Synthetic studies of azido inositols and inositol based glycans 1

72 3 Bioimaging of Azido Inositol Incorporated Lipids and Synthesis of Cyclooctyne Fluorophore Probes .... ix Research Summary This PhD thesis describes synthetic studies of various azi

SYNTHETIC STUDIES OF AZIDO-INOSITOLS AND

INOSITOL-BASED GLYCANS

SANDIP PASARI

NATIONAL UNIVERSITY OF SINGAPORE

2012

SYNTHETIC STUDIES OF AZIDO-INOSITOLS

AND INOSITOL-BASED GLYCANS

SANDIP PASARI

(M.Sc Indian Institute of Technology Madras, India)

A THESIS SUBMITTED FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

DEPARTMENT OF CHEMISTRY

NATIONAL UNIVERSITY OF SINGAPORE

2012

i

Acknowledgements

This PhD thesis has been carried out from August 2007 to January 2012 at the National University of Singapore, Faculty of Science, Department of Chemistry, under the

supervision of Assistant Professor Dr Martin J Lear

I would first like to express my sincere gratitude to my supervisor Asst Prof Martin J

Lear, whose encouragement, valuable guidance and great support from initial to the

final level enabled me to develop an understanding of the project

I am heartily thankful to the members and collaborators, particularly Prof Markus R

Wenk and Shareef M Ismail from Department of Biochemistry and Department of

Biological Sciences, National University of Singapore, not only for providing me with this interesting and challenging project but also for their kind help and support during the course of this work

It is my great opportunity to thank all present and past group members of the Lear

group, particularly Dr Bastien, Munhong, Santosh, Shibaji, Karthik, Ravi, Stanley

and Eugene for valuable discussions and suggestions on my project as well as thesis

I wish to thank Mdm Han Yanhui and Mr Chee Peng for their timely assistance for NMR measurements and Mdm Tan and her co-workers for their assistance in

resolving X-ray structures

I thank all my friends for their kind help and understanding during my stay in

Singapore

Finally, the National University of Singapore is acknowledged for its financial

support (NUS-scholarship)

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Dedication

This thesis is dedicated to my parents, Nimai and Tulsi Pasari, my sister Kabita

Nandi, my brother Sanjoy Pasari and other family members Without their

understanding, encouragement and great support, this work would never have been completed

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Table of Contents:

Research Summary ix

List of Figures: xi

Lists of Schemes: xiii

List of Tables: xxii

List of Abbreviations: xxiii

1 Introduction 1

1.1 Phosphatidyl Inositol and Its Biological Significance 2

1.2 Inositol Structure 4

1.3 Inositol Lipid Biosynthesis and Transport 5

1.4 Inositol Glycolipids and Its Biological Significance 7

1.4.1 Chemical Structures of GPIs 7

1.4.2 Structural Diversity of GPI 9

1.4.3 General Structure of PIMs 10

1.5 Targeting Inositol Lipids 12

1.5.1 Metabolic Labelling Using Inositol Isotopes and Modified Analogues 12

1.5.2 Bioorthogonal Chemistry for Targeting Biomolecules 13

1.6 Design and Hypothesis of Inositol Analogues for Metabolic Engineering 14

1.7 General Synthetic Outline 18

iv

1.8 Synthesis of Myo-Inositol Derivatives Starting from Chiral Sources 21

1.8.1 Inversion Approach Starting from Chiral Sources 21

1.8.2 Ferrier Rearrangement Approach Starting from Chiral Sources 22

1.8.3 SmI2 Mediated Reductive Carbocyclization Approach Starting from Chiral Sources 23

1.9 Synthesis of Myo-Inositol Derivatives Starting from Nonchiral Sources 24

1.9.1 Stereoselective Microbial Oxidation Approach from Nonchiral Sources 24

1.9.2 Chemo-Enzymatic Resolution Approach from Nonchiral Sources 25

1.10 Myo-inositol Starting Material 28

1.10.1 Regioselective Protection of Myo-Inositol 28

1.10.2 Regioselective Protection of Myo-Inositol Based Tetraols 31

1.10.3 Regioselective Protection of Myo-Inositol Based Triols 32

1.10.4 Regioselective Protection of Myo-Inositol Based Diols 33

1.10.5 Regioselective Deprotection of Myo-Inositol Based Protected Derivatives 35 1.11 Goal of Research 36

1.12 References 39

2 Results and Discussion 53

2.1 Synthesis of (±)6-Deoxy-6-Azido Myo-Inositol Analogue 53

2.2 Synthesis of meso 5-Deoxy-5-Azido Myo-Inositol Analogue 58

v

2.3 Synthesis of (±)3-Deoxy-3-Azido Myo-Inositol Analogue 62

2.4 Synthesis of meso 2-Deoxy-2-Azido Myo-Inositol Analogue 67

2.5 Conclusion 70

2.6 References 72

3 Bioimaging of Azido Inositol Incorporated Lipids and Synthesis of Cyclooctyne Fluorophore Probes 74

3.1 Introduction 74

3.2 Incorporation of Synthetic Azido-inositol Surrogates into Inositol Lipids of S Cerevisiae 74

3.3 Inositol Analogue Lipid Profiling in S cerevisiae and Inositol Transporter Dependence 77

3.4 Design of Cyclooctyne Fluorophore Probes for Live-cell Imaging 83

3.5 Synthesis of Cyclooctyne Fluorophore Probes 85

3.6 Conclusion 89

3.8 References 90

4 Synthesis of Glycan Core of GPI of Plasmodium Falciparum 92

4.1 Introduction 92

4.2 Previous Synthesis of GPI Structures 93

4.2.1 Seeberger Approach: (4+2) Strategy 93

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4.2.2 Fraser-Reid Linear Approach: Step-wise Strategy 96

4.3 Desymmetrization Approach: Synthetic Applications Towards Glycan Core 99

4.4 Our Approach: Retro Synthetic Analysis of GPI of Plasmodium Falciparum 101

4.5 Synthetic Approach to Pseudodisaccharide Component 103

4.5.1 Synthesis of Inositol Orthoformate Derivatives 105

4.5.2 Synthesis of Mannoside Building Blocks 107

4.5.2.1 Synthesis of Fully Benzylated Mannoside Schmidt Donor 107

4.5.2.2 Synthesis of Mannoside Schmidt Donor with Accessible C2 Ester Group 109

4.5.3 Synthesis of 2-Deoxy-2-Azido Glucosaminide Schmidt Donor 111

4.5.4 Regioselective and Stereoselective Coupling Studies of Donors and Acceptors 112

4.5.5 Structural Determination (D&L) of Glucosaminated Inositols 118

4.5.6 Synthesis of C4 Differentially Protected Glucosiminide Schmidt Donor 123

4.5.6.1 Azido Nitration Approach of Glucal Derivative 123

4.5.6.2 Copper(II) Catalyzed Diazotransfer Approach of Glucosamine 125

4.5.6.2.1 Thiophenol Glycosylation Approach of Azido Acetate 4-123 125

4.5.6.2.2 p-Methyl Thiophenol Glycosylation Approach of Azido Acetate 4-126 128

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4.5.7 Regioselective Coupling Studies of C4 Modified Glucosiminide Donor 131

4.5.8 Regioselective Cleavage of Orthoformate Moiety 132

4.5.9 Synthesis of Modified C4 Allylated Glucosiminide Donors 136

4.5.10 Regioselective Coupling of Schmidt Donor and orthoformate Functionalization 138

4.5.11 Desymmetrization Studies with Various C4-allylated Glycosaminyl Donors 141

4.6 Synthesis of Mannoside Glycans: Tetramannoside 145

4.6.1 Synthetic Plan of Tetramannoside Glycan 145

4.6.2 Synthesis of Mannoside Building Blocks 146

4.6.3 One Pot Iterative Glycosylation Coupling of 1,2-Orthoesters 148

4.6.4 Synthesis of Alternative Mannoside Schmidt Donor 149

4.6.5 Glycosylation Coupling with Schmidt Donor 150

4.7 Conclusion 151

4.8 References 154

Experimental Procedures for Chapter 2: 161

Appendix 1: NMR Spectra of the Selected Compounds in Chapter 2 186

Experimental Procedures for Chapter 3: 187

Appendix 2: NMR Spectra of the Selected Compounds in Chapter 3 195

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Experimental procedure for Chapter 4 196

Appendix 3: NMR Spectra of the Selected Compounds in Chapter 4 260

References 261

ix

Research Summary

This PhD thesis describes synthetic studies of various azido inositol analogues through

azide installation via double inversion of suitably protected inositol derivatives either

by oxidation-reduction steps or consecutive SN2 approach A final global deprotection

in the presence of azide group by one step acetolysis enabled the synthesis of inositol pentaacetate analogues Methanolysis in the presence of catalytic amount of sodium methoxide eventually facilitated the synthesis of various azido inositols The synthetic efforts towards the synthesis of various azido inositols are described in chapter 2

azido-Chapter 3 summarized metabolic incorporation of various modified azido inositol analogues into various inositol lipids and growth of yeast cells The azido incorporation into inositol lipids was confirmed through mass spectrometry and whole cell imaging of the click product with TAMRA alkyne The azido incorporation into higher order inositol lipids (sphingolipids, glycolipids such as GPIs) is yet to elucidate at the moment The synthesis of a new cyclooctyne fluorophore probe was explored for studying sub cellular location of azido incorporated inositol lipids through a copper free

in vivo imaging with metabolically labelled azido inositols of S cerevisiae This

fluorophore (BODIPY) linked cyclooctyne would facilitate live-cell imaging studies with azide incorporated lipids

Another part of my research work focused on the synthetic studies towards glycan

synthesis glycosylphosphatidyl inositol (GPI) of Plasmodium falciparum through a

(4+2) glycan coupling of phospholipidated pseudodisaccharide and tetramannoside trichloroacetimidate The suitably protected pseudodisaccharide component was achieved through a desymmetrization strategy via a direct regioselective and

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stereoselective glycosylation of a myo-inositol orthoformate derivative with

glucosaminyl donor The natural D inositol and unnatural L inositol configurations of the glucosaminated coupling products were determined by a chemical correlation to the reference intermediates, which were derived from previously reported known mannosylated coupling pair (D & L) The regio- and stereoselective alkylative orthoformate cleavage of the fully protected glucosaminated coupling product to 3,5-acetal derivative was confirmed by comparing experimentally observed NOE correlations in the boat-conformation of the intermediate

Furthermore, a linear convergent glycosylation approach towards the synthesis of tetramannoside glycan with terminally differentiated protecting group was studied After C1 phosphorylation, C2 palmitoylation of the synthesized pseudodisaccharide component and subsequent glycan coupling with a tetramannoside trichloroacetimidate would eventually facilitate the synthesis of fully lipidated glycan core of malaria GPI The synthetic efforts towards the synthesis of glycan portion of malaria GPI are described in chapter 4 This work has yet to be completed, although unexpected but interesting results have been found and described herein

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List of Figures:

Figure 1.1 Types of cell to cell signalling in biological systems 2

Figure 1.2 Hydrolytic cleavage of phosphatidylinositol 3

Figure 1.3 Structure of myo-inositol molecule and arbitrary numbering 4

Figure 1.4 Overview of lipid biosynthesis in Yeast, Mammalian, and Mycobacteria 5

Figure 1.5 The structural domains of protein-bound GPI anchor of P falciparum 9

Figure 1.6 Molecular overview of glycan and lipid structural diversity of selected GPIs 10

Figure 1.7 Structural domains of PIMs 11

Figure 1.8 Structure of different phosphoinositides 15

Figure 1.9 Proposed azido-Inositol analogues for metabolic engineering 17

Figure 1.10 Hypothetical mechanisms for metabolic incorporation into GPIs 18

Figure 1.11 General synthetic considerations 19

Figure 1.12 General contemporary methods for synthesis of inositol derivatives 20

Figure 2.1 X-ray crystal structure of (±)-6-azido-myo-inositol-pentaacetate 1-8a 57

Figure 2.2 X-ray crystal structure of meso-5-azido-myo-inositol-pentaacetate 1-7a 61

Figure 2.3 Favourable transition state of E2 over SN2 66

Figure 2.4 Two dimensional correlation (NOSEY) among axial protons 67

Figure 2.5 Mechanism of one step benzylation 68

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Figure 3.1 Incorporation of deuterated inositol into inositol lipids extracted for

wild type S.cerevisiae 74

Figure 3.2 Analysis of wild type S cerevisiae, BY4741, growth curve in the

presence of 6-azido inositol analogueues 1-8a,b 76

Figure 3.3 Analysis of wild type S cerevisiae, BY4741, growth curve in the

presence of 2-azido inositol analogueues 1-5a,b 76

Figure 3.4 Analysis of wild type S cerevisiae , BY4741, growth curve in the

presence of 5-azido inositol analogueues 1-7a,b 77

Figure 3.5 Mass Spectrometry analysis of lipid extracts of S cerevisiae cultured

in media containing myo-inositol and 5-azido-meso-inositol……… 78

Figure 3.6 Incorporation of deuterated inositol (blue) and 5-azido-inositol (red) in

various species of PtdIns extracted from wild type S cerevisiae, BY4741 79

Figure 3.7 Deuterated inositol (blue) and 5-azido-inositol (red) incorporation in

(a) ∆ITR1 mutants and (b) ∆ITR2 mutants 79

Figure 3.8 Analysis of growth curve of Inositol transport mutant of S

cerevisiae, ∆ITR1, in the presence of azide-modified inositols 80

Figure 3.9 TLC analysis of TAMRA-tagged lipid extracts 81 Figure 3.10 QTOF-MS and MS/MS analysis of TLC fluorescent spots 81 Figure 3.11 Whole-cell imaging of azido-inositol lipids through click-tagging

with TAMRA-alkyne 82

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Figure 3.12 Incorporation of 5-azido-inositol 1-7b into phosphoinositides 82

Figure 3.13 Incorporation of 3-azido-inositol 1-6b into phosphatidylinositol 82

Figure 3.14 Incorporation of 3-azido-inositol 1-6b in various species of PtdIns extracted from wild type S cerevisiae 83

Figure 3.15 Strain promoted various cyclooctyne systems for Cu-free click reactions 84

Figure 3.16 Proposed cyclooctyne fluorophore probe 3-6 85

Figure 4.1 Structure determination of 4-180D by NOESY correlation 139

Lists of Schemes: Scheme 1.1 Synthesis of inositol derivative starting from 1-10 by inversion Approach 21

Scheme 1.2 Synthesis of inositol derivative starting from 1-10 by oxidation-reduction 22

Scheme 1.3 Synthesis of chiral inositol derivative starting from chiral D-glucose 22

Scheme 1.4 Reductive carbocyclization approach starting from D-Xylose 1-12 23

Scheme 1.5 Reductive cyclization approach starting from D-mannitol 23

Scheme 1.6 Microbial oxidation approach starting from benzene 1-19 24

Scheme 1.7 Microbial oxidation approach starting from bromobenzene 1-20 25

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Scheme 1.8 Chemo-enzymatic resolution of (±)1-45 25

Scheme 1.9 Synthesis of chiral azido conduritol (+)1-49 and (-)1-49 26

Scheme 1.10 Synthesis of azido-inositols from azido conduritol (+)1-49 27

Scheme 1.11 Synthesis of azido inositols from conduritol E (±)1-50 27

Scheme 1.12 Regioselective ketal protection among six hydroxyl groups of myo-inositol 1-2 29

Scheme 1.13 Regioselective bis-ketal protection of inositol 1-2 30

Scheme 1.14 Orthoformate protection of inositol 1-2 30

Scheme 1.15 Regioselective protection among hydroxyl groups of tetraols 31

Scheme 1.16 Regioselective monoprotection of tetraol 1-58 31

Scheme 1.17 Regioselective protection of inositol triols 32

Scheme 1.18 Regioselective monoprotection of inositol orthoesters 33

Scheme 1.19 Symmetric and asymmetric di-protection of inositol orthoesters 33

Scheme 1.20 Regioselective monoprotection of inositol di-ketal derivatives 34

Scheme 1.21 Regioselective monoprotection of 4,5-diols 34

Scheme 1.22 Selective cleavage of trans ketal and THP acetal of inositol derivatives 35

Scheme 1.23 Selective hydrolytic cleavage of inositol orthoformate 1-88 36

xv

Scheme 2.1 Schlewer approach for the synthesis of 6-azido inositol derivative 53

Scheme 2.2 Synthesis of dicyclohexylidenemyo-inositol derivatives 54

Scheme 2.3 Regioselective monobenzylation of 1,2,4,5-dicyclohexylidene myo-inositol 55

Scheme 2.4 Stereoselective transformation of myo-alcohol to epi-alcohol 55

Scheme 2.5 Synthesis of 6-azido inositol derivative 56

Scheme 2.6 Comparison between substitution and elimination at C6 position 56

Scheme 2.7 Deprotection sequence to 6-azido inositols 57

Scheme 2.8 Synthesis of fully protected inositol and selective cleavage of trans-ketal 58

Scheme 2.9 Regioselective monobenzylation of 4,5-inositol diol derivative 58

Scheme 2.10 Identification of regioisomeric monobenzylated products 59

Scheme 2.11 Conversion of myo-inositol derivative into neo-inositol derivative 60

Scheme 2.12 Regeneration of myo-configuration by azide displacement 60

Scheme 2.13 Global deprotection into pentaacetate by acetolysis 61

Scheme 2.14 Synthesis of chiro-inositol derivative by efficient SN2 substitution method 62

Scheme 2.15 Synthesis of 2-26 containing C3 free alcohol 63

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Scheme 2.16 Transformation of myo-inositol derivative into chiro-

inositol derivative 63

Scheme 2.17 Deacylation of chiro-inositol derivative 64

Scheme 2.18 Activation of free alcohol 2-29 by sulfonylation 64

Scheme 2.19 Regeneration of myo-configuration from chiro-form 65

Scheme 2.20 Global deprotection of inositol derivative 2-33 67

Scheme 2.21 Synthesis of symmetric 4,6-dibenzyl inositol orthoformate 68

Scheme 2.22 Conversion of myo-inositol derivative into scyllo-inositol derivative 69

Scheme 2.23 Sulfonylation and orthoformate cleavage for synthesis of 2-42 69

Scheme 2.24 Regeneration of myo-configuration 70

Scheme 2.25 Acetolysis of myo-inositol derivative 2-43 70

Scheme 2.26 Highlights of the synthesized azido inositol analogues 1-5a,b to 1-8a,b 71

Scheme 3.1 Synthesis of 3-10 by Sonogashira coupling 85

Scheme 3.2 Synthesis of N-Boc protected Z-alkene 3-12 86

Scheme 3.3 Synthesis of dihydro-dibenzo-azocine 3-14 by reductive amination 86

Scheme 3.4 Synthesis of N-Boc protected cyclooctyne 3-17 87

Scheme 3.5 N-Boc protection of β-alanine 3-18 87

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Scheme 3.6 Synthesis of N-Boc protected dibromide 3-21 88

Scheme 3.7 Synthesis of fluorophore (BODIPY) linked cyclooctyne probe 3-6 88

Scheme 3.8 Highlights of the synthesized cyclooctyne fluorophore probe 3-6 89

Scheme 4.1 Seeberger retrosynthetic analysis of the GPI of P falciparum 94

Scheme 4.2 Seeberger synthesis of glucosamine-inositol disaccharide 4-9 95

Scheme 4.3 Vishwakarma synthesis of pseudodisaccharide precursor 4-14 96

Scheme 4.4 Fraser-Reid retrosynthetic analysis of malaria candidate GPIs 4-15, 4-16 97

Scheme 4.5 Fraser-Reid glycan synthesis of 4-15, 4-16 98

Scheme 4.6 Desymmetrization of inositol derivative and Ley synthesis of pseudodisaccharide 4-34 99

Scheme 4.7 Desymmetrization of inositol derivative and Hung synthesis of PIM2 100

Scheme 4.8 Desymmetrization of inositol diol 4-44 and Hung synthesis of mycothiol 101

Scheme 4.9 Lear-group retro-synthetic approach to the GPI of P Falciparum 102

Scheme 4.10 Proposed strategy to GPI, PIM & LAM glycolipids 103

Scheme 4.11 Chemical correlation strategy for structure determination (D&L) 104

Scheme 4.12 Synthesis of inositol orthoformate 1-66a 105

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Scheme 4.13 Regioselective protection of equatorial hydroxyl group of 1-66a 106

Scheme 4.14 Synthesis of fully benzylated mannoside Schmidt Donor 4-35 108

Scheme 4.15 Glycosylation using remote activation concept (TOPCAT donors) 108

Scheme 4.16 Synthesis of TOPCAT mannosyl donor 4-75 109

Scheme 4.17 Synthesis of mannoside Schmidt donor with accessible C2 benzoate 4-82 110

Scheme 4.18 Synthesis of mannosyl TOPCAT donor 4-83 110

Scheme 4.19 Synthesis of mannoside Schmidt donor with accessible C2 acetate 4-89 111

Scheme 4.20 Synthesis of glucosamine Schmidt donor 4-94 112

Scheme 4.21 Synthesis of glucosaminyl TOPCAT donor 4-95 112

Scheme 4.22 Regioselective coupling between donor 4-35 and acceptor 4-36 113

Scheme 4.23 Iterative glycosylation strategy of orthoester 114

Scheme 4.24 Regioselective coupling of mannoside donor containing accessible C2 esters 115

Scheme 4.25 Palmer glycosylation coupling strategy 116

Scheme 4.26 Regioselective coupling of Schmidt donor 4-94 117

Scheme 4.27 Chemical correlation method for D & L structure determination of unknown pair 119

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Scheme 4.28 Synthetic strategy for chemical correlation with

unknown glucosaminated pair 120

Scheme 4.29 Protection of inositol free hydroxyl group of mannosylated coupling pair 120

Scheme 4.30 MOM-Protection of insitol free hydroxyl group of mannosylated coupling pair 121

Scheme 4.31 Chemical correlation of D & L structures of unknown azido pair 4-103a,b 122

Scheme 4.32 Attempted synthesis of benzylidene 123

Scheme 4.33 Synthesis of differentially protected glucosamine donor 124

Scheme 4.34 Synthesis of 2-deoxy-2-azido tetraacetate glucosaminide 125

Scheme 4.35 Synthesis of 4,6-benzylidene thiglucoside derivatives 4-129 & 4-130 126

Scheme 4.36 Synthesis of differentially protected Schmidt donor 4-135 127

Scheme 4.37 Synthesis of differentially protected Schmidt donor 4-135 128

Scheme 4.38 Synthesis of TOPCAT donor 4-139 128

Scheme 4.39 Facile glycosylation of thioglycosides 129

Scheme 4.40 Synthesis of 4,6-benzylidene thioglycosides 4-145 and 4-146 129

Scheme 4.41 Synthesis of differentially protected thoglycoside 4-149 130

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Scheme 4.42 Synthesis of Totally protected thoglycoside 4-152 130 Scheme 4.43 Regioselective Coupling of various glucosaminyl

donors with meso inositol diol 131

Scheme 4.44 Regioselective and stereoselective reductive

cleavage of orthoformate 136

Scheme 4.50 Attempted Synthesis of allyl modified anomeric alcohol 4-173 137 Scheme 4.51 Synthesis of C4 allylated glucosaminyl Schmidt donor 4-174 137

Scheme 4.52 Synthesis of C4 allylated Glucosaminyl TOPCAT donor 4-175 137

Scheme 4.53 Synthesis of anomeric α-and β-p-methyl thioglycoside donors 138

Scheme 4.54 Regioselective Coupling between 4-174 and 4-64 138 Scheme 4.55 Regioselective alkylative orthoformate cleavage of 4-179D 139 Scheme 4.56 Regioselective and stereoselective nature of orthoformate cleavage 140

promoted β-glycosylation 144

Scheme 4.61 Attempted (R)-BINOL phosphoric acid catalyst

mediated regioselective coupling 144

Scheme 4.62 Seeberger approach: one pot iterative glycosylation strategy 145 Scheme 4.63 Retrosynthetic analysis of tetramannoside 4-200 146 Scheme 4.64 Synthesis of mannoside building block 4-201 147 Scheme 4.65 Synthesis of differentially protected mannoside

building block 4-202 147

Scheme 4.66 Synthesis of dimannoside through iterative glycosylation approach 148 Scheme 4.67 Synthesis of trimannoside through iterative glycosylation approach 149 Scheme 4.68 Synthesis of differentially protected mannoside

Schmidt donor 4-211 150

Scheme 4.69 Synthesis of trimannoside through regular

Schmidt glycosylation approach 150

DMA N,N-dimethylacetamide

p-TSA p-toluenesulfonic acid

s (or) sec secondary

SN1 or SN2 nucleophilic substitution

t (or) tert tertiary

1

1 Introduction

Prelude: Inositol is a carbohydrate that is suited for signalling through structural

modifications such as phosphorylation, lipidation and glycosylation Possibly as a prebiotic molecule, inositol molecules are stable at high temperatures and various pH.1Inositol signalling molecules, particularly inositol triphosphates (IP3), have garnered widespread interest due to their role in calcium (Ca2+) signalling and cell-cell communication.2 Phosphorylated inositol lipids form integral components of cellular membranes and are precursors to signalling molecules Phosphoinositides (PIs) are also involved in cellular signalling, apoptosis, cytosolic calcium balance and possibly in cell cycle modulation.3 Highly glycosylated forms of inositol like glycosylphosphatidylinositol (GPI) anchors, and multiple mannosylated inositol lipids, such as phosphatidyl-myo-inositol mannosides (PIMs) and lipoarabinomannan (LAMs), have been observed on the extracellular membrane These glycolipids are involved in anchoring proteins on the cell surface, endocytic trafficking, and are required for infection4 or similar processes

The different classes of inositol lipids have been widely studied Inositol lipid binding protein domains (PH, ENTH and others) have been linked to fluorescent proteins to study phosphoinositides and to understand changes in their localization and lipid levels.5 Organic synthesis of phosphatidylinositol and phosphoinositides has been used

to study protein interaction.6 Radiolabeled inositol analogues have been used to observe the variation in levels of cellular inositol lipid content.7 Herein, synthetically different azido inositols were prepared to study the variation of metabolic labelling by the incorporation of azide modified analogues of inositol into inositol lipids of yeast cells Azide modified inositol lipids thus enable the tagging of alkyne bearing molecules

2

through click chemistry or Staudinger ligation Incorporation of these modified analogues into higher inositol glycolipids (such as the GPIs) would thus enable the targeting and identification of GPI anchored proteins As such, we are also interested in the total synthesis of various GPIs for further biological study

1.1 Phosphatidyl Inositol and Its Biological Significance

The maintenance of life processes in complex organisms depends on cell-to-cell signaling between individual cells This can be achieved by a variety of ways in Nature

via chemical messengers, e.g., hormones, growth factors and neurotransmitters, and so

forth.8 Usually, the selected messenger depends on the desired function of the cell, which in turn determines the type of receptors found on the individual cell The incoming signals received through the cell membrane are then converted into a response inside the target cell; a process known as signal transduction.9

Figure 1.1 Types of cell to cell signalling in biological systems

3

There are three classes of receptors (Fig 1.1): ion channels, membrane spanning tyrosine kinases, and G-protein coupled receptors Through a cascade of events, G-proteins release guanosine diphosphate (GDP) and, in turn, bind to guanosine triphosphate (GTP) The release of energy activates enzymes to produce secondary messengers for signalling to occur in an intracellular fashion.10 The generation of secondary messengers through G-protein coupled receptors can be categorized into two types: production of cyclic 3’,5’-adenosine monophosphate (cAMP) and the hydrolysis

of inositol phospholipids to diacylglycerol (DAG) and D-1,4,5- inositol triphosphates11(Fig 1.2)

The hydrolytic product (D)-1,4,5-inositol triphosphate efficiently binds to specific receptors in the endoplasmic reticulum, stimulating the release of Ca2+ from intracellular storage sites

Figure 1.2 Hydrolytic cleavage of phosphatidylinositol

Thus, any mechanistic effect leads to changes in the concentration of Ca2+ ions to evoke

a cellular response in which Ca2+ ions are important in many cellular processes In general, the intracellular and extracellular Ca2+ concentrations are maintained to a certain concentration at the level of 10-7 and 10-3 respectively, and are effectively balanced via simple influx and efflux pumps.12

4

1.2 Inositol Structure

Inositols are cyclohexol rings with hydroxyl groups arranged in different spatial

arrangements There are nine possible isomeric inositols with the prefixes scyllo-, myo-,

neo-, epi-, cis-, muco-, allo-, D-chiro-(+)- and L-chiro-(-) The name of myo-inositol

was designated to the first isomer discovered by Scherer in muscle tissue in 1850 It has

a single axial hydroxyl group (arbitrarily assigned as C2 position) while the remaining five hydroxyl groups are all equatorial (Fig 1.3)

Figure 1.3 Structure of myo-inositol molecule and arbitrary numbering

There is a plane of symmetry along the C2 and C5 positions thus resulting in

myo-inositol being a meso-compound As a result, C1 and C3, and C4 and C6, are enantiotopic to each other.11 The naming and numbering of the myo-inositol derivatives

follows an arbitrary system By convention, if the numbering follows an anticlockwise direction, the derivative is assigned as prefix (D) while aclockwise numbering direction will lead to the derivative being assigned as (L) prefix The absolute configuration has

to be determined by other characterization methods such as X-ray crystallography and

optical rotation

5

1.3 Inositol Lipid Biosynthesis and Transport

The biosynthesis of inositol lipids is highly conserved amongst both prokaryotes and eukaryotes, highlighting the importance of the pathway Phosphatidylinositol (PI) forms the precursor to all higher inositol lipids Mostly all species possess the ability to

synthesize L-myo-inositol-1-phosphate from glucose-6-phosphate precursors (Fig 1.4), which are dephosphorylated to give myo-inositol Cytidine-diphosphate-diacylglycerol (CDP-DAG) undergoes head group exchange with myo-inositol to form

phosphatidylinositol (PtdIns, PI) with the release of cytidine-monophosphate (CMP).7,13

Figure 1.4 Overview of lipid biosynthesis in Yeast, Mammalian, and Mycobacteria

Phosphatidylinositol has been identified in many prokaryotic and all eukaryotic cells The free hydroxyl groups of PtdIns are phosphorylated to give various phosphoinositides (PIs) in yeast and higher eukaryotic cells Higher phosphorylated and

organelle membranes The head group of phosphatidylinositol is exchanged with

ceramides in yeast and certain protozoa to give rise to sphingolipids

Inositolphosphoceramide (IPC) is synthesized in yeast through the action of IPC synthase, which is mannosylated to form mannosyl-inositolphosphoceramide (MIPC) and it undergoes a headgroup exchange with another PtdIns to give mannosyl diinositol-phosphoceramide (M(IP)2C) Inositol ceramides in yeast have been shown to

be important for growth, heat stress responses and endocytosis.14 The incorporation of inositol analogues into lipids requires an efficient inositol transport mechanism from the external medium Inositol specific transporters encoded by genes ITR1 and ITR2 have

been identified in yeast species such as S cerevisiae, S pombe, C albicans and

others.15 The SLC216 and SLC517 family of hexose transporters in mammalian cells have H+-myo-inositol transporters (HMIT) and Na+-myo inositol transporters (SMIT),

respectively, which are involved in ion-exchange at the membrane and also in inositol transport

The presence of inositol in most bacteria is limited and an inositol transport mechanism

is not known However, mycobacterial species have a wide range of inositol lipids such

as PtdIns, PIM and LAM They are also known to possess a mechanism of transport

that varies between the different species with inositol auxotrophic mutants of M

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smegmatis requiring lesser concentration of inositol in the media than M tuberculosis.15a, 18 The importance of inositol lipids is such that the absence of PtdIns, PIs, GPIs or inositol sphingolipids (in yeasts) renders the cells unviable.19 The question

of why the molecule inositol is able to play such crucial roles in cell growth, maintenance and function is possibly answered by the structure and stereochemistry of inositol, which enables its modification through multiple phosphorylation, lipidation and glycosylation events

Glycosylphosphatidylinositol (GPI) anchors form essential parts of the plasma

membrane of yeasts, eukaryotes and that of protozoans such as Leishmania and

Trypanosomas and are also observed in free form within cells GPIs catalyze various

extracellular enzymatic reactions and anchored proteins such as Variant Surface

Glycoprotein (VSG) in Trypanosomas is essential for their infectious cycle.4c

1.4 Inositol Glycolipids and Its Biological Significance

1.4.1 Chemical Structures of GPIs

Malaria is one of the three most devastating infectious diseases along with tuberculosis and AIDs Around 300-500 million of the world population suffer from the disease every year causing over 1.2 to 3 million deaths.20 In 1902, Sir Ronaldo Ross discovered that the malaria parasite was transmitted into the human bloodstream through the bite of

an infected female anopheles mosquito There are four major protozoan plasmodium species responsible for malaria in humans, namely Plasmodium falciparum,

Plasmodium vivax, Plasmodium malariae and Plasmodium ovale.21 Out of these,

Plasmodium falciparum is the most lethal species and causes the majority of deaths.22

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This parasite generates specific proteins that are transported to and are embedded in cell

membrane of infected erythrocytes through GPI hosts Hence, in Plasmodium

falciparum, several functionally important cell surface proteins, which are significantly

involved in parasite invasion and survival, are so called GPI anchored Since the biosyntheses of GPIs are essential for cell surface communications of these functional proteins to survive, inhibition of GPI bio-synthesis may prevent the propagation of parasites.23 This provides a general guideline for the development of various GPI-related drugs and vaccine candidates24 because GPI-anchors of Plasmodium falciparum

are apparently crucial for malaria.25 Additionally, in higher eukaryotes, the GPI moiety

is encoded with several functionally diverse proteins that play key roles in signal transduction, immuno-pathogenesis and cell-to-cell contact behaviour, for example, within the blood and connective tissues.26 To date, more than 250 GPI-anchored proteins having a different range of amino acid residue have been identified The glycosyl phosphatidyl-inositol(GPI) moiety, for example, is a glycolipid that is found in all eukaryotic membranes and serves to anchor the C-terminal end of a long list of surface proteins involved in signaling, cell-cell communications and interactions with extracellular molecules in the blood or connective tissue.27

Although postulated by Golgi in 1896, the first GPI structure was characterized by

Ferguson et al in 1988 from the parasite Trypanosoma brucei, which can remarkably

survive in blood due to it being densely covered with GPI anchored proteins and variant surface glyco-proteins (VSG).4c Structurally the GPI anchor is composed of several domains (Fig 1.5): a phosphoethanolamine (PEtN) for protein-attachment, a variably glycosylated poly-mannoside (glycan) attached with D-glucosamine (GluN) residue and

a phosphatidylinositol (PI) lipid (diacyl glycerol) base for membrane-binding.28 The

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structure of GPI anchor can differ significantly from species to species, and can bear an additional or more diversified glycan and lipid branches at various positions

Figure 1.5 The structural domains of protein-bound GPI anchor of P falciparum.28c

1.4.2 Structural Diversity of GPI

Although identified in a wide variety of mammalian tissues, the existence of GPIs in different species including many protozoan parasites can be categorized as to the occurrence of GPIs in prokaryotes, bacteria or algae.4c, 29 The structural diversity of eukaryotic GPI anchors is largely due to the location and nature of branching groups from the glycan residue (Fig 1.6) Additional ethanolamine phosphates (R1) (i.e.,Thy-1 GPI anchor in rat brain)30 appear to be a common feature for higher eukaryotes Most of the structures of GPI anchors contain a diacylglycerol moiety in the lipid residue, (i.e.,

sn-1,2-dimyristoylglycerol in Trypanosoma brucei VSG),31 but alkylacylglycerol lipid

residues are also common (i.e., sn-1-alkyl-2-acylgylcerol in human AChE 32 or rat brain Thy-133) In addition, ceramide-based lipid structures have also been found (i.e., in

Saccharomyces cerevisiae 34) at the lipid branches Human CD52 antigen,35

GPI-10

anchored glycopeptide antigen posses an additional fatty acid attached to the C2

position of inositol.A slight structural difference in the GPI of Toxoplasma gondii (T

gondii)36 has been identified in which a supplementary galactosyl component is attached to the C4 position of the third mannose moiety at the glycan branches GPI glycolipids can be classified into two types of coreglycans14: glycan-A37 contains α(1-

4) GalNAc-linkage to the core mannose adjacent to the non acetylated glucosamine and glycan-B38 contains a novel GlcR(1-4)GalNAc side branch As can be seen in Figure 1.6 each box represents a region of known diversity The location of several domains and nature of the specific branching of several GPIs are depicted in different colours.39

Figure 1.6 Molecular overview of glycan and lipid structural diversity of GPIs28c

1.4.3 General Structure of PIMs

Tuberculosis is a deadly infectious disease due to the transmission of pathogenic

bacteria, Mycobacterium tuberculosis causes around 2 million deaths every year The

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cell wall of M tuberculosis is covered by a ‘glycocalyx’, which is mainly composed of

mycolyl arabinogalactan-peptidoglycan complex (mAGP) and lipoarabinomannan (LAM).40 LAMs contain a domain of phosphatidyl myo-inositol residue that is extended

by adding successive mannose units to form phosphatidyl myo-inositol mono, di, tri and tetramannosides (PIM-1, PIM-2, PIM-3, PIM-4) and can be further extended to corresponding pentamannosides and hexamannosides (PIM-5, PIM-6) by successive α(1-2) mannoside linkages (Fig 1.7) PIMs predominate in the cell-walls of

mycobacteria in the form of PIM-2 and PIM-6, which are the main biosynthetic

precursors of lipomannans (LMs) and lipoarabinomannan (LAMs).4d

Figure 1.7 Structural domains of PIMs

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1.5 Targeting Inositol Lipids

Inositol lipids have been biologically profiled using radiolabelled and synthetic inositol analogues, as well as through the identification of associated proteins Some of these methods are described below

1.5.1 Metabolic Labelling Using Inositol Isotopes and Modified Analogues

Radioisotope labelling of inositol lipids has been developed to study the inositol lipid content and concentration in cells through the labelling of [3H]-myo-inositol and [14C]-

myo-inositol The different classes of inositol lipids have been identified based on their

migration against silica gel plates Scintillation counting also enables the lipid quantification of radiolabeled lipids.41 Nevertheless, the handling difficulties associated with radioactive isotopes and the practical difficulties in their use in analytical

instruments has limited the scope Deuterated and 13-carbon isotopes of myo-inositol

upon incorporation into inositol lipids result in heavier forms of the lipids, which can then be analyzed through mass spectrometry.42

Although, the introduction of modified inositol analogues can greatly inhibit inositol lipid biosynthic pathways and cell growth,43 uptake and incorporation of inositol analogues can be achieved into inositol lipids, especially PtdIns.6b, 44 Further studies, however, have not been reported with regard to the incorporation of modified inositol analogues into PIs, GPIs or inositol phosphoceramides