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 Synthetic Studies of Azido-Inositols and Inositol-based Glycans 2012 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) i Synthetic Studies of Azido-Inositols and Inositol-based Glycans 2012 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 ii Synthetic Studies of Azido-Inositols and Inositol-based Glycans 2012 Table of Contents: Research Summary ix List of Figures: xi Lists of Schemes: xiii List of Tables: .xxii List of Abbreviations: xxiii Introduction 1.1 Phosphatidyl Inositol and Its Biological Significance 1.2 Inositol Structure 1.3 Inositol Lipid Biosynthesis and Transport 1.4 Inositol Glycolipids and Its Biological Significance 1.4.1 Chemical Structures of GPIs 1.4.2 Structural Diversity of GPI 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 iii Synthetic Studies of Azido-Inositols and Inositol-based Glycans 2012 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 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 iv Synthetic Studies of Azido-Inositols and Inositol-based Glycans 2012 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 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 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 v Synthetic Studies of Azido-Inositols and Inositol-based Glycans 2012 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 4126 128 vi Synthetic Studies of Azido-Inositols and Inositol-based Glycans 2012 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 186 Experimental Procedures for Chapter 3: 187 Appendix 2: NMR Spectra of the Selected Compounds in Chapter 195 vii Synthetic Studies of Azido-Inositols and Inositol-based Glycans 2012 Experimental procedure for Chapter 196 Appendix 3: NMR Spectra of the Selected Compounds in Chapter 260 References 261 viii Synthetic Studies of Azido-Inositols and Inositol-based Glycans 2012 2-Deoxy-2-azido-3,6-di-O-benzyl-4-allyl-α-D-glucopyranosyl-(1→6)-2-O-tertbutyldimethylsilyl-4-O-benzyl-D-myo-inositol-1,3,5-orthoformate 4-179D: The C-4 inositol hydroxyl group of the compound 4-178D (50 mg, 0.07 mmol) was benzylated according to the general benzylation procedure to obtain 2-deoxy-2-azido-3,6-di-O-benzyl-4-allyl-αD-glucopyranosyl-(1→6)-2-O-tert-butyldimethylsilyl-4-Obenzyl-D-myo-inositol-1,3,5-orthoformate 4-179D (54 mg, 96%) as colourless viscous liquid 1H NMR (500 MHz, Chloroform-d) δ 7.50 – 7.08 (m, 15H), 5.81 (ddt, J = 16.2, 10.8, 5.6 Hz, 1H), 5.54 (s, 1H), 5.25 – 5.06 (m, 2H), 4.93 (d, J = 3.6 Hz, 1H), 4.72 – 4.56 (m, 3H), 4.55 – 4.41 (m, 4H), 4.39 – 4.29 (m, 3H), 4.23 – 4.11 (m, 3H), 3.96 (td, J = 11.5, 10.7, 6.2 Hz, 2H), 3.71 – 3.56 (m, 3H), 3.49 (t, J = 9.4 Hz, 1H), 3.37 (dd, J = 10.1, 3.6 Hz, 1H), 0.98 (s, 9H), 0.19 (s, 3H), 0.19 (s, 3H) 13C NMR (126 MHz, CDCl3) δ 137.84, 137.72, 137.45, 134.58, 128.54, 128.41, 128.37, 127.93, 127.88, 127.83, 127.78, 127.63, 116.61, 103.06, 98.05, 79.82, 77.97, 75.02, 74.42, 73.67, 73.59, 73.31, 73.01, 72.74, 72.01, 71.60, 69.17, 68.09, 63.11, 61.36, 25.90, 18.34, -4.70, -4.83 2-Deoxy-2-azido-3,6-di-O-benzyl-4-allyl-α-D-glucopyranosyl-(1→6)-2-O-tertbutyldimethylsilyl-4-O-benzyl-3,5-ethylidene-D-myo-inositol 4-180D: To an ice cooled solution of the compound 4-179D (40 mg, 0.049 mmol) in anhydrous DCM (2 ml) under argon was added [2M] AlMe3 (75 μL, eq.) at 0o C and the reaction was allowed to stir at room temperature for h After completion, the reaction was quenched by slow addition of aqueous sodium potassium tartrate, followed by addition of aqueous ammonium chloride and stirred for another h The reaction was diluted with DCM and washed with brine solution The organic 249 Synthetic Studies of Azido-Inositols and Inositol-based Glycans 2012 layer was dried over Na2SO4 and evaporated to dryness The crude mixture was purified by silica gel column chromatography (gradient 20% EA in hexane) to afford the desired product 2-deoxy-2-azido-3,6-di-O-benzyl-4-allyl-α-D-glucopyranosyl-(1→6)-2-O-tertbutyldimethylsilyl-4-O-benzyl-3,5-ethylidene-D-myo-inositol 4-180D (28 mg, 68%) as colourless viscous liquid 1H NMR (500 MHz, Chloroform-d) δ 7.45 – 7.10 (m, 15H), 5.82 (ddt, J = 16.1, 10.7, 5.5 Hz, 1H), 5.44 (q, J = 4.8 Hz, 1H), 5.26 – 5.05 (m, 2H), 5.00 (d, J = 3.4 Hz, 1H), 4.72 (d, J = 11.7 Hz, 1H), 4.67 (t, J = 4.6 Hz, 1H), 4.64 (d, J = 12.0 Hz, 1H), 4.57 – 4.49 (m, 3H), 4.44 – 4.37 (m, 3H), 4.30 (d, J = 10.7 Hz, 1H), 4.24 – 4.14 (m, 2H), 4.12 – 4.05 (m, 1H), 3.99 (dd, J = 12.3, 5.5 Hz, 1H), 3.85 (dd, J = 9.7, 2.7 Hz, 1H), 3.69 (qd, J = 11.0, 2.8 Hz, 2H), 3.60 – 3.47 (m, 2H), 3.40 (d, J = 6.0 Hz, 1H), 3.22 (dd, J = 10.0, 3.7 Hz, 1H), 1.25 (d, J = 4.8 Hz, 3H), 0.97 (s, 9H), 0.18 (s, 3H), 0.17 (s, 3H) 13 C NMR (126 MHz, CDCl3) δ 138.06, 137.95, 137.41, 134.69, 128.50, 128.36, 128.33, 127.85, 127.80, 127.78, 127.67, 127.13, 116.54, 98.25, 90.12, 80.15, 79.59, 78.08, 74.77, 74.62, 73.65, 72.08, 71.82, 71.72, 69.95, 68.67, 68.28, 66.97, 62.99, 25.87, 21.57, 18.30, -4.71, -4.84 MS (ESI): m/z calcd for C44H59N3NaO10Si 840.38 [M+Na]+; found 840.3 2-Deoxy-2-azido-3,6-di-O-benzyl-4-allyl-α-D-glucopyranosyl-(1→6)-1-O-benzoyl-2O-tert-butyldimethylsilyl-4-O-benzyl-3,5-ethylidene-D-myo-inositol 4-186D: To a solution of the compound 4-180D (20 mg, 0.024 mmol) in pyridine (1 ml) was added benzoyl chloride (10 μL, 3.5 eq.) and the reaction was stirred at room temperature for 10 h The solvents were removed under vacuum and the crude mixture was purified by silica gel column chromatography (20% EA in hexane) to give 2-deoxy-2-azido-3,6-di-O-benzyl-4-allyl-α-D-glucopyranosyl-(1→6)-1250 Synthetic Studies of Azido-Inositols and Inositol-based Glycans O-benzoyl-2-O-tert-butyldimethylsilyl-4-O-benzyl-3,5-ethylidene-D-myo-inositol 2012 4- 186D (20 mg, 90%) as white semisolids 1H NMR (500 MHz, Chloroform-d) δ 8.22 – 7.93 (m, 4H), 7.65 – 7.18 (m, 20H), 5.82 (ddt, J = 16.2, 10.5, 5.4 Hz, 1H), 5.49 (q, J = 4.7 Hz, 1H), 5.41 (d, J = 3.4 Hz, 2H), 5.21 – 5.00 (m, 2H), 4.82 – 4.69 (m, 3H), 4.60 (dd, J = 31.8, 11.9 Hz, 2H), 4.53 – 4.43 (m, 2H), 4.40 (d, J = 10.8 Hz, 2H), 4.34 – 4.22 (m, 2H), 4.20 (dd, J = 12.6, 5.6 Hz, 1H), 4.00 (dd, J = 12.4, 5.6 Hz, 1H), 3.83 (dd, J = 9.7, 3.1 Hz, 1H), 3.76 – 3.64 (m, 2H), 3.56 (dq, J = 18.5, 9.1 Hz, 2H), 3.34 (dd, J = 9.9, 3.5 Hz, 1H), 1.22 (d, J = 4.8 Hz, 3H), 0.85 (s, 9H), 0.14 (s, 3H), 0.04 (s, 3H) Allyl-6-O-tert-butyldimethylsilyl-α-D-mannopyranose 4-203: To a solution of the compound 4-66 (665 mg, 3.022 mmol) in DMF (10 ml) was added TBSCl (545 mg, 1.2 eq.), subsequently imidazole (513 mg, 2.5 eq.) and the reaction was stirred at room temperature for 12 h The reaction was diluted with water and extracted with ethyl acetate The organic layer was dried over Na2SO4 and evaporated to dryness The crude mixture was purified by silica gel column chromatography to obtain the desired allyl-6-O-tert-butyldimethylsilyl-α-D- mannopyranose 4-203 as crystalline solids (686 mg, 68%) 1H NMR (500 MHz, Chloroform-d) δ 5.88 (ddt, J = 16.7, 11.0, 5.7 Hz, 1H), 5.42 – 4.98 (m, 3H), 4.83 (d, J = 1.6 Hz, 1H), 4.27 – 4.09 (m, 1H), 4.01 – 3.93 (m, 1H), 3.93 – 3.80 (m, 4H), 3.73 (t, J = 9.4 Hz, 1H), 3.60 (dt, J = 10.1, 5.2 Hz, 2H), 0.90 (s, 9H), 0.09 (s, 6H) 13C NMR (126 MHz, CDCl3) δ 133.69, 117.48, 98.79, 71.76, 71.28, 70.49, 69.99, 67.91, 64.48, 25.86, 18.24, -5.41, -5.44 251 Synthetic Studies of Azido-Inositols and Inositol-based Glycans Allyl-2,3,4-tri-O-benzyl-6-O-tert-butyldimethylsilyl-α-D-mannopyranose 2012 4-204: The triol 4-203 (590 mg, 1.77 mmol) was benzylated according to the general benzylation procedure to obtain allyl-2,3,4-tri-O-benzyl-6-Otert-butyldimethylsilyl-α-D-mannopyranose 4-204 (928 mg, 87%) as a colourless oil H NMR (500 MHz, Chloroform-d) δ 7.51 – 7.16 (m, 15H), 5.88 (ddt, J = 16.4, 10.7, 5.5 Hz, 1H), 5.39 – 5.04 (m, 2H), 5.00 – 4.87 (m, 2H), 4.82 – 4.62 (m, 6H), 4.28 – 4.11 (m, 1H), 4.04 – 3.76 (m, 7H), 3.66 (ddd, J = 7.2, 4.3, 2.2 Hz, 1H), 0.94 (s, 9H), 0.11 (s, 3H), 0.10 (s, 3H) 13 C NMR (126 MHz, CDCl3) δ 138.72, 138.63, 138.48, 133.91, 128.31, 128.29, 128.23, 127.96, 127.67, 127.63, 127.54, 127.46, 117.05, 96.83, 80.28, 75.12, 75.08, 74.97, 73.32, 72.54, 72.18, 67.51, 62.79, 25.90, 18.28, -5.15, -5.31 Allyl-2,3,4-tri-O-benzyl-α-D-mannopyranose 4-205: To a solution of the compound 4-204 (745 mg, 1.233 mmol) in anhydrous THF (6 ml) was added [1M] TBAF in THF (1.47 ml, 1.2 eq.) and the reaction was stirred at room temperature for h After completion, the reaction was concentrated by rotary evaporator and the crude mixture was purified by silica gel column chromatography to obtain allyl-2,3,4-tri-O-benzyl-α-D-mannopyranose 4-205 as white semisolids13 (574 mg, 95%) 1H NMR (500 MHz, Chloroform-d) δ 7.51 – 7.18 (m, 15H), 5.88 (ddt, J = 16.5, 10.7, 5.5 Hz, 1H), 5.37 – 5.11 (m, 2H), 4.99 (d, J = 10.8 Hz, 1H), 4.91 (d, J = 2.1 Hz, 1H), 4.83 (d, J = 12.2 Hz, 1H), 4.76 – 4.68 (m, 4H), 4.21 – 4.11 (m, 1H), 4.09 – 3.93 (m, 3H), 3.91 – 3.80 (m, 3H), 3.72 (dt, J = 8.0, 3.8 Hz, 1H) 13C NMR (126 MHz, CDCl3) δ 139.11, 139.04, 138.88, 134.27, 129.01, 128.99, 128.67, 128.46, 128.32, 128.22, 128.18, 117.94, 98.05, 80.83, 75.84, 75.53, 75.52, 73.58, 72.96, 72.89, 68.49, 62.92 252 Synthetic Studies of Azido-Inositols and Inositol-based Glycans 2012 3,4-Di-O-benzyl-6-O-tert-butyldimethylsilyl-1,2-O-(α-methoxybenzylidene)-β-Dmannopyranose 4-202: To a solution of the compound 4-79 (300 mg, 1.006 mmol) in DMF (5 ml) was added TBSCl (181 mg, 1.2 eq.), subsequently imidazole (171 mg, 2.5 eq.) and the reaction was stirred at room temperature for 12 h The reaction was diluted with water and extracted with ethyl acetate The organic layer was dried over Na2SO4 and evaporated to dryness The crude mixture was purified by silica gel column chromatography to obtain the desired 4-205 (330 mg, 80%) as crystalline solids 1H NMR (500 MHz, Chloroform-d) δ 7.61 (dd, J = 6.8, 3.1 Hz, 2H), 7.43 – 7.29 (m, 3H), 5.59 (d, J = 2.6 Hz, 1H), 4.86 – 4.60 (m, 1H), 3.93 – 3.72 (m, 3H), 3.61 (dd, J = 10.3, 6.6 Hz, 1H), 3.35 (ddd, J = 8.8, 6.7, 5.0 Hz, 1H), 3.30 (d, J = 1.6 Hz, 1H), 3.27 (s, 3H), 2.60 (s, 1H), 1.63 (s, 1H), 0.85 (s, 9H), 0.02 (s, 3H), -0.00 (s, 3H) 13 C NMR (126 MHz, CDCl3) δ 129.10, 128.04, 126.35, 77.65, 73.74, 71.94, 70.53, 64.94, 51.22, 25.78, 18.15, -5.63, -5.66 The diol 4-205 (280 mg, 0.679 mmol) was benzylated according to the general benzylation procedure to obtain 3,4-di-O-benzyl-6-O-tert-butyldimethylsilyl-1,2-O-(αmethoxybenzylidene)-β-D-mannopyranose 4-202 (362 mg, 90%) 1H NMR (500 MHz, Chloroform-d) δ 7.69 (dd, J = 6.7, 2.7 Hz, 2H), 7.53 – 7.05 (m, 13H), 5.48 (d, J = 3.1 Hz, 1H), 4.91 – 4.79 (m, 3H), 4.74 – 4.65 (m, 2H), 3.92 – 3.83 (m, 2H), 3.80 – 3.67 (m, 2H), 3.36 (dt, J = 7.5, 3.6 Hz, 1H), 3.24 (s, 3H), 0.83 (s, 9H), -0.04 (s, 3H), -0.10 (s, 3H) 13 C NMR (126 MHz, CDCl3) δ 138.52, 137.96, 136.31, 128.98, 128.46, 128.34, 127.98, 127.92, 127.87, 127.63, 126.75, 122.27, 97.94, 78.47, 76.12, 76.00, 74.75, 74.50, 72.08, 62.72, 51.36, 25.92, 18.27, -5.43, -5.53 253 Synthetic Studies of Azido-Inositols and Inositol-based Glycans 2012 Allyl-(2,3,4-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-2-O-benzoyl-3,4,6-tri-Obenzyl-α-D-mannopyranoside 4-206: The 2,3,4-tri-O-benzyl-α-Dmannopyranose acceptor 4-201 (347 mg, 0.708 mmol) and the 3,4,6-tri-O-benzyl-1,2-O-(α-methoxybenzylidene)-β-Dmannopyranose donor 4-80 (522 mg, 0.92 mmol, 1.3 eq.) were dried together by azeotropic distillation with anhydrous toluene (3x5 mL) and kept under an argon atmosphere Activated 4Å MS were added under the flash of argon and the starting materials were dissolved in dichloromethane (10 ml) and dibutylphosphate (337 μL, 1.77 mmol, 1.1 eq.) was added to the solution The reaction mixture was stirred at room temperature for h to convert into glycosyl phosphate 4-97 completely (TLC monitoring) Then the reaction mixture was cooled to –30° C, subsequently treated with TMSOTf (256 μL, 1.416 mmol, eq.) and the reaction was stirred for additional h After complete consumption of the alcohol, the reaction was quenched with Et3N (1 ml) and was filtered through celite pad The filtrate was concentrated under vacuum and the residue was purified by silica gel column chromatography (gradient 15% EtOAc/hexanes) to give 4-206 (705 mg, 97%) as a transparent viscous colorless liquid 1H NMR (500 MHz, Chloroform-d) δ 8.14 (d, J = 7.7 Hz, 2H), 7.59 (t, J = 7.4 Hz, 1H), 7.51 – 7.15 (m, 35H), 5.88 (ddt, J = 16.4, 10.7, 5.6 Hz, 1H), 5.84 – 5.78 (m, 1H), 5.27 (dq, J = 17.4, 1.4 Hz, 1H), 5.21 – 5.17 (m, 2H), 5.16 (d, J = 1.9 Hz, 1H), 5.01 – 4.92 (m, 3H), 4.81 (s, 3H), 4.77 (d, J = 11.1 Hz, 2H), 4.69 (s, 2H), 4.61 – 4.50 (m, 4H), 4.20 – 4.13 (m, 3H), 4.04 – 3.72 (m, 11H) 13C NMR (126 MHz, CDCl3) δ 165.47, 138.62, 138.52, 138.45, 138.30, 137.90, 133.65, 132.96, 130.04, 129.96, 128.37, 128.32, 128.29, 128.27, 128.23, 128.16, 128.16, 127.88, 127.82, 127.69, 127.65, 127.61, 127.54, 127.52, 127.48, 127.41, 127.37, 117.43, 98.04, 96.79, 80.28, 77.66, 75.09, 75.02, 74.71, 74.64, 74.20, 73.33, 72.67, 72.04, 71.61, 71.26, 71.08, 254 Synthetic Studies of Azido-Inositols and Inositol-based Glycans 2012 68.97, 68.71, 67.76, 66.66 MS (ESI): m/z calcd for C64H66NaO12 1049.44 [M+Na]+; found 1050.42 Allyl-(2,3,4-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-3,4,6-tri-O-benzyl-α-Dmannopyranoside 4-207: To a solution of the compound 4-206 (705 mg, 0.686 mmol) in ml of DCM/MeOH (1:1) was added K2CO3 (189 mg, 1.37 mmol, eq.) and the solution was stirred for h After completion, the solids were filtered and the filtrate was concentrated The crude residue was purified by silica gel chromatography to afford allyl(2,3,4-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-3,4,6-tri-O-benzyl-α-Dmannopyranoside 4-207 (633 mg, 99%) 1H NMR (500 MHz, Chloroform-d) δ 7.47 – 7.24 (m, 28H), 7.23 – 7.14 (m, 2H), 5.87 (ddt, J = 16.8, 10.9, 5.6 Hz, 1H), 5.30 – 5.21 (m, 1H), 5.21 – 5.15 (m, 1H), 5.11 (d, J = 1.4 Hz, 1H), 4.95 (d, J = 11.1 Hz, 1H), 4.91 (d, J = 1.3 Hz, 1H), 4.86 (d, J = 10.9 Hz, 1H), 4.81 – 4.64 (m, 7H), 4.62 – 4.48 (m, 5H), 4.17 – 4.10 (m, 2H), 4.01 – 3.69 (m, 13H), 3.68 – 3.62 (m, 1H) 13C NMR (126 MHz, CDCl3) δ 138.53, 138.46, 138.31, 138.27, 137.85, 133.66, 128.46, 128.34, 128.33, 128.31, 128.26, 128.22, 127.94, 127.86, 127.80, 127.76, 127.72, 127.65, 127.57, 127.53, 127.48, 117.45, 99.59, 96.83, 80.22, 79.58, 75.05, 74.96, 74.63, 74.21, 73.35, 72.75, 72.10, 71.60, 71.49, 71.04, 68.83, 68.05, 67.79, 66.25 MS (ESI): m/z calcd for C57H62NaO11 945.42 [M+Na]+; found 945.48 2-O-Benzoyl-3,4-di-O-benzyl-6-O-tert-butyldimethylsilyl-α-D-mannopyranosyl trichloroacetimidate 4-211: To a cooled solution (0o C) of the orthoester 4-202 (200 mg, 0.338 mmol) in ml of acetone/H2O (5:1) was added AcOH (40 μL) and the reaction was allowed to stir at 0o C for h After completion, the reaction was diluted with ethyl acetate and washed 255 Synthetic Studies of Azido-Inositols and Inositol-based Glycans 2012 with brine solution The organic solvents was dried over Na2SO4 and concentrated The crude residue was purified by silica gel chromatography (20% EA in hexane) to give 2O-benzoyl-3,4-di-O-benzyl-6-O-tert-butyldimethylsilyl-α-D-mannopyranose 4-210 (172 mg) in 88% yield 1H NMR (500 MHz, Chloroform-d) δ 8.25 – 7.91 (m, 2H), 7.72 – 7.14 (m, 13H), 5.72 – 5.49 ( m, 1H), 5.32 (s, 1H), 4.93 (d, J = 10.9 Hz, 1H), 4.80 (d, J = 11.4 Hz, 1H), 4.67 (d, J = 10.9 Hz, 1H), 4.61 (d, J = 11.3 Hz, 1H), 4.17 (dd, J = 9.0, 3.2 Hz, 1H), 4.06 – 3.92 (m, 3H), 3.87 (d, J = 9.7 Hz, 1H), 3.72 – 3.43 (m, 1H), 0.97 (s, 9H), 0.12 (d, J = 2.3 Hz, 6H) 13C NMR (126 MHz, CDCl3) δ 138.59, 138.07, 133.10, 129.98, 129.95, 128.31, 128.26, 128.00, 127.95, 127.59, 127.54, 92.64, 77.75, 75.15, 74.15, 72.63, 71.61, 69.64, 62.47, 25.98, 18.41, -5.21, -5.38 The anomeric alcohol 4-210 (152 mg, 0.262 mmol) was converted to its corresponding 2-O-benzoyl-3,4-di-O-benzyl-6-O-tert-butyldimethylsilyl-α-D-mannopyranosyl trichloroacetimidate 4-211 (173 mg, 91%) as a faintly yellow semisolid28 according to the general procedure for the preparation of Schmidt donor 1H NMR (500 MHz, Chloroform-d) δ 8.70 (s, 1H), 8.28 – 7.94 (m, 2H), 7.79 – 6.93 (m, 14H), 6.42 (d, J = 2.4 Hz, 1H), 5.77 (t, J = 2.6 Hz, 1H), 4.95 (d, J = 10.7 Hz, 1H), 4.84 (d, J = 11.4 Hz, 1H), 4.73 (d, J = 10.5 Hz, 1H), 4.66 (d, J = 11.7 Hz, 1H), 4.25 (d, J = 9.6 Hz, 1H), 4.23 – 4.11 (m, 2H), 4.09 – 4.02 (m, 1H), 3.97 – 3.83 (m, 2H), 0.98 (s, 10H), 0.13 (d, J = 4.4 Hz, 6H) 13 C NMR (126 MHz, CDCl3) δ 165.45, 160.02, 138.37, 137.63, 133.30, 130.06, 129.58, 128.37, 128.31, 128.24, 128.06, 127.74, 95.77, 77.39, 75.50, 75.28, 73.43, 71.91, 67.80, 61.60, 25.89, 18.25, -5.19, -5.41 256 Synthetic Studies of Azido-Inositols and Inositol-based Glycans Allyl 2012 (2,3,4-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-(3,4,6-tri-O-benzyl-α-D- mannopyranosyl)-(1→2)-2-O-benzoyl-3,4-di-O-benzyl-6-O-tert-butyldimethylsilylα-D-mannopyranoside 4-209: The coupling between the dimannoside alcohol 4-207 (150 mg, 0.164 mmol) and the Schmidt donor 4-211 (155 mg, 0.213 mmol, 1.3 eq.) using BF3.OEt2 (20 μL, 0.098 mmol, 0.6 eq.) was performed according to the general coupling procedure to obtain the desired allyl (2,3,4-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-(3,4,6-tri-O-benzyl-α-Dmannopyranosyl)-(1→2)-2-O-benzoyl-3,4-di-O-benzyl-6-O-tert-butyldimethylsilyl-αD-mannopyranoside 4-209 (210 mg, 87%) 1H NMR (500 MHz, Chloroform-d) δ 8.39 – 7.85 (m, 2H), 7.91 – 6.85 (m, 44H), 5.82 (ddt, J = 16.7, 11.0, 5.6 Hz, 1H), 5.77 (t, J = 2.0 Hz, 1H), 5.23 (d, J = 1.8 Hz, 1H), 5.20 (d, J = 2.1 Hz, 1H), 5.15 – 5.07 (m, 1H), 4.97 (d, J = 1.9 Hz, 1H), 4.94 – 4.84 (m, 4H), 4.77 – 4.40 (m, 14H), 4.18 – 3.58 (m, 19H), 0.96 (s, 9H), 0.10 (d, J = 7.5 Hz, 6H) 13 C NMR (126 MHz, CDCl3) δ 165.49, 138.90, 138.69, 138.57, 138.46, 138.25, 138.23, 138.17, 133.64, 132.96, 130.21, 130.01, 128.34, 128.31, 128.29, 128.25, 128.18, 128.16, 128.08, 128.00, 127.92, 127.89, 127.80, 127.77, 127.69, 127.64, 127.61, 127.56, 127.52, 127.49, 127.46, 127.41, 127.39, 127.31, 117.53, 99.48, 99.09, 96.86, 80.39, 79.31, 78.27, 75.11, 75.00, 74.98, 74.80, 74.65, 74.51, 74.34, 74.01, 73.18, 72.97, 72.70, 72.08, 71.83, 71.68, 71.57, 71.41, 69.34, 69.21, 67.71, 66.56, 62.10, 25.99, 18.34, -5.08, -5.34 MS (ESI): m/z calcd for C90H102NaO17Si 1506.63 [M+Na]+; found 1506.63 257 Synthetic Studies of Azido-Inositols and Inositol-based Glycans Allyl 2012 (2,3,4-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-(3,4,6-tri-O-benzyl-α-D- mannopyranosyl)-(1→2)-3,4-di-O-benzyl-6-O-tert-butyldimethylsilyl-α-Dmannopyranoside 4-212: To a solution of the compound 4-209 (160 mg, 0.107 mmol) in ml of DCM/MeOH (1:1) was added NaOMe (60 mg, 10 eq.) and the solution was stirred for 10 h After completion, the solids were filtered and the filtrate was concentrated The crude residue was purified by silica gel chromatography to afford allyl (2,3,4-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)(3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1→2)-3,4-di-O-benzyl-6-O-tertbutyldimethylsilyl-α-D-mannopyranoside 4-212 (140 mg, 95%) 1H NMR (500 MHz, Chloroform-d) δ 7.57 – 6.90 (m, 47H), 5.85 (ddt, J = 15.3, 10.8, 5.5 Hz, 1H), 5.24 (dd, J = 17.1, 1.8 Hz, 1H), 5.19 (d, J = 1.7 Hz, 1H), 5.18 – 5.15 (m, 1H), 5.00 (d, J = 2.0 Hz, 1H), 4.96 – 4.82 (m, 5H), 4.75 (s, 2H), 4.72 – 4.43 (m, 14H), 4.20 – 4.04 (m, 3H), 4.00 – 3.52 (m, 20H), 0.94 (s, 10H), 0.11 (d, J = 8.0 Hz, 6H) 13C NMR (126 MHz, CDCl3) δ 138.79, 138.73, 138.66, 138.50, 138.45, 138.26, 138.17, 133.64, 128.40, 128.34, 128.29, 128.26, 128.21, 128.17, 127.90, 127.88, 127.83, 127.79, 127.74, 127.65, 127.59, 127.55, 127.51, 127.45, 127.38, 127.27, 117.52, 100.81, 99.10, 96.85, 80.44, 79.92, 79.36, 74.98, 74.94, 74.90, 74.84, 74.68, 74.60, 74.10, 74.01, 73.17, 72.85, 72.77, 72.11, 72.09, 71.86, 71.55, 71.34, 69.19, 68.68, 67.68, 66.60, 62.48, 25.99, 18.35, -5.01, -5.28 258 Synthetic Studies of Azido-Inositols and Inositol-based Glycans Allyl 2012 (2,3,4-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-(3,4,6-tri-O-benzyl-α-D- mannopyranosyl)-(1→2)-(3,4-di-O-benzyl-6-O-tert-butyldimethylsilyl-α-Dmannopyranosyl)-(1→2)-2,3,4,6-tetra-O-benzyl-α-D-mannopyranoside 4-200: The coupling between the trimannoside alcohol 4-212 (40 mg, 0.029 mmol) and the Schmidt donor 4-35 (30 mg, 0.0435 mmol, 1.5 eq.) using BF3.OEt2 (10 μL) was performed according to the general coupling procedure to obtain the desired allyl (2,3,4-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)(3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1→2)-(3,4-di-Obenzyl-6-O-tert-butyldimethylsilyl-α-D-mannopyranosyl)-(1→2)-2,3,4,6-tetra-Obenzyl-α-D-mannopyranoside 4-200 (34 mg, 62%) 1H NMR (500 MHz, Chloroformd) δ 7.61 – 6.88 (m, 66H), 5.81 (dddd, J = 22.2, 16.8, 11.9, 6.7 Hz, 2H), 5.28 – 5.07 (m, 4H), 4.95 – 4.78 (m, 7H), 4.74 – 4.28 (m, 24H), 4.17 (t, J = 2.4 Hz, 1H), 4.14 – 4.01 (m, 3H), 3.94 – 3.61 (m, 23H), 3.56 (t, J = 10.6 Hz, 2H), 0.87 (s, 9H), 0.06 (s, 3H), 0.05 (s, 3H) 259 Synthetic Studies of Azido-Inositols and Inositol-based Glycans 2012 Appendix NMR Spectra: Chapter Appendix containing the NMR (1H & 13 C) spectra (pages 1-89) of the selected compounds in chapter was enclosed in a CD-ROM pasted at the end of this thesis copy 260 Synthetic Studies of Azido-Inositols and Inositol-based Glycans 2012 References (a) Vacca, J P.; Desolms, S J.; Huff, J R.; Billington, D C.; Baker, R.; Kulagowski, J J.; Mawer, I M., Tetrahedron 1989, 45, 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Tetrahedron Lett 2010, 51, 1117-1120 27 Lohman, G J S.; Seeberger, P H., J Org Chem 2004, 69, 4081-4093 28 Jayaprakash, K N.; Lu, J.; Fraser-Reid, B., Angew Chem Int Ed 2005, 44, 5894-5898 263 ... 13 1. 6 Design and Hypothesis of Inositol Analogues for Metabolic Engineering 14 1. 7 General Synthetic Outline 18 iii Synthetic Studies of Azido- Inositols and Inositol- based Glycans. .. Studies of Azido- Inositols and Inositol- based Glycans 2 012 Figure 3 .12 Incorporation of 5 -azido- inositol 1- 7b into phosphoinositides 82 Figure 3 .13 Incorporation of 3 -azido- inositol 1- 6b into... 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