Figure 1.9).

Chan'^' later employed triphenylphosphine sulfide as a reagent for oxirane-thiirane exchange with moderate yields (35-64%). Nucleophilic attack of triphenylphosphine sulfide opens the epoxide ring and attack of the free hydroxyl group to the pentavalent phosphorus atom affords a five-membered heterocyclic ring. The ring then undergoes pseudorotation, and, in the presence of acid, the sulfur atom is protonated, resulting in ring opening. Elimination of triphenylphosphine oxide then affords the thiirane (Figure

1.10).

Ph

"Is

pseudorotation ^^miiR "*-0 Rs Pn^ I U R,'2 Ph Ph

Figure 1.10. Oxirane-thiirane exchange mechanism with triphenylphosphine sulfide 24

A higher yielding oxirane-thiirane exchange reaction has been reported by Takido.'*^ This

method involves the use dimethylthioformamide (DMTF) in the presence of a catalytic

amount of trifluoroacetic acid to form thiiranes in good yields (62- 95%). The reaction

occurs by nucleophilic attack of the sulfur atom in dimethylthioformamide on the least

substituted carbon of the epoxide. Ring closure of the intermediate affords a five-

membered oxonium ring. Elimination of dimethylformamide yields a thiirane (Figure

1.11). rsJMes o n Me H , H*0 S Me R'

/ \ + DMF

Figure 1.11. Oxirane-thiirane exchange mechanism with dimethylthioformamide.

Recently, it has been reported'^^ that thiiranes have been obtained from oxiranes in very high yields (90-95%). The method involves treatment of the epoxide with thiourea in the presence of methanol. However, this method was not suitable for our purposes since our

1,2-anhydrosugars react readily with methanol.

RESULTS AND DISCUSSION

Our work initially focused on model studies for the preparation of thiiranyl acetals via oxirane-thiirane exchange. Rather than employing the exocyclic fucose compound which can be prepared in five steps from a-L-fucose, commercially available iri-(9-benzyl-D- 25

glucal was used as a model system. The glycal was epoxidized quantitatively and

stereospecifically^^ with 3,3-dimethyldioxirane (DMDO). The epoxide was subjected to a

variety of reagents and conditions whereby oxirane-thiirane exchange could be effected

(Figure 1.12)

BnO BnQ

BnOy^ DMDO BnO Oxirane- BnO thiirane DCM, 0°C exchange

BnQ

Figure 1.12. Synthetic scheme for initial model study of oxirane-thiirane exchange.

A number of reaction conditions were employed in order to prepare the endocyclic 1,2-

thioanhydrosugars (Table 1.1). Triphenylphosphine sulfide addition to the epoxide without any sort of promoter resulted in no reaction. Triphenylphosphine sulfide was not nucleophilic enough to open the endocyclic epoxide ring. A variety of promoters were then employed (TFA and Lewis acids) with uiphenylphosphine sulfide in order to open the oxirane. Monitoring these reactions by TLC revealed no change in Rf, but crude

NMR spectra showed loss of starting material (the characteristic doublet at 3.02 ppm for the H-1 proton of the 1,2-anhydrosugar). In order to determine if thiiranes were being formed in these reactions, the crude products were either dissolved in methanol or acetylated. Methanolysis should result in methanol addition to a newly formed thiirane. 26

Acetylation was performed in order to see if S-acetylation rather than 0-acetylation could

be observed since the NMR chemical shifts of 5-acetates differ from those of O- acetates. Neither methanol incorporation nor i'-acetylation was observed in any example indicating only that the epoxide was opened under acidic conditions but no thiirane was formed.

Reagents Temperature ("C) SPPh^, THF 25 SPPh^, THF, ZnCl2 -78 SPPh:^, acetone 25 SPPh^, benzene, TFA 25 SPPh3, benzene, ZnCl2 -78 SPPh3, benzene, BF30Et2 -78 SPPhi, toluene, ZnCl2 -78 SPPh3, THF, Ag0S02CF3 0 KSCN, THF, 0 Ag0S02CF3,18-crown-6 KSCN, benzene, 25 18-crown-6 KSCN, benzene, 80 18-crown-6 KSCN, dioxane, NH4CI, 25 18-crown-6 KSCN, dioxane, -78 Zn(0TfI)2, 18-crown-6 KSCN, acetone, 40 18-crown-6 DMTF, CH2CI2, TFA 25 DMTF, CH2CI2, TFA | 40

Table 1.1. Summary of reactions conditions employed to effect oxirane-thiirane exchange in endocyclic 1,2-anhydrosugars.

Since oxirane-thiirane exchange was not observed with triphenylphosphine sulfide, other potential reagents for thiirane formation were employed. Both potassium thiocyanate and dimethylthioformamide (DMTF) were employed under a variety of 27

conditions (Table I.l). Again, the same strategy for determining thiirane formation by

either trapping with methanol or acetylation was employed. No methanol incorporation or

5-acetylated products were observed in any of these examples. It is believed that no

thiirane products were observed due to strain in the endocyclic system which prevented

the two Walden inversions from occurring.

OBn

DMDO BnO q Oxirane-

DCM, 0°C OBnQ thurane exchange

OBn ^OBn

BhO^'tA—Q "hydride" BnO * o BnO vNf'? 2. DMDO, DCM OBr\J OBn

OBn

OBn

Figure 1.13. Synthetic strategy to C-glycosyl sulfones via oxirane-thiirane exchange of exocyclic 1,2-anhdrosugars.

Since strain in the endocyclic 1,2-anhydrosugar systems may have been a factor for the lack of any thiirane formation, it was hoped that the exocyclic 1,2-anhydrosugars would be less constrained and therefore more able to undergo oxirane-thiirane exchange. 28

The strategy to C-glycosyl sulfones is presented in Figure 1.13. The exocyclic glycol'^ was first epoxidized to give a 3:2 mixture of epoxides.^'* The epoxides were then subjected to oxirane-thiirane exchange. The newly formed thiirane would then be opened with a hydride reagent. Hydride addition should preferentially occur at the anomeric position since it is the most activated center. Methylaiion of the resulting thiol and oxidation of the thiol ether would provide a C-glycosyl sulfone.

The first approach to oxirane-thiirane exchange was to treat the exocyclic tetra-0- benzyl glucose epoxide with triphenylphosphine sulfide in benzene with a stoichiometric amount of trifluoroacetic acid."^^ The exocyclic epoxide reacted cleanly; however attempts to trap the thiirane by addition of methanol or acetylation did not provide evidence for incorporation of sulfur. Attempts to isolate the crude product by flash chromatography failed. The second approach tried was to treat the exocyclic epoxide with dimethylthioformamide in dichloroethane at 600C with a catalytic amount of trifluoroacetic acid.'*^ Again, the epoxide reacted cleanly, but all attempts to trap a possible thiirane failed and isolation of the crude product by column chromatography was not achieved. At this point, the preparation of C-glycosyl sulfones via oxirane-thiirane exchange was abandoned.

A second generation approach to the preparation of C-fucosyl sulfones was undertaken (Figure 1.14) since oxirane-thiirane did not seem to be a synthetically useful approach. This second generation approach involves the preparation of the exocyclic fucose and epoxidation with dimethyldioxirane. A mixture of epoxides (2:1) was obtained. The epoxide was then to be treated with a hydride source to afford a primary alcohol. Subsequent treatment of the primary alcohol under Mitsunobu'^^ conditions should provide the thioacetate. The thioacetate could then be deprotected, methylated and oxidized to give the desired C-fucosyl sulfone. 29

hydride' ^r—Q /^OBn DMDO fiir OBn

SAc OH OBn AcSH /^QBn PPhj, DEAD OBn r OBn OBn OBn

O 11^

n O fir OBn

Figure 1.14. Second generation approach to the preparation of C-fucosyl sulfones.

The question of stereoselectivity of hydride addition to 1,2-anhydrosugars needed to

be examined first. Again, endocyclic model systems were employed to examine the stereoselectivity of hydride addition. Two endocyclic glycals (glucal and allal) were employed since, upon epoxidation, they provided 1,2-anhydrosugars with opposite stereochemistries (Figure 1.15). 30

BnO BnO

BnO BnO O DMDO BnO BnO DCM, G°C

BnO BnO

DMDO BnO pSss=/ DCM, 0°C OBn OBn 1.2 1.3

Figure 1.15. Stereospecific epoxidaiion of glucal (top) and allal (bottom).

The epoxides were then reacted with a hydride source to yield anhydroalditols.'^^ In order to determine the stereochemical outcome of hydride addition to the 1,2-anhydrosugars, the epoxides were also treated with a deuteride source (Figure 1.16). NMR was then employed to confirm the stereochemical outcome of hydride addition. In the case of the glucal epoxide, the NMR spectrum indicated equatorial hydride delivery which was confirmed by addition of LiAlD4 (LAD) to the glucal epoxide. Deuteride addition showed loss of the H-lgq resonance and the collapse of the anomeric doublet of doublets to a doublet (7 = 10.4 Hz) thereby indicating that the two hydrogen atoms were rra/j^-diaxial.

The case of hydride addition to the allal epoxide proved to be more problematic. The

NMR of the hydride addition product to the allal epoxide contained numerous overlapping peaks which made identification of the H-lax and H-lgq difficult. This product was then acetylated in order to more clearly see the anomeric protons. Deuteride addition to the allal epoxide showed loss of the H-lax resonance and the collapse of the

H- Igq resonance at 3.79 ppm to a singlet as seen in the acetylated product. 31

BnO OBn BnO BnO LAH. Et,Q reflux, 2 h 74%

1.4 BnO OBn BnO BnO LAD, EbO reflux, 2 h 61%

1.5

Figure 1.16. Hydride (deuteride) addition to 1,2-anhydrosugars.

After hydride addition to 1.2-anhydrosugars was shown to be highly stereoselective, work was undertaken to optimize reaction conditions. A number of hydride reagents were employed under a variety of conditions. The best hydride source was LiAlH4 while triethylsilane proved to be especially poor as a hydride addition reagent. Table 1.2 provides a summary of the reagents used to prepare anhydroalditols. 32

Reagents Reaction Time Percent Yield TMSOTf, EtsSiH, ISh 0 THF, -780C to r.t. TMSOTf, EtsSiH, 18h 0 MeCN, -30°C to r.t. BH3, Et3SiH, THF, 3h 26 (PC BH3, LiBH4, THF, 3h 26 QOC BF30Et2, Et3SiH, 3h 36 THF, 0°C BH3. LiEt^BH, OOC 2h 60 LiAlH4, Et20, 250c I h 50 LiAlHi, Et20, reflux 2h 74

Table 1.2. Hydride addition to l,2-Anhydro-3,4,6-tri-0-benzyI-D-giucose.

Similar results were obtained with the allal epoxide. Lithium aluminun hydride proved to

be the best reagent for hydride addition while other reagents proved to be less effective.'^

Since hydride addition to the endocyclic 1,2 anhydrosugars proved to be highly stereoselective, hydride addition to exocyclic 1,2-anhydrosugars was investigated as means into C-glycoside sulfones (Figure 1.14). Since LiAlH4 addition proved to be most effective in the endocyclic case, it was employed first in the case of the exocyclic 1,2- anhydrosugars. At this point, D-glucose derivatives were used for model studies since glucose is less expensive than L-fucose. The exocyclic glycal of glucose, which was prepared by Swem oxidation"^^ from commercially available 2,3,4,6-tetra-(9-benzyl-D- glucose and then methylenated by the Tebbe reagent,'*'* was first epoxidized with dimethyldioxirane to yield a 3:2 mixture of epoxides.^'* The epoxide was then treated with a hydride source with anticipated hydride delivery to the anomeric center, which is the most active site in the exocyclic epoxide. Unfortunately in the case where LiAlH4 was 33

used as the hydride source, multiple products were obtained: a and P anomers of the

primary alcohol and a tertiary alcohol (Figure 1.17).

DMDO DCM, 0°C

hydride'

OH

Figure 1.17. Hydride addition to exocyclic 1,2-anhydrosugars.

Attempts to isolate the products obtained in order to determine product ratios proved to be

problematic. Neither flash chromatography nor HPLC afforded pure products. Since

LAH appeared to be too reactive in that hydride addition occurred at both the anomeric center and the least sterically hindered site of the epoxide, other hydride reagents were explored. Brown has shown that diborane is a useful reagent for hydride delivery at the more sterically hindered center of trisubstituted epoxides. Disappointingly, our studies showed that hydride delivery again occurred at both sites of the epoxide.

DIBALH^® and triethylsilane, a mild hydride source, also provided mixtures of products.

Mixed hydrides (LiAlH4/A]Cl3) were not used on these systems. A complete summary of the reagents and conditions employed is shown in Table 1.3. 34

Entry Reagents and Conditions 1 LiAlHt, EtiO, reflux 2 LiAlH4, THF, QoC 3 BH3, NaBH4. THF, OPC 4 BH3, BF^OEtz, THF, (PC 5 BH3, LiBH4, THF, -65

Table 1.3. Summary of reagents used for hydride addition to exocyclic 1,2-

anhydrosugars.

Since the above strategy to C-glycoside sulfones was not synthetically useful, another

strategy needed to be developed. The next strategy involved nucleophilic addition of sulfone or sulfoxide stabilized carbanions to endocyclic epoxides (Figure 1.18). One

advantage of this approach is that nucleophilic addition should be highly stereoselective.

Dimsyl sodium^^ and dimsyl potassium are easily generated, and these carbanions are known to add to epoxides.^^ Sulfonyl carbanions can be readily prepared^^ also and examples are also known of these intermediates adding into epoxides.^'*'^'

Dimsyl salts were the first nucleophiles used in this new strategy for the preparation of

C-glycoside sulfones. A summary of the reagents and reaction conditions employed is provided in Table 1.4. 35

,OBn O t

BnO or BnO O II 1- O

Figure 1.18. Strategy to C-glycoside sulfoxides or sulfones via nucleophilic addition to

endocyclic epoxides.

Entry Reagents and Conditions 1 NaH, DMSO, 65oC 2 KH, DMSO, THF, QOC to 250c 3 KH, DMSO, THF, 250c 4 KH, DMSO, THF, 18-crown-6, 25^ 5 KH, DMSO, THF, 18-crown-6, reflux 6 KH, DMSO, benzene, 18-crown-6, reflux 7 BF30Et2, THF, (K: then KH, DMSO, THF, QOC to 250c 8 ZnCl2. THF, -780C, then KH, DMSO, THF, (PC to 250c

Table 1.4. Reagents and conditions used for addition of dimsyl carbanions to endocyclic

1,2-anhydrosugars. 36

None of the reactions conditions described in Table 1.4 afforded the desired C-glycoside

sulfoxide. 'H NMR spectra of the crude reaction mixtures typically showed unreacted

epoxide. No reaction was observed by TLC when reactions were allowed to run for

longer times. In order to determine if even the dimsyl anion was being formed, the

dimsyl anion was quenched with D2O. NMR revealed that deuterium was

incorporated. Therefore, it is believed that the dimsyl salts were not nucleophilic enough

to open the endocyclic epoxide.

The next reagent that was employed was dimethyl sulfone. It was unclear at the start

whether the methyl sulfonyl carbanion would be nucleophilic enough to open the epoxide.

A summary of the reagents and conditions employed in this study is provided in Table

1.5. Entry Reagents and Conditions 1 Me2S02, BuLi, THF, 250c 2 Me2S02, BuLi, THF, HMPA, 250c 3 Me2S02, BuLi, THF, HMPA, -780C to 250c 4 Me2S02, BuLi, THF, reflux 5 Me2S02. KH, THF, reflux 6 Me2S02, BuLi, HMPA, PhMe, reflux 7 Me2S02, BuLi, THF, BF30Et2 8 Me2S02, KH THF, ZnCl2 -780C to 250c 9 ZnCl2, THF, then Me2S02, BuLi, THF, HMPA, 250c

Table 1.5. Reagents and conditions employed for the attempted addition of dimethyl sulfone carbanion to an endocyclic 1,2-anhydrosugar. 37

As was the case with the dimsyl salts, no addition of dimethyl sulfone to the epoxide was

observed. Again, the methyl sulfonyl anion could be quenched with D2O indicating that

the carbanion was indeed being generated, but that the nucleophilicity of the carbanion

was not sufficient enough to open the epoxide.

Since the nucleophilicity of the dimsyl and methyl sulfonyl anions proved problematic,

an alternative a-sulfonyl nucleophile was sought. The next approach involved the use of

commercially available methyl methanesulfonylacetate. It was hoped that this species,

when deprotonated, would add to the epoxide to form a C-glycoside which upon

decarboxylation would afford the desired C-glycoside sulfone (Figure 1.19).

BnO •OBn BnO MeS02CH2C02Me BnO' BnO BnO Base, THF OH

OBn I. NaOH

2. THF, heat OH

Figure 1.19. Strategy for the preparation of C-glycoside sulfones using methyl methanesulfony lacetate.

Again, the methyl methanesulfonate carbanion proved to be ineffective as a nucleophilic agent TLC showed no reaction and 'H NMR of the crude reaction mixtures indicated the presence of the starting epoxide. A list of the reagents and conditions is provided in Table

1.6. 38

Entry Reagents and Conditions 1 MeS02CH2C02Me, BuLi, THE, -780C to 250C 2 MeS02CH2C02Me, BuLi, THE, HMPA, -780C to 250c 3 MeSOaCHiCOzMe, LDA, THF, (PC 4 MeSC)2CH2C02Me, KH, THF, 250c 5 MeS02CH2C02Me, NaH, THF, reflux 6 MeS02CH2C02Me, KH, THF, l8-crown- 6, reflux 7 ZnCl2, THF, then MeS02CH2C02Me, NaH, THF, -78oC to 250c 8 TMSOTf, THF, then MeS02CH2C02Me NaH, THF, -78oC to 250c 9 BF30Et2, THF, then MeS02CH2C02Me, NaH, THF, -78oC to 250c

Table 1.6. Reagents and conditions employed for the addition of methyl methanesulfonyl acetate carbanion to an endocyclic 1,2-anhydrosugar.

Since nucleophilic addition to the epoxide failed, this approach was abandoned as a means of preparing C-glycoside sulfones. The next strategy employed again started with an exocyclic 1,2-anhydrosugar. The epoxide was treated with trimethylsilyl iodide

(TMSI) in acetonitrile to give an anomeric iodide and a primary alcohol (Figure 1.20).

The anomeric iodide was to be removed via a free radical approach with tributyltin 39

hydride to produce an anomeric hydride. Sulfur was to be incorporated into the molecule

by conversion of the primary alcohol into a thiolacetate via the Mitsunobu reaction.'*^

Deprotection of the S-acetate, aikylation with methyl iodide and oxidation was to provide

the C-glycoside sulfone.

.OBn .OBn

BnO- TMSI BnO- BnO BnO MeCN OH OBn OBn; PhMe, reflux

OBn .OBn BnO' 1. PPhj, DEAD. AcSH BnO BnO- OH 2. NaOMe, MeOH OBn BnO 3. Mel OBn 4. DMDO, DCM, 0°C

Figure 1.20. Approach to C-glycoside sulfones via reaction of exocyclic 1,2- anhydrosugars with TMSI.

The TMSI reacted cleanly with the exocyclic 1,2-anhydrosugar to afford a single product. This product was then treated with tributyltin hydride to remove the anomeric iodide and form the anomeric hydride, but no reaction occurred. Reexamination of the starting material by NMR showed a singlet at 2.06 ppm. This peak was originally thought to be residual acetonitrile. Repurification by flash chromatography and complete characterization of this material by 'H and '^C NMR, IR, and mass spectrometry led to the conclusion that the reaction of the exocyclic epoxide with TMSI in acetoniuile resulted 40

in the formation of a spirocyclic 2-oxazoIine (1.7). Both TLC and NMR indicated the

formation of a single oxazoline; however, the absolute stereochemistry of the spiro- oxazoline has yet to be determined.

The proposed mechanism of oxazoline formation is presented in Figure 1.21.

•OBn OBn

BnO' BnO- TMSI BnO BnO MeCN, -20°C OBn

OBn

OBn N=C—Me

OTMS

.OBn OBn

BnO- BnO N=C—

OBi OBi

IMS 1.7

Figure 1.21. Proposed mechanism of oxazoline formation from an epoxide.

The oxygen of the epoxide is first silylated to form an oxonium ion. The epoxide ring opens generating an oxonium ion on the pyranose ring oxygen. The nitrogen of the acetonitrile solvent attacks the anomeric center forming a C-N bond. The highly 41

electrophilic nitrilium carbon is then subjected to nucleophilic attack from the oxygen to

generate the spiro-oxazoline.

The yields for the formation of the spiro-oxazoline are rather poor (typically, 28-35%

yield). The spiro-oxazoline is the only isolable product after flash chromatography, and

crude NMR spectra looked quite clean. It is unclear at this point what factors are

responsible for the poor yields.

A number of methods have appeared in the literature regarding the synthesis of 2-

oxazolines from epoxides.^^ In 1975 Norman^^ subjected glycidyl tosylate to acid catalyzed ring opening in acetonitrile. The nitrogen of the acetonitrile solvent added into

the epoxide to form an optically active 2-oxazoline in 91% yield (Figure 1.22).

OTs BF^tOEt? MeCN, 0°C

Figure 1.22. Oxazoline synthesis from an epoxide.

Since 1975 a number of other reagents have been used to prepare 2-oxazolines from an epoxide and a nitrile such as Amberlyst A 26 resin,*^® TMS-CN,^^ and silicon tetrafluoride.^^

Since the strategy to C-glycoside sulfones via the reaction of TMSI with the exocyclic

1,2-anhydrosugar provided a spirocyclic oxazoline and not the desired primary alcohol, another strategy to these compounds was needed. In 1970 Igarashi^^ had reported the free radical addition of thiolacetic acid to tri-O-acetyl-D-glucal (Figure 1.23). Radical addition of thiolacetic acid at room temperature using cumene hydroperoxide (CHP) as the free radical initiator resulted in hydrogen radical addition to the anomeric center and thiol 42

radical addition to the 2-position of the glycal to produce a 7:3 ratio of mannoigluco

diastereomers.

AcSH

SAc

Figure 1.23. Free radical addition of thiolacetic acid to tri-O-acetyl-D-glucal.

Radical addition of thiolacetic acid was attempted with our perbenzylated exocyclic

glycal. Rather than using CHP as the radical initiator, 2,2'-azobis(2-methylpropionitrile)

(AIBN) was used. The reaction proceeds smoothly to provide the S-acetyl compound

(1.8) in 83% yield (Figure 1.24).

AcSH, AIBN

benzene, reflux 83%

1.8

Figure 1.24. Free radical addition of thiolacetic acid to perbenzyl exocyclic glycal.

The proposed mechanism of the reaction is as follows (Figure 1.25). Thermolysis of

AIBN generates a free radical in the initiation step. The free radical abstracts hydrogen from thiolacetic acid to give a sulfur radical and isopropyl nitrile in propagation step 1.

The thiolacetyl radical adds to the least substituted end of the glycal to produce an anomeric radical in propagation step 2. In propagation step 3, the anomeric radical 43

abstracts hydrogen from another thiolacetic acid to give the C-glycoside with an 5-acetyl

moiety incorporated and another sulfur radical.

Initiation Step benzene ^ AIBN + N, 80°C CN

Propagation Steps Step 1

Q CN CN o

BnO

BnO O BnO SAc OBn *

AcSH BnO SAc SAc

Figure 1.25. Proposed mechanism for radical addition of thiolacetic acid to an exocyclic glycal.

Both TLC and NMR indicate the formation of a single anomer. It is believed that the product is the P-C-glycoside owing to the anomeric radical effect.Giese contends 44 that the stereoelectronic effect of the ring oxygen results in trans addition of radicals to the anomeric center (Figure 1.26).

PO,

PO \ BugSnH^ PO CN .CN

Figure 1.26. Stereoselectivity for glycosyl radicals.

OBn ,OBn

BnO O l.TEA BnO' BnO Bn BnO 2. C02Me cr

OBn

C,2H25SH B"0 m- BnO Bn

Figure 1.27. Synthesis of P-C-glycosides by hydrogen atom transfer.

Crich*^ has studied hydrogen atom abstraction by C-glycoside radicals. Upon generation of the anomeric radical, r-dodecanethiol is added to quench the radical (Figure

1.27). The 3-C-glycoside is obtained in 92% yield. 45

Cyclohexyl radicals, by comparison, are generally attacked at the less sterically

demanding equatorial position^^-®^ (Figure 1.28).

Bu3SnH

CN CN

Figure 1.28. Stereoselectivity of cyclohexyl radicals.

The C-glycoside thiomethyl compound was the next target after preparation of the C- glycoside thiolacetate (1.8). Deprotection of the 5-acetyl group and alkylation with methyl iodide provided the C-glycoside thiomethyl compound (1.9) (Figure 1.29).

,OBn

BnO- -O 1.MeOH-THF,NaOMe BnO- BnO •SAc SMe OBn 2. Mel, 43-71% 1.8

Figure 1.29. Deprotection of the C-glycoside thiolacetate and methylation.

The C-glycoside thiolacetate was only moderately soluble in methanol so a 1:1 methanol-

THF solution was used to completely solubilize the thiolacetate. Deprotection of the S- acetyl group proceeded rapidly upon addition of a catalytic amount of sodium methoxide.

When TLC indicated complete reaction, methyl iodide was added to the solution to yield the C-glycoside thiomethyl compound. Care must be taken to ensure that the deacylation 46

takes place under anaerobic conditions in order to prevent formation of a disulfide. The

yields for the one pot procedure of deacylation and alkylation are variable (43-71%

yields).

With the C-glycoside sulfide in hand, oxidation of this material to the sulfone was

necessary. Oxidation was readily achieved by treating the sulfide in dichloromediane with

an excess of dimethyldioxirane (Figure 1.30).

DMDO SMe DCM, 0°C 100%

CH3CO2H, H2

Pd(C), 93%

Figure 1.30. Oxidation of C-glycoside sulfide to the sulfone with dimethyldioxirane.

The sulfone (1.10) was prepared in quantitative yield. No purification was necessary since the excess dimethyldioxirane and the acetone by-products were removed in vacuo to afford the C-glycoside sulfone. The benzyl ethers were subsequently removed to provide the deprotected sulfone (1.17).

The C-glycoside sulfoxide (1.11) was also prepared. The sulfoxide was prepared by

treatment of the sulfide with 3-chloroperoxybenzoic acid (m-CPBA) (Figure 1.31) in 34% 47 yield. A 1:1 mixture of diastereomers was obtained based upon integration of the methyl sulfoxide singlets by NMR.

,OBn

m-CPBA BnO BnO DCM, 0°C SMe 34% OBn i 1.11

Figure 1.31. Oxidation of the C-glycoside sulfide to the sulfoxide.

Since the C-glycoside sulfone of glucose was prepared, the synthetic strategy to this molecule was then applied to fucose in an attempt to prepare C-fucosyl sulfones from commercially available a-L-fucose. The synthetic strategy is outlined in Figure 1.32.

The anomeric position was first protected as the methyl glycoside.®^ The methyl glycoside was obtained as a white solid after recrystallization from ethanol in 41% yield.

The remaining three hydroxyl groups were protected as benzyl ethers.^®-^^ The methyl glycoside was readily removed under acidic conditions to provide 2,3,4-tri-<9-benzyl fucose^' in 41% yield after recrystallization from ether-hexanes. The lactol was oxidized to the lactone (1.12) under Swem conditions'^^ to provide the lactone in 86% yield.

Methylenation of the lactone using the Tebbe reagent"^ provided the exocyclic fucose glycal (1.1) in 60% yield. Radical addition of thiolacetic acid afforded the C-fucosyl thioacetate (1.13) in 78% yield. Subsequent deprotection of the 5-acetate and alkylation with methyl iodide proved to be problematic, as was the case with the C-glucosyl thiolacetate. Yields were poor (30-40%) and care had to be taken to perform the reaction under anaerobic conditions to prevent disulfide formation. Oxidation of the sulfide (1.14) to the sulfone (1.15) again proceeded quantitatively upon treatment with

excess of dimethyldioxirane (DMDO). OMe

MeOH ^p-O^OH Bnfir. KOH , Dowex H"*" resin DMSO, 74% f OH reflux, 41% OH

OMe 1. (C(X:i)2, DMSO HCl, HOAc^ OBn PCM, -45°C OBn 90°C,41% 2. TEA, 86% I OBn OBn OBn

p Tebbe Reagent AcSH, AIBN THF, toluene, OBn OBn benzene, reflux pyridine, -45°C 78% OBn 60% OBn 1.12 1.1

SMe SAc 1. NaOMe, MeOH OBn OBn 2. Mel, THF 30-40% OBn OBn OBn OBn 1.14 1.13

DMDO, DCM^ OBn 0°C, 100% OBn OBn 1.15

Figure 1.32. Synthetic scheme to C-fucosyl sulfone. 49

In addition to the C-fucosyl sulfone, the C-fucosyl sulfoxide was prepared. Oxidation of the sulfide with m-CPBA provided the sulfoxide (1.16) in 71% yield (Figure 1.33).

A 1:1 mixture of diastereomers was obtained based upon integration of the sulfoxide methyl singlets. O • SMe m-CPBA

OBn 1.16 1.14

Figure 1.33. Preparation of C-fucosyl sulfoxides by oxidation with m-CPBA.

With the C-fucosyl sulfone (1.15) in hand, model studies were undertaken to determine whether it could be coupled with a sulfonyl chloride (Figure 1.34). If the coupling was successful, this strategy would then be employed to couple the C-fucosyl sulfone with a guanine sulfonyl chloride to provide the fully deprotected synthetic target

(Figure 1.6). There are two sites a to the sulfone that are available for deprotonation.

Both sites should have similar acidities (pKg = 29-31^^). In order to favor deprotonation at the methyl rather than the methylene site, a bulky base (r^rr-butyllithium, lithium hexamethyldisilazide, or lithium diisopropylamide) was employed. All attempted alkylations of tosyl halides failed. 50

I. Base OBn

OBn X—S Me OBn 1.15

OBn

OBn OBn

Figure 1.34. Model study examining the coupling of C-fucosyl sulfone with a tosyl

halide(X = ForCI).

CONCLUSION

A strategy was developed for the highly stereoselective preparation of C-glycosyl sulfones. Both C-glucosyl and C-fucosyl sulfone analogs were prepared. Sulfur incorporation was achieved by free radical addition of thiolacetic acid to exocyclic glycals.

Addition of thiolacetic acid resulted in the formation of P-C-glycosides as the only isolable product.

EXPERIMENTAL SECTION

Starting materials and reagents purchased from commercial suppliers were used without further purification. Solvents were dried by distillation prior to use. Diethyl ether and tetrahydrofuran were distilled from sodium and benzophenone under an argon 51

atmosphere. Dichloromethane, acetonitrile, dimethyl sulfoxide and pyridine were distilled

from calcium hydride under argon. Methanol was distilled from Mg/l2 under argon. All

reactions were performed under an argon atmosphere.

Thin layer chromatography was performed using silica gel 60 F254 plates. Flash

column chromatography was performed using silica gel 60 (230-400 mesh ASTM).

Proton and carbon nuclear magnetic resonance spectra were recorded on either a Bruker

AM-250, Bruker AM-500, or a Varian Unity 300 spectrometer. Chemical shifts are

reported in parts per million relative to the residual solvent peak. 'H NMR data are

reported in the order of chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q

= quartet, m = multiplet, br = broad), the coupling constant in hertz (Hz) and the number of protons. Infrared spectra were recorded using a Nicolet 510P FT-IR spectrometer.

Melting points were obtained using a Fisher-Johns melting point apparatus and are uncorrected. Specific rotations were determined using an Autopol III polarimeter. Mass spectrometry was performed by the Nebraska Center for Mass Spectrometry, Lincoln

NE. Elemental analyses were performed by Desert Analytics, Tucson, AZ.

3,4,6-Tri-O-acetyl-D-allal. Was prepared according to the procedure of Roth.^^ Rf

= 0.50 (50% ethyl acetate/hexanes). NMR (300 MHz, CDCI3) 5 1.85 (s, 3 H), 1.90

(s, 6 H), 4.08 - 4.24 (m, 3 H; H-5, H-6, H-6'), 4.76 (t, J = 5.7 Hz, 1 H, H-2), 4.95

(dd, J = 10.8, 3.6 Hz; 1 H, H-4), 4.26 (m, 1-H, H-3), 6.38 (d, J = 6.0 Hz, 1 H, H-1).

I3C NMR (75 MHz, CDCI3) 5 20.1, 20.2, 20.5 , 61.4, 62.2, 65.9, 70.2, 97.1, 147.4,

168.8, 169.8, 170.0. IR (CDCI3) 420, 548, 604, 648, 734, 761, 828, 916, 963, 1009,

1227, 1373, 1435, 1651, 1716, 1732, 1747, 2965 cm*!. FARMS m/z calcd for

Ci2Hi607Na 295.0793 (M + Na), found 295.0804 . 52

3,4,6-Tri-O-benzyl-D-allal (1.2). 3,4,6-tri-O-acetyl-D-allaI (1.8 g, 6.62 mmol) was dissolved methanol (50 mL). Sodium methoxide (10 mg, 0.19 mmol) was added and the solution was stirred for 3 h. The solvent was removed in vacuo. The crude product was dissolved in dimethyl sulfoxide (40 mL) and the vessel was cooled to lO^C.

Freshly powdered KOH (1.2 g, 21.06 mmol) was added followed by dropwise addition of benzyl bromide (2.5 mL, 21.06 mmol). The reaction vessel was allowed to warm to room temperature over 18 h. The reaction was quenched by addition of 100 mL of H2O.

Dichloromethane (3 x 100 mL) was added. The organic phase was collected, dried

(Na2S04), and concentrated in vacuo. The crude product was purified by flash chromatography (6:1 hexanesrethyl acetate) to give 123 mg (4.4%) as a colorless oil. Rf

= 0.66 (25% ethyl acetate/hexanes). [a]22Q = +226° (c 6.9 in CHCI3). NMR (250

MHz, CDCI3) 5 3.80-3.84 (m, 3 H; H-4, H-6, H-6'), 4.31 (dt, J = 10.4. 3.1 Hz, 1 H,

H-5), 4.45 - 4.74 (m, 6 H; OCHiAr), 4.89 (t, J = 5.8 Hz, 1 H, H-2), 6.47 (d, J = 5.9, 1

H, H-1), 7.22 - 7.38 (m, 15 H, ArH). NMR (63 MHz, CDCI3) 5 65.5, 66.6, 68.9,

70.4, 71.3, 73.0, 73.3, 73.6, 73.9, 92.3, 98.1, 127.6, 127.7, 127.8, 128.0, 128.3,

128.5, 137.9, 138.7, 146.7. IR (CDCI3) 601, 698, 736, 1028, 1116, 1207, 1259,

1329, 1363, 1454, 1496, 1643, 2341, 2364, 2866, 3030, 3063 cm-l. FABMS m/z calcd for C27H2804Na 439.1885 (M + Na), found 439.1882.

Typical procedure for the preparation of 1,2-Anhydrosugars. To a solution of 3,4,6 tri-O-benzyl-D-glucal (100 mg, 0.24 mmol) in dichloromethane (1 mL) cooled to (KC is added dimethyl dioxirane (4.8 ml, 0.24 mmol). The solution was stirred for 20 min, and the solvent was removed in vacuo to yield the product as a white solid.

I,2-Anhydro-3,4,6-tri-0-benzyl-D-allal (L3). Rf = 0 (25% ethyl acetate/hexanes). [a]22D = -333^ (c 0.03 in CHCI3). 'H NMR (300 MHz, CDCI3) 5 53

3.21 (t, J = 2.7 Hz, 1 H, H-1), 3.66 (d, J = 3.3 Hz, 2 H; H-6, H-6'), 3.92 (dd, J = 9.9,

3.3 Hz, 1 H, H-4), 4.2 (dt, J = 6.9, 3.3 Hz, 1 H, H-5), 4.26 (t, / = 2.7 Hz, 1 H, H-2),

4.48 - 4.65 (m, 7 H, OCH2Ar, H-3), 7.22 - 7.37 (m, 15 H, ArH). »3c NMR (75 MHz,

CDCI3) 5 54.2, 68.9, 70.5, 72.3, 72.4, 73.5, 73.7, 73.9, 127.5 127.7, 127.8, 127.9,

128.3, 128.4, 137.8, 138.0. IR (CDCI3) 426, 602, 698, 737, 1028, 1096, 1209, 1095,

1210, 1281, 1369, 1455, 1497, 1638, 2926, 3428, 3630 cm-K FABMS m/z calcd for

C27H2805Na 455.1834 (M + Na), found 455.1833.

Procedures for Hydride Addition to Endocyclic 1,2-Anhydro sugars.

A. To a stirred solution of lithium aluminum hydride (46 mg, 1.2 mmol) in Et20 (2 mL) was added slowly a solution of tri-O-benzyl-D-glucose epoxide (0.24 mmol) dissolved in

Et20 (2 mL). The solution was refluxed for 2 h. The reaction was quenched by sequential addition of H2O (0.6 mL), 10% NaOH (0.6 mL), and H2O (1.8 mL). The solution was filtered and dichloromethane (50 mL) was added. The organic phase was collected, dried (Na2S04), and concentrated in vacuo. The residue was chromatographed

(1:1 hexanesrethyl acetate) to yield 77 mg (73.9%) of product as a colorless oil.

B. To a stirred solution of lithium aluminum hydride (36 mg, 0.96 mmol) in Et20 (4 mL) was added dropwise a solution of tri-O-benzyl-D-glucose epoxide (0.24 mmol) dissolved in Et20 (4 mL). The solution was stirred for 1 h and was quenched by sequential addition of H2O (0.6 mL), 10% NaOH (0.6 mL), and H2O (1.8 mL). The solution was filtered and dichloromethane (50 mL) was added. The organic phase was collected, dried (Na2S04), and concentrated in vacuo. The residue was chromatographed

(1:1 hexanes:ethyl acetate) to yield 53.1 mg (50.4%) as a colorless oil. 54

C. To a solution of borane (240 p.L, 0.24 mmol) and lithium borohydride (120 piL,

0.24 mmol) in THF (2 mL) cooled to 0°C was added dropwise a solution of tri-O-benzyl-

D-glucose epoxide (0.24 mmol) in THF (2 mL). The reaction was quenched after 3 h by the addition of water (I mL). Ethyl acetate (25 mL) was added. The organic phase was collected, dried (Na2S04), and concentrated in vacuo. The residue was chromatographed

(1:1 hexanesrethyl acetate) to yield 27.0 mg (25.9%) as a colorless oil.

D. To a solution of borane (240 |iL, 0.24 mmol) and triethylsilane (115 p.L, 0.72 mmol) in THF (4 mL) cooled to 0°C was added dropwise a solution of tri-O-benzyl-D-glucose epoxide (0.24 mmol) in THF (2 mL). The reaction was quenched after 3 h by the addition of water (1 mL). Ethyl acetate (25 mL) was added. The organic phase was collected, dried (Na2S04), and concentrated in vacuo. The residue was chromatographed

(I;I hexanes:ethyl acetate) to yield 26.9 mg (25.8%) as a colorless oil.

E. To a solution of borane (240 mL, 0.24 mmol) and lithium triethylborohydride (240 jiL, 0.24 mmol) cooled to (PC in THF (2 mL) was added dropwise a solution of tri-<9- benzyl-D-glucose epoxide (0.24 mmol) in THF (2 mL). The reaction was quenched after

2 h by the addition of water (1 mL). Ethyl acetate (25 mL) was added. The organic phase was collected, dried (Na2S04), and concentrated in vacuo. The residue was chromatographed (1:1 hexanes:ethyl acetate) to yield 62.7 mg (60.2%) as a colorless oil.

F. To a solution of tri-O-benzyl-D-glucose epoxide (0.12 mmol) cooled to -65oC in THF

(2 mL) was added BF30Et2 (16 jiL, 0.13 mmol) and triethylsilane (77 |iL, 0.48 mmol).

The solution was warmed to room temperature over 3 h and then quenched by addition of water (1 mL). Ethyl acetate (25 mL) was added. The organic phase was collected, dried 55

(Na2S04), and concentrated in vacuo. The residue was chromatographed (1:1 hexanes:

ethyl acetate) to yield 18.7 mg (35.8%) as a colorless oil.

G. To a solution of tri-O-benzyl-D-glucose epoxide (0.24 mmol) cooled to -30°C in dry

CH3CN was added trimethylsilyl triflate (230 |i.L, 1.2 mmol). The solution was stirred

for 5 min and triethylsilane (190 (iL, 1.2 mmol) was added dropwise. The solution was

allowed to warm to room temperature over 2 h and the solution was stirred at room

temperature for 18 h. TLC indicated no reaction.

3,4,6-tri-O-benzyl-l-deoxy-l-hydrido-D-gIucose (1.4). Method A employed.

Product obtained as a colorless oil (77 mg. 73.9%). Rf = 0.50 (50% ethyl

acetate/hexanes). [a]22D = -83.3° (c 0.48 in CHCI3). NMR (250 MHz. CDCI3) 5

1.88 (br s. 1 H, OH), 3.20 (t. J = 10.9 Hz. 1 H, H-lax), 3.37- 3.47 (m. 2 H; H-6. H-

6'), 3.58 - 3.76 (m, 4 H; H-2. H-3, H-4, H-5). 4.01 (dd. 7 = 11.0. 5.3 Hz. 1 H. H-leq).

4.48 - 4.97 (m, 6 H, OCHiAr), 7.13 - 7.35 (m. 15 H. ArH). NMR (63 MHz,

CDCl3) 5 68.3, 69.0. 69.6, 73.1. 74.4, 74.7. 77.0, 77.5, 86.4. 127.2. 127.3. 127.5.

127.8, 127.9, 128.2, 137.3;. IR (CDCI3) 603, 638, 698, 735. 910. 1028. 1084. 1209.

1323, 1361. 1454, 1496, 2864, 3030, 3063, 3447 cm-l. Anal. Calcd forC27H3o05:

C, 74.62; H, 6.96. Found: C. 74.63; H, 6.93.

3,4,6-tri-O-benzyl-l-deoxy-l-hydrido-D-aItrose. Method A employed.

Product obtained as a colorless oil (18 mg, 57.6%). Rf = 0.50 (50% ethyl acetate/hexanes). [a]22D =-8O.O0 (c 0.25 in CHCI3). ^H NMR (250 MHz. CDCI3) 5

2.25 (br s. 1 H. OH). 3.64 - 3.94 (m. 8 H; H-lax. H-lgq. H-2. H-3. H-4. H-5, H-6. H-

6'). 4.33 - 4.70 (m, 6 H, 0CH2Ph). 7.16 - 7.30 (m. 15 H. ArH). NMR (75 MHz. 56

CDC13) 5 67.7, 68.4, 69.5, 71.4, 72.8, 73.5, 74.0, 75.0, 127.5, 127.6, 127.7, 127.8,

128.3, 137.9, 138.0, 138.3. IR (CDCI3) 492, 604, 698, 737, 802, 1028, 1090, 1262,

1455, 1497, 1645, 2867, 2919, 3031, 3422 cm'K FABMS m/z calcd for CzTHaoOsNa

457.2093 (M + Na), found 457. 1976.

3,4,6-tri-O-benzyl-l-deoxy-l-P-deutero-D-glucose (1.5). Method A employed using LAD. Product obtained as a colorless oil (63 mg, 60.1%). Rf = 0.50

(50% ethyl acetate/hexanes). [a]^^ = -25.0° (c 0.80 in CHCI3). NMR (250 MHz,

CDCI3) 5 2.22 (br s, 1 H, OH), 3.19 (d, 7 = 10.4 Hz, I H, H-lax)> 3.38 - 3.48 (m, 2

H), 3.59 (t, 7 = 9.2 Hz, 1 H), 3.67 - 3.75 (m, 3 H), 4.50 - 4.97 (m, 6 H, 0CH2Ar),

7.14 - 7.37 (m, 15 H, ArH). NMR (63 MHz, CDCI3) 5 68.8, 69.2, 70.0, 73.5,

74.8, 75.1, 77.9, 79.3, 86.8, 127.7, 127.8, 127.9, 128.3, 128.4, 128.6. 137.8, 137.9.

IR (CDCI3) 603, 698, 736, 1026, 1066, 1209, 1261, 1309, 1361, 1454, 1496, 2866,

3030, 3063, 3443 cm-l. FABMS m/z calcd for C27H29D05Na 458.2054 (M + Na), found 458.2062.

3,4,6-tri-O-benzyi-l-deoxy-a-deutero-D-altrose (1.6). Method A was employed using LAD. Product obtained as a colorless oil (10 mg, 32%). Rf = 0.45

(50% ethyl acetate/hexanes). [a]^^ = +90.9° (c 0.22 in CHCI3). ^H NMR (250 MHz,

CDCI3) 5 1.89 (br s, 1 H), 3.67 - 3.69 (m, 3 H), 3.77 - 3.82 (m, 2 H), 3.85 - 3.90 (m,

2 H), 4.36 - 4.74 (ra, 6 H, 0CH2Ar), 7.18-7.31 (m, 15 H, ArH). NMR (63 MHz,

CDCI3) 5 68.3, 69.6, 71.5, 72.9, 73.5, 74.1. 74.9, 127.7, 127.8, 127.9, 128.3, 138.0,

138.4. IR (CDCI3) 698. 738, 850, 910, 1028, 1109, 1207, 1259. 1334. 1400. 1454.

1496. 2870. 3030, 3063. 3433 cm-l. FABMS m/z calcd for C27H29O5 436.2234 (M +

H). found 436.2238. 57

Typical acetyiation procedure. l-deoxy-3,4.6-tri-0-benzyl-D-glucose (1.4, 18 mg,

0.041 mraol) was dissolved in pyridine (1 mL). To this solution was added acetic

anhydride (1 mL) dropwise. The solution was allowed to stir for 3 h. The solvent was

removed by azeotroping in vacuo with toluene (5x5 mL). The crude product was

purified by flash chromatography (3:1 hexanes:ethyl acetate) to yield 20.2 mg (70.6%) of

product as a faintiy yellow oil.

2-0-acetyl-3,4,6-tri-0 -benzyl - 1-deoxy-l-hydrido-D-glucose. Product obtained as a colorless oil (20.2 mg, 70.6%). Rf = 0.67 (50% ethyl acetate/hexanes).

[a]22D = +66.60 (c 0.30 in CHCI3). NMR (250 MHz, CDCI3) 5 1.94 (s, 3 H,

COCH3), 3.15 (t, J = 10.8 Hz, 1 H, H-lax), 3.37 - 3.40 (m, 1 H, H-5), 3.58 - 3.70 (m,

4 H; H-3. H-4,H-6, H-6'), 4.07 (dd, J = II.O, 5.6 Hz, I H. H-leq), 4.49 - 4.82 (m, 6

H, 0CH2Ar), 4.96 (m, 1 H, H-2), 7.15 - 7.35 (m, 15 H, ArH). NMR (63 MHz,

CDCl3) 5 20.4, 66.4. 68.3, 71.1, 73.0, 74.6, 76.0, 79.0, 83.7, 127.2, 127.3. 127.4.

127.5, 127.9, 137.3, 137.4. IR (CDCI3) 603, 698, 738, 860, 908, 1039. 1089. 1236,

1327, 1365. 1454, 1496, 1747. 2864, 3032. 3065. 3088 cm-l. FABMS m/z calcd for

C29H3206Na (M + Na) 499.2078, found 499.2097.

2-0 -acety 1-3,4,6-tri-O-benzyl-l-deoxy-l-hydrido-D-altrose. Product obtained as a faintly yellow oil (18 mg, 92%). Rf = 0.67 (50% ethyl acetate/hexanes).

[a]22D = .76.90 (c 0.39 in CHCI3). 'H NMR (500 MHz, CeDe) 5 2.05 (s, 3 H.

COCH3). 3.83 (dd, J = 10.9, 1.8 Hz, 1 H, H-6), 3.88 - 3.93 (m, 3 H, H-1. H-1'. H-

6'), 4.01 (t, J = 3.2 Hz. 1 H. H-3). 4.06 (dd. J = 9.7, 2.9 Hz. 1 H, H-4). 4.19 (dq. J =

9.8, 1.8 Hz, 1 H, H-5), 4.40 - 4.70 (m. 6 H, OCHiAr). 5.05 (t, J = 1.9 Hz. 1 H. H-2),

7.13 - 7.34 (m, 15 H, ArH). NMR (63 MHz, CDCI3) 5 21.2, 65.2, 69.7, 69.9,

71.4, 71.8, 72.6, 73.0, 74.4, 127.6, 127.7, 127.9, 128.3, 128.4, 138.0, 138.2, 170.3. 58

IR (CDCI3) 605, 690, 736, 908, 1028, 1109, 1236, 1371, 1454, 1740, 2864, 2910,

3030, 3063 cm"^ FABMS m/z calcd for C29H3206Na 499.2199 (M + Na) , found

499.2084.

2-O-acety1-3,4,6-tri-f?-benzyl-l-deoxy -P-l-deutero-D-glucose. Product obtained as a colorless oil (18 mg,, 82%). Rf = 0.67 (50% ethyl acetate/hexanes). [a]22j)

= +8.30 (c 1.2 in CHCI3). IH NMR (500 MHz, CDCI3) 5 1.95 (s, 3 H, COCH3), 3.14

(d, J = 10.5 Hz, 1 H, H-lax), 3.39 - 3.41 (m, 1 H, H-5), 3.59 -3.71 (m, 4 H, H-3, H-4,

H-6, H-6'), 4.51 - 4.99 (m, 6 H, 0CH2Ar), 4.97 (t, J = 9.3 Hz, 1 H, H-2), 7.12 - 7.34

(m, 15 H, ArH). NMR (63 MHz, CDCI3) 6 20.9, 66.6, 68.9, 71.6, 73.5, 75.1,

76.5, 77.9, 79.4, 84.1, 127.7, 127.8, 127.9, 128.0, 128.4, 137.8, 137.9, 138.4,

170.0. IR (CDCI3) 603, 690, 740, 827, 906, 1041, 1084, 1155, 1238, 1361, 1456,

1496, 1747, 2868, 3032, 3065 cm-l. Anal. Calcd for C29H31O6D: C, 72.92; H, 6.97.

Found: C, 72.62; H, 6.62.

2-d?-acetyl-3,4,6-tri-0-benzyl-l-deoxy-l-a-deutero-D-aItrose. Product obtained as a colorless oil (5 mg, 45%). Rf = 0.67 (50% ethyl acetate/hexanes).

= +66.6° (c 0.15 in CHCI3). ^H NMR (500 MHz, CeDe) 5 1.6 (s, 3 H, COCH3), 3.74

(dd, J = 10.8, 1.7 Hz, 1 H, H-6), 3.79 - 3.83 (m, 2 H, H-lgq, H-6'), 3.91 (m, 1 H, H-

3), 3.96 (dd, J = 9.7, 2.8 Hz, 1 H, H-4), 4.09 (m, 1 H, H-5), 4.31 - 4.61 (m, 6 H,

0CH2Ar), 4.95 (dd, J = 3.7, 1.3 Hz, 1 H, H-2), 7.07 -7.33 (m, 15 H, ArH). NMR

(63 MHz, CDCl3)S 21.2, 64.9, 69.6, 69.8, 71.4, 71.7, 72.6, 73.0, 74.3, 127.5,

127.7, 127.8, 127.9, 128.2, 128.3, 137.7, 137.9, 138.0, 170.3. IR (CDCI3) 698, 736,

800, 1026, 1060, 1259, 1340, 1371, 1454, 1496, 1738, 2359, 2860, 2922, 2963, 3030 cm-1. FABMS m/z calcd for C29H32DO6 478.2340 (M + H), found 478.2332. 59

Tetra-0*benzyl-D

3,4,5,7-tetra-(9-benzyl-l-deoxy-D-g/uco-hept-l-enitoI'*'' (51 mg, 0.095 mmol) was dissolved in dichloromethane (1 mL). The solution was cooled to QoC and dimethyldioxirane (1.9 mL, 0.095 mmol) was added. After 20 min the solvent was removed in vacuo to give a 3:2 mixture of epoxides as a colorless oil. The mixture of epoxides was taken up into acetonitrile (3 mL) and cooled to -20oC. Trimethylsilyl iodide

(15 |iL, 0.105 mmol) was added and the solution was wanned to CPC over I h. The solvent was removed in vacuo and the crude product chromatographed (3:1 hexanes;ethyl acetate) to afford the product as a colorless oil (20.0 mg, 35.5%). Rf = 0.30 (25 % ethyl acetate/hexanes). = +66.6° (c 0.45 in CHCI3). NMR (250 MHz, CDCI3) 5

2.06 (s, 3 H, Me), 3.50 (d, J = 9.3 Hz, 1 H, H-3), 3.60 (dd, J = 10.8 Hz, 2.1 Hz, 1 H,

H-7), 3.70 - 3.84 (m, 3 H, H-1, H-5, H-7), 3.96 (d, J = 9.6 Hz, H-T), 4.15 - 4.26 (m,

2 H, H-4, H-6), 4.46 - 4.99 (m, 8 H, 0CH2Ar), 7.26 - 7.31 (m, 20 H, ArH).

NMR (75 MHz, CDCI3) 5 14.7, 68.6, 72.6, 73.4, 74.5, 74.6, 74.9, 75.6, 78.6, 80.1,

84.4, 102.1, 127.4, 127.6, 127.8, 127.9, 128.1, 128.3, 128.4, 138.1, 138.5, 138.6.

IR (CDCI3) 531, 548, 698, 733, 914, 1092, 1223, 1659, 1713, 2253, 2918, 3412 cm-1.

FABMS m/z calcd for C37H40NO6 594.2856 (M + H), found 594.2881.

l-S-Acetyl-2,6-anhydro-l-deoxy-3,4,,5,7-tetra-0-benzyI-D-glycero-D- gulo-heptitol (L8). 2,6-Anhydro-3,4,5,7-tetra-(7-benzyl-l-deoxy-D-g/«co-hept-1- enitol'^'* (98 mg, 0.183 mmol) was dissolved in benzene (3 mL). Freshly distilled thiolacetic acid (65 |XL, 0.914 mmol) was added followed by AIBN (3 mg, 0.0183 mmol). The solution was refluxed for 4 h. The solvent was removed in vacuo and the crude product chromatographed (5:1 hexanes:ethyl acetate) to give the product as a white solid (93 mg, 83.1%). Rf = 0.50 (25% ethyl acetate/hexanes). mp 116-118"C. [a]22Q

= -6.40 (c 4.7 in CHCI3). IH NMR (250 MHz, CDCI3) 5 2.33 (s, 3 H, SCOCH3). 3.06 60

(dd, J = 13.0, 5.8 Hz, 1 H), 3.39 - 3.73 (m, 8 H), 4.52 - 4.90 (m, 8 H, 0CH2Ar), 7.15

- 7.34 (m, 20 H, ArH). NMR (63 MHz, CDCI3) 5 30.0, 69.1, 73.6, 78.5, 78.7,

79.8, 81.2, 87.3, 127.6, 127.7, 127.8, 127.9, 128.0, 128.2, 128.4, 128.6, 138.8,

139.2, 139.5. IR (CDCI3) 629, 669, 698, 737, 955, 1028, 1097, 1221, 1362, 1455,

1497, 1694, 2865, 2920, 3031 cm'K FABMS m/z calcd for C37H41O6S 613.2624 (M +

H), found 613.2632.

1-S-Methy1-2,6-anhydro-l-deoxy-3,4,5,7-tetra-O-benzyl -D-glycero-D-

gulo-heptitol (1.9). l-5-Acetyl-2,6-anhydro-l-deoxy-3,4„5,7-tetra-(9-benzyl-D-

glycero-D-gulo-heptitoI (1.8, 28 mg, 0.0456 mmol) was dissolved in THF (2 mL) and

methanol (2 mL). Sodium methoxide (2 mg, 0.037 mmol) was added. After 30 min,

methyl iodide (2.7 |aL, 0.0435 mmol) was added to the solution. After 1 h the solvent

was removed in vacuo, and the residue was chromatographed (5:1 hexanes:ethyl acetate).

The product was obtained as a colorless oil (18 mg, 70.9%). Rf = 0.60 (25% ethyl

acetate/hexanes). [a]22D = -42.1° (c 0.95 in CHCI3). ^H NMR (250 MHz, CDCI3) 5

2.20 (s, 3 H, SMe), 2.68 (dd, 7 = 14.1, 6.2 Hz, IH, H-1), 2.89 (dd, J = 14.4, 1.8 Hz,

1 H, H-1'), 3.44 - 3.76 (m, 7 H), 4.53 - 4.94 (m, 8 H, OCHzAr), 7.17 - 7.36 (m, 20 H,

ArH). 13c NMR (75 MHz, CDCI3) 8 17.2, 35.7, 69.0, 73.4, 75.0, 78.4, 78.9, 80.2,

80.5, 87.1, 127.6, 127.7, 127.8, 127.9, 128.3, 128.4, 128.5, 138.0, 138.1, 138.2,

138.5. IR (CDCI3) 697, 735, 1028, 1099, 1209, 1309, 1362, 1454, 1497 cm-1.

FABMS m/z calcd for C36H41O5S 585.2675 (M + H), found 585.2696.

l-Methylsulfonyl-2,6-anhydro-l-deoxy-3,4,5,7-tetra-O-benzyl-D-glycero-

D-gulo-heptitol (1.10). l-5-Methyl-2,6-anhydro-I-deoxy-3,4,5,7-tetra-0-benzyl-D- glycero-D-gulo-heptitol (1.9, 10 mg, 0.017 mmol) was dissolved in dichloromethane and cooled to 0°C. Dimethyldioxirane (685 ^L, 0.034 mmol) was added and the reaction 61 stirred for 30 min. The solvent was removed in vacuo to provide the sulfone as a white solid (11 mg, 100%). Rf = 0.11 (25% ethyl acetate/hexanes). mp 125-1280C. [a]22p =

-7I.40 (c 0.28 in CHCI3). NMR (250 MHz, CDCI3) 5 2.92 (s, 3 H, SOiMe). 2.93

(t, J = 10.0 Hz, 1 H), 3.21 (d, J = 14.6 Hz, 1 H), 3.35 (t, J = 9.0 Hz, 1 H), 3.54 - 3.86

(m, 6 H), 4.48 - 4.96 (m, 8 H, 0CH2Ar), 7.16 - 7.39 (m, 20 H, ArH). •3c NMR (63

MHz, CDCI3) 5 43.4, 56.3, 58.6, 68.7, 73.3, 74.5, 75.0, 75.7, 78.1, 78.4, 79.3, 86.9,

127.7, 127.8, 128.0, 128.2, 128.4, 128.5, 128.6, 137.3, 137.6, 137.8, 138.1. IR

(CDCI3) 503, 639, 736, 937, 1028, 1098, 1130, 1210, 1304, 1362, 1395, 1455, 1497.

2870 cm-l. FABMS m/z calcd for C36H4o07SNa 639.2392 (M + Na), found 639.2384.

l-Methylsulfoxyl-2,6-anhydro-l-deoxy-3,4,5,7-tetra-0-ben2yl-D-glycero-

D-gulo-heptitol (1.11). l-5-Methyl-2,6-anhydro-l-deoxy-3,4,5,7-tetra-0-benzyl-D- glycero-D-gulo-heptitol (1.9, 139 mg, 0.238 mmol) was dissolved in dichloromethane (5 mL) and cooled to OoC. 50 - 60% m-CPBA (82.0 mg, 0.238 mmol) was added. After 30 min, the solvent was removed in vacuo and the crude product chromatographed (7:3 benzene/acetone). The sulfoxide was obtained as a colorless oil (48.1 mg, 33.6%) and as

1:1 mixture of diastereomers based upon integration of the methyl sulfoxide singlets. Rf

= 0.20 (30% acetone:benzene). IH NMR (250 MHz, CDCI3) 8 2.55 (s, 3 H, SOCH3),

2.64 (s, 3 H, SOCH3), 2.92 - 3.14 (m 2 H), 3.35 (t, J = 8.9 Hz, 1 H), 3.47 - 3.80 (m, 6

H), 4.49 - 4.98 (m, 8 H, OCH2Ar), 7.18 - 7.40 (m, 20 H, ArH). NMR (63 MHz.

CDCI3) 5 39.2, 39.9, 54.9, 57.5, 68.4, 68.7, 72.9, 73.1, 73.3, 74.9, 75.5, 75.6, 77.9,

78.0, 78.6, 78.9, 80.0, 80.6, 127.7, 127.8, 127.9, 128.0, 128.2, 128.3, 128.4, 137.6,

137.9, 138.2. IR (CDCI3) 706, 751, 1029, 1072. 1080, 1217. 1361. 1463. 1514,

2868, 2919, 3047 cm-1. FABMS m/z calcd for C36H4o06SNa 623.2443 (M + Na), found 623.2442. 62

2,3,4-Tri>{7-benzyl-6-deoxy-L-gaIactopyrano-l,5-lactone (1.12). Oxalyl chloride (435 nL, 5.07 mmol) was dissolved in dichloromethane (25 mL), and the solution was cooled to -50oC. A solution of dimethyl sulfoxide (785 ^iL, 11.1 mmol) in dichloromethane (5 mL) was added dropwise to the oxalyl chloride solution. After 5 min, a solution of 2,3,4-tri-O-benzyl-L-fucose^^ (1.00 g, 2.30 mmol) in dichloromethane (5 mL) was added to oxalyl chloride-DMSO solution. After 1 h, triethylamine (1.60 mL,

11.5 mmol) was added, and the solution was allowed to warm to room temperature. The organic phase was extracted with water (3 x 100 mL) and saturated NaCl (100 mL). The organic phase was collected, dried (Na2S04), concentrated in vacuo and chromatographed (3:1 hexanes/ethyl acetate) to afford the product as a colorless oil (867 mg, 86.3%). Rf = 0.26 (25% ethyl acetate/hexanes). = -80.7° (c 15.0 in

CHCI3). NMR (250 MHz, CDCI3) 5 1.26 (d, J = 6.5 Hz, 3 H, CH3), 3.74 (m, 1 H,

H-4), 3.82 (dd, J = 9.7, 2.16 Hz, 1 H, H-3), 4.23 (qd, J = 6.4, 1.1 Hz, IH, H-5), 4.41

(d, J = 9.7 Hz, 1 H, H-2), 4.60 - 4.75 (m, 4 H, OCHzAr), 4.91 (d, 7 = 11.4 Hz, I H,

OCHzAr), 5.14 (d, 7 = 11.0 Hz, 1 H, OCHiAr), 7.23 - 7.39 (m, 15 H, ArH).

NMR (63 MHz, CDCI3) 5 17.1, 72.9, 74.6, 75.1, 75.4, 75.7, 76.8, 80.3, 127.4,

127.7, 127.8, 128.1, 128.3, 128.4, 137.6, 137.8, 170.5. IR (CDCI3) 586, 700, 744,

864, 912, 1028, 1103, 1363, 1456, 1497, 1736, 2878, 2990, 3032, 3065 cm-l.

FABMS m/z calcd for CzyHzgOsNa 455.1834 (M + Na), found 455.1815.

2,6-Anhydro-3,4,5-tri-0-benzyl-l,7-dideoxy-L-galacto-hept-l-enitol

(1.1). 2,3,4-Tri-0-benzyl-6-deoxy-L-gaIactopyrano-l,5-lactone (1.12, 1.10 g, 2.53 mmol) was dissolved in a solution of toluene (6 mL), THF (2 mL), and pyridine (50 |iL).

The solution was cooled to -450C, and Tebbe reagent (6.60 mL, 3.29 mmol) was added slowly. After 1 h at -45°C, the solution was kept at 0°C for 30 min. After 30 min, the solution was cooled to -15^0 and 10% NaOH (1 mL) was added to the solution 63

dropwise. The solution was warmed to room temperature and diethyl ether was added

(50 raL). The solution was passed over a short pad of Celite and concentrated in vacuo.

The crude product was chromatographed (6:1 hexanes/diethyl ether) to afford the exocyclic methylene as a colorless oil (648 mg, 59.6%). Rf = 0.62 (25% ethyl

acetate:hexanes). [a]22Q = -76.9° (c 5.2 in CHCI3). NMR (250 MHz, CDCI3) 6

1.23 (d, J = 6.4 Hz, 3 H, CH3), 3.62 - 3.71 (m, 3 H, H-4, H-5, H-6), 4.39 (d, J = 9.4

Hz, 1 H, H-3), 4.65 - 4.83 (m, 7 H, H-1, H-T, OCHzAr), 4.97 (d, 7 = 11.6 Hz, 1 H,

OCH2Ar), 7.26 - 7.37 (m, 15 H, ArH). NMR (63 MHz. CDCI3) 5 17.3, 74.2,

74.9, 76.2, 77.1, 77.6, 83.1, 94.2, 127.8, 127.9, 128.2, 128.5, 128.6, 128.7, 138.6,

138.8, 138.9, 158.7. IR (CDCI3) 698, 733, 817, 844, 924, 999, 1028, 1064, 1089,

1126, 1201, 1307, 1361, 1454, 1496, 2880, 2934, 2988, 3030 cm-I. FABMS m/z calcd for C28H3o04Na 453.2042 (M + Na), found 453.2024

l-S-AcetyI-2,6-anhydro-l,7-dideoxy-3,4,5-tri-0-benzyl-L-gIycero-L- galacto-heptitol (1.13). 2,6-Anhydro-3,4,5-tri-0-benzyl-1,7-dideoxy-L-galacto- hept-l-enitol (1.1, 557 mg, 1.30 mmol) was dissolved in benzene (7 mL). Freshly distilled thiolacetic acid (463 |iL, 6.50 mmol) was added to the solution along with AIBN

(21 mg, 0.130 mmol). The solution was heated at reflux for 1 h. The solution was concentrated in vacuo and chromatographed (3:1 hexanes/diethyl ether). The product was obtained as a colorless oil (510 mg, 77.5%). Rf = 0.54 (25% ethyl acetate-.hexanes).

[a]22D = -7.90 (c 7.6 in CHCI3). ^H NMR (300 MHz, CDCI3) 6 1.18 (d, 7 = 6.6 Hz, 3

H, CH3), 2.34 (s, 3 H, SCOCH3), 2.99 (dd, 7 = 13.5, 8.4 Hz, I H, H-1), 3.38 (td, J =

9.3, 2.7 Hz, 1 H, H-2), 3.47 (q, J = 6.3 Hz, 1 H, H-6), 3.60 - 3.69 (m, 3 H, H-T. H-

4, H-5), 3.79 (t, J = 9.3, 1 H, H-3), 4.71 - 4.81 (m. 4 H, 0CH2Ar), 5.00 (t, J = 12.3

Hz, 2 H, OCHiAr), 7.30 - 7.45 (m, 15 H, ArH). NMR (75 MHz, CDCI3) 6 17.1,

30.4, 31.4, 72.4, 74.3, 74.5, 75.3, 76.3, 77.7. 78.5. 84.8, 127.5, 127.6, 127.7, 64

128.1, 128.2, 128.3, 128.4, 138.1, 138.2, 138.5, 195.4. IR (CDCI3) 621, 697, 740,

1114, 1370, 1463, 1506, 1693, 2868, 2936 cm-1. FABMS m/z calcd for C30H35O5S

507.2205 (M + H), found 507.2221.

l-S-Methyl-2,6-anhydro-l,7-dideoxy-3,4,5-tri-0-benzyl-L-glycero-L-

galacto-heptitol (1.14). l-S-Acciyl-2,6-anhydro-l,7-dideoxy-3,4,5-tri-0-benzyl-L-

glycero-L-galacto-heptitol (1.13, 79 mg, 0.16 mmol) was dissolved in methanol (3 mL).

Sodium methoxide (0.84 mg, 0.016 mmol) was added. After 30 min, the methanol was

removed in vacuo. The flask was flushed with argon upon removal of the vacuum. THF

(3 mL) was added to the crude product followed by methyl iodide (9.2 |iL. 0.15 mmol).

After 1 h, the solvent was removed in vacuo and the crude product chromatographed (3:1

hexanes/diethyl ether). The product was obtained as a colorless oil (24 mg, 34%). Rf =

0.58 (25% ethyl acetate:hexanes). = +33.3° (c 0.3 in CHCI3). NMR (250

MHz, CDCI3) 5 1.21 (d, J = 6.3, 3 H, CH3). 2.18 (s, 3 H, SCH3). 2.65 (dd. J =

14.0,8.4 Hz, 1 H, H-1), 2.90 (d, J = 14.0 Hz, 1 H, H-l'), 3.41 - 3.53 (m, 2 H, H-2,

H-6), 3.60 - 3.66 (m, 2 H, H-4, H-5), 3.82 (t, 7 = 9.2 Hz. 1 H, H-3), 4.66 - 4.81 (m, 4

H. 0CH2Ar), 4.96 - 5.03 (m, 2 H, 0CH2Ar), 7.26 - 7.41 (m, 15 H. ArH). NMR

(63 MHz, CDCI3) 5 17.3, 35.8, 72.3, 74.2, 74.5, 75.3, 76.5, 77.5, 80.8, 85.1, 127.5,

127.6, 127.7, 128.1. 128.2, 128.4, 138.3, 138.7. IR (CDCI3) 680. 740. 1098. 1361.

1463, 1489, 1651, 2876, 2953. 3438 cm-1. FABMS m/z calcd for C29H3404SNa

501.2076 (M + Na), found 501.2058.

l-MethyIsuIfonyl-2,6-anhydro-l,7-dideoxy-3,4,5-tri-0-benzyl-L-glycero-

L-galacto-heptitol (1.15). l-5-Methyl-2,6-anhydro-l,7-dideoxy-3.4.5-tri-(9-benzyl-

L-glycero-L-galacto-heptitol (1.14. 18 mg. 0.038 mmol) was dissolved in dichloromethane (I mL) and cooled to 0"C. Dimethyldioxirane (1.8 mL, 0.083 mmol) 65

was added. After 30 min, the solvent was removed in vacuo to provide the sulfone as a colorless oil (19 mg, 99%). Rf = 0.15 (25% ethyl acetaterhexanes). = -90.9° (c

0.22 in CHCI3). NMR (250 MHz, CDCI3) 5 1.13 (d, J = 6.3, 3 H, CH3), 2.94 (s, 3

H, SO2CH3), 3.10 (dd, J = 14.9, 9.3 Hz, 1 H. H-1), 3.27 (d, /= 14.9 Hz, 1 H, H-1').

3.53 - 3.83 (m, 5 H, H-2. H-3, H-4, H-5, H-6), 4.62 - 4.81 (m, 4 H, 0CH2Ar), 4.95 -

5.01 (m, 2 H, 0CH2Ar), 7.28 - 7.39 (m, 15 H, ArH). NMR (63 MHz, CDCI3) 5

17.0, 43.4, 56.5, 72.6, 74.6, 74.8, 75.0, 75.1, 75.8, 76.1, 84.8, 127.7, 127.9, 128.0,

128.2, 128.3, 128.6, 137.6, 138.1. IR (CDCI3) 706, 740, 1029, 1089, 1131, 1310,

1463, 2876, 2927. 3412 cm-1. FARMS m/z calcd for C29H3406SNa 533.1973 (M +

Na), found 533.1949.

l-Methylsulfoxyl-2,6-anhydro-l,7-dideoxy-3,4,5-tri-O-benzyl-L-glycero-

L-galacto-heptitol (1.16). l-5-Methyl-2,6-anhydro-1.7-dideoxy-3,4,5-tri-(9-benzyl-

L-glycero-L-galacto-heptitol (1.14, 21.3 mg, 0.0446 mmol) was dissolved in dichloromethane (2 mL) and cooled to O^C. 50 - 60% m-CPBA (15.4 mg, 0.0446 mmol) was added. After 30 min, the solvent was concentrated in vacuo and the crude product chromatographed (7:3 benzene/acetone). The sulfoxide was obtained as a colorless oil

(15.6 mg, 70.8%) and as a 1:1 mixture of diastereomers based upon integration of the methyl sulfoxide singlets. Rf = 0.40 (30% acetone:benzene). 'H NMR (250 MHz,

CDCI3) 5 1.14 (d, J = 6.4 Hz, 3 H, CH3), 2.52 (s, 3 H, SOCH3), 2.62 (s, 3 H,

SOCH3), 2.91 (dd, 7 = 13.5, 7.2 Hz, 1 H, H-1), 3.06 (d, 7 = 13.1 Hz. 1 H. H-l"), 3.52

(dd, 7 = 12.2, 5.2 Hz, 1 H), 3.62 - 3.78 (m, 3 H), 3.86 (t, 7 = 8.9 Hz, 1 H. H-3), 4.62

- 4.80 (m, 4 H, 0CH2Ar), 4.99 (dd, 7 = 11.4, 3.2 Hz, 0CH2Ar), 7.29 - 7.36 (m, 15 H,

ArH). NMR (63 MHz, CDCI3) 5 16.9, 17.2, 38.9, 39.8, 54.4, 57.7, 72.4, 72.5,

72.8, 73.2, 74.5, 74.7, 74.8, 75.0, 75.1, 76.2, 76.3, 77.2, 84.8, 85.0, 127.6, 127.7, 66

127.8, 128.2, 128.3, 128.4, 128.5, 138.1, 138.4. IR (CDCI3) 706, 740, 1021, 1055,

1098, 1157, 1344, 1463, 1489, 1634, 2885, 3038, 3072, 3455 cm !. FABMS m/z calcd for C29H34C)5SNa 517.2025 (M + Na), found 517.2015.

l-Methylsulfonyl-2,6*anhydro-l-deoxy-D-glycero-D-gulo-heptitoi (1.17). l-MethylsulfonyI-2,6-anhydro-l-deoxy-3,4,5,7-tetra-Obenzyl-D-glycero-D-gulo-heptitol

(1.10, 22 mg, 0.034 mmol) was dissolved in glacial acetic acid (5mL). The system was ev acuated and hydrogen gas added. After 24 h, the system was ev acuated and opened to the atmosphere. The solid was filtered off and the filtrate concentrated in vacuo to provide the product as a colorless oil (8 mg, 94%). 'H NMR (250 MHz, D2O) 6 2.94 (s,

3 H, SO2CH3), 3.22 - 3.68 (m, 9 H). NMR (63 MHz. D2O) 6 43.6, 57.2, 61.3,

61.5, 70.7, 75.7, 78.5, 81.0, 81.7.

(NOTE: FABMS of this compound did not yield the desired molecular ion peak. Further experimental work is needed.) CHAPTER 2 PREPARATION OF SIALIC ACID DERIVATIVES AMENABLE SOLID-PHASE SYNTHESIS 68

INTRODUCTION

Protein and nucleic acid chemistry have experienced rapid advances over the years

due in large part to the development of solid-phase synthesis of peptides^'* and oligonucleotides^^ which has allowed for the facile preparation of these oligomers. The

preparation of oligosaccharides by solid-phase synthesis has been more problematic.

This is due to the fact that there has been a lack of an effective stategy to deal with differential protection/deprotection of the hydroxyl groups and a lack of stereoselective coupling of polyfunctional donors and acceptors that can be achieved in high yield. The

preparation of oligosaccharides via solid-phase or enzymatic methods^^'^^ has provided a more rapid and effective entry into biologically important oligosaccharides than traditional chemical synthesis currendy allows.

Recently, advances have been made in the application of solid-phase and enzymatic methods for the preparation of oligosaccharides. In 1991 Krepinsky^® reported the use of a soluble polyethylene glycol (PEG) polymer-supported resin linked to different carbohydrate hydroxyls through an ester linkage of succinic acid (Su) (Figure 2.1).

Krepinsky's method requires that the polymer-supported carbohydrate syndion be soluble for glycosylation and insoluble for workup of reaction products. Glycosylation is driven to completion by addition of excess glycosylating agent which is then readily removed from the precipitated PEG-bound product. The PEG-bound product is precipitated using diethyl ether and recrystallized from ethanol. The PEG-Su substrates are then cleaved by

DBU-catalyzed methanolysis in dichloromethane. 69

?^^OAc nxnnps

HO BF30Et2 AcO 0C(NH)CCl3 + PEGSu-C OMe DCM OAc NPhth

OAc

OTBDPS AcO O ° ^si^OMe SuPEG NPhlh

Figure 2.1. Krepinsky's solid-phase method for the preparation of oligosaccahrides.

The advantage of this approach is that this method combines the anomeric control of solution-phase chemistry along with the facility of workup of solid-phase preparations.

Kahne®^ has reported using anomeric sulfoxides linked to the Merrified resin for the

preparation of oligosaccharides. Anomeric sulfoxides, which can be activated under mild conditions, are employed as glycosyl donors (Figure 2.2). The glycosyl acceptor, a thioglycoside, is covalendy bound to the resin. The coupling occurs under typical

Knoenigs-Knorr reaction conditions to yield a dimer which can be further coupled with other anomeric sulfoxide glycosyl acceptors. 70

OPiv/OR

PivO l.TMSCI, TEA THF. 0°C 2®CH2C1, DMF 60°C

OPivyOR OPiv. PTr

PivO PivO

OPiv

Hg(0Tf)2. DCM, H2O

OPiVyPTr

OPiv PivO

PivO

Figure 2.2. Kahne methodology for the solid-phase preparation of P-linked oligosaccharides.

This method allows for the preparation of glycosidic linkages other than the 1 -• 6 linkage and the preparation of a and (3 linkages to secondary alcohols stereospecifically and in high yield.

Danishefsky^^ has also recently reported a new strategy for the preparation of oligosaccharides using solid-phase supports. Danishefsky's approach involves the use of a glycal that is attached to a polystyrene copolymer via a silyl ether bond. The glycal is 71

then activated by the addition of an electrophile (3,3-dimethyldioxirane) to form an

epoxide. Glycosylation is achieved stereospecifically by addition of a promoter and a

glycal acceptor (Figure 2.3). The oligosaccharide is then cleaved from the solid-phase

support by addition of tetra-n-butylammonium fluoride (TBAF).

O

Figure 2.3. Danishefsky's protocol for the preparation of oligosaccharides (P =

protecting group, S = solid support).

The advantages of this methodology are that differential protection is more easily achieved in glycals than pyranosyl sugars, the epoxidation with dimethyldioxirane is quantitative, and that the free hydroxyl group at C-2 after glycosylation can allow for the synthesis of branched oligosaccharides. This methodology is also self-correcting in the sense that lack of coupling results in the formation of an anomeric hydroxyl which can be easily 72 separated from the coupled product. The disadvantage of this method is that only P- linked oligosaccharides can be prepared from the glucal epoxide as the glycosyl donor.

Danishefsky^' has employed his solid-phase methodology in the synthesis of the

Lewis^ blood group determinant (Figure 2.4), a mediator in the binding of Heliobacter pylori to human gastric epithelium. Danishefsky was able to prepare the determinant stereospecifically and, because his methodology produces a free hydroxyl at C-2, was able to introduce the necessary branched carbohydrate moiety.

OH

NHAc OH

OR

Figure 2.4. Lewis'' blood group determinant.

While the development and application of solid-phase techniques to oligosaccharide synthesis has greatly improved the potential for preparing these compounds, one of the problems that still remains is that there is no general method available for the synthesis of any glycosidic linkage. One approach to overcome this problem is to use enzymes for glycosylations.^"^"'^-^"^ The advantages of using enzymes are that they exhibit high specificity and that unprotected sugars can be used for glycosylations. The disadvantages of enzymatic synthesis are the high cost or unavailability of the desired glycosyl transferases and the difficulty in work-up and separation. The difficulty in work-up and 73 separation can be partially overcome by performing enzymatic synthesis on a solid support or by using dialysis membranes.

Kopper^^ has reported the use of a preparative scale enzymatic synthesis using a polymer support Kopper's approach involves the binding of the glycosyl acceptor with l-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EIX!I) to a spacer that is attached to a poly(acrylamide) gel (Figure 2.5). Addition of UDP-galactose and galactosyliransferase to the resin effected glycosylation. The disaccharide was then photolytically cleaved from the solid support to yield the free disaccharide. 74

02N^^^\.C02H

gel Gluc-0> ^ '3or4j' EDCI

b^,Iy gel 4 O

UDP-Gal, GalTase

Figure 2.5. Kopper method for the solid-phase preparation of oligosaccharides.

Wong^*^ has developed a method for the solid-phase preparation of glycopeptides and oligosaccharides. Wong's methodology involves the chemical synthesis of peptide bonds and the enzymatic synthesis of glycosidic bonds on an aminopropyl silica support that is 75 compatible with aqueous and organic solvents. Initially, a hexaglycine spacer is attached to the silica support In the second step, the peptide portion of the glycopeptide is linked to the spacer group which is necessary to reduce steric problems between the support and the acceptor residues. Any additional peptide bonds are made and then the glycosidic bonds are made using the appropriate glycosyl transferase (Figure 2.6). The key to

Wong's approach is that the acceptor-spacer attachment must be selectively cleavable in the final release of the glycopeptide from the solid support Wong attaches the peptide to the spacer with a phenylalanyl ester bond which can be selectively cleaved with a- chymotrypsin, which is highly specific for species that contain aromatic amino acid residues. 76

l.TFA,DCM BOCHN. JL. B0C-(Gly)6HN^ ^ Si- • i °n^silicA v y 2.0-(yV-Phe)glycolic acid ' BOP, HOBt, DEA

1.TFA,DCM OH O 2. BOC-Gly-OH, BOP BOCHN^ X HOBt. PEA , 3. TFA, DCM 4. B0C-Asn(GlcNAcp)-0H NHAc BOP. HOBt, DIEA O

1. P-l,4-GalTase, UDP-Gal 2. a-2.3-SiaTasc. CMP-NeuAc , 3. a-chymotrypsin 4. a-l,3-FucTase, GDP-Fuc

BOCHN OH HO HO O NHAc AcHN OH V /

Figure 2.6. Wong's chemical-enzymatic approach to the synthesis of sialyl Lewis*

glycopeptide.

Wong^^ has also used solid-phase synthesis for the preparation of other biologically active oligosaccharides. In this example, Wong used a combined solution- and solid-

phase approach to the synthesis of tetrasaccharide

(NeuAca2-»3Gaipi-»4GlcNAc|3l-»3Gal) that inhibits H. pylori attachment to human gastric mucosa and E-selectin-mediated leukocyte adhesion. In this case a synthetic

primer (GlcNAcpi-» 3GaipOEt) was attached to a controlled pore glass (CPG) via a 77

spacer containing an ester bond. This was followed by enzymatic incorporation of

galactose and sialic acid to produce the desired tetrasaccharide (Figure 2.7). Cleavage of

the tetrasaccharide from the solid-support with hydrazine yielded the desired

tetrasaccharide.

-OH

NHAc \SL-0-~(^C02Cs DMF

^OH OH ^oH HO HO O NHAc OH

1. GalTase, UDP-Gal 2. a-2,3-SiaTase CMP-NeuAc

9^ ^OH

OH HO

AcHN NHAc

Figure 2.7. Synthetic strategy to inhibitors of H. pylori.

In the last few years work has been directed toward the means of preparing oligosaccharides and their analogs more rapidly and efficiently. While solid-phase 78

synthesis has improved the ability to prepare oligosaccharides, solid-phase methods (both

chemical and enzymatic) still are not routine. One new approach is to prepare pepdde

bond-linked carbohydrates as new oligosaccharide ligands.®^ These new materials

(Figure 2.8) have great potential use as novel ligands for use in combinatorial libraries^'-

whereby numerous oligomers can be prepared rapidly and assayed for biological activity. Amide-linked oligosaccharides have potential as drug candidates since they may

not be recognized either by glycosidases (since there is no glycosidic linkage) or proteases (since there is an altered backbone in relation to natural peptides).

OH n

Figure 2.8. Prototypical carbohydrate amino acid.

Several carbohydrate amino acids which could be used in combinatorial libraries have appeared. Paulsen^^ prepared a carbohydrate amino acid in two steps from the benzyl glycoside (Figure 2.9). 79

.OH HO2G HO- 1. O2, Pt HO- HO HO 2. H2, Pd NH I Y OBn OH

OBn

Figure 2.9. Carbohydrate amino acid prepared by Paulsen.

In 1975 Lehmann'^ reported the synthesis of two carbohydrate amino acids (Figure

2.10).

OH OH

Figure 2.10. Carbohydrate amino acids prepared by Lehmann.

Neither Paulsen nor Lehmann prepared amide-linked oligomers from their monomeric units. In 1976 Yoshimura'"^ prepared several disaccharides that were 2-»6 linked via an amide bond (Figure 2.11). However, no further work was done on these types of compounds until the mid-1990s when application of these materials could be made to the development of combinatorial libraries. 80

OH

HO- HO

Hfj OBn O

Figure 2.11. Yoshimura's disaccharide with an amide linkage.

Wessel's research^^ built upon the earlier work of Yoshimura. In this work, Wessel prepared an amide-linked tetramer using a benzhydrylamine polystyrene resin from an

Fmoc-protected uronic acid building block (Figure 2.12). The carbohydrate amino acid was coupled to the solid-support using 0-(7-azabenztriazol-1-yl)-1,1,3.3- tetramethyluronium tetrafluoroborate (TATU) and the subsequent tetramer was cleaved from the resin with TFA.

Q .OH

HO- HO

Fmoc' OBn

Figure 2.12. Monomeric unit for Wessel's tetrasaccharide synthesis.

Wessel has also prepared a carbohydrate amino acid tetramer using solution phase chemistryThe monomeric unit in this case was a normuramic acid. Two dimers were 81 prepared and the dimers were then coupled together in a block synthesis (Figure 2.13).

Coupling was carried out by the diester method using 2-chloro-4,6-dimethoxy-1,3,5- triazine (CDMT) as the activating agent

OH 1. ClCOO/Bu. TEA. MeCN 2. AC2O, pyridine 3. H2, Pd(C) NH; NH OBn OBn

OAc

OAc AcO NH OBn NH OBn

Ph^^O

O MeaCOgC,. .0 1. ClCOO/Bu, TEA, MeCN NH NH2 ^ OBn OBn 2. NaOMe, MeOH

OH

HO .OH HO NH OBn NH OBn

B 82

1. CMDT, DMF A + B AcjO. pyridine 2. NaOMe, MeOH

OH

HO' HO

OBn

Figure 2.13. Wessel's block strategy to a carbohydrate-peptide tetramer.

While Wessel's work has involved the preparation of oligomers of carbohydrate amino acids, most work in this area is still focused on the synthesis of carbohydrate amino acid monomer units. Kessler^^ has synthesized in six steps from D-glucose a monomer unit consisting of a carboxylate group at the 7-position and an amine unit at the

1-position of the C-glycoside (Figure 2.14). 83

I. MeNOj NaOMe

2. MeOH, DCC, DMAP

OMe OH

HO NaOH, H2 HO HO HO NHZ pd(C) NH2 OH OH

Figure 2.14. Kessler's synthesis of a carbohydrate amino acid.

Kessler then examined the effect of the incorporation of this carbohydrate amino acid on linear peptides. In one example, the Gly-Gly unit of natural Leu-enkephalin was replaced with the carbohydrate amino acid, a dipeptide isostere. This enkephalin analog did not possess biological activity, but NMR studies seemed to indicate that the carbohydrate amino acid did induce p-tums in the linear peptide analogs studied (enkephelin and somatostatin).^^ More recently, Kessler^^ has reported preparation of several different carbohydrate amino acid derivatives and the incorpoation of these derivatives into a peptide sequence. The position of the amine and carboxylate functionalities on the carbohydrate backbone determined whether the peptide sequence possessed a linear, (3- tum or y-tum conformation. 84

Merrer^ has synthesized several carbohydrate-like amino acids via a silica gel assisted azidolysis of enantiomerically pure bis-epoxides (Figure 2.15). A mixture of the pyranose and furanose rings are produced with the ftiranose ring system predominating.

The primary alcohol is then oxidized to the carboxylic acid, and the acid is protected as the methyl ester by treatment with diazomethane. The azide is reduced to the amine, and the amine is BCK! protected.

1. Na2Cr207, Et20 2. CH2N2 3. H2, Pd(C) 4. BOCjO, EtOAc

NHBOC

Figure 2.15. Merrer's methodology for the preparation of carbohydrate amino acids.

Lansbury'®° has synthesized twelve new carbohydrate amino acids that could be amenable to solid-phase synthesis for the preparation of carbohydrate oligomers. These compounds were prepared by u-aditional solution phase multistep techniques. However, 85

this multistep approach to the monomeric carbohydrate amino acid units is not especially

advantageous for the development of combinatorial libraries.

Ichikawa'®^ has reported the preparation of carbohydrate amino acids that contain a

C-1 carboxylate and a C-2 amino group (Figure 2.16).

OH

HO CO2R HC

NHR" 2

Figure 2.16. General structure of Ichikawa's carbohydrate amino acid tetramer.

Ichikawa prepared a tetramer in a stepwise fashion from D-glucosamine as the starting material. An anomeric carboxylate was coupled to a free amine at C-2 with benzouiazole- l-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate (BOP) as the coupling reagent in 59% yield (Figure 2.17) to yield a dimer.

NHBOC

AcO AcC CO2R NHBOC

Figure 2.17. Ichikawa su^ategy for the coupling of carbohydrate amino acids. 86

Ichikawa^®^ has also prepared a persulfated P(l-»6)-linked tetramer (Figure 2.18).

O O

NHR"

"OR RO'^^Y^^''"OR OR OR

R = SOsNa

Figure 2.18. Persulfated P( 1 -»6)-linked oligosaccharide synthesized by Ichikawa.

This tetramer was prepared in a stepwise manner from methyl 2,6-anhydro-D-^/yc^ro-D-

^u/o-hepturonate (Figure 2.19)

CO2M6

Figure 2.19. Methyl 2,6-anhydro-D-^/3'cero-D-^w/o-hepturonate.

Solution phase peptide coupling was accomplished with diethylphosphoryl cyanide

(DEPC) as the coupling agent in triethylamine and DMF. The coupling proceeded in 92%

yield. The persulfated tetramer was found to possess an IC50 of l|iM against the Human

Immunodeficiency Virus (HFV). Zidovudine (AZT), by comparison, has a IC50 value of

0.024 - 2.5 ^lM. 87

N-acetyl neuraminic acid (sialic acid) is a naturally occurring carbohydrate that contains

a 5 amino acid functionality (Figure 2.20). The goal of our research in this area is to first

remove the acetyl protecting group on the nitrogen to generate the free amine and then,

through protecting group manipulation, to make sialic acid amenable to amide bond

coupling on solid-phase supports with other sialic acid monomers or with various amino

acids. It is anticipated that the side chain will afford greater solubility of the amide-linked

oligomers.

AcHN

Figure 2.20. N-acetyl neuraminic acid.

RESULTS AND DISCUSSION

The first step in preparing sialic acid amino acid monomer units involves the deprotection of the A^-acetyl protecting group. The first synthetic strategy (Figure 2.21) to this target involved ^-deacylation via 0-ethylation of the amide oxygen with triethyloxonium tetrafluoroborate (Meerwein's salt)'°^and subsequent treatment with saturated NaHCOs to afford the free amine. This method is highly advantageous in that free amines can be prepared under very mild conditions and acetyl and ester functionalities are left unaffected. The peracetylated benzyl ester of sialic acid'°^ was used in our studies. The sialic acid derivative readily reacted with one equivalent of commercially available Meerwein's salt, but attempts to remove the salt to provide the free 88

amine by either hydrolysis with saturated NaHC03 or treatment with Amberlite basic

resin failed.

OAc, OAc AcO Et30"(BF4)-^ C02Bn AcHN DCM AcO

sat'd NaHCOj

or Amberlite basic resin

OAc, OAc OAc AcO COaBn AcO

Figure 2.21. Strategy to sialic acid free amine using Hanessian's methodology.

Since failure was encountered here with sialic acid, attempts were made to repeat

Hanessian's paper in which he used peracetyl glucosamine, but even these attempts to

repeat the literature procedure failed.

The next strategy to remove the N-acetyl group of sialic acid involved tert-

butoxycarbonylation^®^-'®*^ of the A^-acetyl group of sialic acid to form an imide and subsequent removal the A^-acetyl group upon treatment with hydrazineto form a rert-

butyl carbamate (Figure 2.22). 89

OAc AcO BOCjO, DMAP C02Bn AcHN TEA, DCM AcO

OAc AcO N2H4 COaBn BOCAcN MeCN AcO

OAc AcO

BOCAcN

Figure 2.22. Unsuccessful strategy for the removal of the N-acetyl group on sialic acid via rerr-butoxycarbonylation and subsequent hydrazinolysis.

Protection of the nitrogen with di-r^/t-butoxydicarbonate (BOC2O) proceeded smoothly but only in fair yields (typically 40-60%). Treatment with one equivalent of hydrazine in either acetonitrile or methanol at room temperature did not result in loss of the A^-acetyl group, but in the loss of the benzyl ester, which was no longer observed in the 'H NMR spectrum. The product obtained was not fully characterized.

Since hydrazine addition resulted in cleavage of the benzyl ester, another strategy was employed whereby the carboxylate group of sialic acid would be tied up as a 1,4-iactone.

It was hoped that the lactone would be less labile to hydrazinolysis than the benzyl ester of sialic acid. The 1,4-lactone was easily prepared by benzoylation of sialic acid'°®

(Figure 2.23). 90

BzCl, pyridine

AcHN

OBz OBz OBz BOC2O, DMAP BzO OBz'

AcHN- TEA, DCM

OBz OBz BzO ,OBz N2H4

BOCAcN MeOH

OBz BzO

BOCHN

Figure 2.23. Partially successful strategy to A^-deacylated sialic acid via a 1,4-lactone.

A mixture of the 1,4- and 1,7-lactone were obtained and were readily separated by flash

chromatography. The amide nitrogen was then BOC protected to form an imide, and the

imide then reacted with 1.1 equivalents of hydrazine. 'H NMR revealed that indeed the

yV-acetyl group had been removed. The NMR also showed that the lactone had

opened up. It was believed hydrazine first attacks Lhe imidate to form the BOC protected amine and acetyl hydrazine (Figure 2.24). The acetyl hydrazine was sufficiently

nucleophiUc to add into the lactone. 91

• sugar ^sugar XX. N

HzN—NHa

OBz, +• sugar o-— BzO BOCHN HgN—1^-

A

OBz BzO

BOCHN

Figure 2.24. Proposed hydrazinolysis mechanism and subsequent addition of acetyl hydrazine to perbenzoyi 1,4-lactone of sialic acid.

Since hydrazine proved to be too reactive for A^-deacetylation of the imidate, milder conditions were sought. Grieco'°^ reponed that secondary BOC-protected amides could be hydrolyzed to the BOC-protected amine with sodium methoxide in methanol (Figure

2.25). 92

.0 O BOC2O, DMAP NH ^NBOC TEA, DCM

NaOMe HOgC^ ^ ^NHBOC MeOH

Figure 2.25. Grieco's methodology for the methanolysis of lactams.

Our strategy was then to first protect the ^V-acetyl group of sialic acid as an A^-acetyl-

N-BOC imide and then proceed with methanolysis to provide the deactylated A^-BOC sialic acid derivative (Figure 2.26).

OAc AcO, OAc BOC2O, DMAP igBn ^ AcHN TEA, DCM, 60%

OAc OAc AcO. OAc 1. NaOMe, MeOH C02Bn BOCAcN AcO 2. Dowex H"", HjO, 94% 2.1 OH OH HO. OH

BOCHN HO 2.2

Figure 2.26. Strategy to N-BOC sialic acid derivative via imidate methanolysis. 93

The ferr-butoxycarbonylation of the peracetyl benzyl ester of sialic acid typically

proceeded in 40-60% yields with a 2:1 ratio of rotamers formed based upon NMR

integration of the two singlet BOC resonances observed. Treatment of the imide with

sodium methoxide in methanol did cleanly afford the fully deprotected N-BOC sialic acid

derivative in 94% yield. This compound, with a free carboxylic acid and an A'-BOC

protected amine, could then be employed for the preparation of carbohydrate peptide

oligomers.

The deprotected A^-BOC sialic acid derivative was reacetylated so that the material

would be easier to handle in coupling reactions. Upon reacetylation, however, only four

singlet 0-acetyl resonances instead of five were observed. This reacetylation was also

carried out on the anomeric hydride derivative, and again one less 0-acetyl singlet than

expected was observed. It is believed that upon reacetylation of the deprotected sugar,

lactonization between the anomeric carboxylate and one of the free hydroxyls was

occurring (Figure 2.27).

OAc AcO HO AC2O COgH BOCHN BOCHN HO pyridine

OAc

BOCHN BOCHN pyridine HO

Figure 2.27. Lactonization of N-BOC sialic acid derivative upon acetylation. 94

Since reacetylation had proven to be problematic, the A^-BOC sialic acid derivatives

with all hydroxyl groups unprotected needed to be used in couplings for the preparation

of sialic acid oligomers linked through peptide bonds.

Since the free carboxylate, A^-BOC protected sialic acid derivative (2.2) was prepared,

work focused again on the preparation of a free amine, sialic acid ester derivative. Fuchs

and Lehman reported A^-deacylation of an amide by treatment with 2M NaOH (Figure

2.28).

OH

2M NaOH NHAc

Figure 2.28. Methodology employed by Fuchs for A^-deacylation.

The methyl ester methyl glycoside of sialic acid'^® was treated NaOH to provide the free amine in 72% yield (Figure 2.29).

HO HO 2N NaOH COgH 100°C, 72% HO HO 2.3

Figure 2.29. A^-deacylation of the methyl glycoside mediyl ester of sialic acid. 95

The methyl glycoside methyl ester derivative was used in order to prevent a retro-aldol

reaction from occurring under the strongly basic conditions employed. The carboxylic

acid group was then protected as the methyl ester (Figure 2.30). The free amine sialic

acid derivative could then be coupled with other sialic acid derivatives.

OH OMe OH OMe OH HO. HCl, HO.

HCIHoN HO MeOH, reflux HO 93% 2.3 2.4

Figure 2.30. Methyl ester protection of sialic acid free amine.

The sialic acid derivative with the acid function blocked as a methyl ester (2.4) was then coupled to the peracetylated free acid of sialic acid (Figure 2.31).

OAc OMe 9" OH AcO HO. + AcHN AcO HO 2.4

AcO PAc

i^*^OAc OAc PAc 1. BOP, DMF AcO Hunig's Base AcHN OMe AcO AcO 2. AC2O, pyridine, 30%

2.5

Figure 2.31. Preparation of amide-linked sialic acid dimers. 96

Since the initial dimer formed is water soluble and DMF was used as a co-soIvenL, the

crude product was acetylated for the purpose of characterization. The dimer was obtained

in 30% yield overall yield. An attempt to improve the yield for this coupling by using 1-

hydroxy-7-azabenzotriazole (HOBt) as an additive failed to improve the yield. While this coupling reaction has demonstrated that amide-linked sialic acid oligomers can be

prepared through standard peptide coupling techniques, the use of the free acid of

peracetyl sialic acid (with the nitrogen blocked as an A^-acetyl group) does not allow for the facile preparation of sialic acid oligomers.

Since the N-BOC protected sialic acid (2.2) had been prepared, it was attempted to couple this derivative with the methyl glycoside methyl ester free amine of sialic acid

(2.4) (Figure 2.32).

C02Me BOCHN HCIH2N HO HO

AcO

l.BOP, HOBt DMF, Hunig's Base •OMe 2. AC2O, pyridine BOCHN COsMe

Figure 2.32. Attempted coupling of A^-BOC sialic acid derivative with the methyl glycoside methyl ester free amine of sialic acid. 97

No sialic acid dimer units were isolated upon workup and purification of the crude

material. Peracetylated methyl glycoside methyl ester of sialic acid was isolated as the

major product It is believed that no coupling occurred because treatment of the free acid

with BOP and 1-hydroxybenzotriazole (HOBt) resulted in preferential intramolecular

lactone formation from either the 4-OH or the 7-OH.

In order to prepare amide-linked sialic acid oligomers, a revised strategy was

employed. The nitrogen on the free acid monomer would be protected as the N-BOC N-

Ac imide. After coupling to the free acid derivative, the dimer would be deacylated to

provide a dimer with a terminal ^V-BOC group which would be available for elaboration

into longer sialic acid oligomers using standard peptide deprotection/coupling procedures.

The A^-BOC peracetyl benzyl ester of sialic acid (2.1) was prepared by first BOC

protection of amide. Hydrogenolysis of the A^-BOC peracetyl benzyl ester of sialic acid

provided the free acid (2.6) in 85% yield (Figure 2.33).

l.BOCjO, DMAP DCM, TEA, 60% AcHN

BOCAcN AcO 2.6

Figure 2.33. Preparation of free acid monomer unit. 98

The free acid (2.6) was coupled to the sialic acid free amine (2.4) to afford the dimer

(2.7) in 27% yield for both steps (Figure 2.34).'^^

OAc OH OMe OAc OH AcO OAc HO.

BOCAcN AcO HO 2.4 2.6 AcO

1. BOP. DMF AcO Hunig's Base OMe

2. AC2O, pyridine, 27% BOCAcN COaMe

OMe NaOMe, MeOH BOCHN COaMe

Figure 2.34. Revised strategy for the preparation of amide-linked sialic acid oligomers.

The dimer was subsequeniiy deacetylated in 96% yield to provide a terminal N-BOC functionality which is now available for further elaboration. 99

CONCLUSION

A strategy has been developed for the preparation of amide-linked sialic acid oligomers. Sialic acid derivatives possessing either a free amine or a free acid functionality have been synthesized and coupled using standard solution-phase peptide coupling techniques. These compounds may afford greater solubility than previously synthesized amide-linked carbohydrate amino acids. Furthermore, they may give rise to novel helical structures by virtue of the fact that they are derived from sialic acid, glycosidic oligomers of which are known to have helical structures in solution.

EXPERIMENTAL SECTION

Starting materials and reagents purchased from commercial suppliers were used without further purification. Solvents were dried by distillation prior to use.

Dichloromethane, dimethylformamide, triethylamine and pyridine were distilled from calcium hydride under argon. Methanol was distilled from Mg/l2 under argon. All reactions were performed under an argon atmosphere.

Thin layer chromatography was performed using silica gel 60 F254 plates. Flash column chromatography was performed using silica gel 60 (230-400 mesh ASTM).

Proton and carbon nuclear magnetic resonance spectra were recorded on either a Bruker

AM-250 or Varian Unity 300 spectrometer. Chemical shifts are reported in parts per million relative to the residual solvent peak. NMR data are reported in the order of chemical shift, multiplicity (s = singlet, d = doublet, q = quartet, m = multiplet, br = broad), the coupling constant in hertz (Hz) and the number of protons. Infrared spectra were recorded using a Nicolet 510P FT-IR spectrometer. Specific rotations were 100 determined using an Autopol III polarimeter. Mass spectrometry was performed by the

Nebraska Center for Mass Spectrometry, Lincoln, NE.

Benzyl 5-A^-acetyl-5-A^-

(302 mg, 60.3%). A 2:1 mixture of mixture of rotamers was obtained. Rf = 0.50 (50% ethyl acetate:hexanes). [a]22D = -7.1° (c 2.8 in CHCI3). NMR (250 MHz, CDCI3) 5

1.60 (s, 9 H, NCOOCMeB), 1-93 (s, 3 H, OCOCH3), 1.99 (s, 3 H, OCOCH3), 2.00 (s,

3 H, OCOCH3), 2.03 (m, 1 H, H-3ax), 2.05 (s, 3 H, OCOCH3), 2.08 (s, 3 H,

OCOCH3), 2.35 (s, 3 H, NCOCH3), 2.61 (dd, J = 13.3, 5.5 Hz, 1 H, H-3eq), 4.05 -

4.15 (m, 1 H, H-9), 4.34 - 4.48 (m, 1 H, H-9'), 4.85 - 4.99 (m, 1 H, H-5), 5.04 - 5.34

(m, 5 H, H-6, H-7, H-8, OCHiAr), 5.60 - 6.16 (m, 1 H, H-4), &.30 - 7.34 (m, 5 H,

ArH). 13c NMR (63 MHz. CDCI3) 5 20.6, 20.7, 20.8, 26.7, 27.8, 28.2, 36.9, 52.1,

56.2, 61.7, 61.8, 66.0, 66.6, 67.0, 67.3, 67.7, 67.8, 69.7, 70.1, 71.2, 71.9, 84.8,

97.9, 127.8, 128.1, 128.3, 128.4, 134.9, 151.6, 165.6, 169.7, 170.4, 173.7. IR

(CDCI3) 737, 918, 951, 1008, 1036, 1076, 1115, 1145, 1229, 1337, 1372, 1396,

1689, 1705, 1747 cm-I. FABMS m/z calcd for C33H43NOi6Li (M + Li) 716.2742, found 716. 2714. 101 iV-BOC sialic acid (2.2). Peracetyl N-BOC benzyl ester of sialic acid (2.1, 66.0 mg, 0.095 mmol) was dissolved in dry methanol (3 mL). Sodium methoxide (0.5 mg,

0.(X)95 mmol) was added and the solution was stirred for 3 h. The methanol was removed in vacuo. The residue was taken up into water (50 mL) and extracted with ethyl acetate (3 x 50 mL). The aqueous layer was collected. The aqueous phase was acidified to pH 4 with Amberlite IR-120 resin. The resin was filtered and water evaporated to yield the deacetylated and debenzylated A^-BOC derivative of sialic acid as a colorless oil

(33.0 mg, 94.4%). [a]22D = -2.6° (c 7.6 in MeOH). NMR (250 MHz, D2O) 5 1.24

(s, 9 H), 1.84 - 2.04 (m, 2 H), 3.40 - 4.22 (m, 7 H). 13c NMR (63 MHz, D2O) 6 28.8,

29.0, 29.1, 53.6, 64.8, 68.3, 70.2, 70.8, 72.3, 74.5, 81.7, 102.3, 165.8, 176.0. IR

(CD3OD) 1208, 1455, 1591, 1693, 2553, 3404 cm-1. FABMS m/z calcd for

Ci4H250ioNNa 390.1403 (M + Na), found 390.1376.

5-amino-2-C>-methyl-3,5-dideoxy-D-glycero-D-galacto-2- nonulopyranoside (2.3). The sialic acid methyl glycoside methyl ester^'° (300 mg,

0.89 mmol) was dissolved in 2N NaOH (3 mL). The solution was heated at l(X)oC for

48 h. The solution was then acidified to pH 7 with Amberlite IR-120 resin. The solution was diluted with water to a total volume of 50 mL. The Dowex resin was filtered off and the filtrate was extracted three times with ethyl acetate (50 mL). The aqueous phase was collected and evaporated down to yield the free base as a brown oil (180 mg, 72.0%).

[a]22D = -3.90 (c 2.6 in MeOH). ^H NMR (250 MHz, D2O) 6 1.44 (t, 7 = 12.5 Hz, 1

H, H-3ax), 2.14 (dd, J = 13.2, 5.1 Hz, 1 H, H-3eq), 3.00 (s, 3 H, OMe), 3.50 - 3.56

(m, 3 H), 3.63 - 3.90 (m, 6 H). NMR (63 MHz, D2O) 5 39.7. 50.3, 52.5, 63.4,

68.2, 69.1, 70.1, 72.2, 100.5, 181.4. IR (CD3OD) 588, 673, 1244, 1442, 1635, 1726

3377 cm-1. FABMS m/z calcd for C10H20NO8 282.1189 (M + H), found 282.1197. 102

Methyl glycoside methyl ester free amine hydrochloride salt of sialic acid

(2.4). The methyl glycoside free amine of sialic acid (2.3, 253 mg, 0.90 mmol) was dissolved in methanol (10 mL). Anhydrous hydrogen chloride gas was bubbled into the methanol solution for 10 min. The solution was refluxed for 6 h. The solvent was then removed in vacuo to afford the product as a brown oil (248 mg, 93.4%). [a]22j) =

-26.80 (c 1.49 in MeOH). NMR (250 MHz. D2O) 6 1.64 (t, 7 = 12.3 Hz, 1 H, H-

3ax)> 2.24 (dd, J = 13.0, 5.0 Hz, 1 H, H-3eq), 3.10 (s, 3 H, OCH3), 3.51 - 3.73 (m, 8

H), 3.95 - 4.04 (m, 2 H). 13c NMR (63 MHz, D2O) 5 39.1, 46.6, 51.2, 52.1, 53.6,

62.8, 64.5, 67.5, 69.0, 69.5, 99.0, 169.8. IR (CD3OD) 1165, 1446, 1753, 2595,

2927, 3446 cm'l. FABMS m/z calcd for C11H22NO8 296.1354 (M - CI), found

296.1341.

iV-BOC N-kc peracetyl sialic acid free acid (2.6). A^-BOC N-Ac peracetyl sialic acid benzyl ester (2.1, 203 mg, 0.286 mmol) was dissolved in absolute ethyl alcohol (20 mL). Palladium on carbon (25 mg) was added and the flask was placed under a hydrogen atmosphere. After 6 h, the hydrogen was evacuated and the flask opened to the atmosphere. The solids were filtered off and the Titrate was concentrated in vacuo to yield the free acid as a colorless oil (150 mg, 84.8%). [a]22[) = .3.60 (c 11.0 in

CHCI3). IH NMR (250 MHz, CDCI3) 5 1.58 (s, 9 H, OCOCMes), 1.94 (s, 3 H,

OCOCH3), 1.98 - 2.13 (m, 1 H, H-3ax), 2.00 (s, 3 H, OCOCH3), 2.02 (s, 3 H,

OCOCH3), 2.05 (s, 3 H, OCOCH3), 2.16 (s, 3 H, OCOCH3), 2.36 (s, 3 H, NCOCH3),

2.58 - 2.66 (m, 1 H, H-3eq), 4.12 (dd, /= 12.6, 6.1 Hz, 1 H, H-9), 4.33 - 4.47 (m, 1

H, H-9'), 4.81 - 4.94 (m, I H, H-5), 5.02 - 5.21 (m, 3 H, H-6, H-7, H-8), 5.65 (td, J

= 10.2, 5.1 Hz, H-4). 13c NMR (63 MHz, CDCI3) 5 20.4, 20.5, 20.6, 27.7, 36.9,

52.0, 61.8, 65.8, 66.9, 67.7, 70.9, 71.7, 84.8, 97.2, 151.4, 167.5, 168.3, 169.8,

170.1, 170.8, 171.1, 171.8, 173.6. IR (CDCI3) 473, 667, 758, 1035. 1148, 1230, 1336, 1372, 1429, 1690, 1747, 3022 cm-l. FABMS calcd for C26H37NOi6Na

642.2010 (M + Na), found 642.2033.

N-\c sialic acid dinner (2.5). Peracetyl sialic acid free acid^®"^ (24 mg, 0.0463

mmol), diisopropylethylamine (24 |iL, 0.139 mmol) and BOP (30.7 mg, 0.0695 mmol)

were dissolved in a solution of dichloromethane (4(X) |lL) and dimethylformamide (1(X)

p,L). The methyl glycoside methyl ester free amine hydrochloride salt of sialic acid (2.4,

23 mg, 0.0695 mmol) was then added to the solution. After 48 h, the dichloromethane was removed in vacuo. The crude material was extracted with ethyl acetate (50 mL) and water (50 mL). The aqueous layer was collected, and the solvent was removed in vacuo.

The crude product was dissolved in pyridine (1 mL) and acetic anhydride (1 mL) was added. After 18 h, the solvent was concentrated by coevaporating with toluene (5 x 10 mL). The residual solvent was taken up into ethyl acetate (50 mL) and extracted with water (3 x 50 mL). The organic phase was collected, dried (Na2S04) and concentrated in vacuo. The crude product was chromatographed (7:3 benzene/acetone). The dimer was obtained as a colorless oil (11 mg, 24.7%). Rf = 0.11 (30% acetoneibenzene). [a]^2^ =

-30.80 (c 5.2 in CHCI3). NMR (250 MHz, CDCI3) 5 1.71 - 2.05 (m, 2 H), 1.89 (s,

3 H, NHCCX:H3), 2.03 (s, 9 H, OCOCH3), 2.09 (s, 6 H, OCOCH3), 2.10 (s, 6 H,

OCOCH3), 2.14 (s, 3 H, OCOCH3), 2.18 (s 3 H, OCOCH3), 2.47 (dt, J = 13.5, 5.7

Hz, 2 H), 3.28 (s, 3 H, OCH3), 3.80 (s, 3 H, CO2CH3), 3.99 - 4.18 (m, 5 H), 4.26

(d, y = 13.1 Hz, 1 H), 4.45 (dd, J = 13.1, 2.4 Hz, 1 H), 4.76 (dd, J = 13.0, 2.6 Hz, 1

H), 5.06 (br s, 1 H), 5.23 - 5.37 (m, 6 H), 6.99 (d, J = 9.4 Hz, 1 H). NMR (63

MHz, CDCI3) 5 20.8, 20.9, 21.0, 21.1, 29.3, 29.7, 36.4, 37.5, 48.8, 49.7, 51.3, 52.7,

62.1, 62.5, 67.7, 68.3, 68.4, 70.8, 71.3, 71.8, 73.2, 98.8, 166.8, 167.7, 170.1,

170.2, 170.4, 170.5, 170.6, 170.9, 171.1. IR (CDCI3) 612, 740, 1046, 1234, 1378, 104

1446, 1557, 1659, 1761, 2868, 2927, 3429 cm-1. FABMS m/z calcd for

C40H56N2O25Na 987.3070 (M + Na), found 987.3054.

A^-BOC N-Ac sialic acid dimer (2.7). To a flask charged with A^-BOC N-Ac

peracetyl sialic acid free acid (2.6, 65 mg, 0.105 mmol) in DMF (500 ^L) was added

BOP (69 mg, 0.156 mraol), HOBt (21 mg, 0.156 mmol), and diisopropylethylamine (73

HL, 0.420 mmol). The methyl glycoside methyl ester free amine hydrochloride salt of sialic acid (2.4, 82 mg, 0.156 mmol) was dissolved in DMF (500 ^lL) and added to the solution containing the sialic acid free acid solution. After 2 d, pyridine (2 mL) and acetic anhydride (1.8 mL) were added. After 18 h, the solvent was concentrated by coevaporating with toluene (5 x 15 mL). The residual solvent was taken up into ethyl acetate (50 mL) and extracted with water (3 x 50 mL). The organic phase was collected, dried (Na2S04) and concentrated in vacuo. The crude material was chromatographed (7:3 benzene/acetone) to yield the dimer as a colorless oil 29.8 mg, 26.7%). Rf = 0.44 (30% acetonerbenzene). [a]^^ = -20.P (c 1.5 in CHCI3). iH NMR (300 MHz, CDCI3) 5

I.73 - 1.92 (m, 2 H, H-3ax, H-3'ax), 1-95 (s, 3 H, OCOCHs), 2.03 (s, 3 H, OCOCH3),

2.05 (s, 3 H, CX:0CH3), 2.06 (s, 3 H, OCOCH3), 2.07 (s, 3H, OCOCH3), 2.08 (s, 3

H, OCOCH3), 2.11 (s, 3 H, OCOCH3), 2.12 (s, 3 H, OCOCH3), 2.14 (s, OCOCH3),

2.37 (s, 3 H, NCOCH3), 2.45 (dd, J = 12.9, 4.8 Hz, 1 H, H-3eq), 2.55 (dd, J = 14.1,

5.4 Hz, 1 H, H-3'eq), 3.28 (s, 3 H, OCH3), 3.80 (s, 3 H, CO2CH3), 4.00 - 4.26 (m, 4

H), 4.38 - 4.69 (m, 1 H), 4.72 - 4.93 (m, 3 H), 5.05 - 5.43 (m, 5 H), 5.68 (td, J =

II.1, 5.4 Hz, 1 H), 6.93 (d, J = 9.0 Hz. 1 H, NH). NMR (63 MHz, CDCI3) 5

20.7, 20.8, 20.9, 21.0, 21.1, 26.9, 27.9, 29.2, 37.5, 37.7, 49.0, 51.3, 52.5, 52.6,

61.9, 62.5, 66.0, 66.4. 67.4, 68.4, 70.6, 71.3, 72.0, 84.8, 98.7, 167.0, 167.8, 168.2,

169.8, 170.0, 170.6, 170.7, 171.0. IR (CDCI3) 432, 480, 530, 667, 758, 910, 1092, 105

1223, 1363, 1419, 1713, 1747, 2924, 3020, 3412 cm-1. FABMS m/z calcd for

C45H64N2027Li 1071.3857 (M + Li), found 1071.3837.

A^-BOC sialic acid dimer (2.8). Peracetylated A^-BOC yV-Ac dimer (2.7, 39.7 mg,

0.0373 ramol) was dissolved in methanol (2mL) and sodium methoxide (0.2 mg,

0.00373 mmol) was added. After 3 h, the solvent was removed in vacuo to provide the crude product as a colorless oil (23 mg, 95.9%). [a]22j) = -16.6° (c 1.8 in MeOH).

NMR (250 MHz, D2O) 6 1.23 (s, 9 H, NCOOCMes), 1.59 (t, 7= 12.3 Hz, 2 H, H-3ax.

H-3ax'), 2.19 (dd, J = 12.9, 4.2 Hz, 2 H, H-3eq, H-3eq'), 3.07 (s, 3 H, OCH3), 3.13

-3.96 (m, 17 H), 3.66 (s, 3 H, CO2CH3). 13c NMR (75 MHz, D2O) 5 23.8, 39.9,

51.4, 52.1, 52.7, 53.5, 54.0, 63.7, 63.8, 66.4, 68.4, 68.7, 70.3, 70.4, 70.7, 70.8,

96.2, 99.6, 160.6, 171.2, 181.6. IR (CD3OD) 471, 1026, 115, 1603, 1653, 2045,

2226, 2525, 2833, 2945, 3351 cm'l. FABMS m/z calcd for C25H44O17N2 644.2640

(M + H), found 644.2609. 106

CHAPTER 3

CONFORMATIONAL ANALYSIS OF COMPLEX POLYSACCHARIDES 107

INTRODUCTION

Menigococcal meningitis due to Neisseria meningitidis is a common cause of the worldwide cases of meningitis, and it is the only bacterium associated with epidemic meningitis. Neisseria meningitidis resides in the lining of the throat. Transmission occurs generally through airborne droplets or close contact. Illness occurs when the bacterium enters the blood stream and gains access to the meninges, the membranes that cover the brain and spinal cord. The bacteria rapidly grow in these membranes causing the meninges to become inflamed. This inflammation results in fever, headache, neck stiffness, and often, coma. In up to 30% of cases, the pathogen releases an endotoxin that increases the permeability of blood vessels thereby causing the blood pressure of the patient to fall. The ensuing septic shock can result in the loss of skin and parts of limbs.

Meningitis is generally fatal if left untreated.

Neisseria meningitidis is a gram-negative coccus which has been classified into serogroups A, B, C, 29e, W135, X, Y, and Groups H, I, and K were identified by Ding'^^ and group L was identified by Ashton.'*'* Groups A, B, and C are responsible for 90% of the reported cases of meningococcal meningitis. The capsular polysaccharide associated with group A is a partially 0-acetylated (1-»6)-Iinked homopolymer of 2-acetamido-2-deoxy-D-mannopyranosyl phosphate (Figure 3.1) whereas groups B and C are encapsulated with homopolymers of sialic acid. Group B is a-(2-» 8)-linked polysialic acid (Figure 3.2) while group C is a-(2-»9)-Iinked polysialic acid (Figure 3.3). 108

HO AcO

P—O,

Figure 3.1. Group A polysaccharide of Neisseria meningitidis.

OH OH OH HO OH OH CO2H OH AcHN HO AcHN AcHN HO HO

Figure 3.2. Group B polysaccharide of Neisseria meningitidis.

CO2H OH OH

AcHN AcHN HO HO

Figure 3.3. Group C polysaccharide of Neisseria meningitidis. 109

Epidemic meningococcal meningitis, usually caused by group A, can often be controlled by the use of a quadrivalent polysaccharide vaccine (groups A, C, Y, and W135).''^ In the United States, this vaccine is used on military recruits and travelers to epidemic or hyperendemic regions or when outbreaks occur due to groups A, C, Y or W135.

Epidemics of group A meningococcal meningitis recur at 20-30 year intervals. However, in the "meningitis belt" of sub-Saharan Africa, outbreaks occur at about 10 year intervals owing to a combination of endemic, hyperendemic, and epidemic disease.

The development of a polysaccharide vaccine that is effective against all serogroups and all humans remains problematic.^'^ Firstly, group A and C polysaccharide vaccines are not very effective in infants. Secondly, the B serogroup polysaccharide is only weakly immunogenic in humans.'The poor immune response of infants to these polysaccharide vaccines has been investigated extensively.'The immune response of humans to polysaccharides typically involves antibodies of the IgG, IgM, and IgA isotypes. These antibody levels generally remain high for long periods of time. However in infants, the only antibodies produced are those of the IgM isotype.

This immune response is not boostable. Once infants lose the antibodies derived from their mother, they are particularly vulnerable to bacterial infections. The immune response of infants to polysaccharides requires several years to mature.

The weak immunogenicty of group B meningococcal polysaccharide prevents its use as a vaccine against meningitis causcd by this pathogen. In the United States, 50% of cases of endemic meningococcal meningitis are due to the group B strain. Group B meningococcal polysaccharides do produce group B antibodies in humans, but the levels of antibodies produced are low, the antibodies are almost entirely of the IgM isotype, and they exhibit low affinity.

The poor immunogenicity of the group B meningococcal polysaccharide is also believed to be linked to its similarity in structure with oligosaccharides found in the 110

glycopeptides of the human brain.'^-^^^ Polysialosyl chains are linked to a glycoprotein

involved in neural cell adhesion. This glycoprotein is termed a neural cell adhesion

molecule (NCAM). These polysialosyl chains on NCAM are developmental antigens,

and the length of the chains decreases with increasing maturity. These polysialosyl

chains have been shown by Finne to bind group B meningococcal polysaccharide specific

antibodies and to inhibit the serologic reaction of these antibodies. Polysialosyl chains

consist of up to twelve a-(2-^ 8)-linked sialic acid monomer units.Work by

Jennings^^^ and Finne and Makela'^ has shown that the common epitope on the group

B meningococcal polysaccharide and NCAM is an oligosaccharide of at least ten sialic

acid residues. However, this decamer is larger than the maximum size of an antibody

binding site.'^^ This fact led Jennings to propose that the common epitope for the group

B meningococcal polysaccharide and NCAM undergoes conformational changes in order

to fit into the antibody binding site.'^^-'^®

Enhanced immunogenicity has been observed with group B meningococcal

polysaccharide if the polysaccharide is noncovalently complexed with meningococcal

outer membrane proteins.These complexes are believed to be based on

hydrophobic interactions between the protein and a phosphatidyl moiety linked to the

reducing end of a sialic acid residue.However, the enhancement observed in such

complexes was not significant, and the antibodies that were raised in response were only

of the IgM isotype. The modest enhancement in immunogenicity is believed to be due to

the fact that the group B polysaccharide, when it is complexed with a protein, maintains

its structural integrity.Lifely proposed that the structual integrity of the

polysaccharide complexed with the protein is maintained by its abiUty to form interresidue lactones.

Chemical modification of the group B meningococcal polysaccharide has also been attempted as a means of developing an effective vaccine. Jennings'-^"^ showed that both Ill

the carboxylate and amide functionalities are essential for activity. Modification was

achieved at the amide functionality by converting the A/-acetyl group to an N^-propionyl

group (Figure 3.4).

COoNa Base AcHN 100°C

COgH Propionic anhydride EtOCHN

Figure 3.4. Preparation of A'-propionylated group B meningococcal polysaccharide.

The ^-propionylated polysaccharide was shown to be weakly immunogenic in mice. In order to improve immunogenicity, the A'-propionylated polysaccharide was conjugated to a protein, tetanus toxoid. This conjugate was able to produce B polysaccharide-specific

IgG antibodies in mice, and the antiserum produced was also highly bactericidal for group B meningococcal pathogens.This conjugate is serving as a prototype for the development of a vaccine against meningitis caused by group B Neisseria meningitidis.

Along with the seaich for an effective vaccine, work has also been done on the determination of the three dimensional structure of the group B meningococcal polysaccharide by NMR spectroscopy. The goal of this reseach has been to determine how epitope expression of the polymers is associated with their conformations and 112 therefore how to overcome the cross-reactivity between the polysaccharide and neonatal brain tissues.

Jennings^ completed the initial conformational studies on homosialooligosaccharides. Initially, full and assignments were made of the trisaccharide (NeuAc)3 and colominic acid, an a-(2-»8)-linked homopolymer of sialic acid which has an identical spectrum to that of the group B meningococcal polysaccharide (Figure 3.5).

OH OH OH OH HO OH OH OH AcHN HO AcHN AcHN HO HO

Figure 3.5. Colominic acid.

Conformational studies were performed using nuclear Overhauser (NOE) difference experiments. These experiments indicated that both linkages for trisaccharide (NeuAc)3 had different conformations from each other and from the internal linkages in colominic acid. The NOE data also revealed that these conformational differences were observed in both terminal disaccharides of oligomers larger than (NeuAc)5. This was an especially important observation in that it may provide an explanation for the proposed conformational epitope of the group B meningococcal polysaccharide being (NeuAc)io, a size larger than the optimum size for the antibody binding site. Since the two terminal disaccharides differ in conformation from the internal residues, the six internal residues may be the part of the decamer which provide immunological function. The size of the hexamer is consistent with the maximum size of the antibody binding site. 113

Yamasaki'^® completed the first conformational studies on the group B meningococcal

polysaccharide using two-dimensional NMR spectroscopy and molecular modeling.

Once the pure absorption NOESY spectra were obtained at three different mixing times,

theoretical NOE calculations were performed using complete relaxation matrix analysis

(CORMA).^^^ Molecular modeling using MIDAS'^ was then carried out in order to

generate possible conformers of the polysaccharide. In order to simplify the calculations,

tetramers were used as models for the calculations. Based on the calculations, the group

B polysaccharide was determined to have a helical conformation in solution. One turn of

the helix requires 3-4 sialic acid residues with a pitch of 10-11 A. In contrast, the pitch in

double-stranded DNA is 34A''*' indicating that the group B polysaccharide exists in

tighter helical coils than those observed in double-stranded DNA. That also provides an

explanation as to why sialic acid dimers and trimers of a-(2-» 8)-link;ed sialic acid exhibit

weak affinity for anti-B antibodies. These oligomers display much more flexible

structures whereas the polysaccharide is more highly ordered.

Jennings''*^ has also examined the three dimensional structure of the group B

meningococcal polysaccharide by NMR with results that contradict the model proposed

by Yamasaki that the polysaccharide is a rigid helix with 3-4 sialic acid residues per turn.

Based on the NOE data (both ID and 2D experiments), no single conformer was found to satisfy all of the NMR constraints. Potential energy contour maps using MM2 calculations on the NOE data did indeed reveal regions of helicity in the polysaccharide.

Earlier work by Perez and Vergelati'^^ on polysaccharides revealed that polysaccharides often are random coils in solution but they exhibit local regions of well-defined helicity.

The group B meningococcal polysaccharide also displays a number of similarities with polyadenine. Both polymers exhibited similar potential energy contour maps. Both polymers also display similar spatial distribution of charges between the carboxyl group 114

of the polysaccharide and the phosphate group of the poly(A). It is also known that both

poIy(A) and the group B meningococcal polysaccharide react with IgM^OV monoclonal

antibody. Jennings believes the cross-reactivity of the polysaccharide and poly(A)

results from the adoption of similar helices by both polymers. The poly(A) has nine

residues per turn and a pitch of 25A. Based on these similarities, Jennings has

proposed that the local helices of the polysaccharide consist of 8-9 sialic acid residues per

turn with a pitch of 18-22A.

Jennings, unlike Yamasaki, is more ambiguous in terms of the binding of the

polysaccharide with group B meningcoccal polysaccharide antibodies. Jennings does

not offer an exact number of sialic acid residues which are required in order for binding to

occur with group B polysaccharide antibodies. Because the cross reactivity of the

polysaccharide and poly(A) with the IgM^OV monoclonal antibody suggests a conformational epitope that consists of a higher-order helix, large oligosaccharides are

necessary for epitope formation. The group B meningococcal polysaccharide must

undergo topological changes from a random coil to a conformation with more ordered helical regions to form the desired epitope. The epitope is not likely to be expressed in a short oligosaccharide sequence. Jennings also asserts that the poor immunogenicity of the group B polysaccharide is the result of the recognition of the polysaccharide's helical epitope which is also an essential element of the neural cell adhesion molecule

(NCAM).^23

NMR studies on the group B meningococcal polysaccharide have also been performed by Lifely and coworkers.Lifely compared the conformational differences between the group B and C polysaccharide serogroups and found that the group B polysaccharide is much less flexible in solution than the group C polysaccharide and that the group B polysaccharide has a different solution phase conformation. Lifely proposed that the differences in conformation and flexibility are the result of the differing abilities of the 115

group B and C polysaccharides to undergo internal lactone formation. In order to form

an internal lactone in the case of the a-(2-»9)-linked group C polysaccharide between the

8-OH group and the anomeric carbonyl, three bond twists are required, thereby making

this process energetically unfavorable (Figure 3.6).

OH

AcHN AcHN HO HO

9^^ OH OH

AcHN AcHN HO HO

Figure 3.6. Internal esterification of the group C meningococcal polysacccharide.

In the case of the a-(2-» 8)-linked group B meningococcal polysaccharide, internal esterification is much more facile. Only one bond needs to be rotated so that the 9-OH and the anomeric carbonyl are in sufficient proximity to form internal lactones (Figure

3.7). 116

HC

CO2H AcHN AcHN AcHN

^O, HO.

AcHN CO2H AcHN AcHN

n

Figure 3.7. Internal esterification of the group B meningococcal polysaccharide.

Internal esterification of these polysaccharides does have immunochemical consequences.''*^ Under physiological conditions only the weakly immunogenic group

B polysaccharide would be expected to undergo internal esterification due to the more favorable energy required for lactone formation than for the immunogenic group C polysaccharide. The antigenicity of the group B polysaccharide is decreased by about

20% upon lactone formation. The formation of lactones results in a decreased ability of the group B polysaccharide to react with antibodies. Low molecular weight group B polysaccharide (both before and after lactonization) has been shown to be poorly antigenic which is indicative of a conformational determinant on the polysaccharide.

In summation, the group B meningococcal polysaccharide is a weakly immunogenic a-(2-» 8)-linked homopolymer of sialic acid produced by Neisseria meningitidis that causes meningitis. NMR studies by Jennings'"*^ suggest that the polysaccharide exists as a random coil with local regions of helicity. Lifely has determined that the group B 117

polysaccharide readily forms interresidue lactones under mildly acidic conditions.

The research that is to be discussed below will probe the solution phase conformation of

the polylactone of the group B meningococcal polysacharide.

This research is part of a program directed at identifying new helical structures.

Helical, polyanionic compounds such as aurintricarboxylic acid and dextran sulfate

(Figure 3.8) are known to interrupt the binding of the glycoprotein gpl20 of the Human

Immunodeficiency Virus (HIV) to a CD4+ T-cell host.^'*^'^^^

OH

OH OH

OH

OH HO OH OH COgH

OH HO' •OH

HO OH

HO

Figure 3.8. Dextran sulfate (top) and aurintricarboxylic acid (bottom). 118

Helicity, rather than electrostatics, in these structures is believed to be the more important

feature in interrupting HIV entry. This research will probe the solution-phase

conformation of this polylactone by NMR techniques to determine if indeed it does

possess helicity and, if so, to address the pitch and number of residues per turn.

RESULTS AND DISCUSSION

The polylactone was prepared according to the procedure reported by Lifely.^''® In order to determine the solution phase conformation of the polylactone, initial work focused on the complete ^H and assignment of the polysaccharide. The spectrum was obtained at 25oC; however the peaks were not very well resolved at this temperature.

The ^H spectrum was subsequently reacquired at 970C in the hope of obtaining a more well resolved spectrum (Figure 3.9).

^—P— — I— —-—•—T— 1 r 6 S 4 3 2 1

Figure 3.9. 300 MHz NMR spectrum of colominic acid polylactone at 91°C. 119

The spectral region between 3.3 and 3.8 ppm was resolved at 97°C which proved to be

crucial in making the full assignment. The only peaks that were readily identifiable

from the one-dimensional spectrum at 91°Q were the NH, the methyl protons of the

yV-acetyl group, and the axial and equatorial H-3 protons. Full assignment could only

be made by using double quantum filtered correlation spectroscopy (DQF-COSY).'^^

Although the H-6 and H-7 cross peaks were not well resolved in the spectrum, H-8 was

assigned from cross peaks to H-9 and H-9' and the remaining cross peak to H-8 allowed

for the assignment of H-7 at 3.51 ppm.

Hydrogen Atom Chemical Shift (ppm) H-3ax 1.39 H-3ea 2.29 H-4 4.20 H-4 (OH) 4.89 H-5 3.55 H-6 3.40 H-7 3.51 H-7 (OH) 5.14 H-8 3.99 H-9, 9' 4.47, 4.59 .V-Ac (CH3), N-H 1.91, 8.00

Table 3.1. Complete ^H chemical shift assignment of the colominic acid polylactone.

The full assignment of the carbon resonances of the polylactone had been previously reported; however, during the course of our experiments, we discovered that the C-4 and C-9 resonances were misassigned.'^^ The one-dimensonal spectrum only allowed for assignment of the anomeric, carboxy carbonyl, and the acetamido resonances

(Figure 3.10). 120

-I-

lES isa 28

Figure 3.10. 75 MHz NMR spectrum of colominic acid polylactone.

Several 2D heteronuclear techniques were required to fully assign all of the carbon resonances. Heteronuclear y-resolved spectroscopy (HET2DJ)^^^ allowed for the ready identification of the C-3 and C-9 resonances as triplets occuring at 40.33 and 67.60 ppm. respectively (Figure 3.11). 121

F2 (ppmJl 40i

60i

60i

150-

120 60 40 0 -40 -50 -140 PI (Hz) Figure 3.11. 75 MHz HET2DJ spectrum of colominic acid polylactone.

The HET2DJ spectrum also allowed for the determination of the ^/C.H coupling constants (Table 3.2). 122

Carbon Peak Multiplicity Vc,H (Hz) (ppm) 22.00 ql 117 40.33 t 166 51.59 d 158 65.94 d 175 67.60 t 189 68.92 d 190 69.33 d 162 72.37 d 158 95.92 s 0 165.02 s 0 171.88 s 0

Table 3.2. Carbon multiplicity and Vc,h coupling constant data derived from the

HET2DJ spectrum.

Heteronuclear correlation spectroscopy (HETCOR)'^^ was then employed which clearly showed that the C-9 was coupled through to the H-9 and H-9' resonances at 4.47 and

4.59 ppm (Figure 3.12). 123

2 ^ - T

t.t S.S s.t 4.S «.• 3.5 3.1 e.S 2.1 l.S l.| ».$

Hgure 3.12. 75 MHz HETCOR spectrum of colominic acid polylactone.

A summary of the complete assignment is given in Table 3.3.

Carbon Atom Chemical Shift (ppm) C-1 165.03 C-2 95.72 C-3 40.33 C-4 65.94 C-5 51.59 C-6 72.37 C-1 68.92 C-8 69.33 C-9 67.60 A^-Ac (CHi) 22.00 N-\c (C=0) 171.88

Table 3.3. Complete chemical shift assignment of colominic acid polylactone.

Once the full and assignment had been made, work next focused on the determination of the three dimensional structure of the polylactone. Two dimensional 124

nuclear Overhauser effect (NOESY) experiments were run in States mode'^ with mixing

times of 25, 50, 75, 90, 100, 110, 125, 150, and 200 ras. Data processing was done

with the FELIX 2.30 software (Biosym) on a Sun SPARCstation. The NOE crosspeaks

were footprinted and a macro was established to automatically calculate the volumes of

the cross-peaks in each experiment

Once the volumes of the NOE crosspeaks were calculated, NOE buildup curves for

each NOE observed were plotted by Abby Parrill of the Gervay group as a function of

mixing time (Figure 3.13).'^'

1200T 3a,3e y = 599.67 + 200.50* R»2 = 0.976 3a,4 y = 80.000 + 98.500X R^2 = 0.994 3e, 4 y = 204.67 + 9 l.OOOx R''2 = 0.984 3e,5 y = 115.33+ 21.OOOx R''2 = 0.838 3a.5 y = 113.33+ 78.500* R'^2 = 1.000 3a.6 y = 55.567 + 20.300X R'^2 = 0.985 4.5 y = 116.00 + 39.000* R''2 = 1.000 4.6 y = 168.00 + 41.500* R'^2 = 0.996 06oa 4. NH y = 20.533 + 48.300* R'^2 = 0.992 5. NH y = -9.0667 + 38.800* R'^2 = 0.986 7.8 y = 351.67 + 99.000* R'^2 = 0.998 7.9 y = 111.00 + 79.000* R"2 = 0.998 7. 9* y = 113.33 + 78.500* R'^2 = 1.000

.025 .050 .075 Mixing Time (sec)

Figure 3.13. NOE buildup curves of the colominic acid polylactone.

NOE buildup curves were used rather than an NOE volume at a single mixing time in

order to achieve a better correlation between distances.^®^ NOE buildup curves prevent errors in volume due to spin-diffusion effects at longer mixing times. Spin diffusion effects were observed at mixing times longer than 75 ms. Therefore, mixing times of 25,

50 and 75 ms were used in the NOE buildup curves. 125

In order to determine interproton distances from NOE buildup curves, either the

rotational correlation function must be determined or a standard curve must be used. A standard curve which requires known distances was employed. A sialyllactone dimer

(Figure 3.14) was used in order to computationally determine interproton distances.

AcHN AcHN

Figure 3.14. Sialyllactone dimer used in conformational search.

A conformational search by the Monte Carlo method in Macromodel 4.5 was performed by Abby Parrill on the disialic acid lactone in order to determine the the low energy conformations of the pyranose rings. Three conformations were obtained: a boat conformation which exhibited hydrogen bonding from the amide hydrogen to the carbonyl oxygen of the lactone (Figure 3.15) and two chair conformations (Figures 3.16 and 3.17) one of which exhibited hydrogen bonding from the amide hydrogen to the 7-

OH position. 126

OH OH OH HO CO2H

HO AcHN HO

Figure 3.15. Boat conformation of the disialyllactone.

OH OH HO. OH OH

AcHN AcHN HO HO

Figure 3.16. Chair conformation of the disialyllactone with no hydrogen bonding.

OH HO H HO. OH

AcHN HO HO

Figure 3.17. Chair conformation of the disialyllactone exhibiting intramolecular hydrogen bonding.

Interproton distances for each of the three low energy conformations were then measured.

These distances were used with the NOE buildup rates to determine which conformation best fit the NMR data. The NOE buildup rates were plotted versus distance to the reciprocal sixth power (Figure 3.18). 127

300

• Cliair H-Bond y = 40.556 + 5383.8X = 0.868 0 Chair y = 37.306 + 5475.6x R''2 = 0.909 X Boat y = 38.334 + 5195.0X 0.862

D 0 0.00 0.01 0.02 0.03 0.04 l/Distance'^6

Figure 3.18. Linear regression for the three low energy pyranose conformations of the sialyllactone dimer.

The conformation which best fit the data was the chair conformation in which there was no intramolecular hydrogen bonding.

All non-adjacent proton pair distances in the ring of this chair conformation (Figure

3.16) were then measured. The NOE volumes were plotted against distance to the reciprocal sixth power to give a final calibration plot (Figure 3.19) which was then used to determine distances for remaining observed NOE's. 128

y = 14.082 + 1 J235e44x R'^2 = 0.916 70 -

60 •

z SO -

30 •

0.000 0.001 0.002 0.003 0.004 1/Distance'^ 6

Figure 3.19. Final calibration plot relating NOE to distance for the sialyllactone dimer.

The distances calculated from the NOE buildup rates from the model sialyllactone dimer were used by Abby Parrill as constraints for a conformational search using the genetic algorithm WIZARD Little regularity was observed in the conformations found by WIZARD III. Molecular dynamics calculations using MacroModel

(MacroModel, New York) were performed to search the local conformational space using the initial conformations generated by WIZARD IE. The calculation in MacroModel was performed with SHAKE constraints applied to bond distances and selected conformations were examined to determine which local regions of the poiyiactone best fit the distance constraints. Local conformations were then applied across the poiyiactone. Based on molecular dynamics, two helical conformations for the poiyiactone were proposed

(Figure 3.20). Conformation A Conformation B

Figure 3.20. Two helical conformations of the colominic acid polylactone consistent with the NOE data. 130

The differences in the two conformers arises from the dihedral angle defined by

C7-H7. The dihedral angle in conformation A is 180° whereas in conformation B it is

60°. Ab initio calculations on the O1-C2-C3-C4 exocyclic unit of C-glycosides have shown a 0.3 kcal/mol preference for a gauche conformation over an anti conformation'^ when there is no additional alkyl substituent at C-3 (Figure 3.21). The 0.3 kcal/mol gauche preference would favor conformation B over conformation, assuming equivalent interactions among both conformations.

HO

H

Figure 3.21. Gauche conformation of a l-»4-linked C-disaccharide.

The decamer of conformation A has a length of 58A, a distance of 12A per turn, and

2.1 residues per turn. The decamer of conformation B has a length of 34 A, a distance of

SA per turn, and 2.3 residues per turn. The group B meningococcal polysaccharide, by comparison, is believed to be a randon coil possessing local regions of helicity.''*^ These local helices contain 8-9 sialic acid residues per turn with a pitch of 18-22A.

CONCLUSION

It is believed that the group B polysaccharide of N. meningitidis forms lactones in vivo which accounts for its poor immunogenicity. The three dimensional structure of a polylactone has been investigated by NMR. Based upon the measured NOE data and molecular dynamics, two conformations have been proposed (Figure 3.20). Both 131

proposed conformers are helical with the difference being in the distance per turn (12A for conformation A and SA for conformation B) owing to the Hg-Cg-Cy-Hy dihedral angle. Conformation B with a H6-C6-C7-H7 dihedral angle of 60° is believed to be the preferred conformation. Since the polylactone does possess helicity, further studies may be directed to examining any role it may have in the interruption of gpI20 binding to

CD4+T-ceIls.

EXPERIMENTAL SECTION

Polysialyllactone (20 mg) was prepared according to the procedure of Lifely^'^^ and was dissolved in 1 mL of DMSO-^ig.

All NMR spectra were obtained on a Varian Unity 300 spectrometer (for at 299.96

MHz and for at 75.43 MHz) at 91°C. Chemical shifts (^H and '^C) are reported in ppm relative to the residual DMSO peaks. The 2D double-quantum filtered homonuclear correlation (DQCOSY) spectrum was acquired as 2K x 2K data points with a spectral width of 4(XX) Hz. The data was processed with a phase-shifted sine bell and was zero- filled in the tj dimension. The 2D heteronuclear (^H and chemical shift correlation

(HETCOR) spectrum was obtained as 512 x 2K data points. The ^H spectral width was

4000 Hz and the spectral width was 18(X)1.8 Hz. The data was processed with a phase-shifted sinebell in the tj and t2 dimensions and was zero-filled in the ti dimension.

The 2D heteronuclear (^H and '^C) y-resolved spectrum (HET2DJ) was acquired as 128

X 2K data points. The ^H spectral width was 500 Hz and the ^^C spectral width was

18001.8 Hz. The data was processed with both a phase-shifted sinebell and a Gaussian window function in the ti and t2 dimensions and was zero-filled in the ti dimension.

Since decoupler gating was used, the actual J value is twice that of the measured J value

(•^act = 2 X 7meas)- Two dimensional nuclear Overhauser effect (NOESY) experiments 132 were run in States mode with mixing times of 25, 50, 75, 90, 100, 110, 125, 150, and

200 ms. The relaxation delay between acquisitions was 2 s, and the sweep width was

4000 Hz. Martrices of 256 x 1024 were recorded with 32 scans per ti increment. Data processing was done with the FtLIX 2.30 software (Biosym) on a Sun SPARCstation.

A sinebell window function (512, 90), a baseline correction (0.2), and zerofill to 1024 were applied to the FIDs. The NOE crosspeaks were footprinted and a macro was established to automatically calculate the volumes of the cross-peaks in each experiment 133

APPENDIX A NMR AND IR SPECTRA IH NMR of 3,4,6-tri-C)-acetyl-D-allal 135

NMR of 3A6-tri-Oacetyl-I>aIial MAVENUMBERS IH NMR of 3.4,6-tri-O-benzyI-D-aIlal (1.2)

\ 184.4 »20-

JOO-

80-

60-

S

40-

18.0 —I r T r 40l>0

4600.0 HAVENUMBERS 140

IH NMR of l.2,aiihydro-4A6-tri-0-benzyI-D-allal (1.3) 1 ri I ^'

D 13c NMR of l,2,anhydro-4A6-tri-0-beiizyl-I>allal (1.3) % TflANSMlTTANCE

o o

3427.94

2926.39 m

m

o

1637.77

1369.63 1280.90 1209.52

601.87

e

ZP\ 143

NMR of l-deoxy-3,4,6-tri-0-benzyI-D-glucose (1.4)

5

•- m 144

NMR of l-deoxy-3,4,6-lri-0-benz>1-D-glucose (1.4) WAVENUMBERS X

cL re s

> 5.

I _yvJL

7.0 6.0 5.0 4.0 3.0 2.0 1.0 PPM NMR of l-deoxy-3,4,6-lri-0-benzyl-I>altrose 173.7

160

140-•

OUl 120-' -

M 1004 cn

60-

40

21.8 4000 3000 2000

WAVENUMBEflS 149

IH NMR of p-deutero-I-deoxy-3 A6-tri-

o X OJ

o m 2 OS •a Ta

o in

o

CD

o \ Ut n

2, •TO k I ?

X OiJ > 9^ 5. I 9 ST

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NMR of I-1-D-gIucose

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1454.03

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REFERENCES

(1) Etzioni, A. "Adhesion Molecules- Their Role in Health and Disease" Pediatric Res. 1996,39, 191-198.

(2) Curamings, R. D.; Smith, D. F. "The Selectin Family of Carbohydrate-Binding Proteins: Structure and Importance of Carbohydrate Ligands for Cell Adhesion" BioEssays 1992,14, 849-856.

(3) Drickamer, K. "Two Distinct Classes of Carbohydrate-Recognition Domains in Animal Lectins" J. Biol. Chem. 1988, 263. 9557-9560.

(4) Gallatin, M.; St. John, T.; Siegelman, M.; Reichert, R.; Butcher, E.; Weissman, I. "Lymphocyte Homing Receptors" Cell 1986, 44, 673-680.

(5) Watson, S. R.; Fennie, C.; Lasky, L. A. "Neutrophil Influx into an Inflammatory Site Inhibited by a Soluble Homing Receptor-IgG Chimera" Nature 1991,349, 164-167.

(6) Lasky, L. A. "Lectin Cell Adhesion Molecules (LEC-CAMs): A New Family of Cell Adhesion Proteins involved with Inflammation" J. Cell Biochem. 1991, 45, 139-146.

(7) Bevilacqua, M. P.; Stengelin, S.; Gimbrone, M. A.; Seed, B. "Endothelial Leukocyte Adhesion Molecule 1: An Inducible Receptor for Neutrophils Related to Complement Regulatory Proteins and Lectins" Science 1989, 243, 1160-1165.

(8) Kishimoto, T. K.; Jutila, M. A.; Berg, E. L.; Butcher, E. C. "Neutrophil Mac-1 and MEL-14 Adhesion Proteins Inversely Regulated by Chemotactic Factors." Science 1989,245, 1238-1241.

(9) Lawrence, M. B.; Springer, T. A. "Leukocytes Roll on a Selectin at Physiologic Flow Rates: Distinction from and Prerequisite for Adhesion through Integrins" Cell 1991,65, 859-873.

(10) Hsu-Lin, S. C.; Berman, C. L.; Furie, B. C.; August, D.; Furie, B. "A Platelet Membrane Protein Expressed During Platelet Activation" J. Biol. Chem. 1984, 259, 9121-9126.

(11) McEver, R. P. "Selectins: Novel Receptor that Mediate Leukocyte Adhesion During Inflammation" Thromb. Haemostas 1991, 65, 223-228.

(12) Patel, K. D.; Zimmerman, G. A.; Prescott, S. M.; McEver, R. P.; Mclntyre, T. M. "Oxygen Radicals Induce Human Endothelial Cells to Express GMP-140 and Bind Neutrophils" J. Cell Biol. 1991, 112, 749-759.

(13) Smith, C. W.; Marlin, S. D.; Rothlein, R.; Toman, C.; Anderson. D. C. "Cooperative Interactions of LFA-1 and Mac-1 with Intercellular Adhesion Molecule-1 in Facilitating Adherence and Transendothelial Migration of Human Neutrophils in vitro" J. Clin. Invest. 1989, 5i. 2008-2017. 237

(14) Rosen, S. D.; Singer, M. S.; Yednock, T. A.; Stoolamn, L. M. "Involvement of Sialic Acid on Endothelial Cells in Organ-Specific Lymphocyte Recirculation" Science 1985, 22S, 1005-1007.

(15) Lowe, J. B.; Stoolman, L. M.; Nair, R. P.; Berhend, T. L.; Marks, R. M. "ELAM- 1 Dependent Cell Adhesion to Vascular Endothelium Determined by a Transfected Human Fucosyltransferase cDNA" Cell 1990,63, 475-484.

(16) Phillips, M. L.; Nudelman, E.; Gaeta, F. C. A.; Perez, M.; Singhal, A. K.; Hakamori, S.; J.C., P. "ELAM-1 Mediates Cell Adhesion by Recognition of a Carbohydrate Ligand Sialyl Le*" Science 1990,250, 1130-1131.

(17) Zhou, Q.; Moore, K. L.; Smith, D. F.; Varki, A.; McEver, R. P.; Cummings, R. D. "The Selectin GMP-140 Binds to Sialylated, Fucosylated Lactosamminoglycans on Both Myeloid and Nonmyeloid Cells" J. Cell. Biol. 1991,115, 557-564.

(18) Stoolman, L. M.; Ebling, H. "Adhesion Molecules of Cultured Hematopoietic Malignancies: A Calcium-Dependent Lectin is the Principle Mediator of Binding to the High Endothelial Venule of Lymph Nodes" J. Clin. Invest. 1990, 84, 1196-1205.

(19) Magnani, J. L. "The Tumor Markers of SLe^ and SLe* Bind ELAM-1" Glycobiology 1991,1, 318-320.

(20) Ogawa, J.; Inoue, H.; Koide, S. "Expression of a-l,3-Fucosyltransferase Type IV and VII Genes is Related to Poor Prognosis in Lung Cancer" Cancer Res. 1995, 56, 325-329.

(21) Yamada, N.; Chung, Y. S.; Maeda, K.; Sawada, T.; Ikehara, T.; Nishino, H.; Okuno, M.; Sowa, M. "Increased Expression of Sialyl Lewis^ and Sialyl Lewis* in Liver Metastases of Human Colorectal Carcinoma" Invasion & Metastasis 1995, 15, 95-102.

(22) Fukushima, K.; Hirota, M.; Terasaki, P. I.; Wakisaki, A.; Togashi, H.; Chia, D.; Suyama, N.; Fukishi, Y.; Nudelman, E.; Hakamori, S. "Characterization of Sialosylated Lewis* as a Tumor-associated Antigen" Cancer Res. 1984, 44, 5279-5285.

(23) Polley, M. J.; Phillips, M. L.; Wayner, E.; Nudelman, E.; Singhal, A. K.; Hakamori, S.; Paulson, J. C. "CD62 Edothelial Cell-Leukocyte Adhesion Molecule 1 (ELAM-l) Recognise the Same Carbohydrate Ligand, Sialyl-Lewis'^" Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 6224-6228.

(24) Walz, G.; Aruffo, A.; Kolanus, W.; Bevilacqua, M.; Seed, B. "Recognition of ELAM-1 of the Sialyl-Le'^ Determinant on Myeloid and Tumor Cells" Science 1990, 250, 1132-1135.

(25) Schneider, J. S.; Pope, A.; Simpson, K.; Taggart, J.; Smith, M. G.; DiStefano, L. "Recovery from Experimental Parkinsonism in Primates with GMj Ganglioside Treatment" Science 1992, 256, 843-846. 238

(26) Morin, M. J.; Bemacki, R. J. "Biochemical Effects and Therapeutic Potential of Tunicamycin in Murine L1210 leukemia" Cancer Res. 1983,43, 1669-1674.

(27) Gil-Fernandez, G.; Perez, S.; Vilas, P.; Perez, C.; de las Heras, F. G.; Gancedo, A. G. "Antiviral Activity of Uridine 5'-Diphosphate Glucose Analogues Against Some Enveloped Viruses in Cell Culture" Antiviral Res. 1987, S, 299-310.

(28) Vaghefi, M. M.; Bernacki, R. J.; Hennen, W. J.; Robins, R. K. "Synthesis of Certain Nucleoside Methylenediphosphonate Sugars as Potential Inhibitors of Glycosyltransferases" 7. A/ei/. C/iem. 1987, iO, 1391-1399.

(29) Huang, Z.; Schneider, K. C.; Benner, S. A. "Building Blocks for Oligonucleotide Analogues with Dimethylene Sulfide, Sulfoxide, and Sulfone Groups Replacing Phosphodiester Linkages" J. Org. Chem. 1991, 56, 3869-3882.

(30) Nambiar, K. P.; Stackhouse, J.; Stauffer, D. M.; Kennedy, W. P.; Eldredge, J. K.; Benner, S. A. "Total Synthesis and Cloning of a Gene Coding for the Ribonuclease S ?xo\e.m" Science 1984, 22J, 1299-1301.

(31) Miller, P. S.; Yano, J.; Yano, E.; Carroll, C.; Jayaraman, K.; Ts'o, P. O. "Nonionic Nucleic Acid Analogues: Synthesis and Characterization of Dideoxyribonucleoside Methylphosphonates" Biochemistry 1979,18, 5134-5143.

(32) Halcomb, R. L.; Danishefsky, S. J. "On the Direct Epoxidation of Glycals: Application of a Reiterative Strategy for the Synthesis of p-Linked Oligosaccharides" J. Anu Chem. Soc. 1989, 111, 6661-6666.

(33) Klein, L. L.; McWhorter, W. W.; Ko, S. S.; PfafT, K.-P.; Kishi, Y.; Uemura, D.; Hirata, Y. "Stereochemistry of Palytoxin. 1. C85-C115 Segment" J. Am. Chem Soc. 1982,104, 7362-7364.

(34) Nicotra, F.; Panza, L.; Russo, G. "Epoxidation of Exocyclic Glycals: A New Access to Glycosides of Ketosugars" Tetrahedron Lett. 1991, 32, 4035-4038.

(35) Murray, R. W.; Jeyaraman, R. "Dioxiranes: Synthesis and Reactions of Methyldioxiranes" J. Org. Chem. 1985, 50, 2847-2853.

(36) Singh, S.; Ramamurthy, V. "Upper Excited State Reactions of Thioketenes: D'x-tert- butylthioketene" J. Org. Chem. 1984, 49, 393-395.

(37) Singh, S.; Nimmesgem, H.; Schaumann, E.; Ramamurthy, V. "Photochemical Behavior of Thioketenes in Solution; Reaction from S2" J. Org. Chem. 1985, 50, 4799- 4805.

(38) Beiner, J. M.; Lecadet, D.; Paquer, D.; Thuillier, A.; Vialle, J. "Composes Organiques Sulferes XLIl, Reaction du Diazomethane Avec des Composes Thicarbonyles; Preparation de Thiirannes. I." Bull. Chim. Soc. Fr. 1973, 6, 1979- 1983. 239

(39) Beiner, J. M.; Lecadet, D.; Paquer, D.; Thuillier, A. "Composes Organiques Sulfures. Reaction du Diazoethane et du Diazo-2-propane sur des Composes Thiocarbonyles; Preparation de Thiirannes (2 partie)" Bull. Chinu Soc. Fr. 1973, 6, 1983-1987.

(40) Price, C. C.; Kirk, P. F. "Some Observations on the Reaction of Alkali Thiocyanates with Epoxides" J. Am. Chem. Soc. 1953, 75, 2396-2400.

(41) Chan, T. H.; Finkenbine, J. R. "Facile Conversion of Oxiranes to Thiiranes by Phosphine Sulfides. Scope, Stereochemistry, and Mechanism" J. Am. Chem. Soc. 1972, 94, 2880-2882.

(42) Takido, T.; Kobayashi, Y.; Itabashi, K. "A Convenient Synthesis of Thiirans by the Use of Dimethylthioformamide" Synthesis 1986, 779-780.

(43) Carlsen, P. H. J.; Rist, O.; Maldal, T.; Vikane, O.; Gilje, E. "Synthesis and Properties of Alkane-I,2-disulfonate Surfactants" Acta Chem. Scand. 1996, 50, 158- 163.

(44) RajanBabu, T. V.; Reddy, G. S. "1-MethyIene Sugars as C-Glycoside Precursors" J. Org. Chem. 1986,57,5458-5461.

(45) Mitsunobu, O. "The Use of Diethyl Azodicarboxylate and Triphenylphosphine in Synthesis and Transformation of Natural Products" Synthesis 1981, 1-28.

(46) Flaherty, T. M.; Gervay, J. "Hydride Addition to 1,2-Anhydrosugars: A Highly Stereoselective Route to Anhydroalditols" Tetrahedron Lett. 1996,37,961-964.

(47) Mancuso, A. J.; Swem, D. "Activated Dimethyl Sulfoxide: Useful Reagents for Synthesis" Synthesis 1981, 165-185.

(48) Brown, H. C.; Yoon, N. M. "Reaction of Diborane in Tetrahydrofuran with Styrene Oxide and Related Epoxides in the Presence of Boron Trifluoride. A Convenient Anti- Markovnikov Reductive Opening of Such Epoxides" Chem, Commun. 1968, 1549- 1550.

(49) Brown, H. C.; Yoon, N. M. "The Borohydride-Catalyzed Reaction of Diborane with Epoxides. The Anti-Markovnikov Opening of Trisubstituted Epoxides" J. Am. Chem. Soc. 1968, 90, 2686-2688.

(50) Winterfeldt, E. "Applications of Diisbutylaluminum Hydride (DIBAH) and Triisobutylaluminum (TTBA) as Reducing Agents in Organic Synthesis" Synthesis 1975, 617-630.

(51) Corey, E. J.; Chaykovsky, M. "Methylsulfinylcarbanion" J. Am. Chem. Soc. 1962, 84, 866-867.

(52) Takano, S.; Tomita, S.; Iwabuchi, Y.; Ogasawara, K. "A Facile Synthesis of (/?)-l- Benzyloxy-3-buten-2-or' Synthesis 1988,610-611. 240

(53) Bartlett, P. A.; Green, F. R.; Rose, E. H. "Synthesis of Acetylenes from Carboxylic Acid Derivatives via P-Keto Sulfones" J. Am, Chem. Soc. 1978,100, 4852-4858.

(54) Julia, M.; Uguen, D. "Syntheses a I'aide de Sulfones" Bull. Chim. Soc. Fr. 1976, 513-518.

(55) Carretero, J. C.; Ghosez, L. "A Practical Route Towards a,P-Unsaturated 5- Lactones Based on a [3+3] Strategy. Synthesis (-)-Argentilactone" Tetrahedron Lett. 1988, 29, 2059-2062.

(56) Nakata, T.; Saito, K.; Oishi, T. "Synthetic Studies on (+)-Aplasmomycin. 2. Stereoselective Synthesis of Corey's Key Intermediate, A Formal Total Synthesis" Tetrahedron Lett. 1986, 27, 6345-6348.

(57) Nwaukwa, S. O.; Lee, S.; Keehn, P. M. "Epoxide Ring Opening by a-Sulfonyl Carbanions. Synthesis of y-Hydroxy Sulfones and Triflones." Synth. Commun. 1986, 16, 309-329.

(58) Cant, T. G.; Myers, A. I. "The Chemistry of 2-Oxazolines" Tetrahedron 1994, 50, 2297-2360.

(59) Smith, J. R. L.; Norman, R. O. C.; Stillings, M. R. "Synthesis of Oxazolines from Epoxides" J. Chem. Soc., Perkin I 1975, 1200-1202.

(60) Bongini, A.; Cardillo, G.; Orena, M.; Sandri, S.; Tomasini, C. "lodocyclofunctionalization of (E)-Trichloroacetimidoalk-2-enes. Synthesis of erythro- Sphinganine Triacetate" J. Chem. Soc., Perkin Trans. I 1986, 1345.

(61) Legraverend, M.; Huel, C.; Guilhem, J.; Bisagni, E. "Synthesis of 2-oxa-4- azabicyclo[3.2. l]-octane and -octene systems via X-ray-analyzed rra/zj-3-dibenzamido- cyclopentene-1-oxide" Carbohydr. Res. 1992, 228, 21.

(62) Shimizu, M.; Yoshioka, H. "Regiospecific Preparation of 2-Oxazolines Promoted by Silicon Tetrafluoride" Heterocycles 1988,27, 2527-2529.

(63) Igarashi, K.; Honma, T. "Addition of Glycals. IV. The Free-Radical Addition of Thiolacetic Acid to D-Glucal Triacetate" J. Org. Chem. 1970, 35,606-609.

(64) Giese, B. "Stereoselective Syntheses with Carbohydrate Radicals" Pure Appi. Chem. 1988.60, 1655-1658.

(65) Dupuis, J.; Giese, B.; Ruegge, D.; Fischer, H.; Korth, H.-G.; Sustmann, R. "Conformation of Glycosyl Radicals: Radical Stabilization by P-CO Bonds" Angew. Chem., Int. Ed Engl. 1984, 23, 896-898.

(66) Crich, D.; Lim, L. B. L. "Diastereoselective Free-Radical Reactions. Part 2. Synthesis of 2-Deoxy-P-C-pyranosides by Diasteroselective Hydrogen-Atom Transfer" J. Chem. Soc. Perkin Trans. I 1991, 2205-2208. 241

(67) Damm, W.; Giese, B.; Hartung, J.; Hasskerl, T.; Houk, K. N.; Huter, O.; Zipse, H. "Diasterofacial Selectivity in Reactions of Substituted Cyclohexyl Radicals. An Experimental and Theoretical Study" J. Am. Chem. Soc. 1992,114, 4067-4079.

(68) Curran, D. P.; Porter, N. A.; Giese, B. Stereochemistry of Radical Reactions', VCH Publishers: New York, 1996.

(69) Zehavi, U.; Sharon, N. "The Synthesis of Methyl 2,4-Diacetamido-2,4,6-trideoxy Hexopyranosides" 7. Org. Chem. 1972, J7, 2141-2145.

(70) Decoster, E.; Lacombe, J.-M.; Strebler, J.-L.; Ferrari, B.; Pavia, A. A. "Une Autre Approache a la Synthese de Derives Benzyles des Monasaccharides Reducteurs. Etude des Equilibres Pyrannose-Furannose par R.M.N, du Carbone-13" J. Carbohydr. Chem. 1983, 2, 329-341.

(71) Dejter-Juszynski, M.; Flowers, H. M. "Studies on the Koenigs-Knorr Reaction. Pan H. Synthesis of an a-L-Linked Disaccharide from Tri-O-Benzyl-a-L-Fucopyranosyl Bromide" Carbohydr. Res. 1971, 18, 219-226.

(72) Matthews, W. S.; Bares, J. E.; Bartmess, J. E.; Bordwell, F. G.; Comforth, F. J.; Druker, G. E.; Margolin, Z.; McCallum, R. J.; McCollum, G. J.; Vanier, N. R. "Equilibrium Acidities of Carbon Acids. VI. Establishment of an Absolute Scale of Acidities in Dimethyl Sulfoxide Solution" J. Am. Chem. Soc. 1975, 97, 7006-7014.

(73) Roth, W.; Pigman, W. In Methods in Carbohydrate Chemistry; R. L. Whistler and M. L. Wolfram, Ed.; Academic Press: New York, 1962; Vol. 2; pp 405-407.

(74) Merrifield, R. B. "Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide" J. Am. Chem. Soc. 1963, 85, 2149-2154.

(75) Caruthers, M. H. In Genetic Engineering-, J. Stelow and A. Holaender, Ed.; Plenum: New York, 1982; Vol. 4.

(76) Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. "Enzymes in Organic Synthesis-Application to the Problems of Carbohydrate Recognition. 2." Angew. Chem., Int. Ed. Engl. 1995, 34, 521-546.

(77) Wong, C.-H. "Enzymatic and Chemo-Enzymatic Synthesis of Carbohydrates" Pure Appl.Chem. 1995,67,1609-1616.

(78) Wong, C.-H.; R.L.. H.; Ichikawa, Y.; Kajimoto, T. "Enzymes in Organic Synthesis-Application to the Problems of Carbohydrate Recognition. 1." Angew. Chem., Int. Ed. Engl. 1995, 34, 412-432.

(79) Wang, P. G.; Fitz, W.; Wong, C.-H. "Making Complex Carbohydrates via Enzymatic Means" Chemtech 1995, 25, 22-32.

(80) Douglas, S. P.; Whitfield, D. M.; Krepinsky, J. J. "Polymer-Supported Solution Synthesis of Oligosaccharides" J. Am. Chem. Soc. 1991, 113, 5095-5097. 242

(81) Yan, L.; Taylor, C. M.; Goodnow, R.; Kahne, D. "Glycosylation on the Merrifield Resin Using Anomeric Sulfoxides" J. Am. Chem. Soc. 1994,116, 6953-6954.

(82) Danishefsky, S. J.; McClure, K. F.; Randolph, J. T.; Ruggeri, R. B. "A Strategy for the Solid-Phase Synthesis of Oligosaccharides" Science 1993,260, 1307-1309.

(83) Randolph, J. T.; Danishefsky, S. J. "An Interactive Strategy for the Assembly of Complex, Branched Oligosaccharide Domains on a Solid Support: A Concise Synthesis of the Lewis'' Domain in Bionconjugatable Form" Angew. Chem., Int. Ed. Engl. 1994, 33, 1470-1473.

(84) Toone, E. J.; Simon, E. S.; Bednarski, M. D.; Whitesides, G. M. "Enzyme- Catalyzed Synthesis of Carbohydrates" Tetrahedron 1989, 45, 5365-5422.

(85) Kopper, S. "Polymer-Supported Enzymatic Synthesis on a Preparative Scale" Carbohydr. Res. 1994, 265, 161-166,

(86) Schuster, M.; Wang, P.; Paulson, J. C.; Wong, C.-H. "Solid-Phase Chemical- Enzymatic Synthesis of Glycopeptides and Oligosaccharides" J. Am. Chem Soc. 1994, 116, 1135-1136.

(87) Halcomb. R. L.; Huang, H.; Wong, C.-H. "Solution- and Solid-Phase S>mthesis of Inhibitors of H. p\lori Attachment and E-Selectin-Mediated Leukocyte Adhesion" J Am. Chem. Soc 1994,7/6, 11315-11322.

(88) Nicolaou, K. C.; Florke, H.; Egan, M. G.; Barth, T.; Estevez, V. A. "Carbonucleotides and Carbopeptoids: New Carbohydrate Oligomers" Tetrahedron Lett. 1995, i6, 1775-1778.

(89) Terrett, N. K.; Gardner, M.; Gordon, D. W.; Kobylecki, R. J.; Steele, J. "Combinatorial Synthesis-The Design of Compound Libraries and Their Application to Drug Discovery" Tetrahedron 1S>95, 51, 8135-8173.

(90) Gallop, M. A.; Barrett, R. W.; Dower, W. J.; Fodor, S. P. A.; Gordon. E. M. "Applications of Combinatorial Technologies to Drug Discovery. 1. Background and Peptide Combinatorial Libraries" J. Med. Chem 1994, 37, 1233-1251.

(91) Gordon, E. M.; Barrett, R. W.; Dower. W. J.; Fodor, S. P. A.; Gallop, M. A. "Applications of Combinatorial Libraries to Drug Discovery. 2. Combinatrial Organic Synthesis, Library Screening Strategies, and Future Directions" J. Med Chem 1994, 37, 1385-1401.

(92) Heyns, K.; Paulsen. H. "Synthese der D-glucosaminuronsaure (2-amino-2-desoxy- D-glucuronsaure) und einige ihrer derivate (IX. mitteU. uber oxydative umwandlungen an kohlenhydraten)" Chem. Ber. 1955,88, 188-195.

(93) Fuchs, E.-F.; Lehmann, J. L. "Synthese von 7-amino-2.6-anhydro-7-desoxy-D- ^/vcero-L-ma/7/70-heptonsaure und 7-amino-2,6-anhydro-7-desoxy-D-^?/vcerf7-D-^?u/<7- heptonsaure" Chem. Ber. 1975. 108, 2254-2260. 243

(94) Yoshimura, J.; Ando, H.; Sato, T.; Tsuchida, S.; Hashimoto, H. "Aminosugars. XXVI. Synthesis of Amido-bonded Disaccharides Containing Hexsaminuronic Acids" Bull. Chenu Soc. Jpn. 1976, 49, 2511-2514.

(95) Muller, C.; Kitas, E.; Wessel, H. P. "Novel Oligosaccharide Mimetics by Solid- Phase Synthesis" J. Chenu Soc., Chem. Commun. 1995, 2425-2426.

(96) Wessel, H. P.; Mitchell, C. M.; Lobato, C. M.; Schmid, G. "Saccharide-Peptide Hybrids as Novel Oligosaccharide Mimetics" Angew. Chem., Int. Ed. Engl. 1995,34, 2712-2713.

(97) Graf van Roedern, E.; Kessler, H. "A Sugar Amino Acid as a Novel Peptidomimetic" Angew. Chem., Int. Ed. Engl. 1994, 33, 687-689.

(98) Graf van Roedern, E.; Lohof, E.; Hessler, G.; Hoffmann, M.; Kessler, H. "Synthesis and Conformational Analysis of Linear and Cyclic Peptides Containing Sugar Amino Acids" J. Am. Chem. Soc. 1996,118, 10156-1()167.

(99) Poitout, L.; Merrer, Y. L. "Synthesis of Sugar-Like Amino-Carboxylic Acids from D-Mannitol" Tetrahedron Lett. 1995, 38, 6887-6890.

(100) McDevitt, J. P.; Lansbury, P. T. "Glycosamino Acids: New Building Blocks for Combinatorial Synthesis" J. Am. Chem. Soc. 1996, 118, 3818-3828.

(101) Sahara, Y.; Hildreth, J. E. K.; Ichikawa, Y. "Synthesis of a New Carbohydrate Mimetics: "Carbopeptoid" Containing a C-1 Carboxylate and a C-2 Amino Group" Tetrahedron Lett. 1996. i7, 1575-1578.

(102) Suhara, Y.; Ichikawa, M.; Hildreth, ]. E. K.; Ichikawa, Y. "Synthesis of 3-1,6- Linked Oligosaccharide Mimetics: A Novel Potent Inhibitor of HIV Replication" Tetrahedron Lett. 1996, 37, 2549-2552.

(103) Hanessian, S. "Selective Hydrolysis of Amide Bonds in Acetamido Deoxy Sugars. O-Ethyl Acetamidium Ruoroborates" Tetrahedron Lett. 1967,16, 1549-1552.

(104) Ratcliffe, M. R.; VenoL, A. P.; Abbas, Z. S. "Sialic Acid Glycosides, Antigens, Immunoadsorbents, and Methods for their Preparation" Eur. Patent Appl. 1989, EP 0 319 253 A2.

(105) Flynn, D. L.; Zelle, R. E.; Grieco, P. A. "A Mild Two-Step Method for the Hydrolysis/Methanolysis of Secondary Amides and Lactams" J. Org. Chem. 1983,48, 2424-2426.

(106) Grehn, L.; Gunnarson, K.; Ragnarsson, U. "A Simple Method for the tert- Butoxycarbonylation of Amides" Acta Chem Scand. 1986, 40, 745-750.

(107) Grehn, L.; Gunnarsson, K.; Ragnarsson, U. "Removal of Forrayl, Acetyl, and Benzoyl Groups from Amides with Conversion into the Corresponding r-Butyl Carbamates" J. Chem. Soc., Chem. Commun. 1985, 1317-1318. 244

(108) Sato, S.; Furuhata, K.; Ogura, H. "Studies on Sialic Acids. XIV. Lactone Derivatives of //-Acetylneuraminic Acid" Chem, Pharnu Bull. 1988,1988,4678-4688.

(109) Fuchs, E.-F.; Lehmann, J. "Selective Fling-Opening of 3,4,5-Tri-0-Acetyl-2,6- Anhydro-D-g/yccro-L-/mjn/io-Heptaric Anhydride with Ammonia: The Synthesis of 7- Amino-2,6-Anhydro-7-Deoxy-L-^/ycero-L-^a/acro-Heptonic Acid" Carbohydr. Res. 1975,45, 135-141.

(110) Yu, R. K.; Ledeen, R. "Configuration of the Ketosidic Bond of Sialic Acid" 7. Biol. Chenu 1969,244, 1306-1313.

(111) Gervay, J.; Raherty, T. M.; Nguyen, C. P. Tetrahedron Lett. 1997, 38, accepted for publication.

(112) Gotschlich, E. C. In Bacterial Vaccines; E. Germanier, Ed.; Academic: New York, 1984; 237-255.

(113) Ding, S.-Q.; Ye, R.-B.; Zhang, H.-C. "Three New Serogroups of Neisseria Meningitidis" J. Biol. Stand. 1981, 9, 307-315.

(114) Ashton, F. E.; Ryan, A.; Jennings, H. J. "A New Serogroup (L) of Neisseria meningitidis" J. Clin. Microbiol. 1983,17, 722-727.

(115) Cadoz, M.; Armand, J.; Arminjon, F.; Gire, F.; C., L. "Tetravalent (Ak C Y W135) Meningococcal Vaccine in Children: Immunogenicity and Safety" Vaccine 1985. 3, 340-342.

(116) Jennings, H. J. "Capsular Polysaccharides as Vaccine Candidates" Curr. Top. Microbiol. Immunol. 1990, 150, 97-127.

(117) Goldschneider, I.; Gotschlich, E. C.; Artenstein, M. S. "Human Immunity to the Meningococcus. I. TTie Role of Humoral Antibodies" J. Exp. Med. 1969,129, 1307- 1326.

(118) Gotschlich, E. C.; Goldschneider, I.; Lepow, M. L.; Gold, R. In Antibodies in Human Diagnosis and Therapy, E. Haber and R. M. Krause, Ed.; Raven: New York, 1977; pp 391-402.

(119) Wyle, F. A.; Artenstein, M. S.; Brandt, B. L.; Tramont, E. C.; Kasper, D. L.; Altieri, P. L.; Berman, S. L.; Lowenthal, J. P. "Immunologic Response of Man to Group B Meningococcal Polysaccharide Vaccines" J. Infect. Dis. 1972,126, 514-521.

(120) Petola, H.; Kayhty, H.; Sivonen, A.; Makela, P. H. "Haemophilus influenza Type B Capsular Polysaccharide Vaccine in Children: A Double Blind Field Study of 100,000 Vaccines 3 Months to 5 Years of Age in Finland" Pediatrics 1977, 60, 730-737.

(121) Austrian, R. "Polysachharide Vaccines" Ann. Inst. Pasteur Microbiol. 1985, 136B, 295-307. 245

(122) Kayhty, H.; Schneerson, R.; Sutton, A. "Class-Specific Antibody Response to Haemophilus inluenza Type B Capsular Polysaccharide Vaccine" J. Infect. Dis. 1983, 148, 767.

(123) Finne, J.; Finne, U.; Deagostini-Bazin, H.; Goridis, C. "Occurance of a2-8 Linked Polysialosyl Units in a Neural Cell Adhesion Molecule" Biochenu Biophys. Acta 1983, 112, 482-487.

(124) Finne, J.; Leinonen, M.; Makela, P. H. "Antigenic Similarities Between Brain Components and Bacteria Causing Meningitis" Lancet 1983,2(8346), 355-357.

(125) Finne, J.; Makela, P. H. "Cleavage of the Polysialosyl Units of Brain Glycoproteins by a Bacteriophage Endosialidase" J. Biol. Chem. 1985, 260, 1265- 1270.

(126) Jennings, H. J.; Roy, R.; Michon, F. "Determinant Specificities of the Groups B and C Polysaccharides of Neisseria meningitidis" J. Immunol. 1985,134, 2651-2657.

(127) Kabat, E. A. "The Nature of the Antigenic Determinant" J. Immunol. 1966, 97, l-ll.

(128) Jennings, H. J.; Katzenellenbogen, E.; Lugowski, C.; Michon, F.; Roy, R.; Kasper, D. L. "Structure Conformation and Immunology of Sialic Acid Containing Polysaccharides of Human Pathogenic Bacteria" Pure Appl. Chem. 1984, 56, 893-905.

(129) Frasch, C. E.; Peppier, M. S. "Protection Against Group B Neisseria Meningococcal Disease: Preparation of soluble Protein and Protein-Polysaccharide Immunogens." Infect. Immunol. 1982, 37, 271-280.

(130) Moreno, C.; Lifely, M. R.; Esdaile, J. "Immunity ajid Protection of Mice Against Neisseria meningitidis Group B by Vacination using Polysaccharide Complexed with Outer Membrane Proteins: A Comparison with Purified B Polysaccharide" Infect. Immunol. 1985, 47, 527-533.

(131) Zollinger, W. D.; Mandrell, R. E.; Griffis, J. M.; Atieri, P.; Berman, S. "Complex of Meningococcal Group B Polysaccharide and Type 2 Outer Membrane Protein Immunogenic in Man" J. Clin. Invest. 1979,63, 836-848.

(132) Lifely, M. R.; Moreno, C.; Lindon, J. C. "An Integrated Molecular and Immunological Approach towards a Meningococcal Group B Vaccine" Vaccine 1987,5, 11-26.

(133) Gotschlich, E. C.; Fraser, B. A.; Nishimura, O.; Robbins, J. B.; Liu, T.-Y. "Lipid on Capsular Polysaccharides of Gram-Negative Bacteria." J. Biol. Chem. 1981, 256, 8915-8921.

(134) Jennings, H. J.; Roy, R.; Gamian, A. "Induction of Meningococcal Group B Polysaccharide-Specific IgG Antibodies in Mice by using an /V-propionylated B Polysaccharide-Tetanus Toxoid Conjugate Vaccine" J. Immunol. 1986,137, 1708- 1713. 246

(135) Jennings, H. J.; Roy, R.; Gamian, A. 'W-Propionylated Group B Meningococcal Polysaccharide Mimics a Unique Epitope on Group B Neisseria meningitidis" J. Exp. Med 1987,755, 1207-1211.

(136) Yamasaki, R. In Polysialic Acid\ J. Roth; U. Rutishauser and F. A. Troy, Ed.; Birkhauser Verlag; Basel, 1993; 1-10.

(137) Michon, F.; Brisson, J.-R.; Jennings, H. J. "Conformational Differences between Linear a(2-8)-Linked Homosialooligosaccharides and the Epitope of the Group B Meningococcal Polysaccharide" Biochemistry 1987,26, 8399-8405.

(138) Yamasaki, R.; Bacon, B. "Three-Dimensional Structural Analysis of the Group B Polysaccharide of Neisseria meningitidis 6275 by Two-Dimensional NMR: The Polysaccharide is Suggested to Exist in Helical Conformations in Solution" Biochemistry 1991, iO, 851-857.

(139) Borgias, B. A.; James, T. L. "COMATOSE, A Method for Constrained Refinement of Macromolecular Structure Based n Two-Dimensional Nuclear Overhauser Effect Spectra" J. Magn. Reson. 1988, 79, 493-512.

(140) Ferrin, T. E.; Huang, C. C.; Jarvis, L. E.; Langridge, R. "The MIDAS Database System" J. Mol. Graphics 1988, 6, 2-12.

(141) Dickerson, R. E.; Comer, B. N.; Kopka, M. L.; Drew, H. R. In Nucleic Acid Research', K. Mizubichi; I. Watanabe and J. D. Watson, Ed.; Academic Press: New York, 1983; pp 35-59.

(142) Brisson, J.-R.; Baumann, H.; Imberty, A.; Perez, S.; Jennings, H. J. "Helical Epitope of the Group B Meningococcal a(2-8)-Linked Sialic Acid Polysaccharide" Biochemistry 1992, 31, 4996-5004.

(143) Perez, S.; Vergelati, C. "Unified Representation of Helical Parameters: Application to Polysaccharides" 1985,24, 1809-1822.

(144) Kabat, E. A.; Liao, J.; Osserman, E. F.; Gamian, A.; Michon, F.; Jennings, H. J. J. Exp. Med 1988, 168, 699-711.

(145) Saenger, W.; Riecke, J.; Suck, D. "A Structural Model for the Polyadenylic Acid Single Helix" J. Mol. Biol 1975, 93, 529-534.

(146) Lindon, J. C.; Vinter, J. G.; Lifely, M. R.; Moreno, C. "Conformational and Dynamic Differences Between N. meningitidis Serogroup B and C Polysaccharides, using N.M.R. Spectroscopy and Molecular Mechanics Calculations" Carbohydr. Res. 1984, 133, 59-74.

(147) Lifely, M. R.; Gilbert, A. S.; Moreno, C. "Rate, Mechanism, and Immunochemical Studies of Lactonisation in Serogroup B and C Polysaccharides of Neisseria meningitidis" Carbohydr. Res. 1984, 134, 229-243. 247

(148) Lifely, M. R.; Gilbert, A. S.; Moreno, C. "Sialic Acid Polysaccharide Antigens of Neisseria memingitidis and Escherichia coli: Esterification Between Adjacent Residues" Carbohydr. Res. 1981, 94, 193-203.

(149) Yoshida, O.; Nakashima, H.; Toshida, T.; Kanedo, Y.; Matsuzaki, K.; Uryu, T.; Yamamoto, N. "Sulfatation of the Iramunomodulating Polysaccharide Lentinan: A Novel Strategy for Anti-virals to Human Immunodeficiency Virus (HTV)" Biochem. Pharmacol. 1988, 37, 2887-2889.

(150) Ueno, R.; Kuno, S. "Dextran sulfate, a Potent anti-HIV Agent in vitro Having Synergism with Zidovudine" Lancet 1987, /, 1379.

(151) Leydet, A.; Barthelemy, P.; Boyer, B.; Lamaty, G.; Roque, J. P.; Bousseau, A.; Evers, M.; Henin, Y.; Snoeck, R.; Andrei, G.; Ikeda, S.; Reymen, D.; DeClerq, E. "Polyanion Inhibitors of Human Immunodeficiency Virus and Other Viruses. Part 1. Polymerized Anionic Surfaces." J. Med. Chenu 1996, 38, 2433-2440.

(152) Leydet, A.; El Hachemi, H.; Boyer, B.; Lamaty, G.; Roque, J. P.; Schols, D.; Snoeck, R.; Andrei, G.; Dceda, S.; Neyts, J.; Reymen, D.; Este, J.; Witvrouw, M.; DeClerq, E. "Polyanion Inhibitors of Human Immunodeficiency Virus and Other Viruses. Part 2. Polymerized Anionic Surfactants Derived from Amino Acids and Dipeptides." J. Med.Chem. 1996,39, 1626-1634.

(153) Ito, M.; Baba, M.; Pauwela, R.; DeClerq, E.; Shigeta, S. "Inhibitory Effect of Dexfran Sulfate and Heparin on the Replication of Human Immunodeficiency Virus (HFV) in vitro" Antiviral Res. 1987, 7, 361-367.

(154) Baba, M.; Schols, D.; DeClerq, E.; Pauwels, R.; Nagy, M.; Gyorgyi-Edelenyi, J.; Low, M.; Gorog, S. "Novel Sulfated Polymers as Highly Potent and Selective Inhibitors of Human Immunodeficiency Virus Replication and Giant Cell Formation." Antimicrob. Agents Chemother. 1990, 134-138.

(155) Baba, M.; Nakajima, H.; Schols, D.; Pauwels, R.; Balzarini, J.; DeClerq, E. "Pentosan Sulfate, a Sulfated Oligosaccharide, is a Potent and Selective anti-HIV agent in vitro" Antiviral Res. 1988, 9, 335-343.

(156) Piantini, U.; Sorensen, O. W.; Ernst, R. R. "Multiple Quantum Filters for Elucidating NMR Coupling Networks" J. Am. Chenu Soc. 1982, 104, 68{X)-6801.

(157) Flaherty, T. M.; Gervay, L "2D NMR Analysis of the Polylactone Derivative of Colominic Acid. Complete ^H and ^^C NMR Chemical Shift Assignments" Carbohydr. Res. 1996,281, 173-177.

(158) Freeman, R.; Keeler, J. "Suppresion of Artifacts in Two-Dimensional J Spectra" J. Magn. Reson. 1981,45,484-487.

(159) Bax, A.; Morris, G. A. "An Improved Method for Heteronuclear Chemical Shift Correlation by Two-Dimensional NMR" J. Magn. Reson. 1981, 42, 501-505. 248

(160) States, D. J.; Haberkorn, R. A.; Ruben, D. J. "A Two-Dimensional Nuclear Overhauser Experiment with Pure Absorption Phase in Four Quadrants" J. Magn. Reson. 1982, 48, 286-292.

(161) Parrill, A. L. Ph.D. Thesis, The University of Arizona, 1996.

(162) Wuthrich, K. NMR of Proteins and Nucleic Acids; John Wiley & Sons: New York, 1986.

(163) Walters, W. P. Ph.D. Thesis, The University of Arizona, 1995.

(164) Houk, K. N.; Eksterowicz, J. E.; Wu, Y.-D.; Fuglesang, C. D.; Mitchell, D. B. "Conformational Preferences of the 0-C-C-C Unit in Acyclic and Cyclic Systems. The Exo-Deoxoanomeric Effect and Related Phenomena" J. Am. Chem. Soc. 1993,115, 4170-4177.