PART 1: DEVELOPMENT TOWARDS A POTENTIAL ANTI-PERTUSSIS GLYCOCONJUGATE VACCINE; PART 2: BINDING AND MITIGATION OF CYTOTOXICITY OF AMYLOID BETA AND TAU OLIGOMERS BY HEPARIN By Peng Wang A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Chemistry-Doctor of Philosophy 2018 ABSTRACT PART 1: DEVELOPMENT TOWARDS A POTENTIAL ANTI-PERTUSSIS GLYCOCONJUGATE VACCINE; PART 2: BINDING AND MITIGATION OF CYTOTOXICITY OF AMYLOID BETA AND TAU OLIGOMERS BY HEPARIN By Peng Wang Despite massive vaccination, the world has been experiencing a resurgence of pertussis, a highly contagious respiratory disease. Current acellular vaccines lack sufficient bactericidal activity and there is an urgent need of better vaccines aiming at clearing the pathogen, Bordetella pertussis. Oligosaccharide on the surface of the bacteria has been proven to be a promising protective antigen that elicits antibody-mediated complement-dependent cytotoxicity against Bordetella pertussis. However, obtaining the saccharide on a large scale with high purify remains one of the main obstacles. Herein, we report the first total synthesis of a pentasaccharide antigenic determinant from Bordetella pertussis. Immunization of mice with a conjugate of the pentasaccharide with a carrier protein, bacteriophage Qβ, elicited high titers of IgG antibodies. An IgG subclass study showed that it induced Th1-weighted immune response. The antibodies were able to bind Bordetella pertussis in flow cytometry and induced complement-dependent cytotoxicity. Further study will be focused on the epitope mapping on the pentasaccharide and optimization of the antigen structure. Accumulation of amyloid β in the brain is believed to play a key role in the pathology of Alzheimer’s disease and it is one of the most important biomarkers in the early diagnosis of AD. Glycosaminoglycans have been found to participate in the process of Aβ aggregation. Herein, we report the study on the interaction between Aβ and superparamagnetic iron oxide nanoparticles coated with heparin, a member of the GAG family. The interaction between Aβ and nanoparticle was studied through enzyme-linked immunosorbent assay, gel electrophoresis and thioflavin T assay. Furthermore, the nanoparticle showed no toxicity against neurons and effectively protected neurons from Aβ, which made it a potential tool in the detection of Aβ in vivo. Neurofibrillary tangles formed by intracellular aggregation of tau proteins are another important hallmark of Alzheimer’s disease. However, recent studies have suggested that tau oligomers, rather than neurofibrillary tangles, are playing a key role in the progression of the disease. Glycosaminoglycans can mediate the intercellular propagation of tau proteins. Herein, we report the synthesis of heparin-like oligosaccharides with different lengths and sulfation patterns. Binding assays with tau oligomers revealed that longer backbones and higher sulfation degrees resulted in stronger binding affinity. The oligosaccharides promoted aggregation of tau oligomers and effectively protected SH-SY5Y cells against tau oligomers. Copyright by PENG WANG 2018 ACKNOWLEDGEMENT There are so many people that I must thank for their support along the way to the completion of my phD dissertation. The person that I have the most gratitude to is my research advisor, Prof. Xuefei Huang. I would never have accomplished such a huge task without his generous guidance and help. He has been a role model to me, not only because of his enthusiasm and dedication for scientific research, but also the way he gets along with research fellows and graduate students. Whenever I am encountered with difficult challenges and failures in my research, he has never failed to inspire me with his great patience and insightful suggestions. He consistently encourages me to think over the projects and develop new ideas, which has been a huge boost on my way of becoming a real scientist. I also would like to thank my committee members, Prof. Kevin Walker, Prof. William Wulff and Prof. Norbert Kaminski. Their valuable feedback and effective questions for my 2nd-year report and final dissertation have been of great help in realizing my weaknesses and inspired me to pursue the knowledge for a better career. Support from the research staff has also been vital in my research. I would like to thank Dr. Daniel Holmes for his training and tireless technical guidance in acquiring important NMR data. The mass spectrum facility team, including Prof. Daniel, Dr. Lijun Chen, Dr. Tony Schilmiller and Dr. Scott Smith, has also been so kind in teaching me how to run the high-resolution mass analysis. The Huang group has also been extremely important during my academic journey. I would like to express my gratitude to all previous group members, Dr. Zhaojun Yin, Dr. Bo Yang, Dr. Hovig Kouyoumdjian, Dr. Xiaowei Lu, Dr. Steven Dulaney, Dr. Herbert Kavunja and Dr. v Suttipun Sungsuwan, for all they have done for me, which has helped me greatly in adapting to the experiments and the new life in the United States. I also want to thank the current members, Qian, Weizhun, Xuanjun, Jicheng, Zeren, Mehdi, Sherif, Jia, Shuyao, Zibin, Changxin, Yuetao, Kedar, Tianlu, Zahra, Mengxia, Hunter, Setare, Shivangi, Kunli. As I said before, I would never have made it through the difficult times without your company and friendship over the years. It is also true for all my friends at MSU including Yubai, Xiaopeng, Yi, Jun, Wei, Xinliang, Hadi, Yukari, Xiaoxiao, Wenjing, Chengpeng, Yongle and many more people. Finally, I want to thank my family, especially my parents, for their unconditional support and love throughout my life. I am so excited to have a chance to go back to China and share my success with them, before I depart again from home and step onto the next stage in my career. Thank you, Peng Wang vi TABLE OF CONTENTS LIST OF TABLES .......................................................................................................................... x LIST OF FIGURES ....................................................................................................................... xi LIST OF SCHEMES................................................................................................................. xxvii KEY TO ABBREVIATIONS .................................................................................................. xxviii Chapter 1. A Review of Developing Vaccines against Bordetella pertussis .................................. 1 1.1. Introduction .......................................................................................................................... 1 1.2. Hypotheses for Resurgence of Pertussis .............................................................................. 4 1.3. Acellular Pertussis Vaccines ................................................................................................ 5 1.4. Pertussis Lipooligosaccharide .............................................................................................. 9 1.4.1. Structure Elucidation of Pertussis LOS ....................................................................... 10 1.4.2. Immunology of Pertussis LOS .................................................................................... 15 1.5. Pertussis LOS-based Glycoconjugate Vaccines ................................................................. 19 1.6. Future Outlook ................................................................................................................... 21 REFERENCES .......................................................................................................................... 23 Chapter 2. Synthesis and Immunological Evaluation of Pertussis Pentasaccharide Bearing Multiple Rare Sugars as Potential Anti-pertussis Vaccines .......................................................... 30 2.1. Retrosynthetic Analysis and Rare Sugar Building Block Preparation ............................... 30 2.2. Stereochemical Challenges in Formation of the C-D Linkage .......................................... 36 2.3. Assembly of Pertussis Pentasaccharide and Deprotection ................................................. 44 2.4. Bioconjugation of Pertussis-like Pentasaccharide with Bacteriophage Qβ Carrier ........... 46 2.5. Immunization Study ........................................................................................................... 47 2.6. Conclusions and Future Plans ............................................................................................ 51 2.7. Experimental Section ......................................................................................................... 52 2.7.1. Synthesis of the Glycan-Carrier Protein Conjugates ................................................... 52 2.7.2. Quantification of Glycan Number on the Carrier Protein ........................................... 52 2.7.3. Immunization Studies .................................................................................................. 54 2.7.4. Enzyme-linked Immunosorbent Assay (ELISA) ......................................................... 55 vii 2.7.5. Cell Culture of B. pertussis.......................................................................................... 56 2.7.6. Flow Cytometry Experiment ....................................................................................... 56 2.7.7. Complement Assay ...................................................................................................... 56 2.7.8. Product Preparation and Characterization Data ........................................................... 57 APPENDIX ............................................................................................................................. 109 REFERENCES ........................................................................................................................ 202 Chapter 3. Heparin Nanoparticles for β Amyloid Binding and Mitigation of β Amyloid Associated Cytotoxicity .............................................................................................................. 206 3.1. Introduction ...................................................................................................................... 206 3.2. Results and Discussion ..................................................................................................... 207 3.2.1. Preparation and Characterization of Hep-SPION. ..................................................... 207 3.2.2. Assessment of Binding between Aβ and Hep-SPION by ELISA. ............................ 209 3.2.3. Effect of Hep-SPION on Aβ Aggregation. ................................................................ 210 3.2.4. Effect of Hep-SPION on Aβ-Induced Cytotoxicity .................................................. 213 3.3. Conclusions ...................................................................................................................... 215 3.4. Experimental Section ....................................................................................................... 215 3.4.1. Materials and Instrumentation ................................................................................... 215 3.4.2. Synthesis of Hep-SPION ........................................................................................... 216 3.4.3. Transmission Electron Microscopy (TEM) Procedure .............................................. 218 3.4.4. Preparation of Aβ....................................................................................................... 218 3.4.5. Native-PAGE Gel Electrophoresis ............................................................................ 218 3.4.6. Thioflavin T Assay .................................................................................................... 219 3.4.7. ELISA Assay ............................................................................................................. 219 3.4.8. Cell Viability Assay................................................................................................... 220 REFERENCES ........................................................................................................................ 221 Chapter 4. Mitigation of Neurotoxicities of Toxic Tau Oligomers by Heparin Like Oligosaccharides ......................................................................................................................... 225 4.1. Introduction ...................................................................................................................... 225 4.2. Results and Discussion ..................................................................................................... 227 4.2.1. Preparation of Heparin Oligosaccharide Backbones ................................................. 227 4.2.2. Deprotection and Sulfation ........................................................................................ 230 viii 4.2.3. Binding Assay with Tau Oligomers .......................................................................... 233 4.2.4. Heparin Oligosaccharides Mitigates Cytotoxicity of Tau Oligomers (Done by Dr. Rakez Kayed lab) ................................................................................................................. 234 4.3. Conclusions ...................................................................................................................... 238 4.4. Experimental Section ....................................................................................................... 238 4.4.1. General Procedure for Preactivation Based Glycosylation. ...................................... 238 4.4.2. General Procedure for TBS Removal ........................................................................ 239 4.4.3. General Procedure for Removal of Levulinoyl Esters ............................................... 240 4.4.4. General Procedure for Oxidation of 6-OH ................................................................ 240 4.4.5. General Procedure for Methyl Ester Formation after Oxidation ............................... 240 4.4.6. General Procedure for Benzyl Ester Formation after Oxidation ............................... 241 4.4.7. General Procedure for Transesterification ................................................................. 241 4.4.8. General Procedure for 1, 3-Propanedithiol Mediated Azide Reduction .................... 241 4.4.9. General Procedure for Selective N-Sulfation ............................................................ 241 4.4.10. General Procedure for Simultaneous O, N-Sulfation .............................................. 242 4.4.11. General Procedure for Global Debenzylation ......................................................... 242 4.4.12. General Procedure for Methyl Ester Saponification ................................................ 242 4.4.13. Preparation of Tau Oligomers ................................................................................. 243 4.4.14. BLI Binding Assay of Heparin and Tau Oligomers ................................................ 243 4.4.15. Preparation of Tau Oligomers in the Presence of Heparin-like Oligosaccharides .. 244 4.4.16. Atomic Force Microscopy (AFM) ........................................................................... 244 4.4.17. Cell Toxicity Assays ................................................................................................ 244 4.4.18. Product Preparation and Characterization Data ....................................................... 245 APPENDIX ............................................................................................................................. 278 REFERENCES ........................................................................................................................ 331 ix LIST OF TABLES Table 2.1. Investigation of protecting group schemes for β-selective fucosylation. 39 Table 2.2. The relative energies and geometry of the most stable 20 conformers. 42 x LIST OF FIGURES Figure 1.1. Reported pertussis incidence (per 100,000 persons) by age group in the United States from 1990–2016. 1 Figure 1.2. The number of pertussis cases reported to CDC from 1922 to 2016. 3 Figure 1.3. Common structure of lipopolysaccharide of Gram-negative bacteria. 9 Figure 1.4. Structure of the major molecular species present in B. pertussis lipid A. 11 Figure 1.5. Proposed structure of the heptasaccharide present at the reducing end of LOS-1 isolated from B. pertussis endotoxin. 12 Figure 1.6. Structure of the trisaccharide present in the distal pentasaccharide from B. pertussis. Some short interproton distances that could be observed in the NOE experiments and used to determine the absolute configuration of the sugars are shown. 14 Figure 1.7. Complete structure of the LOS of B. pertussis strain 1414. 15 Figure 1.8. Structure and molecular models of B. pertussis BP1414 LOS. 18 Figure 1.9. Scheme of conjugation of B. pertussis and B. bronchiseptica core OS. 21 Figure 2.1. Structures of the pentasaccharide 1 cleaved by deamination and our synthetic target pentasaccharide 2. 31 Figure 2.2. Two stable conformers of the postulated oxocarbenium intermediate. 43 Figure 2.3. Immulogical evaluation of Qβ-glycan conjugate vaccine. 49 Figure 2.4. Flow cytometry indicated that the mouse antisera had no significant binding with the bacteria. Only whole-cell vaccine control sera had strong binding against B. pertussis. 50 Figure 2.5. Complement-dependent cytotoxic assay of mouse antisera. The bactericidal activity of mouse antisera was assessed by counting the colony-forming units of B. pertussis. 50 Figure 2.6. ESI-MS analysis of the Qβ-glycan conjugate 49. 53 Figure 2.7. MALDI-TOF MS analysis of the BSA-glycan conjugate 53. 54 xi Figure 2.8. 1H-NMR of 9 (500 MHz CDCl3). 110 Figure 2.9. 13C-NMR of 9 (125 MHz CDCl3). 110 Figure 2.10. 1H-1H gCOSY of 9 (500 MHz CDCl3). 111 Figure 2.11. 1H-13C gHSQCAD of 9 (500 MHz CDCl3 . 111 Figure 2.12. 1H-NMR of 10 (500 MHz CDCl3). 112 Figure 2.13. 13C-NMR of 10 (125 MHz CDCl3). 112 Figure 2.14. 1H-1H gCOSY of 10 (500 MHz CDCl3). 113 Figure 2.15. 1H-13C gHSQCAD of 10 (500 MHz CDCl3). 113 Figure 2.16. 1H-NMR of 6 (500 MHz CDCl3). 114 Figure 2.17. 13C-NMR of 6 (125 MHz CDCl3). 114 Figure 2.18. 1H-1H gCOSY of 6 (500 MHz CDCl3). 115 Figure 2.19. 1H-13C gHSQCAD of 6 (500 MHz CDCl3). 115 Figure 2.20. 1H-NMR of 11 (500 MHz CDCl3). 116 Figure 2.21. 13C-NMR of 11 (125 MHz CDCl3). 116 Figure 2.22. 1H-1H gCOSY of 11 (500 MHz CDCl3). 117 Figure 2.23. 1H-13C gHSQCAD of 11 (500 MHz CDCl3). 117 Figure 2.24. 1H-NMR of 12 (500 MHz CD3OD). 118 Figure 2.25. 13C-NMR of 12 (125 MHz CD3OD ). 118 Figure 2.26. 1H-1H gCOSY of 12 (500 MHz CD3OD). 119 Figure 2.27. 1H-13C gHSQCAD of 12 (500 MHz CD3OD). 119 Figure 2.28. 1H-NMR of 13 (500 MHz CDCl3). 120 xii Figure 2.29. 13C-NMR of 13 (125 MHz CDCl3). 120 Figure 2.30. 1H-1H gCOSY of 13 (500 MHz CDCl3). 121 Figure 2.31. 1H-13C gHSQCAD of 13 (500 MHz CDCl3). 121 Figure 2.32. 1H-NMR of 14 (500 MHz CDCl3). 122 Figure 2.33. 13C-NMR of 14 (125 MHz CDCl3). 122 Figure 2.34. 1H-1H gCOSY of 14 (500 MHz CDCl3). 123 Figure 2.35. 1H-13C gHSQCAD of 14 (500 MHz CDCl3). 123 Figure 2.36. 1H-NMR of 16 (500 MHz CDCl3). 124 Figure 2.37. 13C-NMR of 16 (125 MHz CDCl3). 124 Figure 2.38. 1H-1H gCOSY of 16 (500 MHz CDCl3). 125 Figure 2.39. 1H-13C gHSQCAD of 16 (500 MHz CDCl3). 125 Figure 2.40. 1H-NMR of 17 (500 MHz CDCl3). 126 Figure 2.41. 13C-NMR of 17 (125 MHz CDCl3). 126 Figure 2.42. 1H-1H gCOSY of 17 (500 MHz CDCl3). 127 Figure 2.43. 1H-13C gHSQCAD of 17 (500 MHz CDCl3). 127 Figure 2.44. 1H-NMR of 15 (500 MHz CDCl3). 128 Figure 2.45. 13C-NMR of 15 (125 MHz CDCl3). 128 Figure 2.46. 1H-1H gCOSY of 15 (500 MHz CDCl3). 129 Figure 2.47. 1H-13C gHSQCAD of 15 (500 MHz CDCl3). 129 Figure 2.48. 1H-NMR of 18 (500 MHz CD3OD). 130 Figure 2.49. 13C-NMR of 18 (125 MHz CD3OD). 130 xiii Figure 2.50. 1H-1H gCOSY of 18 (500 MHz CD3OD). 131 Figure 2.51. 1H-13C gHSQCAD of 18 (500 MHz CD3OD). 131 Figure 2.52. 1H-NMR of 19 (500 MHz CDCl3). 132 Figure 2.53. 13C-NMR of 19 (125 MHz CDCl3). 132 Figure 2.54. 1H-1H gCOSY of 19 (500 MHz CDCl3). 133 Figure 2.55. 1H-13C gHSQCAD of 19 (500 MHz CDCl3). 133 Figure 2.56. 1H-NMR of 5 (500 MHz d6-DMSO). 134 Figure 2.57. 1H-NMR of 5 (500 MHz d6-DMSO, VT at 90 ℃). 134 Figure 2.58. 13C-NMR of 5 (125 MHz d6-DMSO). 135 Figure 2.59. 1H-1H gCOSY of 5 (500 MHz d6-DMSO). 135 Figure 2.60. 1H-13C gHSQCAD of 5 (500 MHz d6-DMSO). 136 Figure 2.61. 1H-NMR of 21 (500 MHz CDCl3). 137 Figure 2.62. 13C-NMR of 21 (125 MHz CDCl3). 137 Figure 2.63. 1H-1H gCOSY of 21 (500 MHz CDCl3). 138 Figure 2.64. 1H-13C gHSQCAD of 21 (500 MHz CDCl3). 138 Figure 2.65. 1H-NMR of 22 (500 MHz CDCl3). 139 Figure 2.66. 13C-NMR of 22 (125 MHz CDCl3). 139 Figure 2.67. 1H-1H gCOSY of 22 (500 MHz CDCl3). 140 Figure 2.68. 1H-13C gHSQCAD of 22 (500 MHz CDCl3). 140 Figure 2.69. 1H-NMR of 23 (500 MHz CDCl3). 141 Figure 2.70. 13C-NMR of 23 (125 MHz CDCl3). 141 xiv Figure 2.71. 1H-1H gCOSY of 23 (500 MHz CDCl3). 142 Figure 2.72. 1H-13C gHSQCAD of 23 (500 MHz CDCl3). 142 Figure 2.73. 1H-NMR of 3 (500 MHz CDCl3). 143 Figure 2.74. 13C-NMR of 3 (125 MHz CDCl3). 143 Figure 2.75. 1H-1H gCOSY of 3 (500 MHz CDCl3). 144 Figure 2.76. 1H-13C gHSQCAD of 3 (500 MHz CDCl3). 144 Figure 2.77. 1H-NMR of S3 (500 MHz CDCl3). 145 Figure 2.78. 13C-NMR of S3 (125 MHz CDCl3). 145 Figure 2.79. 1H-1H gCOSY of S3 (500 MHz CDCl3). 146 Figure 2.80. 1H-13C gHSQCAD of S3 (500 MHz CDCl3). 146 Figure 2.81. 1H-NMR of S4 (500 MHz CDCl3). 147 Figure 2.82. 13C-NMR of S4 (125 MHz CDCl3). 147 Figure 2.83. 1H-1H gCOSY of S4 (500 MHz CDCl3). 148 Figure 2.84. 1H-13C gHSQCAD of S4 (500 MHz CDCl3). 148 Figure 2.85. 1H-NMR of S5 (500 MHz CDCl3). 149 Figure 2.86. 13C-NMR of S5 (125 MHz CDCl3). 149 Figure 2.87. 1H-1H gCOSY of S5 (500 MHz CDCl3). 150 Figure 2.88. 1H-13C gHSQCAD of S5 (500 MHz CDCl3). 150 Figure 2.89. 1H-NMR of 4 (500 MHz CDCl3). 151 Figure 2.90. 13C-NMR of 4 (125 MHz CDCl3). 151 Figure 2.91. 1H-1H gCOSY of 4 (500 MHz CDCl3). 152 xv Figure 2.92. 1H-13C gHSQCAD of 4 (500 MHz CDCl3). 152 Figure 2.93. 1H-NMR of 24 (500 MHz CDCl3). 153 Figure 2.94. 13C-NMR of 24 (125 MHz CDCl3). 153 Figure 2.95. 1H-1H gCOSY of 24 (500 MHz CDCl3). 154 Figure 2.96. 1H-13C gHSQCAD of 24 (500 MHz CDCl3). 154 Figure 2.97. 1H-NMR of 25 (500 MHz CDCl3). 155 Figure 2.98. 13C-NMR of 25 (125 MHz CDCl3). 155 Figure 2.99. 1H-1H gCOSY of 25 (500 MHz CDCl3). 156 Figure 2.100. 1H-13C gHSQCAD of 25 (500 MHz CDCl3). 156 Figure 2.101. 1H-NMR of 26 (500 MHz CDCl3). 157 Figure 2.102. 13C-NMR of 26 (125 MHz CDCl3). 157 Figure 2.103. 1H-1H gCOSY of 26 (500 MHz CDCl3). 158 Figure 2.104. 1H-13C gHSQCAD of 26 (500 MHz CDCl3). 158 Figure 2.105. 1H-NMR of 27 (500 MHz CDCl3). 159 Figure 2.106. 13C-NMR of 27 (125 MHz CDCl3). 159 Figure 2.107. 1H-1H gCOSY of 27 (500 MHz CDCl3). 160 Figure 2.108. 1H-13C gHSQCAD of 27 (500 MHz CDCl3). 160 Figure 2.109. 1H-NMR of 28 (500 MHz CDCl3). 161 Figure 2.110. 13C-NMR of 28 (125 MHz CDCl3). 161 Figure 2.111. 1H-1H gCOSY of 28 (500 MHz CDCl3). 162 Figure 2.112. 1H-13C gHSQCAD of 28 (500 MHz CDCl3). 162 xvi Figure 2.113. 1H-NMR of 29 (500 MHz CDCl3). 163 Figure 2.114. 13C-NMR of 29 (125 MHz CDCl3). 163 Figure 2.115. 1H-1H gCOSY of 29 (500 MHz CDCl3). 164 Figure 2.116. 1H-13C gHSQCAD of 29 (500 MHz CDCl3). 164 Figure 2.117. 1H-NMR of 31 (500 MHz CDCl3). 165 Figure 2.118. 13C-NMR of 31 (125 MHz CDCl3). 165 Figure 2.119. 1H-1H gCOSY of 31 (500 MHz CDCl3). 166 Figure 2.120. 1H-13C gHSQCAD of 31 (500 MHz CDCl3). 166 Figure 2.121. 1H-NMR of 32 (500 MHz CDCl3). 167 Figure 2.122. 13C-NMR of 32 (125 MHz CDCl3). 167 Figure 2.123. 1H-1H gCOSY of 32 (500 MHz CDCl3). 168 Figure 2.124. 1H-13C gHSQCAD of 32 (500 MHz CDCl3). 168 Figure 2.125. 1H-NMR of 33 (500 MHz CDCl3). 169 Figure 2.126. 13C-NMR of 33 (125 MHz CDCl3). 169 Figure 2.127. 1H-1H gCOSY of 33 (500 MHz CDCl3). 170 Figure 2.128. 1H-13C gHSQCAD of 33 (500 MHz CDCl3). 170 Figure 2.129. 1H-NMR of 34 (500 MHz CDCl3). 171 Figure 2.130. 13C-NMR of 34 (125 MHz CDCl3). 171 Figure 2.131. 1H-1H gCOSY of 34 (500 MHz CDCl3). 172 Figure 2.132. 1H-13C gHSQCAD of 34 (500 MHz CDCl3). 172 Figure 2.133. 1H-NMR of 35 (500 MHz CDCl3). 173 xvii Figure 2.134. 13C-NMR of 35 (125 MHz CDCl3). 173 Figure 2.135. 1H-1H gCOSY of 35 (500 MHz CDCl3). 174 Figure 2.136. 1H-13C gHSQCAD of 35 (500 MHz CDCl3). 174 Figure 2.137. 1H-NMR of 36α (500 MHz CDCl3). 175 Figure 2.138. 13C-NMR of 36α (125 MHz CDCl3). 175 Figure 2.139. 1H-1H gCOSY of 36α (500 MHz CDCl3). 176 Figure 2.140. 1H-13C gHSQCAD of 36α (500 MHz CDCl3). 176 Figure 2.141. 1H-NMR of 36β (500 MHz CDCl3). 177 Figure 2.142. 1H-13C gHSQCAD of 36β (500 MHz CDCl3). 177 Figure 2.143. 1H-NMR of 37α (500 MHz CDCl3). 178 Figure 2.144. 13C-NMR of 37α (125 MHz CDCl3). 178 Figure 2.145. 1H-1H gCOSY of 37α (500 MHz CDCl3). 179 Figure 2.146. 1H-13C gHSQCAD of 37α (500 MHz CDCl3). 179 Figure 2.147. 1H-NMR of 37β (500 MHz CDCl3). 180 Figure 2.148. 13C-NMR of 37β (125 MHz CDCl3). 180 Figure 2.149. 1H-1H gCOSY of 37β (500 MHz CDCl3). 181 Figure 2.150. 1H-13C gHSQCAD of 37β (500 MHz CDCl3). 181 Figure 2.151. 1H-NMR of 38 (500 MHz CDCl3). 182 Figure 2.152. 13C-NMR of 38 (125 MHz CDCl3). 182 Figure 2.153. 1H-1H gCOSY of 38 (500 MHz CDCl3). 183 Figure 2.154. 1H-13C gHSQCAD of 38 (500 MHz CDCl3). 183 xviii Figure 2.155. 1H-NMR of 39 (500 MHz CDCl3). 184 Figure 2.156. 13C-NMR of 39 (125 MHz CDCl3). 184 Figure 2.157. 1H-1H gCOSY of 39 (500 MHz CDCl3). 185 Figure 2.158. 1H-13C gHSQCAD of 39 (500 MHz CDCl3). 185 Figure 2.159. 1H-NMR of 40 (500 MHz CDCl3). 186 Figure 2.160. 13C-NMR of 40 (125 MHz CDCl3). 186 Figure 2.161. 1H-1H gCOSY of 40 (500 MHz CDCl3). 187 Figure 2.162. 1H-13C gHSQCAD of 40 (500 MHz CDCl3). 187 Figure 2.163. 1H-NMR of 41 (500 MHz CDCl3). 188 Figure 2.164. 13C-NMR of 41 (125 MHz CDCl3). 188 Figure 2.165. 1H-1H gCOSY of 41 (500 MHz CDCl3). 189 Figure 2.166. 1H-13C gHSQCAD of 41 (500 MHz CDCl3). 189 Figure 2.167. 1H-NMR of 42 (500 MHz CDCl3). 190 Figure 2.168. 13C-NMR of 42 (125 MHz CDCl3). 190 Figure 2.169. 1H-1H gCOSY of 42 (500 MHz CDCl3). 191 Figure 2.170. 1H-13C gHSQCAD of 42 (500 MHz CDCl3). 191 Figure 2.171. 1H-NMR of 43 (500 MHz CDCl3). 192 Figure 2.172. 13C-NMR of 43 (125 MHz CDCl3). 192 Figure 2.173. 1H-1H gCOSY of 43 (500 MHz CDCl3). 193 Figure 2.174. 1H-13C gHSQCAD of 43 (500 MHz CDCl3). 193 Figure 2.175. 1H-NMR of 44 (500 MHz CDCl3). 194 xix Figure 2.176. 13C-NMR of 44 (125 MHz CDCl3). 194 Figure 2.177. 1H-1H gCOSY of 44 (500 MHz CDCl3). 195 Figure 2.178. 1H-13C gHSQCAD of 44 (500 MHz CDCl3). 195 Figure 2.179. 1H-NMR of 45 (500 MHz CDCl3). 196 Figure 2.180. 13C-NMR of 45 (125 MHz CDCl3). 196 Figure 2.181. 1H-1H gCOSY of 45 (500 MHz CDCl3). 197 Figure 2.182. 1H-13C gHSQCAD of 45 (500 MHz CDCl3). 197 Figure 2.183. 1H-NMR of 2 (500 MHz D2O, PRESAT). 198 Figure 2.184. 13C-NMR of 2 (125 MHz D2O). 198 Figure 2.185. 1H-1H gCOSY of 2 (500 MHz D2O). 199 Figure 2.186. 1H-13C gHSQCAD of 2 (500 MHz D2O). 199 Figure 2.187. 1H-1H TOCSY of 2 (500 MHz D2O). 200 Figure 2.188. ESI-MS of 2. 200 Figure 2.189. 1H-NMR of 47 (500 MHz CD3OD). 201 Figure 2.190. ESI-MS of 47. 201 Figure 3.1. (A) TEM characterization of Hep-SPION; (B) TGA of SPION and Hep-SPION. 209 Figure 3.2. (A) Aβ binding to plate decreased with increasing concentrations of Hep-SPION. The bound Aβ was detected by an anti-Aβ IgG mAb 6E10, followed by addition of HRP-conjugated anti-IgG secondary antibody and the TMB substrate. (B) ELISA curve for Aβ incubated with increasing concentrations of SPION. SPIONs without heparin coating showed little effect on Aβ binding to the plate. 210 Figure 3.3. (A) PAGE gel of Aβ only (lane 1) or Aβ (25 μM) incubated with 0.0078 mg/mL (lane 2), 0.0156 mg/mL (lane 3), 0.0312mg/mL (lane 4) and 0.125 mg/mL (lane 5) of Hep-SPION. (B) Percentage of low-molecular-weight Aβ oligomer in total Aβ in presence of various xx concentrations of Hep-SPION. The percentage was calculated by dividing the intensity of the low molecular weight oligomer band by the sum of the intensities of all bands in the specific lane. 211 Figure 3.4. The intensities of ThT fluorescence at 489 nm (λex = 440 nm). 212 Figure 3.5. Cell viability assay of SH-SY5Y cells. 214 Figure 4.1. Sensograms of heparin like oligosaccharide binding with tau oligomers. 234 Figure 4.2. Biophysical characterization of Tau oligomers alone and in the presence of heparin-like oligosaccharides. 235 Figure 4.3. Viability and Cytotoxicity assays of Tau oligomers alone and in the presence of heparin-like oligosaccharides on human SH-SY5Y neuroblastoma cell line. 237 Figure 4.4. 1H-NMR of 3 (500 MHz CDCl3). 279 Figure 4.5. 13C-NMR of 3 (125 MHz CDCl3). 279 Figure 4.6. 1H-1H gCOSY of 3 (500 MHz CDCl3). 280 Figure 4.7. 1H-13C gHSQCAD of 3 (500 MHz CDCl3). 280 Figure 4.8. 1H-NMR of 7 (500 MHz CDCl3). 281 Figure 4.9. 13C-NMR of 7 (125 MHz CDCl3). 281 Figure 4.10. 1H-1H gCOSY of 7 (500 MHz CDCl3). 282 Figure 4.11. 1H-13C gHSQCAD of 7 (500 MHz CDCl3). 282 Figure 4.12. 1H-NMR of 8 (500 MHz CDCl3). 283 Figure 4.13. 13C-NMR of 8 (125 MHz CDCl3). 283 Figure 4.14. 1H-1H gCOSY of 8 (500 MHz CDCl3). 284 Figure 4.15. 1H-13C gHSQCAD of 8 (500 MHz CDCl3). 284 Figure 4.16. 1H-NMR of 9 (500 MHz CDCl3). 285 xxi Figure 4.17. 13C-NMR of 9 (125 MHz CDCl3). 285 Figure 4.18. 1H-1H gCOSY of 9 (500 MHz CDCl3). 286 Figure 4.19. 1H-13C gHSQCAD of 9 (500 MHz CDCl3). 286 Figure 4.20. 1H-NMR of 10 (500 MHz CDCl3). 287 Figure 4.21. 1H-NMR of 11 (500 MHz CDCl3). 288 Figure 4.22. 13C-NMR of 11 (125 MHz CDCl3). 288 Figure 4.23. 1H-1H gCOSY of 11 (500 MHz CDCl3). 289 Figure 4.24. 1H-13C gHSQCAD of 11 (500 MHz CDCl3). 289 Figure 4.25. 1H-NMR of 12 (500 MHz CDCl3). 290 Figure 4.26. 13C-NMR of 12 (125 MHz CDCl3). 290 Figure 4.27. 1H-1H gCOSY of 12 (500 MHz CDCl3). 291 Figure 4.28. 1H-13C gHSQCAD of 12 (500 MHz CDCl3). 291 Figure 4.29. 1H-NMR of 13 (500 MHz CDCl3). 292 Figure 4.30. 13C-NMR of 13 (125 MHz CDCl3). 292 Figure 4.31. 1H-1H gCOSY of 13 (500 MHz CDCl3). 293 Figure 4.32. 1H-13C gHSQCAD of 13 (500 MHz CDCl3). 293 Figure 4.33. 1H-NMR of 14 (500 MHz CDCl3). 294 Figure 4.34. 13C-NMR of 14 (125 MHz CDCl3). 294 Figure 4.35. 1H-1H gCOSY of 14 (500 MHz CDCl3). 295 Figure 4.36. 1H-13C gHSQCAD of 14 (500 MHz CDCl3). 295 Figure 4.37. 1H-NMR of 15 (500 MHz CDCl3). 296 xxii Figure 4.38. 13C-NMR of 15 (125 MHz CDCl3). 296 Figure 4.39. 1H-1H gCOSY of 15 (500 MHz CDCl3). 297 Figure 4.40. 1H-13C gHSQCAD of 15 (500 MHz CDCl3). 297 Figure 4.41. 1H-NMR of 16 (500 MHz CDCl3). 298 Figure 4.42. 13C-NMR of 16 (125 MHz CDCl3). 298 Figure 4.43. 1H-1H gCOSY of 16 (500 MHz CDCl3). 299 Figure 4.44. 1H-13C gHSQCAD of 16 (500 MHz CDCl3). 299 Figure 4.45. 1H-NMR of 17 (500 MHz CDCl3). 300 Figure 4.46. 13C-NMR of 17 (125 MHz CDCl3). 300 Figure 4.47. 1H-1H gCOSY of 17 (500 MHz CDCl3). 301 Figure 4.48. 1H-13C gHSQCAD of 17 (500 MHz CDCl3). 301 Figure 4.49. 1H-NMR of 18 (500 MHz CDCl3). 302 Figure 4.50. 13C-NMR of 18 (125 MHz CDCl3). 302 Figure 4.51. 1H-1H gCOSY of 18 (500 MHz CDCl3). 303 Figure 4.52. 1H-13C gHSQCAD of 18 (500 MHz CDCl3). 303 Figure 4.53. 1H-NMR of 19 (500 MHz CDCl3). 304 Figure 4.54. 13C-NMR of 19 (125 MHz CDCl3). 304 Figure 4.55. 1H-1H gCOSY of 19 (500 MHz CDCl3). 305 Figure 4.56. 1H-13C gHSQCAD of 19 (500 MHz CDCl3). 305 Figure 4.57. 1H-NMR of 20 (500 MHz CDCl3). 306 Figure 4.58. 13C-NMR of 20 (125 MHz CDCl3). 306 xxiii Figure 4.59. 1H-1H gCOSY of 20 (500 MHz CDCl3). 307 Figure 4.60. 1H-13C gHSQCAD of 20 (500 MHz CDCl3). 307 Figure 4.61. 1H-NMR of 21 (500 MHz CDCl3). 308 Figure 4.62. 13C-NMR of 21 (125 MHz CDCl3). 308 Figure 4.63. 1H-1H gCOSY of 21 (500 MHz CDCl3). 309 Figure 4.64. 1H-13C gHSQCAD of 21 (500 MHz CDCl3). 309 Figure 4.65. 1H-NMR of 22 (500 MHz CDCl3). 310 Figure 4.66. 13C-NMR of 22 (125 MHz CDCl3). 310 Figure 4.67. 1H-1H gCOSY of 22 (500 MHz CDCl3). 311 Figure 4.68. 1H-13C gHSQCAD of 22 (500 MHz CDCl3). 311 Figure 4.69. 1H-NMR of 23 (500 MHz CDCl3). 312 Figure 4.70. 13C-NMR of 23 (125 MHz CDCl3). 312 Figure 4.71. 1H-NMR of 24 (500 MHz CDCl3). 313 Figure 4.72. 13C-NMR of 24 (125 MHz CDCl3). 313 Figure 4.73. 1H-NMR of 25 (500 MHz CD3OD). 314 Figure 4.74. 13C-NMR of 25 (125 MHz CD3OD). 314 Figure 4.75. 1H-1H gCOSY of 25 (500 MHz CD3OD). 315 Figure 4.76. 1H-13C gHSQCAD of 25 (500 MHz CD3OD). 315 Figure 4.77. 1H-NMR of 26 (500 MHz D2O). 316 Figure 4.78. 13C-NMR of 26 (125 MHz D2O). 316 Figure 4.79. 1H-1H gCOSY of 26 (500 MHz D2O). 317 xxiv Figure 4.80. 1H-13C gHSQCAD of 26 (500 MHz D2O). 317 Figure 4.81. ESI-MS of 26. 318 Figure 4.82. 1H-NMR of 27 (500 MHz D2O). 319 Figure 4.83. 13C-NMR of 27 (125 MHz D2O). 319 Figure 4.84. 1H-1H gCOSY of 27 (500 MHz D2O). 320 Figure 4.85. 1H-13C gHSQCAD of 27 (500 MHz D2O). 320 Figure 4.86. ESI-MS of 27. 321 Figure 4.87. 1H-NMR of 28 (500 MHz D2O). 322 Figure 4.88. 13C-NMR of 28 (125 MHz D2O). 322 Figure 4.89. 1H-1H gCOSY of 28 (500 MHz D2O). 323 Figure 4.90. 1H-13C gHSQCAD of 28 (500 MHz D2O). 323 Figure 4.91. ESI-MS of 28. 324 Figure 4.92. 1H-NMR of 29 (500 MHz D2O). 325 Figure 4.93. 13C-NMR of 29 (125 MHz D2O). 325 Figure 4.94. 1H-1H gCOSY of 29 (500 MHz D2O). 326 Figure 4.95. 1H-13C gHSQCAD of 29 (500 MHz D2O). 326 Figure 4.96. ESI-MS of 29. 327 Figure 4.97. 1H-NMR of 30 (500 MHz CDCl3). 328 Figure 4.98. 1H-NMR of 31 (500 MHz D2O). 328 Figure 4.99. 1H-NMR of 32 (500 MHz D2O). 329 Figure 4.100. 1H-13C gHSQCAD of 32 (900 MHz D2O). 329 xxv Figure 4.101. ESI-MS of 32. 330 xxvi LIST OF SCHEMES Scheme 2.1. Retrosynthetic analysis of target pentasaccharide 2. 32 Scheme 2.2. Preparation of building block 6. 33 Scheme 2.3. Preparation of fucosamine building block by following the previously reported route.5-6 34 Scheme 2.4. Preparation of building block 5. 35 Scheme 2.5. Preparation of building block 3. 36 Scheme 2.6. Undesired α-isomer 29 isolated from glycosylation between 28 and 25. 37 Scheme 2.7. Selective formation of β-fucoside. 44 Scheme 2.8. Complete synthesis of the target pentasaccharide 2. 45 Scheme 2.9. Preparation of protein-glycan conjugates by reaction of activated NHS ester compound 47 with 1) Qβ, 2) KLH and 3) BSA. 47 Scheme 2.10. Preparation of building block 4. 71 Scheme 3.1. Synthesis of heparin coated magnetic nanoparticles by A) the thermal decomposition and ligand exchange method; and B) the co-precipitation method. 208 Scheme 4.1. Synthesis of non-reducing end disaccharide module 3. 228 Scheme 4.2. Construction of heparin tetrasaccharide backbones. 229 Scheme 4.3. Constructions of heparin hexa- and deca-saccharide backbones. 230 Scheme 4.4. Deprotection of heparin oligosaccharides. 231 Scheme 4.5. Sulfation and deprotection of tetrasaccharides. 232 Scheme 4.6. Sulfation and deprotection of hexa- and deca-saccharide. 233 xxvii 7-AAD AAT ACT Ac2O AcOH AD AFM AgOTf aP APC ATCC BAIB BF3·Et2O Bn BSA Bz Cbz CDC COSY KEY TO ABBREVIATIONS 7-aminoactinomycin D 2-acetamido-4-amino-2,4,6-trideoxygalactose adenylate cyclase toxin acetic anhydride acetic acid Alzheimer’s disease Atomic force microscopy silver trifluoromethanesulfonate acellular pertussis antigen-presenting cells American Type Culture Collection bis(acetoxy)iodobenzene boron trifluoride etherate benzyl bovine serum albumin benzoyl benzyloxycarbonyl Centers for Disease Control and Prevention correlation spectroscopy xxviii CSA camphorsulfonic acid DBU DCM DDQ DIAD DIPEA DLS DMAP DMEM DMF DMSO DTPads DTT 1, 8-diazabicyclo[5.4.0]undec-7-ene dichloromethane 2, 3-dichloro-5, 6-dicyanobenzoquinone Diisopropylazodicarboxylate diisopropylethylamine dynamic light scattering 4-dimethylaminopyridine Dulbecco's Modified Eagle Medium dimethylformamide dimethyl sulfoxide diphtheria, tetanus toxoids and whole-cell pertussis vaccines on alum dithiothreitol EDC·HCl 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ELISA ESI-MS Et3N FAB FACS FBS Fe(acac)3 enzyme-linked immunosorbent assay electrospray-ionization mass spectrometry trimethylamine fast atom bombardment fluorescence-activated cell sorting fetal bovine serum ferric acetylacetonate xxix FeCl2·4H2O FeCl3·6H2O FHA FPLC ferrous chloride tetrahydrate ferric chloride hexahydrate filamentous hemagglutinin fast protein liquid chromatography Fuc2NAc4NMe 2-acetamido-4-N-methyl-2,4,6-trideoxy-galactose GAGs GlcA GlcN GlcNAc HCl glycosaminoglycans glucuronic acid glucosamine 2-acetamidoglucose hydrochloric acid Hep-SPION heparin-coated superparamagnetic iron oxide nanoparticles H2O2 HPLC HRMS HRP HS HSF-LPF-IAP HSQC IACUC IdoA hydrogen peroxide high performance liquid chromatography high resolution mass spectrometry horseradish peroxidase heparan sulfate histamine-sensitizing, islet-activating lymphocyte-leukocyte-promoting and heteronuclear single quantum correlation Institutional Animal Care and Use Committee iduronic acid xxx IgG kDa Kdo KLH KPB LDH LevOH LiAlH4 LiOH LOS LPS mAb Immunoglobulin G kilo-dalton 3-deoxy-D-manno-2-octulosonic acid keyhole limpet hemocyanin potassium phosphate buffer lactate dehydrogenase levulinic acid lithium aluminum hydride lithium hydroxide lipooligosaccharide lipopolysaccharide monoclonal antibody MALDI-TOF matrix assisted laser desorption ionization-time of flight ManHep mannoheptose Man2NAc3NAcA 2,3-diacetamido-2,3-dideoxy-mannuronic acid MD MeI MPLA MS NaN3 NFTs molecular dynamics methyl iodide monophosphoryl lipid A mass spectrometry sodium azide neurofibrillary tangles xxxi NH4OH NHS NIS NMO NMR OD OsO4 PAGE PBS PBST Pd(OH)2 PGs Ph PHF PMB PRN PT p-TolSCl p-TolSH Py/Pyr Qβ ammonium hydroxide N-hydroxysuccinimide N-iodoxuccinimide 4-methylmorpholine N-oxide nuclear magnetic resonance spectroscopy Optical density osmium tetraoxide polyacrylamide gel electrophoresis phosphate buffered saline PBS/0.5% Tween-20 palladium hydroxide proteoglycans phenyl paired helical filament p-methoxybenzyl pertactin pertussis toxin p-toluenesulfenyl chloride p-toluenethiol pyridine bacteriophage Qbeta xxxii SA sat. SDS SEC SPION SSM STD-NMR TauO TBAF TBAI TBDPS TBS t-Bu TCT TEM TEMPO TFA Tf2O TGA Th THF streptavidin saturated sodium dodecyl sulfate size exclusion chromatography superparamagnetic iron oxide nanoparticles Stainer-Scholte media saturation transfer difference NMR tau oligomers tetrabutylammonium fluoride tetrabutylammonium iodide t-butyldiphenylsilyl t-butyldimethylsilyl t-butyl tracheal cytotoxin transmission electron microscopy 2, 2, 6, 6-tetramethyl-1-piperidinyloxyl trifluoroacetic acid trifluoromethanesulfonic anhydride thermogravimetric analysis helper T cell tetrahydrofuran xxxiii thioflavin T thin layer chromatography 3,3’,5,5’-tetramethylbenzidine total correlation spectroscopy trichloroethyl chloroformate tetanus toxoid 2,4,6-tri-t-butylpyrimidine World Health Organization whole-cell pertussis ThT TLC TMB TOCSY TrocCl TT TTBP WHO wP xxxiv Chapter 1. A Review of Developing Vaccines against Bordetella pertussis 1.1. Introduction Pertussis, commonly known as whooping cough or 100-day cough, is a highly contagious acute respiratory disease of which the most characteristic symptom is uncontrollable violent coughing. Severe coughing fits followed by gasping for breath of pertussis patients result in the “whooping” sound, hence its name. Although severity of syndromes of pertussis is usually mild and not life-threating to adults, this disease can be particularly harmful and fatal for infants.1-2 According to the surveillance and reporting by the Centers for Disease Control and Prevention (CDC), most pertussis incidents have been reported for babies less than one year old for the last 20 years in the United States, with an incident rate at about 60 per 100,000 persons in 2016. Figure 1.1. Reported pertussis incidence (per 100,000 persons) by age group in the United States from 1990–2016. Infants aged <1 year, who are at greatest risk for serious disease and death, continue to have the highest reported rate of pertussis. This figure is adapted and reproduced from reference3 (free of copyright restrictions). 1 The etiological agent of pertussis, Bordetella pertussis, was isolated and identified by Bordet and Gengou in 1906.4 It is a Gram-negative, aerobic encapsulated coccobacillus of the genus Bordetella. As defined by World Health Organization (WHO), the course of pertussis is consisted of the catarrhal, paroxysmal and convalescent phases. The infection of pertussis is through aerosol transmission of droplets emitted in coughs or sneezes, which mostly happens during the catarrhal phase. After being inhaled into the respiratory system, B. pertussis colonizes ciliated cells of mucosa and produces an array of virulence factors that play a key role in the establishment of infection, including pertussis toxin (PT), adenylate cyclase toxin (ACT), filamentous hemagglutinin (FHA) and pertactin (PRN).5 PT is known to be main exotoxin which elicits a number of deleterious consequences including leukocytosis, splenomegaly and histamine sensitization, while both FHA and pertactin are presumed as adhesins. The best way to prevent infection of B. pertussis is vaccination and it is recommended for routine use by the WHO and CDC. The first vaccine was prepared as early as in 1929 by T. Madsen, which was composed of a suspension of B. pertussis in rabbit blood.6 Efficacy of the vaccine was proven by the decreased death rate of vaccinees compared to that of nonvaccinees, according to a two-year period of surveillance. The more well-known tri-functional vaccine, which combined whole-cell pertussis vaccines with diphtheria and tetanus toxoids onto alum (DTPads) was then invented and applied throughout the United States. The number of deaths caused by B. pertussis infection dropped dramatically since the introduction and standardization of whole-cell pertussis vaccines.7 However, the vaccine itself raised safety concerns as it occasionally results in serious adverse effects such as local reactions, fever and seizures.8 2 With the increasing public scrutiny on vaccine safety, a safer acellular vaccine composed of pertussis toxin and filamentous hemagglutinin was invented in Japan and now adopted in most developed countries.9 Although widespread vaccination has greatly reduced the morbidity, the world has been experiencing a resurgence of pertussis in recent decades with more and more reported cases of pertussis patients, especially after the introduction of acellular vaccine (Figure 1.2). Figure 1.2. The number of pertussis cases reported to CDC from 1922 to 2016. Following the introduction of pertussis vaccines in the 1940s when case counts frequently exceeded 100,000 cases per year, reports declined dramatically to fewer than 10,000 by 1965. During the 1980s pertussis reports began increasing gradually, and by 2015 more than 20,000 cases were reported nationwide. This figure is adapted and reproduced from reference3 (free of copyright restrictions). 3 1.2. Hypotheses for Resurgence of Pertussis Although the introduction of acellular pertussis vaccine appeased safety concerns of vaccination, it has been noticed that a widespread resurgence of pertussis is occurring. Around the world, there are about 260,000 deaths due to pertussis infection annually. Even in developed countries where the vaccination rates are high, the number of reported cases of pertussis has markedly risen during recent decades,10-11 reaching a 60 year high in countries such as US and Australia.12The infection rate of pertussis was reported to be 1-6%,13-14 which became the most prevalent vaccine-preventable disease in developed countries. The reasons proposed for the resurgence of pertussis includes pathogen adaption and conversion from whole-cell vaccines to acellular vaccines. Virulence factors contained in the acellular pertussis vaccines, including pertussis toxin, filamentous hemagglutinin, pertactin and other components, can elicit protective antibodies against the pathogen. However, strain differences and mutations in those virulence factors have resulted in the antigenic divergence between vaccine strains and the currently circulating strains, which rendered the vaccines ineffective. The dramatically increasing fraction of pertactin-deficient strains have been discovered in the recent outbreak of pertussis,15-17 presumably due to the selection of bacteria under the immune pressure. It turned out to be advantageous for B. pertussis since the deficiency in the expression of pertactin conferred more invasive infection on patients.18 Polymorphism in pertactin region 1 also limited the efficacy of vaccines since the antibodies were found to be type-specific and shown little cross reactivity.19 The emergence of strains that overproduced pertussis toxin and led to more severe syndromes 4 further proved the importance of pathogen adaption of B. pertussis.20 A phylogenetic analysis of a worldwide collection of 343 B. pertussis strains isolated from 1920 and 2010 was performed by Marieke J. Bart and coworkers.21 Comparative genomics indicated that the genotype population and diversity of B. pertussis had increased a lot since the introduction of vaccination, consistent with the suggestion that the vaccine was the main driving force of pathogen adaption. Several changes in the gene coding of the proteins included in acellular vaccines were also discovered, which might have contributed to the “vaccine escape” in the resurgence of pertussis. 1.3. Acellular Pertussis Vaccines It was proposed by Margaret Pittman of National Institute of Health (NIH) in 1979 to name the histamine-sensitizing, lymphocyte-leukocyte-promoting and islet-activating (HSF-LPF-IAP) antigen as “pertussis toxin” that had caused harmful effects and prolonged immunity of pertussis.22 She also suggested that an antitoxin against pertussis toxin would be a good defense against the disease. An acellular pertussis vaccine (aP vaccine) composed of pertussis toxin and filamentous hemagglutinin was invented in Japan in 1981 and evaluation of the vaccine showed excellent efficacy as well as much lower reactogenicity compared to whole-cell pertussis vaccine (wP vaccine).23 The current commercial aP vaccine may contain up to five virulence factors from B. pertussis, including pertussis toxin, filamentous hemagglutinin, pertactin, and fimbriae 2&3. Although debates are still going on about the necessity of including more virulence factors, there has been agreement that pertussis toxin is an essential component in the aP vaccines.24 Early clinical trials of aP vaccines appeared to be comparably effective as wP vaccines.25-26 5 However, recent studies revealed that aP vaccines are actually not as durable as initially thought. Epidemiological data show that aP vaccines induce shorter term immunity compared to wP vaccines. The anti-pertussis immune responses from acellular vaccines tend to drop with reduced efficacy after 3-5 years and only 10% of the children vaccinated with aP vaccines would still have protection by the time of the next adolescent booster.27 To better understand the different protection stimulated by wP and aP vaccines, it is essential to know the difference in the immunological responses. It was found that wP and aP vaccines induce different skewing of immune responses. Similar to natural infection, wP vaccines mainly induce a cellular-immunity-related Th1-dependent immune response, eliciting IgG2 antibody subclass as well as IgG1 and IgG3. On the contrary, aP vaccines induce Th2/Th1 mixed or more Th2-skewed responses.28-30 Although Th2 cells were thought to be important for promoting antibody responses against extracellular pathogens such as bacteria, Kingston H. G. Mills and coworkers31 recently found that this immune response was actually dispensable for protection against B. pertussis. Knockout of the gene of the cytokine IL4 that was important for inducing differentiation of naïve T helper cells to Th2 cells did not affect clearance of bacteria. On the contrary, much slower clearance of bacteria was observed in IL17-/- mutant mice. They also found that the clearance of bacteria was mainly mediated by Th17 cells that were associated with recruiting macrophages and neutrophils to the lungs and promoting the killing of B. pertussis. The immune protection can be improved by substituting the commonly used adjuvant alum in aP vaccines with an adjuvant that promotes Th1 cells. The redundant Th2 component in immune responses against aP vaccines may have even caused the rare type hypersensitivity reactions seen 6 in children after a fourth or fifth injection.32-33 Protection by wP vaccines mainly depends on the production of IFN-γ by Th1 cells. Although the role of Th17 response upon immunization with wP vaccines is not well understood yet, the induction of Th17 cells via IL-1 might explain the better protection provided by wP vaccines. To better understand the mechanism of immune responses as in humans, a recent study by Tod J. Merkel and coworkers was performed on nonhuman primate model using baboons.34 The baboons were immunized with wP or aP vaccines on the same schedule as infants before being subjected to direct challenge with B. pertussis. Although post-vaccination serum analysis indicated comparable levels of antibodies against four main virulence factors and aP also successfully prevented leukocytosis, more persistent colonization of bacteria were found in aP vaccinated baboons according to the analysis of nasopharyngeal washes. To mimic the transmission through cough illness in human disease, they either cohoused challenged animals with aP vaccinated animal or naïve animals with challenged animals that had been pre-immunized with the aP vaccine. Surprisingly, transmission of bacteria was observed in both cases despite vaccination. Their results indicated that aP vaccines failed to prevent colonization of B. pertussis in the respiratory tract or transmission to healthy individuals. This compromised herd immunity might be another mechanism leading to the resurgence of pertussis. The endotoxins included in the aP vaccines have different biological functions and contribute synergistically to the pathology of pertussis. Pertussis toxin secreted by B. pertussis impedes the antigen processing and presentation by inhibiting the migration and phagocytosis of antigen-presenting cells (APC).30, 35 Both FHA and pertactin carries the RGD motif and are 7 presumed as adhesins that help cell attachment.36-37 Although people expected by targeting those antigens, especially the outer-membrane bound proteins, to confer complement-mediated bactericidal killing, it has been found that immunization with aP vaccines did not improve bactericidal activity.38 In a survey of 34 pairs of pre- and post-immunization serum samples from adults by Alison A. Weiss and coworkers, no significant increases in bactericidal activity at high serum concentrations were observed after vaccination in spite of elevated titers of IgG against all three exotoxins (pertussis toxin, FHA and pertactin) in the vaccine.39 Those results suggested that antibodies induced by aP vaccines might neutralize the exotoxins and reduce related syndromes, rather than directly kill the pathogens. The failure of aP vaccines in providing prolonged protection urges the development of better vaccines for pertussis. However, returning to wP vaccines will not be an acceptable choice due to the potential side effects for adolescents and adults. Improvement of the current pertussis vaccines should be focused on discovery of antigens that are able to elicit bactericidal antibodies and optimal adjuvant that correctly tunes the adaptive immune arm. Kingston H. G. Mills and coworkers identified and characterized an endogenous lipoprotein BP1569 from B. pertussis, which was able to activate macrophages and dendritic cells via TLR2.40 A synthetic lipoprotein of the N-terminus was capable of activating both Th1 and Th17 responses and conferring protection against B. pertussis. The same group also demonstrated the capability of infection clearance by activating TLR4, which is the receptor of lipopolysaccharide.41 8 1.4. Pertussis Lipooligosaccharide Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria, which protects the bacteria from the surroundings by serving as a physical barrier. It is commonly comprised of three parts: lipid A, core oligosaccharide and O-specific antigen. Polysaccharide O-Specific Chain Region III Outer Core II b Inner Core II a Lipid Lipid A I Figure 1.3. Common structure of lipopolysaccharide of Gram-negative bacteria. Unlike two other bacteria in the same genus, B. bronchiseptica and B. parapertussis, which both contain a homopolymer of 2,3-dideoxy-2,3-diacetamidogalactosaminuronic acid, the LPS of B. pertussis is devoid of such O-specific chain, thus commonly referred to as lipooligosaccharide (LOS). The LOS of B. pertussis was first isolated by Eldering in 1941 with an acid method, and later a more commonly used hot phenol-water method was applied to the extraction of B. pertussis LOS, as reported by MacLennan.42 SDS-PAGE analysis of B. pertussis LOS depicts two separate bands. The more abundant, slowly migrating band, which is referred to as band A, represents a LOS containing a dodecasaccharide core, while the minor, fast-migrating band B lacks a distal trisaccharide. The biological activity of B. pertussis LOS is similar to those of the endotoxins isolated from other Gram-negative bacteria. It was found that LOS worked synergistically with tracheal 9 cytotoxin (TCT) inducing epithelial NO production exclusively in non-ciliated cells, which eventually caused the disruption of ciliated cells in respiratory mucosa.43 LOS also protects B. pertussis from the innate immunity in respiratory tract mediated by surfactant proteins A and D, which bind to the lipid A region and core saccharide region respectively, inducing aggregation and acting as opsonins for macrophages and neutrophils.44-45 The interaction was shielded mainly by the distal trisaccharide in LOS, as mutants lacking the trisaccharide can be aggregated and permeabilized by the innate defense mechanism. 1.4.1. Structure Elucidation of Pertussis LOS Detailed elucidation of the structure of pertussis LOS was pioneered by Martine Caroff and coworkers.46-48 They extracted the LOS from B. pertussis strain 1414 following the hot phenol-water method and cleaved lipid A from LOS with the mild sodium dodecyl sulfate (SDS) condition. Selective hydrolysis of fatty acids from the major component of lipid A with hydroxylamine or sodium hydroxide pinpointed the linkage of fatty acids, while the distribution of fatty acids on the β-(1→6)-linked glucosamine disaccharide backbone was resolved by analyzing the fragmentation pattern in fast atom bombardment (FAB) data.46 The ensemble of those results defined the only structure for lipid A, as shown in Figure 1.4. A minor species was less in molecular weight by 226 Da, presumably by losing a hydroxytetradecanoic acid. 10 O PO HO OO OH O O NH OH O HOO O O O O O OH O NH O P O OH O OH C10OH C14OH C14OH C14OH C14 Figure 1.4. Structure of the major molecular species present in B. pertussis lipid A. Structural characterization of the heptasaccharide that is adjacent to lipid A was first attempted by Richard Chaby and coworkers.49-52 By hydrolysis with hydrochloric acid of different concentrations, di- or trisaccharide subunits at the non-reducing end of the heptasaccharide were harvested in low yields. These di- or trisaccharides were further digested into monosaccharides, which were then subjected to chemical modifications such as reduction or acetylation and comparison with standard samples in HPLC. The structure of the heptose was proven to be L-glycero-D-mannoheptose by chemical degradation. Enzymolysis by stereochemistry-specific enzymes revealed the correct configuration for glycosidic linkage. Eventually, the substitution positions of glucosamine and glucuronic acid on heptose were determined to be 7 and 2 respectively by analyzing fragments from the reduction with NaB3H4 11 and cleavage with NaIO4.49-50 Similarly, the structure of another trisaccharide purified from the acidic hydrolysis was characterized as the 4-O-(2-amino-2-deoxy-α-D-glucopyranosyl) -6-O-(2-amino-2-deoxy-α-D-galactopyranuronyl)-D-glycopyranose configuration.51 Due to the lability of 3-deoxy-D-manno-2-octulosonic acid (Kdo) in strong acid, they applied a nitrous acid-cleavage protocol and successfully separated an Kdo-containing oligosaccharide that was claimed to be a tetrasaccharide with the configuration of D-glucopyranosyl-β-(1→3)-D-glycopyranuronyl-β-(1→2)-L-glycero-D-mannoheptopyranosyl-α- (1→5)-3-deoxy-D-manno-2-octulosonic acid. By overlapping the shared monosaccharide units in all fragments, they proposed the structure of the reducing end heptasaccharide as in Figure 1.5: GalNA HO COOH O OH HO HO GlcN O OH O NH2 HO Hep OH O OH O HO HO O COOH O O OH GlcA OH O COOH OH Kdo GlcN HO HO NH2O HO O OH O OH O NH2 OH Glc 12 Figure 1.5. Proposed structure of the heptasaccharide present at the reducing end of LOS-1 isolated from B. pertussis endotoxin. However, exactness of the structure of the last claimed-to-be tetrasaccharide relied heavily on the colorimetric estimation of monosaccharides. They failed to notice that the ratio of hexoses to heptoses was actually 1:2 rather than 1:1 and therefore the wrong structure was proposed. After sequential dephosphorylation with hydrofluoric acid, deamination with nitrous acid and hydrolysis promoted by sodium dodecyl sulfate, Ladislas Szabo and coworkers isolated a hexasaccharide from the B. pertussis endotoxin.47 Methylation analysis revealed another mannoheptose in the structure, which was 3, 4-disubstituted and not detected in the previous research. Although anomeric configuration was not easy to assign in the hexasaccharide due to overlapped signals, exhaustive Smith degradation with NaIO4 truncated all but the non-reducing end disaccharide and the glycosidic linkage was determined to be α by NMR analysis. The anomeric stereochemistry of the glucuronic acid was also determined to be α by NMR, which was incorrectly assigned to be β by cleavage reaction with a commercial β-D-glucuronidase. Nitrous acid deamination afforded the distal pentasaccharide from the LOS of B. pertussis stain 1414. Relative structures of the five subunits in the pentasaccharide were resolved by NMR analysis, which suggested the existence of α-2-acetamidoglucose (α-GlcNAc), β-2-acetamido-4-N-methyl-2,4,6-trideoxy-galactose (β-Fuc2NAc4NMe), β-2,3-diacetamido-2,3-dideoxy-mannuronic acid (β-Man2NAc3NAcA), α-mannoheptose (α-ManHep) and anhydromannitol. By hydrolyzing the pentasaccharide with hydrochloric acid, the α-GlcNAc and α-Hep subunits were isolated and the absolute configuration was determined to be D and L, D by GC-MS.48 Potential energy calculation in combination with NOE measurements helped determining the absolute structures of the other three monosaccharides. 13 Based on all data obtained, the complete structure of B. pertussis LOS was proposed as in Figure 1.7. GlcNAc OH O AcHN HO HO Fuc2NAc4NMe O O NHAc MeNH O H HOOC O AcHN H NHAc O H Man23NAcA R Figure 1.6. Structure of the trisaccharide present in the distal pentasaccharide from B. pertussis. Some short interproton distances that could be observed in the NOE experiments and used to determine the absolute configuration of the sugars are shown. 14 GlcN HO O OH HO GlcA COOH O OH HO HO O NH2 GalNA HO O COOH O OH HO NH2 O OH O OH O Hep HO HO OH O OHHO HO O Hep O OH O NH2 GlcN O O OH OH Glc Fuc2NAc4NMe O NHAc HO OH HO Man23NAcA HOOC O MeHN O O AcHN NHAc O NHAc GlcNAc OH O O HO Hep HO HO O O [PPEA]n=0,1 Kdo COOH O GlcN O O O H2O3PO HN O HO O O O HO O O O HO GlcN O HN OPO3H2 O OH C10OH C14OH C14OH C14OH C14 Figure 1.7. Complete structure of the LOS of B. pertussis strain 1414. 1.4.2. Immunology of Pertussis LOS Early failure in wP vaccines gave impetus to research that aims at identifying the key factors in the protective immunogenicity for pertussis. Jean M. Dolby and coworkers characterized fractions of antibodies in the B. pertussis antisera and identified a complement-mediated bactericidal antibody capable of killing B. pertussis.53-54 However, the bactericidal activity pattern did not correlate well with any of the titers for agglutinin, antihaemagglutinin or anti-histamine sensitizing factors, suggesting that the antibody was stimulated by a complete 15 different antigen, which was referred to as “the bactericidal antigen”. Extraction of the endotoxin LOS from six strains of B. pertussis and immunization at multiple doses of the LOS coupled with carrier protein elicited bactericidal antibodies, which suggested that the antigen was actually LOS.54 There has been a significant amount of research done to further identify antigenic determinants on the lipooligosaccharide of B. pertussis. Bernard R. Brodeur and coworkers described the preparation of both monoclonal antibodies specific for band A or band B by the hybridoma cell line protocol.55-56 The LOS band A-specific antibodies were found to react well with strains of LOS AB phenotype but not the atypical strain 134 of LOS B phenotype. At least two antigenic epitopes were discovered, with five out of the seven LOS band A-specific antibodies found to recognize the same epitope.55 Although band B-specific antibody BL-8 bound to strain 134 and led to moderate lytic activities, it failed to affect predominant strains that express both LOS band A and B, presumably due to the limited expression and accessibility of epitopes on the cell membrane.56 Structure elucidation of the pertussis LOS significantly helped epitope mapping for antigen design. Richard Chaby and coworkers immunized mice with strain 1414 which carried a majority of band A LOS.57 Three monoclonal antibodies were tested against LOS from Bp1414, BpA100, B. bronchiseptica, B. parapertussis and different subparts of band A LOS. All three monoclonal antibodies bound to the terminal pentasaccharide, which was located far from lipid A, but recognized GlcNAc-Man2NAc3NAcA, Fuc2NAc4NMe-GlcN and Hep-GlcN respectively. The carbohydrate region which was proximal to lipid A was considered as poorly immunogenic 16 because of the failure in generating an antibody that bound to strain A100. Tomasz Niedziela and coworkers conjugated the pentasaccharide cleaved from band A LOS with nitrous acid to tetanus toxoid for mouse immunization to generate polyclonal antibodies.58 Those antibodies failed to bind with strain 606 in western blot, which carries only band B LOS and was included in the wP vaccines in Poland. It implies that the immunogenic epitopes were mainly located on the distal trisaccharide. Saturation transfer difference NMR experiments (STD-NMR) were also employed to investigate the structural elements on the pentasaccharide that contributed to the binding epitope. Although information provided by STD-NMR supported that major components of antigenic epitopes were on the trisaccharide, weak signals on the ManHep suggested that the heptose unit may also play a role in the immunogenicity of LOS. It was further confirmed by the research done by John Robbins and coworkers, in which they found that repetitive expression of the distal trisaccharide from a B. brochiseptica mutant led to a decreased binding affinity with anti-LOS antibodies.59 Molecular modeling of the LOS from B. pertussis strain 1414 also revealed that those epitopes were exposed at the extremities regardless of the existence of PPEA on Kdo, as illustrated in Figure 1.8. 17 Figure 1.8. Structure and molecular models of B. pertussis BP1414 LOS. (A) Structure of the LPS where ManX represents Man2NAc3NAcA and FucX represents Fuc2NAc4NMe. (B) Molecular model with PPEA showing lipid A. Models for (C) and (D) did not include PPEA and lipid A. Conformers in (C) and (D) show the flexibility about the GlcN(1–7)Hep and Fuc (1– 6)GlcN linkages. The dihedral angle (O7–C7–C6–O6) of the (1–7)Hep residue is -78° for models (B) and (C) and 55° for model (D). The dihedral angle (O6–C6–C5–O5) of the (1–6)GlpN residue is 64° for model (B) and -60° for models (C) and (D). The residues are colored gray for Kdo, green for Hep and ManX, blue for Glc and GlcNAc, orange for GlcN, purple for GlcA, yellow for GalNA, red for FucX, mauve for PPEA and PO4 of lipid A and tan for lipid A. Hydrogen atoms are not shown in the molecular models. This figure is adapted and reproduced with permission from reference.60 18 1.5. Pertussis LOS-based Glycoconjugate Vaccines Despite the antibodies elicited from LOS in wP vaccines, it is not feasible to directly use B. pertussis LOS as a vaccine. Many lipopolysaccharides from Gram-negative bacteria have been reported to be highly immunogenic, but B. pertussis LOS was poorly immunogenic when injected alone, probably due to the low molecular weight. Unlike proteins, the LOS is only a T-independent antigen and triggers B cells to secret predominantly IgM with low binding affinity. Moreover, LOS was suspected to be the main culprit for side effects of wP vaccines because of its endotoxin activity.61 Therefore, it is necessary to remove the lipid A part, which is the endotoxin determinant. Covalent conjugation of the immunogenic carbohydrate region with a carrier protein results in uptake by B cells and antigen presentation through MHC II to CD4+ T cells. Cytokines secreted by the activated T cells will stimulate the maturation of B cells and cause the class-switching from IgM to high-affinity IgG, which helps achieve the optimal immune responses. Although the relative abundance of two bands of oligosaccharides may vary across different strains, such as band B being the major component in strain A100, the structure of B. pertussis core saccharide remains relatively conserved, which is different from exotoxins included in the current aP vaccines. This conclusion was supported by an investigation on the structure of B. pertussis LOS from pre- and post- vaccination era,60 suggesting that those antigens may be good potential vaccine components. Tomasz Niedziela and coworkers cleaved the pentasaccharide from B. pertussis LOS by nitrous acid and conjugated it with tetanus toxoid (TT) through reductive amination with the free aldehyde group on the reducing-end anhydromannose. They found that the mouse polyclonal 19 antibodies against such a conjugate bound strongly with the whole wild-type LOS of B. pertussis as well as the live B. pertussis bacteria. It was reported previously that LOS worked synergistically with tracheal cytotoxin and induced the release of NO from secretory epithelial cells.43 Such effect was significantly inhibited by the polyclonal antibodies against the pentasaccharide-TT conjugate along with lower production of IL6 and TNF-α, which are both proinflammatory cytokines involved in the inflammatory process stimulated by LOS. Instead of choosing the most immunogenic distal pentasaccharide as the antigen, John R. Robbins and coworkers cleaved the whole dodecasaccharide from LOS with 1% acetic acid and conjugated it with BSA. A mutant of B. bronchiseptica which lacked the complete wbm locus and did not express the O-specific chain was used as an alternative source of LOS, due to the ease in culturing compared with B. pertussis. The purified dodecasaccharide shared the same structure as that in B. pertussis, except that ~50% of the non-reducing end GlcNAc was replaced by GalNAc. The glycan-BSA conjugate successfully induced antisera that were bactericidal against B. pertussis Tohama I strain. 20 BSA-NH2 O N O O O O Br N H LOS OS-Kdo4P-Lipid A Succinimidyl 3-(bromoacetamido)-propionate (SBAP) 1% acetic acid O O BSA N H Br N H HS NH2 O O-(3-thiopropyl)hydroxylamine OS-anhydro-Kdo O O OS HOOH O OH O O BSA N H S N H O NH2 PBS, pH 7.4, 16h, r.t. O O BSA N H N H S O N O OS HOOH O OH Figure 1.9. Scheme of conjugation of B. pertussis and B. bronchiseptica core OS. BSA-ONH2/OS conjugate 1.6. Future Outlook The failure of aP vaccines in inducing durable protection urges the development of new vaccines that are able to elicit long-term robust bactericidal immune responses. Carbohydrates present on the surface of bacteria have been used as immunogens to develop carbohydrate-based 21 vaccines against many pathogens such as Streptococcus pneumoniae, Neisseria meningitides and Haemophilus influenza.62 Conjugates of B. pertussis LOS with a carrier protein elicit bactericidal antibodies, which were reported to overcome the BrkA protein-induced resistance against complement-dependent bactericidal pathway.24, 63 This along with the highly conserved structure of LOS across strains renders LOS an appealing target for vaccine development. However, isolating LOS in abundance from the highly aerosol transmissible pathogen remains a big obstacle, while the alternative way of expressing LOS in a mutant B. bronchiseptica results in heterogeneity in the desired structure. The obtainable sequences of oligosaccharides limited by LOS processing methods also impose restrictions on screening for the optimal immunogenic epitope and understanding the adaptive immunity against LOS. Synthetic carbohydrate chemistry provides the flexibility in accessible antigen structures and can add to the arsenal for combating bacteria with higher quality control compared to natural sources. 22 REFERENCES 23 REFERENCES 1. Kerr, J. R.; Matthews, R. C., Bordetella pertussis infection: pathogenesis, diagnosis, management, and the role of protective immunity. Eur. J. Clin. Microbiol. Infect. Dis. 2000, 19 (2), 77-88. 2. Kilgore, P. E.; Salim, A. M.; Zervos, M. J.; Schmitt, H.-J., Pertussis: microbiology, disease, treatment, and prevention. Clin. Microbiol. Rev. 2016, 29 (3), 449-486. 3. CDC National Notifiable Disease Surveillance System and Supplemental Pertussis Surveillance System, https://www.cdc.gov/pertussis/surv-reporting.html. 4. Cherry, J. D., Epidemiological, clinical, and laboratory aspects of pertussis in adults. Clin. Infect. Dis. 1999, 28 (Supplement_2), S112-S117. 5. Marzouqi, I.; Richmond, P.; Fry, S.; Wetherall, J.; Mukkur, T., Development of improved vaccines against whooping cough: current status. Hum. Vaccin. 2010, 6 (7), 543-553. 6. Madsen, T., Vaccination against whooping cough. J. Am. Med. Assoc. 1933, 101 (3), 187-188. 7. Warfel, J. M.; Edwards, K. M., Pertussis vaccines and the challenge of inducing durable immunity. Curr. Opin. Immunol. 2015, 35 (Supplement C), 48-54. 8. Linnemann, C. C.; Perlstein, P. H.; Ramundo, N.; Minton, S. D.; Englender, G. S.; McCormick, J. B.; Hayes, P. S., Use of pertussis vaccine in an epidemic involving hospital staff. The Lancet 1975, 306 (7934), 540-543. 9. Robbins, J. B.; Schneerson, R.; Keith, J. M.; Miller, M. A.; Kubler-Kielb, J.; Trollfors, B., Pertussis vaccine: a critique. Pediatr. Infect. Dis. J. 2009, 28 (3), 237-241. 10. Celentano, L. P.; Massari, M.; Paramatti, D.; Salmaso, S.; Tozzi, A. E., Resurgence of pertussis in Europe. Pediatr. Infect. Dis. J. 2005, 24 (9), 761-765. 11. Control, C. f. D.; Prevention, Summary of notifiable diseases-United States, 2010. MMWR. Morb. Mortal. Wkly. Rep. 2012, 59 (53), 1-111. 12. Rumbo, M.; Hozbor, D., Development of improved pertussis vaccine. Hum. Vaccines Immunother. 2014, 10 (8), 2450-2453. 13. de Melker, H. E.; Versteegh, F. G. A.; Schellekens, J. F. P.; Teunis, P. F. M.; Kretzschmar, M., The incidence of Bordetella pertussis infections estimated in the population from a combination of serological surveys. J. Infect. 53 (2), 106-113. 24 14. Ward, J. I.; Cherry, J. D.; Chang, S.-J.; Partridge, S.; Keitel, W.; Edwards, K.; Lee, M.; Treanor, J.; Greenberg, D. P.; Barenkamp, S.; Bernstein, D. I.; Edelman, R., Bordetella pertussis infections in vaccinated and unvaccinated adolescents and adults, as assessed in a national prospective randomized acellular pertussis vaccine trial (APERT). Clin. Infect. Dis. 2006, 43 (2), 151-157. 15. Lam, C.; Octavia, S.; Ricafort, L.; Sintchenko, V.; Gilbert, G. L.; Wood, N.; McIntyre, P.; Marshall, H.; Guiso, N.; Keil, A. D., Rapid increase in pertactin-deficient Bordetella pertussis isolates, Australia. Emerging Infect. Dis. 2014, 20 (4), 626-633. 16. Pawloski, L.; Queenan, A.; Cassiday, P.; Lynch, A.; Harrison, M.; Shang, W.; Williams, M.; Bowden, K.; Burgos-Rivera, B.; Qin, X., Prevalence and molecular characterization of pertactin-deficient Bordetella pertussis in the United States. Clin.Vaccine Immunol. 2014, 21 (2), 119-125. 17. Otsuka, N.; Han, H.-J.; Toyoizumi-Ajisaka, H.; Nakamura, Y.; Arakawa, Y.; Shibayama, K.; Kamachi, K., Prevalence and genetic characterization of pertactin-deficient Bordetella pertussis in Japan. PLoS One 2012, 7 (2), e31985. 18. Martin, S. W.; Pawloski, L.; Williams, M.; Weening, K.; DeBolt, C.; Qin, X.; Reynolds, L.; Kenyon, C.; Giambrone, G.; Kudish, K.; Miller, L.; Selvage, D.; Lee, A.; Skoff, T. H.; Kamiya, H.; Cassiday, P. K.; Tondella, M. L.; Clark, T. A., Pertactin-negative Bordetella pertussis strains: evidence for a possible selective advantage. Clin. Infect. Dis. 2015, 60 (2), 223-227. 19. He, Q.; Mäkinen, J.; Berbers, G.; Mooi, F. R.; Viljanen, M. K.; Arvilommi, H.; Mertsola, J., Bordetella pertussis protein pertactin induces type-specific antibodies: one possible explanation for the emergence of antigenic variants? J. Infect. Dis. 2003, 187 (8), 1200-1205. 20. Mooi, F. R., Bordetella pertussis and vaccination: The persistence of a genetically monomorphic pathogen. Infect., Genet. Evol. 2010, 10 (1), 36-49. 21. Bart, M. J.; Harris, S. R.; Advani, A.; Arakawa, Y.; Bottero, D.; Bouchez, V.; Cassiday, P. K.; Chiang, C.-S.; Dalby, T.; Fry, N. K.; Gaillard, M. E.; van Gent, M.; Guiso, N.; Hallander, H. O.; Harvill, E. T.; He, Q.; van der Heide, H. G. J.; Heuvelman, K.; Hozbor, D. F.; Kamachi, K.; Karataev, G. I.; Lan, R.; Lutyńska, A.; Maharjan, R. P.; Mertsola, J.; Miyamura, T.; Octavia, S.; Preston, A.; Quail, M. A.; Sintchenko, V.; Stefanelli, P.; Tondella, M. L.; Tsang, R. S. W.; Xu, Y.; Yao, S.-M.; Zhang, S.; Parkhill, J.; Mooi, F. R., Global population structure and evolution of Bordetella pertussis and their relationship with vaccination. mBio. 2014, 5 (2). 22. Pittman, M., Pertussis toxin: the cause of the harmful effects and prolonged immunity of whooping cough. A hypothesis. Rev. Infect. Dis. 1979, 1 (3), 401-412. 23. Aoyama, T., Acellular pertussis vaccines developed in Japan and their application for disease 25 control. J. Infect. Dis. 1996, 174 (Supplement_3), S264-S269. 24. Robbins, J. B.; Schneerson, R.; Kubler-Kielb, J.; Keith, J. M.; Trollfors, B.; Vinogradov, E.; Shiloach, J., Toward a new vaccine for pertussis. Proc. Natl. Acad. Sci., U. S. A. 2014, 111 (9), 3213-3216. 25. Taranger, J.; Trollfors, B.; LagergÅrd, T.; Lind, L.; Sundh, V.; Zackrisson, G.; Bryla, D. A.; Robbins, J. B., Unchanged efficacy of a pertussis toxoid vaccine throughout the two years after the third vaccination of infants. Pediatr. Infect. Dis. J. 1997, 16 (2), 180-184. 26. Lugauer, S.; Heininger, U.; Cherry, J. D.; Stehr, K., Long-term clinical effectiveness of an acellular pertussis component vaccine and a whole cell pertussis component vaccine. Eur. J. Pediatr. 2002, 161 (3), 142-146. 27. McGirr, A.; Fisman, D. N., Duration of pertussis immunity after DTaP immunization: a meta-analysis. Pediatrics 2015, peds. 2014-1729. 28. Ryan; Murphy; Nilsson; Shackley; Gothefors; Øymar; Miller; Storsaeter; Mills, Distinct T-cell subtypes induced with whole cell and acellular pertussis vaccines in children. Immunology 1998, 93 (1), 1-10. 29. Edwards, K. M.; Berbers, G. A. M., Immune responses to pertussis vaccines and disease. J. Infect. Dis. 2014, 209 (Supplement_1), S10-S15. 30. Higgs, R.; Higgins, S.; Ross, P.; Mills, K., Immunity to the respiratory pathogen Bordetella pertussis. Mucosal Immunol. 2012, 5 (5), 485-500. 31. Ross, P. J.; Sutton, C. E.; Higgins, S.; Allen, A. C.; Walsh, K.; Misiak, A.; Lavelle, E. C.; McLoughlin, R. M.; Mills, K. H. G., Relative contribution of Th1 and Th17 cells in adaptive immunity to Bordetella pertussis: Towards the rational design of an improved acellular pertussis vaccine. PLoS Path. 2013, 9 (4), e1003264. 32. Ryan, E. J.; Nilsson, L.; Kjellman, N. I. M.; Gothefors, L.; Mills, K. H. G., Booster immunization of children with an acellular pertussis vaccine enhances Th2 cytokine production and serum IgE responses against pertussis toxin but not against common allergens. Clin. Exp. Immunol. 2000, 121 (2), 193-200. 33. Rennels, M. B.; Black, S.; Woo, E. J.; Campbell, S.; Edwards, K. M., Safety of a fifty dose of diphtheria and tetanus toxoid and acellular pertussis vaccine in children experiencing extensive, local reactions to the fourth dose. Pediatr. Infect. Dis. J. 2008, 27 (5), 464-465. 34. Warfel, J. M.; Zimmerman, L. I.; Merkel, T. J., Acellular pertussis vaccines protect against disease but fail to prevent infection and transmission in a nonhuman primate model. Proc. Natl. Acad. Sci., U. S. A. 2014, 111 (2), 787-792. 26 35. Meade, B.; Kind, P.; Ewell, J.; McGrath, P.; Manclark, C., In vitro inhibition of murine macrophage migration by Bordetella pertussis lymphocytosis-promoting factor. Infect. Immun. 1984, 45 (3), 718-725. 36. Relman, D.; Tuomanen, E.; Falkow, S.; Golenbock, D. T.; Saukkonen, K.; Wright, S. D., Recognition of a bacterial adhesin by an integrin: Macrophage CR3 (αMβ2, CD11bCD18) binds filamentous hemagglutinin of Bordetella pertussis. Cell 1990, 61 (7), 1375-1382. 37. Leininger, E.; Roberts, M.; Kenimer, J. G.; Charles, I. G.; Fairweather, N.; Novotny, P.; Brennan, M. J., Pertactin, an Arg-Gly-Asp-containing Bordetella pertussis surface protein that promotes adherence of mammalian cells. Proc. Natl. Acad. Sci., U. S. A. 1991, 88 (2), 345-349. 38. Weingart, C. L.; Keitel, W. A.; Edwards, K. M.; Weiss, A. A., Characterization of bactericidal immune responses following vaccination with acellular pertussis vaccines in adults. Infect. Immun. 2000, 68 (12), 7175-7179. 39. Weiss, A. A.; Patton, A. K.; Millen, S. H.; Chang, S.-J.; Ward, J. I.; Bernstein, D. I., Acellular pertussis vaccines and complement killing of Bordetella pertussis. Infect. Immun. 2004, 72 (12), 7346-7351. 40. Dunne, A.; Mielke, L.; Allen, A.; Sutton, C.; Higgs, R.; Cunningham, C.; Higgins, S.; Mills, K., A novel TLR2 agonist from Bordetella pertussis is a potent adjuvant that promotes protective immunity with an acellular pertussis vaccine. Mucosal Immunol. 2015, 8 (3), 607-617. 41. Higgins, S. C.; Jarnicki, A. G.; Lavelle, E. C.; Mills, K. H., TLR4 mediates vaccine-induced protective cellular immunity to Bordetella pertussis: role of IL-17-producing T cells. J. Immunol. 2006, 177 (11), 7980-7989. 42. Maclennan, A. P., Specific lipopolysaccharides of Bordetella. Biochem. J. 1960, 74 (2), 398-409. 43. Flak, T. A.; Goldman, W. E., Signalling and cellular specificity of airway nitric oxide production in pertussis. Cell. Microbiol. 1999, 1 (1), 51-60. 44. Schaeffer, L. M.; McCormack, F. X.; Wu, H.; Weiss, A. A., Interactions of pulmonary collectins with Bordetella bronchiseptica and Bordetella pertussis lipopolysaccharide elucidate the structural basis of their antimicrobial activities. Infect. Immun. 2004, 72 (12), 7124-7130. 45. Schaeffer, L. M.; McCormack, F. X.; Wu, H.; Weiss, A. A., Bordetella pertussis lipopolysaccharide resists the bactericidal effects of pulmonary surfactant protein A. J. Immunol. 2004, 173 (3), 1959-1965. 46. Caroff, M.; Deprun, C.; Richards, J.; Karibian, D., Structural characterization of the lipid A of Bordetella pertussis 1414 endotoxin. J. Bacteriol. 1994, 176 (16), 5156-5159. 27 47. Lebbar, S.; Caroff, M.; Szabó, L.; Mérienne, C.; Szilégyi, L. s., Structure of a hexasaccharide proximal to the hydrophobic region of lipopolysaccharides present in Bordetella pertussis endotoxin preparations. Carbohydr. Res. 1994, 259 (2), 257-275. 48. Caroff, M.; Brisson, J.-R.; Martin, A.; Karibian, D., Structure of the Bordetella pertussis 1414 endotoxin. FEBS Lett. 2000, 477 (1-2), 8-14. 49. Chaby, L-glycero-D-manno-heptose. Eur. J. Biochem. 1976, 70 (1), 115-122. Szabo, R.; L., 7-O-(2-Amino-2-deoxy-α-D-glucopyranosyl) L., 50. Chaby, 2-O-(β-D-Glucuronyl)-7-O-(2-amino-2-deoxy-α-D-glucopyranosyl)-L-glycero-D-manno-heptose :a constituent of the Bordetella pertussis endotoxin. Eur. J. Biochem. 1977, 76 (2), 453-460. Moreau, SzabÓ, M.; R.; 51. Moreau, M.; Chaby, R.; Szabo, L., trisaccharide containing 2-amino-2-deoxy-D-galacturonic acid from the Bordetella pertussis endotoxin. J. Bacteriol. 1982, 150 (1), 27. Isolation of a 52. Moreau, M.; Chaby, R.; Szabo, L., Structure of the terminal reducing heptasaccharide of polysaccharide 1 isolated from the Bordetella pertussis endotoxin. J. Bacteriol. 1984, 159 (2), 611-617. 53. Dolby, J. M.; Vincent, W., Characterization of the antibodies responsible for thebactericidal activity patterns' of antisera to Bordetella pertussis. Immunology 1965, 8 (5), 499. 54. Ackers, J.; DOLBY, J. M., The antigen of Bordetella pertussis that induces bactericidal antibody and its relationship to protection of mice. Microbiology 1972, 70 (2), 371-382. 55. Archambault, D.; Rondeau, P.; Martin, D.; Brodeur, B. R., Characterization and comparative bactericidal activity of monoclonal antibodies to Bordetella pertussis lipo-oligosaccharide A. Microbiology 1991, 137 (4), 905-911. 56. Martin, D.; Peppler, M.; Brodeur, B., Immunological characterization of lipooligosaccharide B band of Bordetella pertussis. Infect. Immun. 1992, 60 (7), 2718-2725. the 57. Le Blay, K.; Caroff, M.; Blanchard, F.; Perry, M. B.; Chaby, R., Epitopes of Bordetella pertussis lipopolysaccharides as potential markers for typing of isolates with monoclonal antibodies. Microbiology 1996, 142 (4), 971-978. 58. Niedziela, T.; Letowska, I.; Lukasiewicz, J.; Kaszowska, M.; Czarnecka, A.; Kenne, L.; Lugowski, C., Epitope of the vaccine-type Bordetella pertussis strain 186 lipooligosaccharide and antiendotoxin activity of antibodies directed against the terminal pentasaccharide-tetanus toxoid conjugate. Infect. Immun. 2005, 73 (11), 7381-7389. 28 59. Kubler-Kielb, J.; Vinogradov, E.; Lagergård, T.; Ginzberg, A.; King, J. D.; Preston, A.; Maskell, D. J.; Pozsgay, V.; Keith, J. M.; Robbins, J. B.; Schneerson, R., Oligosaccharide conjugates of Bordetella pertussis and bronchiseptica induce bactericidal antibodies, an addition to pertussis vaccine. Proc. Natl. Acad. Sci., U. S. A. 2011, 108 (10), 4087-4092. 60. AlBitar-Nehme, S.; Basheer, S. M.; Njamkepo, E.; Brisson, J.-R.; Guiso, N.; Caroff, M., Comparison of lipopolysaccharide structures of Bordetella pertussis clinical isolates from pre- and post-vaccine era. Carbohydr. Res. 2013, 378 (Supplement C), 56-62. 61. Dias, W. O.; van der Ark, A. A. J.; Sakauchi, M. A.; Kubrusly, F. S.; Prestes, A. F. R. O.; Borges, M. M.; Furuyama, N.; Horton, D. S. P. Q.; Quintilio, W.; Antoniazi, M.; Kuipers, B.; van der Zeijst, B. A. M.; Raw, I., An improved whole cell pertussis vaccine with reduced content of endotoxin. Hum. Vaccin. Immunother. 2013, 9 (2), 339-348. 62. Khatun, F.; Stephenson, R. J.; Toth, I., An overview of structural features of antibacterial glycoconjugate vaccines that influence their immunogenicity. Chem. Eur. J. 2017, 23 (18), 4233-4254. 63. Weiss, A. A.; Mobberley, P. S.; Fernandez, R. C.; Mink, C. M., Characterization of human bactericidal antibodies to Bordetella pertussis. Infect. Immun. 1999, 67 (3), 1424-1431. 29 Chapter 2. Synthesis and Immunological Evaluation of Pertussis Pentasaccharide Bearing Multiple Rare Sugars as Potential Anti-pertussis Vaccines 2.1. Retrosynthetic Analysis and Rare Sugar Building Block Preparation In Chapter 1 we demonstrated that the pertussis pentasaccharide could be a key component in designing a new potential bactericidal vaccine. The pertussis pentasaccharide has multiple unique structural features, which include three rare monosaccharides, i.e., 2,3-dideoxy-2,3-diamino-D-mannuronic acid (B), 2,3,6-trideoxy-4-methylamino-2 -acetamido-L-galactose (C), and L-glycero-D-manno-heptose (E). In addition, it contains three 1,2-cis glycosyl linkages and a 1,2-trans linkage between units C and D, which is found to be surprisingly challenging to form (vide infra). The structure of the pentasaccharide cleaved from pertussis LOS is illustrated in Figure 2.1. The reducing end, 2,5-anhydro-mannose, is converted from glucosamine by reacting with nitrous acid, and the deamination reaction also adds an additional nitroso on the methylamino group of the unit C. To best represent the native structure of the epitopes on pertussis LOS, we choose compound 2 as our target structure with a C3 linker at the reducing end for carrier protein conjugation. Unit E is not a free glucosamine as in the pertussis LOS since converting it to anhydromannose did not affect the antibody binding and saturation transfer difference NMR (STD-NMR) did not locate the key epitopes on it.1 30 HO HO OH O AcHN HOOC O AcHN NHAc O AcHN O HO O HO HO HO OH 1 O N NO O O HO O CHO A OH O AcHN HO HO HO O HO E HO HO OH B NHAc O HOOC O AcHN AcHN O O O O NHAc O HO 2 D O NH C NH2 Figure 2.1. Structures of the pentasaccharide 1 cleaved by deamination and our synthetic target pentasaccharide 2. The synthetic design of the target pentasaccharide 2 was based on several considerations (Scheme 2.1). For the CDE branching trisaccharide, we envision the E unit should be installed onto D first before C as the 4-OH of D may be too hindered if the 6-OH of D is glycosylated first. To facilitate the formation of the 1,2-trans linkage between C and D, Troc, which is known to be a participating neighboring group, was used as the N-protecting group for 2-amine of unit C (building block 5). The 1,2-cis linkage between amino-mannuronic acid B and fucosamine C is challenging to form directly. Instead, we opted for an indirect route using the 3-amino glucose derivative 6, the 2-O stereochemistry of which could be stereospecifically inverted following glycosylation. The 2-O Bn and 2-N3 bearing building blocks 3 and 7 were designed to facilitate the formation of α- glycosyl linkages. 31 A HO HO OH O AcHN B NHAc O HOOC O AcHN O NH C AcHN 6 O O 4 O HO HO O HO HO HO OH E O O NHAc NH2 D 2 AcO AcO BnO BnO E OBn O STol 3 4 HO AcO 6 OTBDPS D O O NHTroc 4 Cbz N Bn C N HO Cbz Ph O 5 TrocHN TolS A STol BnO BnO B O 2 LevO O O N3 6 OAc O N3 7 STol Scheme 2.1. Retrosynthetic analysis of target pentasaccharide 2. Our synthesis commenced with preparation of building blocks for the rare sugars (3, 5 and 6) in the pentasaccharide. Acid-catalyzed ketal hydrolysis of the known 3-azido glucose derivative 82 followed by global acetylation and treatment with p-toluenethiol (p-TolSH) and boron trifluoride etherate (BF3·Et2O) gave the thioglycoside 9 in 54% yield over 3 steps (Scheme 2.2). All O-acetyl groups of 9 were removed with NaOMe and 4, 6-benzylidene was installed to afford 10 in 81% yield. Protection of the free 2-OH with levulinic acid (LevOH) aided by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and 4-dimethylaminopyridine (DMAP) yielded the first rare sugar building block 6.3-4 32 O O O O N3 O 8 O O N3 Ph O OH STol 10 1. 10% HCl 2. Ac2O, DMAP, Py 3. BF3 ·Et2O, p-TolSH, DCM 54% for 3 steps AcO N3 LevOH, EDC·HCl, DMAP, DCM Ph 91% 9 O O N3 1. NaOMe, DCM/MeOH 2. PhCH(OMe)2, CSA, MeCN 81% OAc O OAc STol O 6 STol OLev Scheme 2.2. Preparation of building block 6. Preparation of 2-acetamido-4-amino-2,4,6-trideoxygalactose (AAT) presented significant difficulties. The first route we undertook started with bis-triflation of α-rhamnoside 115-6 to be followed by SN2 displacement of O-triflates with sodium azide (Scheme 2.3). However, triflation resulted in a low yield of the desired bis-triflate with a major side product isolated containing the STol migrated to C-2 presumably due to the 1,2-trans arrangement of the a-STol and the purported intermediate bearing 2-OTf. To overcome this, the corresponding β-rhamnoside 12 was prepared, which was selectively protected at the 3-OH with benzoyl (Bz) and triflated at the 2- and 4-OH. Sequential displacement of the two triflates by sodium azide and potassium phthalimide gave the fucosamine analog 14 in a moderate 30-50% yield. Deprotection of the phthalimide and Bz groups with hydrazine produced the free amine 15, but with a low 30% yield. 33 STol O HO AcO OH a-rhamnoside 11 HO O STol HO OH β-rhamnoside 12 BzCl, Me2SnCl2 DIPEA, THF , 89% HO BzO O STol OH 13 1.Tf2O, Py, DCM 2.TBAN3(1.0eq), CH3CN, -30°C 3.PhthNK, DMF, r.t. 30-50% O N3 PhthN OBz 14 STol N2H4, THF, reflux 30% O N3 STol OH H2N 15 Scheme 2.3. Preparation of fucosamine building block by following the previously reported route.5-6 To improve the yield, we explored trichloroacetimidate as the protecting group7-8 of 3-OH of β-rhamnoside 12 rather than Bz so that C4 inversion could be achieved in situ upon triflation (Scheme 2.4). The thioglycoside 12 was treated with trichloroacetonitrile and 1, 8-diazabicyclo[5.4.0]undec-7-ene (DBU), giving 16 in a yield of 90%. The imidate 16 was subjected to trifluoromethanesulfonic anhydride (Tf2O) and pyridine at -30 ℃, which formed 3, 4-oxazoline ring spontaneously following nucleophilic displacement of 4-OTf by the neighboring imidate. Displacement of the 2-OTf with sodium azide (NaN3) in DMF at 50 ℃ afforded 17 in an excellent yield of 85% over 2 steps. Refluxing 17 in 4M hydrochloric acid (HCl) opened the oxazoline ring and gave 15 in 89% yield with the two N moieties on the ring differentiated. Among multiple methods investigated, monomethylation of 4-NH2 was best performed by refluxing 15 in ethyl formate and trimethylamine (Et3N) followed by treatment 34 with lithium aluminum hydride (LiAlH4), which was accompanied by simultaneous reduction of the 2-N3 to the amine. Selective protection of the equatorial primary 2-NH2 with trichloroethyl chloroformate (TrocCl) yielded 19, which was then treated with benzyl chloroformate giving the fucosamine building block 5 in 75% yield. HO O STol CCl3CN, DBU, THF 0 °C → r.t. HO 12 OH 90% HO HN O STol OH CCl3 O 16 HCl, DCM/MeOH /H2O, reflux 89% O OH 15 H2N STol N3 1. HCO2Et, Et3N, reflux 2. LiAlH4, THF, -30 C→reflux STol NHTroc CbzCl, Na2CO3, THF/H2O 0 °C→r.t. 75% O OH NH 19 70% O OH 5 N Cbz STol NHTroc 1. Tf2O, Py, DCM, -30 °C → r.t. 2. NaN3, DMF, 50 °C STol N3 O N O CCl3 17 85% O OH NH 18 STol NH2 TrocCl, Py, THF, 0 °C 88% Scheme 2.4. Preparation of building block 5. Preparation of the mannoheptose building block with the desired (L, D) configuration9-13 started from Swern oxidation of the mannosyl thioglycoside 20 (Scheme 2.5). The resulting aldehyde was not stable, which was directly subjected to Wittig olefination to give the product 21 in 83% yield. Treatment of the olefin 21 with osmium tetraoxide (OsO4) and 4-methylmorpholine N-oxide (NMO) at 0 ℃ furnished diols 22 and 23 in 2.4:1 ratio (22: H-6 3.93 ppm, C-7 63.1 ppm; 23: H-6 3.98 ppm, C-7 65.0 ppm12), which were separated by column chromatography. The D, D diastereoisomer 22 was then epimerized to the desired L, D-isomer 23. Acetylation of the diol afforded the desired mannoheptose building block 3 in 91% yield. 35 HO BnO BnO OBn O STol 20 (COCl)2, DMSO, DCM, -50 °C, Et3N; then (PPh3Me)Br, n-BuLi, THF, -40 °C→r.t. 83% OBn O BnO BnO STol 21 OsO4, NMO, Acetone/H2O 68% D:L = 2.4:1 Ac2O, DMAP, Py 91% AcO AcO BnO BnO OBn O STol 3 HO HO HO BnOBnO OBn O + HO BnOBnO OBn O STol 22 STol 23 84% over 4 steps 1. TBDPSCl, Py 2. p-NO2PhCO2H, PPh3, DIAD, THF 3. K2CO3, DCM/MeOH 4. TBAF, THF Scheme 2.5. Preparation of building block 3. 2.2. Stereochemical Challenges in Formation of the C-D Linkage With all monosaccharide building blocks in hand, we began the oligosaccharide assembly. Reaction of donor 3 with acceptor 4 proceeded smoothly promoted by p-TolSCl and AgOTf affording the DE disaccharide 24 in 80% yield (JH1-C1 = 171.0, 159.5 Hz). Removal of the t-butyldiphenylsilyl (TBDPS) of 24 exposed 6-OH on glucosamine, which was poised to be glycosylated by a fucosamine donor (Scheme 2.6.1). To test the formation of BC disaccharide, glycosylation of acceptor 5 by donor 6 under the activation of p-TolSCl and AgOTf at -78 ℃ produced disaccharide 26 in 70% yield (JH1-C1 = 163.0, 161.0 Hz). Removal of 2-O-levulinoyl group exposed free hydroxyl in 27, which was then triflated and substituted by azide to furnish the mannose configuration in BC disaccharide 28. The glycosylation of 28 and disaccharide acceptor 25 was explored next. While tetrasaccharide 36 29 was formed in good yield (73%), surprisingly, the newly formed glycosyl linkage was α only (JH1-C1 = 177.0, 171.0, 163.5, 166.0 Hz) despite the presence of the 2-N-Troc capable of neighboring group participation (Scheme 2.6.2). 1) AcO AcO BnO BnO AgOTf, p-TolSCl, 4Å MS, DCM/Et2O, -78 °C→r.t. AcO AcO BnO BnO OBn O STol 3 TBDPSO HO AcO then 4 80% R = O OR NHTroc Bn N Cbz OBn O O AcO 24 OTBDPS O OR NHTroc HF· Py, Py, 0 °C→r.t. 85% AcO AcO BnO BnO OBn O O AcO 25 OH O OR NHTroc 2) Ph O O N3 6 O OLev STol AgOTf, p-TolSCl, 4Å MS, DCM/Et2O, -78°C→r.t. Ph STol NHTroc then O OH N 5 70% Cbz O O O N3 TrocHN TolS OLev O 26 Cbz O N ·AcOH, Py, N2H4 AcOH, DCM Ph 93% Cbz O N O O N3 TrocHN TolS O OH 1. Tf2O, Py, DCM, -30°C→r.t. Ph 2. NaN3, DMF, 60°C 84% for 2 steps N3 O O O N3 TrocHN TolS 28 O Cbz O N 25, AgOTf, p-TolSCl, 4Å MS, DCM/Et2O, -78°C→r.t. 73% O 27 Cbz O N a linkage O O OR NHTroc Ph OBn O AcO AcO BnOBnO N3 O O O N3 TrocHN O O AcO 29 Scheme 2.6. Undesired α-isomer 29 isolated from glycosylation between 28 and 25. To form the desired β-isomer for CD linkage, we tested a series of reactions by varying protecting groups on the fucosamine donor with 3-azido propanol 30 as the model acceptor (Table 2.1). Glycosylation of donor 31 with acceptor 30 with p-TolSCl/AgOTf promoter gave a 3:2 α:β ratio using dichloromethane (DCM) as the reaction solvent (entry 1). Switching the promoter to NIS/TfOH gave a small improvement of the β selectivity (entry 2). Acetonitrile is well known to favor the formation of equatorial glycosides. However, 10% acetonitrile as the 37 co-solvent did not impact stereoselectivity much (entry 3). Increasing the amount of acetonitrile to 50% gave very low yield of the glycoside product (entry 4). The 2-picoloyl was pioneered by the Demchenko group as a remote participating group, which can form a hydrogen bond with the acceptor and direct the addition of the acceptor to the activated glycosyl donor with high syn selectivity.14 However, in our system, introduction of picoloyl onto the 3-OH of the fucosamine donor gave worse β selectivity (entry 5). Switching the 4-NCbz to 4-NPico or leaving the 3-OH unprotected in the fucosamine donor did not change the stereoselectivity much (entries 6, 7 and 8). The 3-Lev bearing donor 34 favored the formation of the β-isomer with 3-azidopropanol. Unfortunately, the α-glycoside became the major product (α:β = 2:1) when glycosylating disaccharide acceptor 25. One possible reason for the difficulty in forming β-glycoside is the epimerization of β-glycoside during the reaction as the α-glycoside should be more stable due to the anomeric effect. To test this, the β-glycoside product was subjected to the glycosylation condition with the addition of 1 eq of TfOH. No appreciable amounts of the α-glycoside were found suggesting the β-glycoside once formed was stable under the reaction condition. 38 Table 2.1. Investigation of protecting group schemes for β-selective fucosylation N3 STol O O O O [condition] N3 + R R + HO N3 30 O R Entry Substrate Reaction Condition Yield α : β 1 2 3 4 5 6 7 8 9 STol NHTroc STol NHTroc STol NHTroc STol NHTroc STol NHTroc STol NHTroc STol NHTroc STol NHTroc STol NHTroc Cbz Cbz Cbz Cbz Cbz Pico Pico Cbz Cbz O N OAc 31 O N OAc 31 O N OAc 31 O N OAc 31 O N OPico 32 O N OPico 33 O N OPico 33 O N OH 5 O N OLev 34 [p-TolSCl]a, DCM 89% 3 : 2 [NIS]b, DCM 92% 2 : 3 [NIS], DCM/MeCN = 9/1 90% 2 : 3 [NIS], DCM/MeCN = 1/1 25% α only [NIS], DCM 60% 5 : 1 [NIS], DCM <5% N/A [p-TolSCl], DCM 30% α only [NIS], DCM 80% 3 : 1 [NIS], DCM 91% 1 : 2 39 Table 2.1. (cont’d) 10 11 O NH OTBS 35 O NH OTBS 35 STol NHTroc STol NHTroc a. [p-TolSCl]: p-TolSCl, AgOTf, 4Å MS, -78℃ b. [NIS]: NIS, TfOH, 4Å MS, -78℃ c. Ratio determined by crude NMR [NIS], DCM <5% N/A [p-TolSCl], DCM 60% 1 : 2.5c To better understand the preference for α-glycoside formation using donors 31-34, molecular modeling of the postulated oxocarbenium intermediate of donor 31 was performed. To obtain conformers covering a wide range of the configuration space, plain molecular dynamics (MD) simulations were performed at 900 K. From the resulting MD frames, 100 conformers with equal time intervals were extracted, and further optimized using the B3LYP/6-31G* method. Then, the optimized 100 conformers were sorted by their energies and the 20 most stable conformers were selected for geometrical comparison. Only 4 out of those 20 conformers exhibited the anticipated neighboring group participation by the carbonyl oxygen of 2-NHTroc with a distance of 1.58-1.59 Å between the carbonyl oxygen and C1 (d2 in Table 2.2). Intriguingly, most of the stable conformers revealed an unexpected remote group participation by the carbonyl oxygen of 4-NMeCbz (d3 = 1.51-1.55 Å in Table 2.2). This information provided a potential explanation why an α/β mixture was generated when the intermediate was reacted with a small aliphatic alcohol 30. As a non-participating group in the subsequent SN-2 like reaction, the Cbz carbonyl oxygen was closer to the anomeric carbon than Troc (2.74-2.76 Å vs 2.89-3.31 Å). Moreover, the 40 orientation of the Cbz group in conformer No. 9, 10, 12 and 13 blocked the SN-2 like attack (Figure 2.2B), while the non-participating Troc group, such as in conformer No. 4, pointed away from the backside (Figure 2.2A). The difference in steric hindrance could explain why a larger amount of α stereoisomer was formed when the bulkier disaccharide acceptor 25 was used for glycosylation (Scheme 2.7.1). 41 Table 2.2. The relative energies and geometry of the most stable 20 conformers d1 O Me OAc O N O d2 O H N O d3 CCl3 d2 (Å) 3.0308 3.0307 3.0075 3.0115 2.8922 2.9113 3.2624 3.2151 1.5893 1.5893 2.9889 1.5848 1.5849 2.9811 3.2133 3.3137 2.9887 2.9888 2.9886 2.9887 d3 (Å) 1.5152 1.5152 1.5226 1.522 1.5363 1.5343 1.5132 1.5138 2.7571 2.7574 1.5325 2.7395 2.7393 1.5304 1.5259 1.5491 1.5302 1.5302 1.5301 1.5302 Conformer No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Relative E (kcal/mol) 0.000 0.000 1.373 1.746 1.788 1.834 2.045 2.056 2.153 2.153 2.268 2.382 2.382 2.404 2.412 2.441 2.504 2.504 2.504 2.504 d1 (Å) 1.3625 1.3626 1.3606 1.361 1.3569 1.3564 1.3626 1.3618 1.3333 1.3333 1.3584 1.3349 1.335 1.359 1.3618 1.3483 1.3597 1.3597 1.3598 1.3597 42 A B SN2-like SN2-like Figure 2.2. Two stable conformers of the postulated oxocarbenium intermediate. A) Remote group participation by the carbonyl oxygen of Cbz in conformer No. 4. B) Neighboring group participation by the carbonyl oxygen of Troc in conformer No. 9. SN-2 like backside attack of conformer 9 by an acceptor is more sterically hindered than that of conformer 4 due to the existence of Cbz. This information led us to design donor 35, where the 4-methyl amino group was unprotected. Donor 35 failed to be activated by NIS/TfOH to glycosylate 3-azidopropanol presumably due to neutralization of TfOH by the free secondary amine in the donor. Changing the promoter to p-TolSCl/AgOTf gave the β anomer (β:α = 2.5:1) as the major product (entry 11). Interestingly, the reaction of 35 with acceptor 25 gave excellent β-selectivity. Following Cbz 43 protection, the two anomers were separated providing 37α (10%) and 37β (73%) for the 2 steps. Although selective β-glycosylation of 2,4-diamino fucose has been reported in the synthesis of O-specific polysaccharides of Shigella sonnei15-17 and Providencia alcalifaciens18, this is the first time that such a β-linked fucosamine glycoside bearing methyl amine has been produced to the best of our knowledge. 1) STol NHTroc O N OLev 34 Cbz 25, AgOTf, p-TolSCl, 4Å MS, DCM/MeCN, -78 °C→r.t. 85% = 2:1 a:β 2) STol NHTroc O NH OTBS 35 1. 25, AgOTf, p-TolSCl, 4Å MS, DCM/MeCN, -78 °C→r.t. 2. CbzCl, Na2CO3, THF/H2O, r.t. 83% for 2 steps = 1:7.3 a:β AcO AcO BnO BnO TrocHN OBn O O O AcO 36a N Cbz LevO O O OR NHTroc AcO AcO BnO BnO TrocHN OBn O O AcO 37a TBSO N Cbz O O O OR NHTroc R = + + AcO AcO BnO BnO AcO AcO BnO BnO Bn N Cbz OBn O TrocHN O LevO O N Cbz O OR NHTroc O AcO 36β OBn O TrocHN O TBSO N Cbz O O OR NHTroc O AcO 37β Scheme 2.7. Selective formation of β-fucoside. 2.3. Assembly of Pertussis Pentasaccharide and Deprotection With the challenge of β-fucosamine formation overcome, we next focused on assembling the full pentasaccharide. After removal of TBS from 37β, the acceptor 38 was glycosylated with donor 6 yielding tetrasaccharide 39. The 2-O-Lev group was removed with hydrazine hydrate in 84% yield. The liberated 2-OH on 40 was then subjected to triflation, whereupon nucleophilic displacement of 2-O-triflate by NaN3 afforded mannose configuration on 41 in 86% yield over 2 steps. The benzylidene group of 41 was cleaved with trifluoroacetic acid (TFA) in presence of 44 H2O to give the diol 42 in 86% yield. Selective oxidation of the 6-OH followed by methylation with methyl iodide (MeI) produced the tetrasaccharide acceptor 43 in 66% yield over 2 steps. The last glycosylation with donor 7 completed the backbone construction and gave the fully protected pentasaccharide 44 in 63% yield. The deprotection of 44 was carried out by first converting all azido and NHTroc groups to acetamido with zinc power, acetic acid and acetic anhydride in THF in 65% yield. The major side product isolated was found to carry one dichloroethyloxycarbonyl group due to partial reduction by zinc. Saponification of 45 with lithium hydroxide (LiOH) followed by hydrogenolysis over palladium hydroxide (Pd(OH)2) gave the deprotected 2, completing the first synthesis of pertussis like pentasaccharide. AcO AcO BnO BnO OBn O TrocHN O TBSO N Cbz O O OR NHTroc O AcO 37β HF· Py, Py, 0 °C→r.t. 85% AcO AcO BnO BnO OBn O TrocHN O N Cbz HO O 6, AgOTf, I-TolSCl, 4Å MS, DCM/MeCN, -78 °C→r.t. O OR NHTroc O AcO 38 65% Ph BnO O AcO AcO BnO OBn O O O N3 TrocHN O OLev O O N Cbz O OR NHTroc O AcO 39 Ph N2H4 ·H2O, DCM/AcOH/Py, r.t. 84% BnO O AcO AcO BnO OBn 1. BAIB, TEMPO, DCM/t-BuOH/H2O, r.t. 2. MeI, K2CO3, DMF, r.t. 66% for 2 steps O OH O O N3 TrocHN O O N Cbz O 1. Tf2O, Py, DCM, -30 °C→r.t. 2. NaN3, DMF, 50 °C O OR NHTroc O AcO 40 86% for 2 steps MeOOC HO N3 O N3 TrocHN O O N Cbz BnO BnO O BnO O AcO AcO BnO OBn O AcO 43 O OR NHTroc AgOTf, p-TolSCl, 4Å MS, DCM/Et2O, -78 °C→r.t. 63% N3 O HO HO N3 TrocHN O O N Cbz O DCM/TFA/H2O, r.t. 86% BnO O AcO AcO BnO OBn O AcO 42 O OR NHTroc Ph BnO O AcO AcO BnO OBn OAc O N3 7 STol N3 O O O N3 TrocHN O O O N Cbz O AcO 41 O OR NHTroc BnO BnO OAc O N3 O MeOOC N3 O N3 TrocHN O O O OR NHTroc BnO O AcO AcO BnO OBn O AcO 44 O N Cbz Zn, AcOH, Ac2O, THF, r.t. 65% BnO BnO OAc O AcHN MeOOC O AcHN NHAc O AcHN O O O OR NHAc BnO O AcO AcO BnO OBn O AcO 45 O N Cbz 1) LiOH, THF/H2O, 0 °C→r.t. 2) H2, Pd(OH)2/C, THF/AcOH/H2O, r.t. 68% for 2 steps HO HO OH O AcHN HO O HO HO HO OH HOOC O AcHN NHAc O O NH AcHN O O O O NHAc O HO 2 NH2 R = Bn N Cbz Scheme 2.8. Complete synthesis of the target pentasaccharide 2. 45 2.4. Bioconjugation of Pertussis-like Pentasaccharide with Bacteriophage Qβ Carrier As carbohydrates are typically T cell independent B cell antigens, to generate high titers of anti-glycan IgG antibodies, it is important to link the carbohydrates to an immunogenic carrier capable of activating helper T cells. We have previously demonstrated that bacteriophage Qβ is a superior carrier to deliver tumor associated carbohydrate antigens. To elicit powerful anti-pertussis antibody responses, we investigated the conjugation of pertussis pentasaccharide 2 with Qβ. For bioconjugation, pertussis pentasaccharide 2 was first treated with CSCl2 to convert the free amine to a thiocyanate moiety. However, the highly reactive CSCl2 also reacted with the secondary amine on Unit C, resulting in split methyl peaks on Unit C in NMR and higher molecular weight in ESI-MS. We next investigated NHS activated ester chemistry by treating 2 with adipic acid di-NHS ester 46 to produce pertussis pentasaccharide activated ester 47. The doublet for 6-methyl on Unit C of 47 remained unchanged in 1H-NMR compared to 2. 47 was then incubated with bacteriophage Qβ in PBS buffer at pH 7.4, which successfully introduced pertussis pentasaccharide onto Qβ (Scheme 2.9.1). On average, there were 235 copies of the glycan per Qβ particle according to the analysis via electrospray-ionization mass spectrometry (ESI-MS). To compare the effect of carrier protein in eliciting anti-glycan antibodies, we adopted the commonly used keyhole limpet hemocyanin (KLH). Similar conjugation reaction with 47 resulted in 1180 copies of the glycan per KLH (Scheme 2.9.2), as determined by the anthrone-sulfuric acid assay.19 For the enzyme-linked immunosorbent assay (ELISA) analysis of 46 serum antibodies, a bovine serum albumin (BSA)-glycan conjugate with 14 copies of glycan was prepared (Scheme 2.9.3). 1) HO HO OH O AcHN HOOC O AcHN NHAc O O NH HO O HO HO HO OH AcHN O O O O NHAc O HO 2 NH2 NH2 720 48 KPBS buffer, pH 7.4, overnight O O N O O 46 O O N O O HO HO OH O AcHN HOOC O AcHN NHAc O O NH DIPEA, DMF 90% HO O HO HO HO OH O HO AcHN O O O O NHAc 47 O N H O O N O O HO HO OH O AcHN HO O HO HO HO OH HOOC O AcHN NHAc O O NH AcHN O O O O NHAc O HO O N H H N O 235 2) 3) 50 52 NH2 6000 47, KPBS buffer, pH 7.4, overnight NH2 59 47, KPBS buffer, pH 7.4, overnight 49 HO HO OH O AcHN HOOC O AcHN NHAc O O NH HO O HO HO HO OH HO HO OH O AcHN HO O HO HO HO OH AcHN O O O O NHAc O HO 51 HOOC O AcHN NHAc O O NH AcHN O O O O NHAc O HO 53 O N H O N H H N O 1180 H N O 14 Scheme 2.9. Preparation of protein-glycan conjugates by reaction of activated NHS ester compound 47 with 1) Qβ, 2) KLH and 3) BSA. 2.5. Immunization Study With the Qβ-glycan conjugate 49 in hand, we then investigated its ability to generate 47 anti-glycan antibodies. C57BL/6 mice were immunized subcutaneously with three biweekly injections of 49 containing 2 μg or 8 μg glycan or 51 containing 2 μg glycan, along with MPLA as the adjuvant. Sera were collected from the mice one week after each injection. Unconjugated Qβ was used for immunization for the control group of mice. ELISA analysis of post-immunization mouse sera showed good titers for anti-glycan antibodies, compared to those of the control group of mice immunized by unconjugated Qβ. Although immunization with 49 at different doses did not lead to much difference in antibody level, the carrier protein Qβ elicited much higher IgG antibody response than did KLH (Figure 2.3A). The IgG antibody level reached maximum on day 35 and remained at a high level over 250 days (Figure 2.3B). A study on the subclasses of IgG antibodies indicated a higher level for IgG2 compared with IgG1 and IgG3, which suggested a more Th1-weighted immune response (Figure 2.3C). Booster injection on day 261 was able to raise the antibody level (Figure 2.3D). Since aP vaccines mainly elicited a Th2-skewed immune response,20-23 our synthetic glycoconjugate vaccine might be a good complement to the current treatment schemes. 48 A C B D Figure 2.3. Immunological evaluation of Qβ-glycan conjugate vaccine. A) Comparison of serum IgG titers from mice immunized at difference doses or with different carrier proteins. Qβ-2/8 μg: 49 containing 2/8 μg glycan. Qβ-wt: 48 only. KLH-2 μg: 51 containing 2 μg glycan. B) Serum IgG titer profile over 250 days. Mice were immunized at the dose of 2 μg with 49 on day 0, 14 and 28. C) The level of anti-glycan IgG subclasses measured by ELISA. D) The level of IgG titers as determined by ELISA after another booster injection on day 261. Sera from mice which received the first three injections but not the fourth booster were tested as a control. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns: not significant. To test the binding of our mouse antisera against B. pertussis, the strain Tohama I was used in the flow cytometry experiments (This part was done by the Dr. Jennifer Maynard lab). However, only slightly better binding was observed for the antisera than the negative control despite the high titers against the synthetic glycan, while a much stronger binding was observed for the sera from whole cell vaccine (Figure 2.4, the blue area centered ~ 103). 49 Complement-dependent cytotoxicity was observed for mouse 1, 7 and 9 (Figure 2.5) but overall it was much weaker compared to the sera from whole cell vaccine (data not shown). Figure 2.4. Flow cytometry indicated that the mouse antisera had no significant binding with the bacteria. Only whole-cell vaccine control sera had strong binding against B. pertussis. M1-5 were immunized at the dose of 2 μg, and M6-10 were immunized at the dose of 8 μg. s U F C e g a r e v A 2 0 0 1 5 0 1 0 0 5 0 0 M 1 M 2 M 3 M 4 M 5 M 6 M 7 M 8 M 9 M 10 Figure 2.5. Complement-dependent cytotoxic assay of mouse antisera. The bactericidal activity of mouse antisera was assessed by counting the colony-forming units of B. pertussis. 50 2.6. Conclusions and Future Plans In this study, we successfully synthesized the pentasaccharide from B. pertussis in a linear synthetic route. The key challenge in selective β-fucosylation was solved and well explained by molecular modeling. Conjugation of the pentasaccharide to the carrier protein Qβ elicited a Th1-weighted immune response as well as high titers of anti-glycan antibodies. However, the mouse antisera showed little binding affinity against B. pertussis strain Tohama I and low complement-dependent cytotoxicity. The main difficulty in designing a more convergent synthetic route resides in the choice of protecting groups on the fucose. The remote group participation hampers selective β-fucosylation and therefore any carbamate protection groups have to be ruled out. The current protection group-free protocol helped formation of the β-isomer when the fucose building block was used as the donor in glycosylation reactions. However, previous trials using the same fucose building block as acceptor gave no desired product. Novel protecting schemes for the methylamino group remain to be developed to facilitate the design of a convergent synthetic route for the pentasaccharide or the whole dodecasaccharide. It was considered that the reducing-end glucosamine was not important in immune reactions since conversion to anhydromannose by treatment with nitrous acid still led to antibodies that bound to B. pertussis. However, our synthetic glycan failed to elicit strong-binding antibodies or high bactericidal activity. A previous study on the LOS monoclonal antibodies24 also indicated that the glucosamine played a role in the epitope recognition. Since the activity of antibodies relies on the recognition of epitopes, it is necessary to analyze the epitopes on the synthetic 51 glycan recognized by mouse antisera. Redesigning the carbohydrate antigen accordingly may help the development of a better vaccine against B. pertussis. 2.7. Experimental Section 2.7.1. Synthesis of the Glycan-Carrier Protein Conjugates To the carrier protein (Qβ/BSA/KLH) suspended in potassium phosphate buffer (KPB, 0.1 M, pH 7.0, 1mL) was added compound 47 (4 eq per NH2) in DMSO (0.1 mL). The reaction mixture was rotated on a rotating mixer at room temperature overnight. The solution was then subjected to ultracentrifugation in Millipore 100k MWCO centrifugal filter tube to remove excess glycan. The conjugates were purified by size exclusion chromatography (SEC) on an AKTApure 25L system equipped with Superose 6 Increase 10/300 GL column. 2.7.2. Quantification of Glycan Number on the Carrier Protein For Qβ conjugate, the particle was treated with dithiothreitol (DTT) at 90 ℃ for 30 min. The average number of conjugated glycan on each viral capsid subunit was estimated from the intensity of peaks in the deconvoluted mass spectra from LC-MS analysis. Results are shown in Figure 2.6. For BSA conjugate, the number of conjugated glycan was calculated from the mass obtained from MALDI-TOF MS analysis. Results are shown in Figure 2.7. For KLH conjugate, the number of conjugated glycan was measured with a previously reported anthrone-sulfuric acid assay.19 52 Figure 2.6. ESI-MS analysis of the Qβ-glycan conjugate 49. 53 Figure 2.7. MALDI-TOF MS analysis of the BSA-glycan conjugate 53. 2.7.3. Immunization Studies Pathogen-free C57BL/6 female mice age 6-10 weeks were obtained from Jackson Laboratory and maintained in the University Laboratory Animal Resources facility of Michigan State University. All animal care procedures and experimental protocols have been approved by the Institutional Animal Care and Use Committee (IACUC) of Michigan State University. Groups of 5 mice were injected subcutaneously under the scruff on day 0 with 0.2 mL of Qβ conjugate (contain 2 μg or 8 μg glycan) or KLH conjugate (contain 2 μg glycan) with lipid A 54 monophosphoryl (MPLA, from Salmonella enterica serotype minnesota Re 595, Sigma-Aldrich, 20 μg) as the adjuvant. Boosters at the same dose were given subcutaneously under the scruff on day 14 and 28. Serum samples were collected on day 0 (before immunization), 7, 21 and 35. 2.7.4. Enzyme-linked Immunosorbent Assay (ELISA) A Nunc MaxiSorp® flat-bottom 96 well plate was first coated with BSA-glycan (10 μg/mL) in NaHCO3/Na2CO3 buffer (0.05 M, pH = 9.6) overnight at 4 °C. The coated plate was then washed 4 times with PBS/0.5% Tween-20 (PBST), followed by the addition of 1% (w/v) BSA in PBS to each well and incubation at room temperature for one hour. The plate was washed again 4 times with PBST. 100 μl of the dilution of mouse sera in 0.1% BSA/PBS were added to each well. The plate was incubated for two hours at 37 °C and washed. A 1:2000 diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, IgG1, IgG2b, IgG2c, or IgG3 antibody (Jackson ImmunoResearch Laborator) in 0.1% BSA/PBS was added to each well, respectively. The plate was incubated for one hour at 37 ℃, washed, and a solution of 3,3’,5,5’-tetramethylbenzidine (TMB) was added (200 μL). The color was allowed to develop for 15 min, and then a solution of 0.5 M H2SO4 (50 μL) was added to stop the reaction. The optical density was measured at 450 nm using a microplate autoreader (BioRad). Each experiment was repeated at least four times, and the average of the quadruplicate was used to calculate the titer. The titer was determined by linear regression analysis with reciprocal of dilution plotted with optical density (background subtracted). The titer was calculated as the highest dilution that gave OD = 0.1. 55 2.7.5. Cell Culture of B. pertussis B. pertussis strain Tohama I was grown on 15% BG blood agar plates (Bordet-Gengou agar supplemented with 15% defibrinated sheep’s blood). The plates were placed at the top of 37 ℃ incubator for 3 days. 2.7.6. Flow Cytometry Experiment B. pertussis was scraped from blood plates into FACS buffer in 1.5 mL tubes. They were centrifuged, washed 3 times and adjusted with FACS buffer to O.D. ~1.0 at 600 nm. Primary antibodies or mouse antisera were diluted to proper concentration in 100 μL FACS buffer and 10 μL of B. pertussis was added. The mixture was incubated on ice for 30 min and washed twice. The residue was resuspended in 100 μL 1:50 secondary antibodies (AF647 anti-human Fc or AF647 anti-mouse Fc) and incubated on ice for 30 min. After washing twice, the residue was resuspended in 700 μL FACS buffer and measured by flow cytometry (Thresholds: FSC-500, SSC-200; Voltages: FSC-595, SSC-160). 2.7.7. Complement Assay The entire plate was inoculated in 25mL Stainer-Scholte media (SSM) in a 125mL polycarbonate filter-top Erlenmeyer flask. The culture was then put in a 37 ℃ incubator, shaking at 210rpm until the O.D. reached 0.22 (~3-4 hours). Note: the culture was not allowed to grow past 5 hours regardless of the optical density to avoid Brk- mutations and complement resistance. In this time, the reaction plate was set up. Duplicate wells were established in a non-treated 96-well assay plate with lid (Costar, 3370) for all of the antibodies to be tested and the controls. A total volume of 40uL of SSM or SSM + 1.25 μg antibody was added to the duplicate wells. 56 Once at the proper O.D., the culture was serially diluted ten-fold twice to get a working stock solution. 5uL of this bacterial working stock was added to all wells and mixed thoroughly. The plate was covered and allowed to equilibrate at 37 ℃ for 30 min. 5 μL was then added to each of the wells: naïve sera to all of the antibody samples and the duplicate naïve control wells, infected sera to those controls, and 5uL of SSM to the media only control. This reaction was covered and allowed to incubate 1 hour at 37 ℃. Blood plates were brought to room temperature at this time. After the hour, 15 μL from each well was diluted into 135 μL of PBS and then 50 μL was taken from this first tube into a second tube of 450 μL PBS to achieve two ten-fold dilutions. 7.5 μL was then taken three times from the plate to create three spaced out drops across the top of a blood plate, forming row one. Three drops were then added below this from the first tube and then below that from the second tube. This was repeated for all the wells and the plates were allowed to dry thoroughly. The plates were then covered, inverted, and placed on the top rack of a 37 ℃ incubator for 3 days. After 3 days, the number of colonies per drip was counted, and the colony number from the second row (first dilution tube) was reported as an average of the three drops over the two duplicate sample plates. 2.7.8. Product Preparation and Characterization Data OAc AcO N3 O OAc STol p-Tolyl 2, 4, 6-tri-O-acetyl-3-azido-3-deoxy-1-thio-β-D-glucopyranoside (9) 10% HCl (25 mL) was added to 3-azido-1,2:5,6-di-O-isopropylidene-3-deoxy-α-D-allofuranose2 57 (1.52 g, 5.3 mmol) and the mixture was stirred at r.t. for 16 hours. The solution was then concentrated, diluted with pyridine (10 mL) and cooled to 0℃. Acetic anhydride (Ac2O, 12 mL) and DMAP (100 mg, 0.8 mmol) were added. The solution was allowed to warm up slowly to r.t. and stirred overnight. Upon completion, excess Ac2O was quenched by the slow addition of MeOH. The reaction mixture was concentrated under vacuum, diluted with EtOAc and washed successively with 1M HCl, saturated Na2CO3 solution and saturated brine. The organic layer was then dried over anhydrous Na2SO4, filtered and concentrated. The product was mixed with p-toluenethiol (1.02 g, 8.2 mmol), dissolved in DCM and cooed to 0℃. Then BF3·Et2O (3.0 mL, 24.6 mmol) was added and the reaction was stirred for 6 hours. The mixture was washed with saturated NaHCO3 solution, dried and concentrated. Compound 9 was purified from column chromatography (Hexanes/EtOAc = 3/1) as a white powder in 54% yield. 1HNMR (500 MHz, CDCl3): δ = 2.08 (s, 3H), 2.11 (s, 3H), 2.18 (s, 3H), 2.35 (s, 3H), 3.62-3.66 (m, 2H), 4.16-4.17 (m, 2H), 4.58 (d, 1H, J = 10 Hz), 4.87 (t, 1H, J = 10 Hz), 4.92 (t, 1H, J = 10 Hz), 7.11-7.13 (m, 2H), 7.38-7.40 (m, 2H). 13CNMR (125 MHz, CDCl3): δ = 20.85, 20.96, 21.06, 21.38, 62.37, 65.99, 68.44, 70.19, 76.52, 86.59, 128.02, 129.86, 133.79, 138.93, 169.25, 169.37, 170.81. HRMS: m/z calc. for C19H27N4O7S: 455.1600; found: 455.1614 [M + NH4]+. Ph O O N3 O OH STol p-Tolyl 3-azido-4, 6-O-benzylidene-3-deoxy-1-thio-β-D-glucopyranoside (10) Compound 9 (1.5 g, 3.4 mmol) was dissolved in a mixture of DCM/MeOH (7/7 mL) and the pH 58 was adjusted to 11 with 30% NaOMe. The reaction was stirred for 1 h and then neutralized with H+ resin. The mixture was then filtered through celite and concentrated. The residue was diluted with MeCN (40 mL) and then benzaldehyde dimethyl acetal (0.77 mL, 5.1 mmol) and (1S)-(+)-10-camphorsulfonic acid (CSA, 230 mg, 1.0 mmol) was added to the solution, which was allowed to stir at r.t. overnight. Upon completion as judged by TLC, the reaction was quenched with Et3N and concentrated. The residue was diluted with DCM and washed with brine. Compound 10 was obtained through recrystallization in Hexanes/EtOAc (4/1) as a white powder in 81% yield. 1HNMR (500 MHz, CDCl3): δ = 2.37 (s, 3H), 2.62 (d, 1H, J = 2.5 Hz), 3.35 (dt, 1H, J = 2.5, 9.5 Hz), 3.47 (t, 1H, J = 9.5 Hz), 3.54 (dt, 1H, J = 5, 9.5 Hz), 3.72 (t, 1H, J = 9.5 Hz), 3.76 (t, 1H, J = 9.5 Hz), 4.39 (dd, 1H, J = 5, 10.5 Hz), 4.56 (d, 1H, J = 10 Hz), 5.55 (s, 1H), 7.15-7.17 (m, 2H), 7.35-7.39 (m, 3H), 7.41-7.43 (m, 2H), 7.46-7.49 (m, 2H). 13CNMR (125 MHz, CDCl3): δ = 21.41, 65.97, 68.74, 71.64, 71.7, 79.33, 89.34, 101.71, 126.17, 126.71, 128.52, 129.37, 130.2, 134.12, 136.78, 139.41. HRMS: m/z calc. for C20H22N3O4S: 400.1331; found: 400.1323 [M + H]+. Ph O O N3 O OLev STol p-Tolyl 3-azido-4, 6-O-benzylidene-3-deoxy-2-levulinoyl-1-thio-β-D-glucopyranoside (6) Compound 10 (1.0 g, 2.5 mmol) was dissolved in DCM (40 mL) followed by the addition of levulinic acid (0.77 mL, 7.5 mmol), EDC·HCl (1.58 g, 8.3 mmol) and DMAP (31 mg, 0.25 59 mmol). The reaction was stirred at r.t. overnight and then washed with saturated NaHCO3 solution. Compound 6 was obtained through column chromatography (Hexanes/EtOAc = 2/1) as a white solid in 91% yield. 1HNMR (500 MHz, CDCl3): δ = 2.23 (s, 3H), 2.35 (s, 3H), 2.67-2.74 (m, 2H), 2.80-2.90 (m, 2H), 3.52 (dt, 1H, J = 5.0, 9.5 Hz), 3.57 (t, 1H, J = 9.5 Hz), 3.76-3.81(m, 2H), 4.39 (dd, 1H, J = 4.5, 10.5 Hz), 4.66 (d, 1H, J = 9.5 Hz), 4.85 (t, 1H, J = 9.5 Hz), 5.57 (s, 1H), 7.12-7.16 (m, 2H), 7.34-7.41 (m, 5H), 7.45-7.49 (m, 2H). 13CNMR (125 MHz, CDCl3): δ = 21.33, 28.09, 30.0, 37.94, 64.74, 68.58, 70.69, 71.34, 79.17, 87.3, 101.54, 126.07, 127.91, 128.44, 129.3, 129.9, 133.79, 136.63, 138.92, 171.3, 206.12. HRMS: m/z calc. for C25H27N3NaO6S: 520.1518; found: 520.1517 [M + Na]+. HO AcO STol O OH p-Tolyl 3-O-acetyl-1-thio-α-L-rhamnopyranoside (11) Compound 11 was prepared by following the previously reported protocol.25 1HNMR (500 MHz, CDCl3): δ = 1.35 (d, 3H, J = 6.0 Hz), 2.18 (s, 3H), 2.33 (s, 3H), 2.42 (br, 2H), 3.71 (t, 1H, J = 9.5 Hz), 4.23 (dq, 1H, J = 6.5, 9.5 Hz), 4.29 (dd, 1H, J = 1.5, 3.0 Hz), 5.05 (dd, 1H, J = 3.0, 9.5 Hz), 5.38 (d, 1H, J = 1.5 Hz), 7.12 (d, 2H, J = 8.0 Hz), 7.35 (d, 2H, J = 8.5 Hz). 13CNMR (125 MHz, CDCl3): δ = 17.58, 21.25, 21.27, 70.0, 71.21, 71.69, 75.02, 88.11, 129.94, 130.04, 132.33, 138.04, 171.6. HRMS: m/z calc. for C15H20NaO5S: 335.0929; found 335.0937 [M + Na]+. 60 HO O STol HO OH p-Tolyl 1-thio-α-L-rhamnopyranoside (12) Compound 12 was prepared by following the previously reported protocol.26 1HNMR (500 MHz, CD3OD): δ = 1.31 (d, 3H, J = 6.0 Hz), 2.31 (s, 3H), 3.25 (dq, 1H, J = 6.0, 9.0 Hz), 3.37 (t, 1H, J = 9.0 Hz), 3.44 (dd, 1H, J = 3.5, 9.5 Hz), 4.04 (dd, 1H, J = 1.0, 3.5 Hz), 4.86 (d, 1H, J = 1.0 Hz), 7.12 (d, 2H, J = 7.5 Hz), 7.36 (d, 2H, J = 8.0 Hz). 13CNMR (125 MHz, CD3OD): δ = 18.24, 21.05, 73.64, 74.19, 75.91, 77.88, 89.04, 130.62, 131.87, 133.3, 138.2. HRMS: m/z calc. for C13H18NaO4S: 293.0823; found 293.0833 [M + Na]+. HO BzO O STol OH p-Tolyl 3-O-benzoyl-1-thio-β-L-rhamnopyranoside (13) Compound 13 was prepared by following the previously reported protocol.25 1HNMR (500 MHz, CDCl3): δ = 1.44 (d, 3H, J = 6.0 Hz), 2.28 (br, 1H), 2.35 (s, 3H), 2.43 (br, 1H), 3.46 (dq, 1H, J = 6.0, 9.0 Hz), 3.89 (t, 1H, J = 9.5 Hz), 4.39 (s, 1H), 4.89 (d, 1H, J = 1.0 Hz), 4.98 (dd, 1H, J = 3.0, 10.0 Hz), 7.12-7.16 (m, 2H), 7.40-7.49 (m, 4H), 7.56-7.62 (m, 1H), 8.06-8.11 (m, 2H). 13CNMR (125 MHz, CDCl3): δ = 18.08, 21.29, 70.83, 70.98, 77.13, 77.8, 87.46, 128.69, 129.44, 129.84, 130.02, 130.04, 132.6, 133.77, 138.3, 166.83. HRMS: m/z calc. for C20H22NaO5S: 397.1086; found 397.1089 [M + Na]+. 61 O N3 OBz PhthN STol p-Tolyl 2-azido-3-O-benzoyl-2, 4-dideoxy-4-phthalimido-1-thio-β-L-rhamnopyranoside (14) Compound 14 was prepared by following the previously reported protocol.6 1HNMR (500 MHz, CDCl3): δ = 1.18 (d, 3H, J = 6.5 Hz), 2.37 (s, 3H), 3.94-4.00 (m, 1H), 4.65 (d, 1H, J = 10.5 Hz), 4.89 (t, 1H, J = 10.0 Hz), 5.02 (dd, 1H, J = 3.0, 7.0 Hz), 5.36 (dd, 1H, J = 7.0, 9.5 Hz), 7.14-7.19 (m, 2H), 7.27-7.33 (m, 2H), 7.43-7.51 (m, 2H), 7.52-7.57 (m, 2H), 7.66-7.79 (m, 3H), 7.82-7.87 (m, 2H). 13CNMR (125 MHz, CDCl3): δ = 17.07, 21.37, 51.7, 62.15, 73.19, 73.59, 89.45, 123.78, 128.58, 128.72, 128.8, 129.77, 129.84, 129.91, 130.03, 130.07, 133.12, 133.66, 134.24, 138.4, 165.19. HRMS: m/z calc. for C28H25N4O5S: 529.1546; found: 529.1548 [M + H]+. HO HN O STol O OH CCl3 p-Tolyl 1-thio-3-trichloroacetimidate-β-L-rhamnopyranoside (16) p-Tolyl 1-thio-β-L-rhamnopyranoside 12 (11.5 g, 42.7 mmol) was dissolved in THF (150 mL) followed by the addition of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 1.3 mL, 8.5 mmol). To this mixture, trichloroacetonitrile (5.1 mL, 51 mmol) in THF (30 mL) was added over a period of 1 h at 0 ℃. The reaction was allowed to warm up and stirred at r.t. overnight. It was concentrated and purified through column (DCM/EtOAc = 3/1). The impurities (not characterized, might be from extra side reactions on 2- and 4-OH) were dissolved in DCM/MeOH/AcOH (40/40/5 mL) 62 and purified with the same condition, which gave compound 16 as a white solid in a combined yield of 90%. 1HNMR (500 MHz, CDCl3): δ = 1.43 (d, 3H, J = 6.5 Hz), 2.34 (s, 3H), 2.81 (br, 3H), 3.46 (dq, 1H, J = 1.0, 6.5 Hz), 4.18 (dd, 1H, J = 1.0, 6.5 Hz), 4.54 (t, 1H, J = 6.5 Hz), 4.90-4.94 (m, 2H), 7.13 (d, 2H, J = 7.5 Hz), 7.44 (m, 2H). 13CNMR (125 MHz, CDCl3): δ = 18.89, 21.28, 71.91, 76.28, 80.19, 82.7, 84.66, 103.51, 117.13, 129.92, 130.95, 131.91, 138.01. HRMS: m/z calc. for C15H18Cl3NNaO4S: 435.9920; found: 435.9928 [M + Na]+. O N O CCl3 STol N3 p-Tolyl 2-azido-2, 4-dideoxy-1-thio-3, 4-trichlorooxazoline-β-L-fucopyranoside (17) Compound 16 (16.0 g, 38.5 mmol) was dissolved in anhydrous DCM (200 mL) and cooled to -30 ℃. Pyridine (31.0 mL, 0.38 mol) and trifluoromethanesulfonic anhydride (19.4 mL, 0.12 mol) were added and the solution was allowed to warm up to r.t. over 3 h. Upon completion by TLC, the reaction was quenched and washed with saturated NaHCO3 solution. The organic layer was dried with Na2SO4, concentrated and redissolved in DMF (60 mL). NaN3 (7.5 g, 0.12 mol) was added and the reaction was stirred at 50 ℃ overnight. The mixture was diluted with EtOAc, washed with brine, dried and concentrated. Compound 17 was obtained through column chromatography (Hexanes/DCM/EtOAc = 3/1/1) as a colorless solid in a yield of 85%. 1HNMR (500 MHz, CDCl3): δ = 1.58 (d, 3H, J = 6.0 Hz), 2.33 (s, 3H), 3.29 (dd, 1H, J = 7.0, 10.0 Hz), 3.89 (dq, 1H, J = 3.0, 6.5 Hz), 4.10 (dd, 1H, J = 3.0, 8.0 Hz), 4.34 (d, 1H, J = 10.5 Hz), 63 4.88 (dd, 1H, J = 6.5, 8.0 Hz), 7.14 (m, 2H), 7.46 (m, 2H). 13CNMR (125 MHz, CDCl3): δ = 18.16, 21.16, 62.31, 68.78, 73.03, 84.86, 85.37, 86.31, 126.71, 129.83, 134.12, 138.9, 162.64. HRMS: m/z calc. for C15H16Cl3N4O2S: 421.0060; found: 421.0072 [M + H]+. O OH H2N STol N3 p-Tolyl 4-amino-2-azido-2, 4-dideoxy-1-thio-β-L-fucopyranoside (15) Compound 17 (13.9 g, 33.0 mmol) was dissolved in DCM/MeOH/H2O/conc. HCl (40/50/20/30 mL) and the mixture was refluxed at 90 ℃ overnight. The reaction was then concentrated, diluted with DCM and washed with saturated NaHCO3 solution. The organic layer was collected and dried with Na2SO4. Compound 15 was obtained through column chromatography (DCM/MeOH = 10/1) as a colorless syrup in a yield of 89%. 1HNMR (500 MHz, CDCl3): δ = 1.28 (d, 3H, J = 6.5 Hz), 2.35 (s, 3H), 2.82 (d, 1H, J = 4.0 Hz), 3.04 (t, 1H, J = 10.0 Hz), 3.51 (dd, 1H, J = 4.5, 9.5 Hz), 3.61 (q, 1H, J = 6.5 Hz), 4.28 (d, 1H, J = 10.5 Hz), 7.15 (d, 2H, J = 8.5 Hz), 7.48 (d, 2H, J = 8.0 Hz). 13CNMR (125 MHz, CDCl3): δ = 17.16, 21.21, 54.31, 63.35, 73.61, 75.07, 86.04, 127.56, 129.77, 133.98, 138.72. HRMS: m/z calc. for C13H19N4O2S: 295.1229; found: 295.1242 [M + H]+. O OH NH STol NH2 p-Tolyl 2-amino-2, 4-dideoxy-4-methylamino-1-thio-β-L-fucopyranoside (18) Compound 15 (6.1 g, 20.7 mmol) was dissolved in ethyl formate (50 mL) and Et3N (3 mL) was 64 added. The mixture was refluxed overnight and concentrated. The obtained syrup was diluted in anhydrous THF (60 mL) and cooled to -30 ℃. To the solution was added LiAlH4 in THF (37.2 mL, 2M). The mixture was refluxed overnight and quenched with 15% NaOH, followed by filtration through celite and concentration. Compound 18 was obtained through column chromatography (DCM/MeOH = 8/1 with 1% Et3N) as a colorless oil in a yield of 70%. 1HNMR (500 MHz, CD3OD): δ = 1.31 (d, 3H, J = 6.5 Hz), 2.32 (s, 3H), 2.51 (s, 3H), 2.54 (dd, 1H, J = 1.0, 4.0 Hz), 2.61 (t, 1H, J = 10.0 Hz), 3.43 (dd, 1H, J = 4.5, 9.5 Hz), 3.68 (dq, 1H, J = 1.0, 6.5 Hz), 4.37 (d, 1H, J = 10.0 Hz), 7.14 (d, 2H, J = 8.0 Hz), 7.43 (d, 2H, J = 8.5 Hz). 13CNMR (125 MHz, CD3OD): δ = 16.85, 19.73, 37.59, 52.36, 63.5, 74.1, 75.2, 89.15, 129.26, 129.45, 132.15, 137.68. HRMS: m/z calc. for C28H44N4NaO4S2: 587.2702; found: 587.2707 [2M + Na]+. O OH NH p-Tolyl STol NHTroc 2,4-dideoxy-4-methylamino-1-thio-2-(2,2,2-trichloroethyloxycarbonylamino)- β-L-fucopyranoside (19) Compound 18 (3.7 g, 13.1 mmol) was dissolved in THF (100 mL) and pyridine (3.2 mL, 39.2 mmol) was added. The mixture was cooled in ice bath and trichloroethyl chloroformate (1.7 mL, 12.4 mmol) in THF (50 mL) was added slowly over 1 h. The reaction was concentrated, diluted with DCM and washed with saturated NaHCO3 solution. Compound 19 was obtained through column chromatography (DCM/MeOH = 6/1 with 1% Et3N) as a white solid in a yield of 88%. 65 1HNMR (500 MHz, CDCl3): δ = 1.37 (d, 3H, J = 6.5 Hz), 2.32 (s, 3H), 2.60 (s, 3H), 2.64 (dd, 1H, J = 1.5, 4.5 Hz), 3.20 (q, 1H, J = 10.0 Hz), 3.57 (dd, 1H, J = 4.0, 9.5 Hz), 3.70 (q, 1H, J = 6.5 Hz), 4.61 (d, 1H, J = 10.5 Hz), 4.68 (d, 1H, J = 12.0 Hz), 4.81 (d, 1H, J = 12.0 Hz), 5.29 (br, 1H), 7.10 (d, 2H, J = 8.0 Hz), 7.37 (d, 2H, J = 7.5 Hz). 13CNMR (125 MHz, CDCl3): δ = 17.93, 21.13, 38.39, 55.43, 63.07, 71.52, 74.55, 75.68, 87.3, 95.54, 129.63, 129.67, 132.55, 137.95, 154.51. HRMS: m/z calc. for C17H24Cl3N2O4S: 457.0522; found: 457.0523 [M + H]+. Cbz STol NHTroc O N OH p-Tolyl 4-[N-(methyl)-benzyloxycarbonylamino]-2,4-dideoxy-1-thio-2- (2,2,2-trichloroethyloxycarbonylamino)-β-L-fucopyranoside (5) Compound 19 (3.0 g, 6.55 mmol) was dissolved in THF/H2O (40/10 mL) and cooled to 0℃. To the solution was added benzyl chloroformate (1.1 mL, 7.86 mmol) and sodium carbonate (1.39 g, 13.1 mmol). The reaction was allowed to warm up to room temperature and stirred overnight. The reaction was concentrated, diluted with DCM and washed with saturated NaHCO3 solution. Compound 5 was obtained through column chromatography (Hexanes/EtOAc = 1/1) as a white solid in a yield of 75%. 1HNMR (500 MHz, d6-DMSO): δ = 1.05&1.07 (d, 3H, J = 6.0 Hz, H-6), 2.27&2.28 (s, 3H, STol-Me), 2.82&2.88 (s, 3H, N-Me), 3.64-3.73 (m, 1H, H-2), 3.75-3.88 (m, 2H, H-3, H-5), 4.27&4.35 (dd, 1H, J = 2.5, 6.0 Hz, H-4), 4.66&4.69 (d, 1H, J = 10.0 Hz, H-1), 4.76 (d, 1H, J = 12.5 Hz, Troc-CH2), 4.91&4.92 (d, 1H, J = 12.5 Hz, Troc-CH2), 4.98&5.10 (d, 1H, J = 13.0 Hz, 66 Cbz-CH2), 5.08 (s, 1H, Cbz-CH2), 5.45&5.53 (d, 1H, J = 6.0 Hz, OH), 7.12-7.17 (m, 2H), 7.27-7.39 (m, 7H), 7.73 (d, 1H, J = 9.5 Hz, Troc-NH). 13CNMR (125 MHz, d6-DMSO): δ = 16.8, 16.95, 20.64, 32.56, 32.98, 54.01, 54.11, 56.87, 56.92, 66.17, 66.29, 69.66, 69.74, 73.49, 73.51, 73.9, 74.04, 86.32, 96.26, 127.16, 127.27, 127.61, 127.68, 128.31, 128.4, 129.46, 129.49, 129.52, 129.59, 131.63, 131.8, 136.9, 136.99, 137.13, 137.2, 154.46, 156.97, 157.19. HRMS: m/z calc. for C25H30Cl3N2O6S: 591.0890; found: 591.0878 [M + H]+. BnO BnO OBn O STol p-Tolyl 2, 3, 4-tri-O-benzyl-6,7-dideoxy-1-thio-α-D-mannohept-6-enopyranoside (21) To a solution of oxalyl chloride (202 μL, 2.36 mmol) in DCM (4 mL) was added a solution of DMSO (200 μL, 2.83 mmol) in DCM (6 mL) at -65℃. After 15 min, a solution of p-Tolyl 2, 3, 4-tri-O-benzyl-1-thio-α-D-mannopyranoside 20 (875 mg, 1.57 mmol) in DCM (5 mL) was added to the above solution via syringe. The reaction was allowed to stir at -50 ℃ for 2 h. Et3N was added and the above mixture was warmed up to r.t. over a period of 4 h before it was quenched with water and extracted with DCM. The organic layer was washed with brine, dried and concentrated to give the crude aldehyde which was used without further purification. To a suspension of methyl triphenylphosphonium bromide (843 mg, 2.36 mmol) in THF (6 mL) at -40 ℃ was added n-BuLi (0.94 mL, 2.36 mmol, 2.5 M solution in hexane) and after 0.5 h, the above aldehyde in THF (5 mL) was added and the mixture was stirred at the same temperature for 1 h. The reaction was slowly warmed up to r.t. over 4 h before quenched with saturated 67 NH4Cl solution and extracted with EtOAc. The organic layer was washed with brine, dried and concentrated. Compound 21 was obtained through column chromatography (Hexanes/EtOAc = 10/1) as a yellow syrup in a yield of 83%. 1HNMR (500 MHz, CDCl3): δ = 2.33 (s, 3H), 3.81 (t, 1H, J = 9.5 Hz), 3.88 (dd, 1H, J = 3.0, 9.5 Hz), 4.00 (dd, 1H, J = 1.5, 3.0 Hz), 4.56 (dd, 1H, J = 6.5, 9.5 Hz), 4.60-4.68 (m, 4H), 4.73 (d, 1H, J = 12.5 Hz), 4.88 (d, 1H, J = 11.0 Hz), 5.28-5.31 (m, 1H), 5.43-5.49 (m, 2H), 6.04 (ddd, 1H, J = 6.0, 10.5, 17.0 Hz), 7.10 (d, 2H, J = 8.0 Hz), 7.26-7.38 (m, 17H). 13CNMR (125 MHz, CDCl3): δ = 21.25, 72.19, 72.47, 73.87, 75.4, 76.57, 78.97, 79.86, 86.26, 118.43, 127.8, 127.81, 127.87, 127.94, 128.11, 128.19, 128.46, 128.51, 128.53, 129.93, 130.72, 132.29, 135.13, 137.77, 138.04, 138.38, 138.54. HRMS: m/z calc. for C35H40NO4S: 570.2678; found: 570.2681 [M + NH4]+. HO HO BnOBnO OBn O STol p-Tolyl 2, 3, 4-tri-O-benzyl-D-glycero-1-thio-α-D-mannoheptopyranoside (22) To a solution of compound 21 (2.57 g, 4.65 mmol) in Acetone/water (27/3 mL) at 0 ℃ were added 4-methylmorpholine N-oxide (NMO, 1.09 g, 9.3 mmol) and OsO4 (2.3 mL, 0.18 mmol, 2.5%wt in t-BuOH). The reaction was allowed to stir at r.t. for 6 h before it was quenched with saturated NaHSO3 solution. After 15 min, the mixture was concentrated and extracted with EtOAc. The organic layer was washed with brine, dried and concentrated. Column chromatography (Hexanes/EtOAc = 2/1) afforded compound 22 (1.3 g, 48%) and compound 23 68 (0.52 g, 20 %). 1HNMR (500 MHz, CDCl3): δ = 2.04 (br, 1H), 2.33 (s, 3H), 3.06 (br, 1H), 3.57 (dd, 1H, J = 4.0, 11.5 Hz), 3.65 (dd, 1H, J = 5.0, 12.0 Hz), 3.90 (dd, 1H, J = 3.0, 9.0 Hz), 3.93 (q, 1H, J = 4.5 Hz), 3.99 (dd, 1H, J = 2.0, 3.0 Hz), 4.05 (t, 1H, J = 9.5 Hz), 4.22 (dd, 1H, J = 5.0, 9.5 Hz), 4.58 (d, 1H, J = 11.5 Hz), 4.61 (d, 1H, J = 11.5 Hz), 4.63-4.69 (m, 3H), 5.04 (d, 1H, J = 11.0 Hz), 5.39 (d, 1H, J = 1.5 Hz), 7.12 (d, 2H, J = 8.0 Hz), 7.27-7.38 (m, 17H). 13CNMR (125 MHz, CDCl3): δ = 21.14, 63.09, 71.93, 72.24, 72.3, 72.66, 75.08, 75.87, 76.63, 80.25, 86.25, 127.86, 127.9, 127.93, 128.05, 128.18, 128.47, 128.52, 128.57, 129.57, 129.96, 132.75, 137.57, 137.68, 137.72, 138.25. HRMS: m/z calc. for C35H38NaO6S: 609.2287; found: 609.2278 [M + Na]+. HO HO BnOBnO OBn O STol p-Tolyl 2, 3, 4-tri-O-benzyl-L-glycero-1-thio-α-D-mannoheptopyranoside (23) Compound 22 (1.28 g, 2.18 mmol) was dissolved in pyridine (8 mL) and t-butyldiphenylsilyl chloride (TBDPSCl, 1.13 mL, 4.26 mmol) was added. The reaction was stirred at r.t. overnight before it was diluted with DCM and washed with brine. The organic layer was dried and concentrated to give the crude silyl ether which was taken forward without purification. A solution of the silyl ether, PPh3 (1.14 g, 4.26 mmol) and p-nitrobenzoic acid (0.73 g, 4.36 mmol) in THF (30 mL) was treated with DIAD (858 μL, 4.36 mmol) at r.t. and stirred for 5 h. The reaction was the concentrated, diluted with DCM and washed with brine. The organic layer was 69 concentrated and diluted with DCM/MeOH (10/30 mL) and K2CO3 (0.5 g, 3.57 mmol) was added. The reaction was stirred at r.t. for 3 h before concentrated, diluted with EtOAc and washed with brine. The organic layer was concentrated, dissolved in THF (30 mL) and treated with TBAF (3.5 mL, 3.5 mmol, 1 M in THF) overnight. The mixture was then concentrated, diluted with EtOAc and washed with brine. Column chromatography (Hexanes/EtOAc = 2/1) gave compound 23 in an 84% yield over 4 steps. 1HNMR (500 MHz, CDCl3): δ = 2.33 (s, 3H), 3.56 (m, 2H), 3.88 (dd, 1H, J = 3.0, 9.0 Hz), 3.97 (m, 2H), 4.05 (dd, 1H, J = 1.0, 9.5 Hz), 4.20 (t, 1H, J = 9.5 Hz), 4.62 (d, 1H, J = 12.0 Hz), 4.65 (d, 1H, J = 12.0 Hz), 4.69 (s, 2H), 4.71 (d, 1H, J = 10.5 Hz), 4.98 (d, 1H, J = 10.5 Hz), 5.47 (d, 1H, J = 1.5 Hz), 7.12 (d, 2H, J = 8.5 Hz), 7.24 (d, 2H, J = 8.0 Hz), 7.27-7.37 (m, 15H). 13CNMR (125 MHz, CDCl3): δ = 21.29, 65.21, 69.25, 72.37, 72.52, 74.0, 74.51, 75.56, 76.05, 80.08, 86.36, 127.93, 127.97, 127.99, 128.0, 128.05, 128.26, 128.61, 129.54, 130.21, 132.47, 137.96, 138.18, 138.37, 138.41. HRMS: m/z calc. for C35H42NO6S: 604.2733; found: 604.2745 [M + NH4]+. AcO AcO BnO BnO OBn O STol p-Tolyl 6, 7-di-O-acetyl-2, 3, 4-tri-O-benzyl-L-glycero-1-thio-α-D-mannoheptopyranoside (3) Compound 23 (1.64 g, 2.80 mmol) was dissolved in pyridine (10 mL) and treated with Ac2O (1.1 mL, 11.2 mmol) and DMAP (17 mg, 0.14 mmol) at 0 ℃. The reaction was allowed to warm up to r.t. and stirred overnight. It was then concentrated, diluted with EtOAc and washed with 1 M 70 HCl, brine, saturated NaHCO3 and brine in order. The organic layer was dried and concentrated. Column chromatography (Hexanes/EtOAc = 3/1) gave compound 3 as a colorless syrup in a yield of 91%. 1HNMR (500 MHz, CDCl3): δ = 1.92 (s, 3H), 2.12 (s, 3H), 2.32 (s, 3H), 3.86-3.92 (m, 2H), 3.99 (dd, 1H, J = 1.5, 3.0 Hz), 4.04-4.13 (m, 2H), 4.24 (d, 1H, J = 13.0 Hz), 4.47 (d, 1H, J = 9.5 Hz), 4.58 (s, 2H), 4.64 (d, 1H, J = 12.5 Hz), 4.76 (d, 1H, J = 12.5 Hz), 4.90 (d, 1H, J = 10.0 Hz), 5.63 (d, 1H, J = 1.5 Hz), 5.66 (ddd, 1H, J = 1.5, 6.0, 7.5 Hz), 7.09 (d, 2H, J = 8.5 Hz), 7.23 (d, 2H, J = 8.5 Hz), 7.27-7.39 (m, 15H). 13CNMR (125 MHz, CDCl3): δ = 20.65, 20.95, 21.07, 62.29, 68.24, 70.77, 71.77, 72.0, 73.67, 75.36, 75.47, 80.36, 85.54, 127.8, 127.85, 127.91, 127.96, 128.38, 128.45, 128.48, 128.57, 129.91, 130.0, 131.25, 137.72, 137.79, 137.88, 170.31, 170.41. HRMS: m/z calc. for C39H46NO8S: 688.2944; found: 688.2937 [M + NH4]+. AcO AcO AcO S1 O STol NHTroc S2, AgOTf, p-TolSCl, 4Å MS, DCM/MeCN, -78°C Bn N Cbz HO S2 84% AcO AcO AcO O OR NHTroc S3 1. NaOMe, DCM/MeOH 2. PhCH(OMe)2, CSA, DMF 78% for 2 steps Ph O O HO S4 O OR NHTroc 1. Ac2O, Py, DMAP 2. CSA, MeOH/DCM 84% for 2 steps HO HO AcO O OR NHTroc S5 TBDPSCl, Py, 16h 95% TBDPSO HO AcO O OR NHTroc 4 R = Bn N Cbz Scheme 2.10. Preparation of building block 4. 71 AcO AcO AcO O O NHTroc Bn N Cbz N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 3,4,6-tri-O-acetyl-2-deoxy-2- (2,2,2-trichloroethyloxycarbonylamino)-β-D-glucopyranoside (S3) A solution of p-tolyl 3,4,6-tri-O-acetyl-2-deoxy-2- (2,2,2-trichloroethyloxycarbonylamino) -1-thio-β-D-glucopyranoside S1 (2.12 g, 3.61 mmol), N-(benzyl)-benzyloxycarbonyl- 3-aminopropanol S2 (1.0 g, 3.33 mmol) and freshly activated 4 Å molecular sieves (1.5 g) in DCM (50 mL) was stirred at r.t. for 20 min and then cooled to -78 ℃. To the above solution was added silver trifluoromethanesulfonate (AgOTf, 2.23 g, 8.68 mmol) in MeCN (2.5 mL). The mixture was stirred for 10 min and p-TolSCl (480 μL, 3.61 mmol) was added directly into it via microsyringe. The reaction was allowed to stir at the same temperature for 2 h before quenched with Et3N (0.5 mL). The mixture was then filtered through celite and concentrated. Column chromatography gave compound S3 in a yield of 84%. 1HNMR (500 MHz, CDCl3): δ = 1.73 (m, 2H), 2.04 (s, 3H), 2.05 (s, 3H), 2.08 (s, 3H), 2.98 (m, 1H), 3.20-3.50 (m, 2H), 3.56 (m, 1H), 3.75 (q, 1H, J = 9.5 Hz), 3.83 (m, 1H), 3.92 (m, 1H), 4.10 (d, 1H, J = 12.0 Hz), 4.24 (d, 0.5H, J = 4.5 Hz), 4.27 (d, 0.5H, J = 5.0 Hz), 4.34 (d, 1H, J = 9.0 Hz), 4.52-4.78 (m, 3H), 5.07 (m, 1H), 5.13-5.22 (m, 3H), 6.29 (d, 1H, J = 9.0 Hz), 7.17 (d, 1H, J = 7.0 Hz), 7.27-7.44 (m, 9H). 13CNMR (125 MHz, CDCl3): δ = 20.66, 20.75, 20.77, 27.31, 43.03, 50.08, 56.13, 61.99, 67.07, 67.49, 68.64, 71.74, 73.09, 74.31, 101.19, 127.2, 127.47, 127.95, 128.03, 128.53, 128.64, 136.76, 137.45, 154.79, 156.5, 169.46, 170.64, 170.75. HRMS: m/z calc. for C33H39Cl3N2NaO12: 783.1466; found: 783.1440 [M + Na]+. 72 Ph O O HO O O NHTroc Bn N Cbz N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 4,6-O-benzylidene-2-deoxy-2- (2,2,2-trichloroethyloxycarbonylamino)-β-D-glucopyranoside (S4) Compoud S3 (2.13 g, 2.80 mmol) was dissolved in DCM/MeOH (10/10 mL) and the solution was cooled to -10 ℃. NaOMe was added to adjust the pH to 11 and the solution was stirred at the same temperature for 2 h. After neutralizing with H+ resin, the solution was filtered, concentrated and diluted in DMF (10 mL). Benzaldehyde dimethyl acetal (611 μL, 4.07 mmol) and CSA (166 mg, 0.72 mmol) were added and the mixture was heated at 50 ℃ overnight. Upon completion by TLC the reaction was quenched with Et3N, diluted with EtOAc and washed with brine. The organic layer was dried and concentrated. Column chromatography (Hexanes/EtOAc = 1/1) gave compound S4 in a 78% yield over 2 steps. 1HNMR (500 MHz, CDCl3): δ = 1.69-1.77 (m, 2H), 2.94 (d, 1H, J = 14.0 Hz), 3.27-3.39 (m, 2H), 3.46-3.61 (m, 2H), 3.69-3.82 (m, 2H), 3.83-3.94 (m, 2H), 4.21-4.32 (m, 2H), 4.34 (d, 1H, J = 8.5 Hz), 4.42-4.66 (m, 2H), 4.70 (d, 1H, J = 16.0 Hz), 4.81 (d, 1H, J = 12.0 Hz), 5.13-5.27 (m, 2H), 5.54 (s, 1H), 6.93 (br, 1H), 7.17 (d, 2H, J = 7.5 Hz), 7.26-7.43 (m, 11H), 7.46-7.54 (m, 2H). 13CNMR (125 MHz, CDCl3): δ = 27.45, 42.92, 43.34, 50.05, 50.76, 59.02, 59.04, 66.03, 66.13, 66.91, 67.38, 67.56, 68.6, 72.98, 74.61, 81.43, 95.6, 101.32, 101.89, 126.38, 127.2, 127.5, 127.89, 128.15, 128.23, 128.34, 128.37, 128.53, 128.62, 128.67, 129.25, 136.65, 137.1, 137.37, 156.38, 156.65. HRMS: m/z calc. for C34H38Cl3N2O9: 723.1643; found: 723.1631 [M + H]+. 73 HO HO AcO O O NHTroc Bn N Cbz N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 3-O-acetyl-2-deoxy-2- (2,2,2-trichloroethyloxycarbonylamino)-β-D-glucopyranoside (S5) To a solution of S4 (1.66 g, 2.30 mmol) in pyridine (6 mL) was added acetic anhydride (1 mL, 10 mmol)) and DMAP (20 mg, 0.16 mmol) at 0 ℃. The solution was stirred at r.t. overnight before it was quenched with MeOH, concentrated, diluted with EtOAc and washed with brine. The organic layer was dried, concentrated and dissolved in DCM/MeOH (8/8 mL). CSA (150 mg, 0.65 mmol) was added and the solution was stirred at r.t. overnight, followed by quenching with Et3N. The mixture was concentrated, diluted with EtOAc, washed with brine, dried and concentrated again. Column chromatography (DCM/EtOAc = 1/2) gave compound S5 in an 84% yield over 2 steps. 1HNMR (500 MHz, CDCl3): δ =1.71-1.77 (m, 2H), 2.10 (s, 3H), 2.64 (br, 1H), 3.05-3.16 (m, 2H), 3.21-3.37 (m, 2H), 3.39-3.45 (m, 1H), 3.60-3.74 (m, 2H), 3.76-3.93 (m, 3H), 4.27-4.35 (m, 1H), 4.37 (d, 1H, J = 8.5 Hz), 4.48-4.63 (m, 2H), 4.64-4.68 (m, 1H), 4.95-4.99 (m, 1H), 5.13-5.22 (m, 2H), 6.18 (d, 1H, J = 8.5 Hz), 7.11-7.20 (m, 2H), 7.25-7.42 (m, 8H). 13CNMR (125 MHz, CDCl3): δ = 21.14, 27.65, 28.16, 43.38, 43.63, 50.31, 56.12, 62.26, 67.42, 67.65, 69.71, 74.44, 75.73, 76.27, 95.83, 101.41, 127.34, 127.60, 128.04, 128.21, 128.66, 128.78, 136.70, 137.59, 154.99, 156.65, 172.24. HRMS: m/z calc. for C29H35Cl3N2NaO10: 699.1255; found: 699.1227 [M + Na]+. 74 TBDPSO HO AcO O O NHTroc Bn N Cbz N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 3-O-acetyl-6-tert-butyldiphenylsilyl-2-deoxy-2- (2,2,2-trichloroethyloxycarbonylamino)-β-D-glucopyranoside (4) To a solution of S5 (0.86 g, 1.27 mmol) in pyridine (6 mL) was added TBDPSCl (0.65 mL, 2.5 mmol) and the mixture was allowed to stir at r.t. overnight. Then it was concentrated, diluted with EtOAc and washed with brine. The organic layer was dried, concentrated and purified through column chromatography to afford compound 4 in a 95% yield. 1HNMR (500 MHz, CDCl3): δ = 1.07 (s, 9H), 1.70-1.75 (m, 2H), 2.12 (s, 3H), 2.97-3.05 (m, 1H), 3.09-3.16 (m, 1H), 3.18-3.41 (m, 3H), 3.65-3.85 (m, 4H), 3.86-3.95 (m, 2H), 4.22-4.33 (m, 1H), 4.40-4.57 (m, 1H), 4.59-4.72 (m, 2H), 4.91-5.02 (m, 1H), 5.10-5.24 (m, 2H), 6.14 (d, 1H, J = 9.0 Hz), 7.12-7.19 (m, 2H), 7.26-7.48 (m, 14H), 7.63-7.73 (m, 4H). 13CNMR (125 MHz, CDCl3): δ = 19.32, 21.16, 22.78, 25.4, 26.91, 27.03, 27.49, 28.33, 31.71, 34.79, 43.46, 50.32, 50.97, 55.99, 64.79, 66.66, 67.4, 67.54, 71.28, 74.4, 74.61, 74.86, 74.98, 76.08, 95.87, 100.64, 101.14, 127.33, 127.53, 127.91, 127.95, 128.0, 128.06, 128.16, 128.63, 128.73, 130.04, 132.74, 132.93, 135.67, 135.76, 136.83, 137.7, 137.91, 154.28, 155.01, 156.51, 156.82, 171.92. HRMS: m/z calc. for C45H57Cl3N3O10Si: 932.2879; found: 932.2835 [M + NH4]+. 75 AcO AcO BnO BnO OBn O O AcO OTBDPS O O NHTroc Bn N Cbz N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6, 7-di-O-acetyl-2, 3, 4-tri-O-benzyl-L-glycero -α-D-mannoheptopyranosyl-(1→4)-3-O-acetyl-6-tert-butyldiphenylsilyl-2-deoxy-2- (2,2,2-trichloroethyloxycarbonylamino)-β-D-glucopyranoside (24) A solution of compound 3 (160 mg, 0.24 mmol), 4 (197 mg, 0.21 mmol) and freshly activated 4 Å molecular sieves (300 mg) in DCM (5 mL) was stirred at r.t. for 20 min and then cooled to -78 ℃. To the above solution was added AgOTf (153 mg, 0.60 mmol) in Et2O/DCM (6/1 mL). The mixture was stirred for 10 min and p-TolSCl (31.5 μL, 0.24 mmol) was added directly into it via microsyringe. The reaction was allowed to warm up to r.t. over a period of 2 h before quenched with Et3N. The mixture was then filtered through celite and concentrated. Column chromatography gave compound 24 in a yield of 80%. 1HNMR (500 MHz, CDCl3): δ = 1.03 (s, 9H), 1.71-1.76 (m, 2H), 1.88 (s, 3H), 1.92 (s, 3H), 2.07 (s, 3H), 2.95-3.00 (m, 1H), 3.24-3.36 (m, 3H), 3.55-3.68 (m, 3H), 3.72-3.80 (m, 4H), 3.82-3.92 (m, 3H), 4.06 (dd, 1H, J = 7.5, 11.5 Hz), 4.23 (d, 1H, J = 8.0 Hz), 4.28 (d, 1H, J = 15.0 Hz), 4.43 (d, 1H, J = 10.5 Hz), 4.46-4.54 (m, 1H), 4.55-4.58 (m, 2H), 4.62-4.69 (m, 4H), 4.77-4.84 (m, 1H), 5.04-5.10 (m, 1H), 5.14-5.22 (m, 3H), 5.43-5.46 (m, 1H), 6.16 (d, 1H, J = 8.5 Hz), 7.14-7.19 (m, 2H), 7.22-7.41 (m, 29H), 7.62-7.71 (m, 4H). 13CNMR (125 MHz, CDCl3): δ = 19.42, 20.86, 21.07, 26.93, 27.04, 27.46, 43.34, 50.31, 50.99, 56.68, 63.11, 63.29, 66.53, 67.62, 68.7, 72.02, 72.29, 72.43, 73.82, 74.42, 74.72, 75.19, 75.50, 75.58, 75.86, 79.8, 99.21, 100.75, 76 127.34, 127.58, 127.64, 127.79, 127.85, 127.87, 127.98, 128.14, 128.44, 128.51, 128.57, 128.61, 128.65, 128.77, 129.8, 129.91, 133.02, 133.42, 135.68, 135.89, 136.85, 137.71, 138.13, 138.22, 155.06, 156.55, 170.28, 170.48, 170.54. Two C1-H1 coupling constants (171.0, 159.5 Hz) confirmed the stereochemistry. HRMS: m/z calc. for C77H91Cl3N3O18Si: 1478.5132; found: 1478.5088 [M + NH4]+. AcO AcO BnO BnO OBn O O AcO OH O O NHTroc Bn N Cbz N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6, 7-di-O-acetyl-2, 3, 4-tri-O-benzyl-L-glycero -α-D-mannoheptopyranosyl-(1→4)-3-O-acetyl-2-deoxy-2-(2,2,2-trichloroethyloxycarbonylamin o)-β-D-glucopyranoside (25) To a solution of 24 (190 mg, 0.13 mmol) in pyridine (4 mL) in a plastic centrifuge tube, HF pyridine complex (2 mL) was added at 0 ℃. The mixture was allowed to warm up to r.t. and stirred overnight. It was then diluted with DCM, washed with saturated CuSO4 solution, 1M HCl and brine. The organic layer was dried and concentrated. Column chromatography (Hexanes/EtOAc = 1/1) gave 25 in a yield of 85%. 1HNMR (500 MHz, CDCl3): δ = 1.72-1.76 (m, 2H), 1.94 (s, 3H), 2.05 (s, 3H), 2.11 (s, 3H), 2.44 (br, 1H), 3.06-3.14 (m, 1H), 3.23-3.31 (m, 2H), 3.35-3.42 (m, 1H), 3.60-3.77 (m, 4H), 3.77-3.90 (m, 6H), 4.26-4.36 (m, 3H), 4.37-4.43 (dd, 1H, J = 4.5, 11.5 Hz), 4.47 (d, 1H, J = 11.5 Hz), 4.57-4.72 (m, 6H), 4.77-4.84 (m, 1H), 5.14-5.23 (m, 4H), 5.60-5.65 (m, 1H), 6.12 (m, 1H), 77 7.14-7.18 (m, 2H), 7.24-7.44 (m, 23H). 13CNMR (125 MHz, CDCl3): δ = 20.92, 21.02, 21.15, 27.58, 29.77, 43.41, 50.21, 56.59, 61.33, 63.06, 67.32, 67.58, 68.85, 72.0, 72.2, 72.78, 73.75, 74.09, 74.36, 74.88, 74.99, 75.11, 75.64, 79.56, 95.73, 99.22, 101.17, 127.26, 127.52, 127.57, 127.64, 127.79, 127.95, 128.09, 128.45, 128.49, 128.52, 128.57, 128.71, 136.73, 137.57, 137.91, 138.09, 138.14, 154.87, 156.56, 170.49, 170.59, 170.76. HRMS: m/z calc. for C61H70Cl3N2O18: 1223.3689; found: 1223.3668 [M + H]+. Ph O O O N3 TrocHN TolS OLev O Cbz O N p-Tolyl 3-azido-4,6-O-benzylidene-3-deoxy-2-levulinoyl-β-D-glucopyranosyl-(1→3)-4-[N-(methyl)-ben zyloxycarbonylamino]-2,4-dideoxy-2-(2,2,2-trichloroethyloxycarbonylamino)-β-L-fucopyranosi de (26) A solution of compound 6 (788 mg, 1.58 mmol) and freshly activated 4 Å molecular sieves (1.6 g) in DCM (20 mL) was stirred at r.t. for 20 min and then cooled to -78 ℃. To the above solution was added AgOTf (1.02 g, 3.96 mmol) in Et2O/DCM (20/2 mL). The mixture was stirred for 10 min and p-TolSCl (209 μL, 1.58 mmol) was added directly into it via microsyringe. After activation completed as indicated by disappearance of orange color and by TLC, acceptor 4 (797 mg, 1.35 mmol) in DCM (5 mL) was added slowly along the wall of the flask. Another 3 mL of DCM was used to rinse once. The reaction was allowed to warm up to r.t. over a period of 2 h before quenched with Et3N. The mixture was then filtered through celite and concentrated. 78 Column chromatography gave compound 26 in a yield of 70%. 1HNMR (500 MHz, CDCl3): δ = 1.20-1.28 (m, 3H), 2.13&2.21 (s, 3H), 2.33&2.34 (s, 3H), 2.40-2.82 (m, 4H), 2.86&2.94 (s, 3H), 3.03 (t, 1H, J = 10.0 Hz), 3.37-3.73 (m, 5H), 3.77-3.88 (m, 1.5H), 4.22 (dd, 0.5H, J = 5.0, 10.5 Hz), 4.32 (d, 1H, J = 8.0 Hz), 4.37-4.47 (m, 1H), 4.53 (0.5H, dd, J = 2.5, 7.0 Hz), 4.59 (d, 0.5H, J = 12.0 Hz), 4.64&4.66 (d, 0.5H, J = 10.0 Hz), 4.68 (d, 0.5H, J = 7.5 Hz), 4.71-4.84 (m, 3H), 5.01 (d, 1H, J = 12.0 Hz), 5.14 (d, 0.5 H, J = 12.0 Hz), 5.23 (d, 1H, J = 12.5 Hz), 5.35 (d, 0.5H, J = 12.0 Hz), 5.49&5.54 (s, 1H), 7.07-7.13 (m, 2H), 7.28-7.44 (m, 10H), 7.45-7.52 (m, 2H). 13CNMR (125 MHz, CDCl3): δ = 21.1, 27.61, 29.82, 32.73, 37.59, 37.82, 51.55, 53.18, 62.78, 63.06, 67.22, 67.68, 71.25, 71.56, 85.48, 96.84, 99.94, 101.57, 125.95, 126.01, 127.92, 128.19, 128.38, 128.55, 128.9, 128.95, 129.23, 129.26, 129.67, 129.7, 133.85, 134.16, 136.44, 138.49, 138.69, 156.49, 157.6. Two C1-H1 coupling constants (163.0, 161.0 Hz) confirmed the stereochemistry. Most peaks were split due to the secondary amides on the fucose. HRMS: m/z calc. for C43H49Cl3N5O12S: 964.2164; found: 964.2117 [M + H]+. Ph O O N3 TrocHN TolS O OH Cbz O N O p-Tolyl 3-azido-4,6-O-benzylidene-3-deoxy-β-D-glucopyranosyl-(1→3)-4-[N-(methyl)-benzyloxycarbon ylamino]-2,4-dideoxy-2-(2,2,2-trichloroethyloxycarbonylamino)-β-L-fucopyranoside (27) Compound 26 (366 mg, 0.379 mmol) was dissolved in DCM/Pyridine (10/0.05 mL) followed by addition of N2H4·AcOH (50 mg, 0.543 mmol). The mixture was stirred at r.t. for 4 h before it 79 was diluted with DCM and washed with brine. The organic layer was dried and concentrated. Column chromatography (Hexanes/DCM/EtOAc = 1/1/1) gave 27 as white solid in a yield of 93%. 1HNMR (500 MHz, CDCl3): δ = 1.31 (d, 3H, J = 6.5 Hz), 2.33 (s, 3H), 2.98 (s, 3H), 3.40 (t, 1H, J = 9.5 Hz), 3.42-3.52 (m, 2H), 3.61 (t, 1H, J = 9.5 Hz), 3.76 (t, 1H, J = 9.5 Hz), 3.88 (dd, 1H, J = 3.0, 6.5 Hz), 4.14 (dd, 1H, J = 5.5, 11.5 Hz), 4.32 (dd, 1H, J = 5.0, 10.5 Hz), 4.40 (d, 1H, J = 7.5 Hz), 4.59 (dd, 1H, J = 3.0, 5.5 Hz), 4.76 (d, 1H, J = 12.5 Hz), 4.81 (d, 1H, J = 12.0 Hz), 4.86 (d, 1H, J = 2.0 Hz), 4.91 (d, 1H, J = 10.5 Hz), 5.09 (d, 1H, J = 12.5 Hz), 5.20 (d, 1H, J = 12.5 Hz), 5.34 (d, 1H, J = 7.5 Hz), 5.52 (s, 1H), 7.08-7.13 (m, 2H), 7.32-7.45 (m, 10H), 7.46-7.52 (m, 2H). 13CNMR (125 MHz, CDCl3): δ = 17.3, 21.33, 33.2, 53.4, 56.21, 64.49, 67.58, 68.27, 68.58, 73.32, 74.35, 74.63, 79.02, 81.7, 86.27, 95.62, 101.73, 106.53, 125.42, 126.18, 127.75, 128.15, 128.35, 128.46, 128.49, 128.78, 129.16, 129.37, 129.8, 134.42, 136.23, 136.67, 138.92, 154.22, 159.87. Two C1-H1 coupling constants (161.0, 156.5 Hz) confirmed the stereochemistry. HRMS: m/z calc. for C38H43Cl3N5O10S: 866.1796; found: 866.1745 [M + H]+. Ph N3 O O O N3 TrocHN TolS O Cbz O N p-Tolyl 2,3-diazido-4,6-O-benzylidene-2,3-dideoxy-β-D-mannopyranosyl-(1→3)-4-[N-(methyl)-benzylo xycarbonylamino]-2,4-dideoxy-2-(2,2,2-trichloroethyloxycarbonylamino)-β-L-fucopyranoside 80 (28) Compound 27 (307 mg, 0.35 mmol) was dissolved in anhydrous DCM (10 mL) and cooled to -30 ℃. Pyridine (285 μL, 3.54 mmol) and Tf2O (179 μL, 1.06 mmol) were added and the mixture was allowed to warm up to r.t. over a period over 4 h. It was then quenched with MeOH, diluted with DCM and washed with brine. The organic layer was dried, concentrated and dissolved with DMF (10 mL). NaN3 (140 mg, 2.17 mmol) was added and the mixture was heated at 50 ℃ overnight. After diluting with EtOAc and washing with brine, compound 28 was purified through column chromatography (Hexanes/DCM/EtOAc = 3/2/1) in a yield of 84% over 2 steps. 1HNMR (500 MHz, CDCl3): δ = 1.31 (d, 3H, J = 6.5 Hz), 2.33 (s, 3H), 2.96 (s, 3H), 3.26 (d, 1H, J = 3.5 Hz), 3.32 (dt, 1H, J = 5.0, 9.5 Hz), 3.46 (dd, 1H, J = 4.0, 10.0 Hz), 3.60-3.69 (m, 1H), 3.74-3.89 (m, 3H), 4.12-4.20 (m, 1H), 4.26 (dd, 1H, J = 4.5, 10.5 Hz), 4.56-4.62 (m, 2H), 4.72 (d, 1H, J = 1.5 Hz), 4.80 (d, 1H, J = 10.5 Hz), 4.95 (d, 1H, J = 12.0 Hz), 5.06 (d, 1H, J = 12.5 Hz), 5.14 (d, 1H, J = 6.5 Hz), 5.20 (d, 1H, J = 12.5 Hz), 5.56 (s, 1H), 7.08-7.14 (m, 2H), 7.32-7.51 (m, 12H). 13CNMR (125 MHz, CDCl3): δ = 17.3, 21.32, 32.88, 52.83, 54.13, 59.96, 62.38, 67.74, 67.94, 68.37, 74.39, 74.64, 76.07, 76.73, 86.75, 95.69, 98.6, 101.61, 101.76, 125.92, 125.99, 128.05, 128.43, 128.61, 128.72, 128.98, 129.26, 129.3, 129.74, 129.77, 134.19, 134.53, 136.63, 136.67, 138.74, 154.22, 158.48. Two C1-H1 coupling constants (163.5, 163.0 Hz) confirmed the stereochemistry. Most peaks were split due to the secondary amides on the fucose. HRMS: m/z calc. for C38H42Cl3N8O9S: 891.1861; found: 891.1813 [M + H]+. 81 Ph N3 O O O N3 TrocHN OBn O O Cbz O N O AcO AcO BnOBnO O AcO O O NHTroc N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl Bn N Cbz 2,3-diazido-4,6-O-benzylidene-2,3-dideoxy -β-D-mannopyranosyl-(1→3)-4-[N-(methyl)-benzyloxycarbonylamino]-2,4-dideoxy-2-(2,2,2-tric hloroethyloxycarbonylamino)-α-L-fucopyranosyl-(1→6)-[6,7-di-O-acetyl-2,3,4-tri-O-benzyl-L-g lycero-α-D-mannoheptopyranosyl-(1→4)]-3-O-acetyl-2-deoxy-2-(2,2,2-trichloroethyloxycarbon ylamino)-β-D-glucopyranoside (29) A solution of compound 28 (50 mg, 0.0561 mmol), 25 (55 mg, 0.0449 mmol) and freshly activated 4 Å molecular sieves (100 mg) in DCM (5 mL) was stirred at r.t. for 20 min and then cooled to -78 ℃. To the above solution was added AgOTf (36 mg, 0.14 mmol) in Et2O/DCM (3/0.5 mL). The mixture was stirred for 10 min and p-TolSCl (7.4 μL, 0.0561 mmol) was added directly into it via microsyringe. The reaction was allowed to warm up to r.t. over a period of 2 h before quenched with Et3N. The mixture was then filtered through celite and concentrated. Column chromatography (Hexanes/DCM/EtOAc = 2/2/1) gave compound 29 in a yield of 73%. 1HNMR (500 MHz, CDCl3): δ = 1.20 (d, 3H, J = 7.0 Hz), 1.65-1.77 (m, 2H), 1.86 (s, 3H), 2.06 (s, 3H), 2.12 (s, 3H), 2.85-2.94 (m, 1H), 3.19 (s, 3H), 3.25-3.31 (m, 1H), 3.34-3.49 (m, 4H), 3.53 (dd, 1H, J = 4.0, 10.0 Hz), 3.60-3.72 (m, 3H), 3.73-3.91 (m, 8H), 4.00-4.08 (m, 1H), 4.12 (dd, 1H, J = 4.0, 11.0 Hz), 4.15-4.32 (m, 5H), 4.33-4.41 (m, 1H), 4.47 (d, 2H, J = 10.0 Hz), 4.55-4.65 (m, 5H), 4.67-4.77 (m, 4H), 4.77-4.88 (m, 3H), 5.01-5.09 (m, 2H), 5.11 (d, 1H, J = 12.5 Hz), 82 5.14-5.23 (m, 3H), 5.35 (d, 1H, J = 4.0 Hz), 5.40 (d, 1H, J = 7.5 Hz), 5.58 (s, 1H), 5.60-5.65 (m, 1H), 6.46 (d, 1H, J = 8.5 Hz), 7.12-7.19 (m, 2H), 7.26-7.52 (m, 33H). 13CNMR (125 MHz, CDCl3): δ = 16.69, 16.74, 20.95, 21.0, 21.06, 27.37, 29.81, 33.21, 42.84, 50.05, 51.12, 54.54, 56.33, 59.92, 62.56, 63.17, 65.0, 65.82, 66.86, 67.69, 67.88, 68.25, 68.42, 68.64, 72.08, 72.23, 72.67, 73.78, 74.02, 74.39, 74.44, 74.6, 75.27, 75.42, 75.52, 76.69, 77.36, 77.63, 79.58, 95.92, 97.66, 99.08, 100.75, 101.75, 125.95, 126.03, 127.26, 127.58, 127.61, 127.66, 127.89, 127.96, 128.02, 128.1, 128.12, 128.39, 128.47, 128.58, 128.6, 128.68, 128.76, 129.1, 129.16, 129.21, 136.62, 136.76, 137.38, 137.76, 137.98, 138.04, 154.41, 155.17, 156.65, 158.49, 170.37, 170.42, 170.55. Four C1-H1 coupling constants (177.0, 171.0, 166.0, 163.5 Hz) confirmed the stereochemistry. Most peaks were split due to the secondary amides on the fucose and the linker. HRMS: m/z calc. for C92H106Cl6N11O27: 2006.5391; found: 2006.5326 [M + NH4]+. Cbz STol NHTroc O N OAc p-Tolyl 3-O-acetyl-4-[N-(methyl)-benzyloxycarbonylamino]-2,4-dideoxy-1-thio-2-(2,2,2-trichloroethylo xycarbonylamino)-β-L-fucopyranoside (31) Compound 5 (102 mg, 0.172 mmol) was dissolved in pyridine (3 mL) followed by addition of DMAP (10 mg) and acetic anhydride (200 μL) at 0 ℃. The reaction was stirred at room temperature overnight. Upon completion by TLC, the reaction was diluted with EtOAc, washed with 1 M HCl, sat. NaHCO3 solution and brine. Column chromatography (Hexanes/DCM/EtOAc 83 = 1/1/1) gave compound 31 in a yield of 85%. 1HNMR (500 MHz, CDCl3): δ = 1.22&1.27 (d, 3H, J = 6.5 Hz, H-6), 1.76 (s, 3H, Ac), 2.32 (s, 3H, STol-Me), 2.87&2.92 (s, 3H, N-Me), 3.83-3.92 (m, 1H, H-5), 3.92-4.01 (m, 1H, H-2), 4.48-4.52&4.56-4.60 (m, 1H, H-4), 4.59&4.64 (d, 1H, J = 10.5 Hz, H-1), 4.73 (d, 1H, J = 11.5 Hz, Troc-CH2), 4.80 (d, 1H, J = 12.0 Hz Troc-CH2), 5.02&5.04 (d, 1H, J = 12.5 Hz, Cbz-CH2), 5.08&5.17 (d, 1H, J = 12.5 Hz, Cbz-CH2), 5.12 (dd, 1H, J = 5.5, 11.0 Hz, H-3), 5.20-5.30 (m, 1H, Troc-NH), 7.08-7.14 (m, 2H), 7.27-7.38 (m, 5H), 7.41-7.47 (m, 2H). 13CNMR (125 MHz, CDCl3): δ = 17.07, 17.08, 20.45, 20.55, 21.3, 32.78, 33.17, 51.74, 51.86, 54.64, 54.78, 67.47, 67.73, 71.71, 71.83, 74.59, 74.64, 86.48, 86.95, 95.65, 127.55, 127.9, 128.13, 128.33, 128.35, 128.6, 128.64, 129.7, 129.74, 134.4, 134.74, 136.37, 136.82, 138.81, 154.14, 154.35, 157.31, 158.07, 170.51. HRMS: m/z calc. for C27H32Cl3N2O7S: 633.0996; found: 633.1012 [M + H]+. Cbz STol NHTroc O N OPico p-Tolyl 4-[N-(methyl)-benzyloxycarbonylamino]-2,4-dideoxy-3-O-picoloyl-1-thio-2-(2,2,2-trichloroethy loxycarbonylamino)-β-L-fucopyranoside (32) Compound 5 (134 mg, 0.226 mmol) was dissolved in DCM (5 mL), followed by addition of picolinic acid (84 mg, 0.678 mmol), EDC·HCl (143 mg, 0.747 mmol) and DMAP (5.5 mg, 0.045 mmol). The reaction was stirred at room temperature overnight before concentrated and washed with sat. NaHCO3 solution. The organic phase was dried over Na2SO4, concentrated and purified 84 through column (Hexanes/DCM/EtOAc = 2/2/3) to obtain compound 32 in a yield of 94%. 1HNMR (500 MHz, CDCl3): δ = 1.26&1.29 (d, 3H, J = 6.5 Hz, H-6), 2.30&2.33 (s, 3H, STol-Me), 2.96&3.00 (s, 3H, N-Me), 3.96-4.02&4.10-4.15 (m, 1H, H-5), 4.15-4.24 (m, 1H, H-2), 4.53&4.90 (d, 1H, J = 12.5 Hz, Cbz-CH2), 4.58&4.69 (d, 1H, J = 12.0 Hz, Troc-CH2), 4.62 (s, 1H, Troc-CH2), 4.65&4.84 (dd, 1H, J = 3.0, 6.5 Hz, H-4), 4.80&4.93 (d, 1H, J = 10.5 Hz, H-1), 4.82&4.86 (d, 1H, J = 12.5 Hz, Cbz-CH2), 5.56&5.77 (dd, 1H, J = 6.0, 11.0 Hz, H-3), 5.96&4.82 (d, 1H, J = 9.0 Hz, Troc-NH), 7.03-7.30 (m, 6H), 7.35-7.90 (m, 5H), 8.69-8.79 (m, 1H). 13CNMR (125 MHz, CDCl3): δ = 17.12, 17.15, 21.3, 32.94, 33.28, 51.67, 51.74, 54.97, 55.02, 67.28, 67.72, 73.44, 73.8, 74.34, 74.51, 74.54, 86.0, 86.14, 95.54, 95.65, 125.17, 125.46, 126.91, 126.99, 127.21, 127.32, 127.7, 127.96, 128.09, 128.13, 128.39, 128.49, 129.6, 129.71, 134.83, 135.0, 135.96, 136.65, 137.14, 137.51, 138.77, 139.07, 146.81, 147.11, 150.17, 150.36, 154.49, 154.68, 157.39, 157.98, 163.63, 163.85. HRMS: m/z calc. for C31H33Cl3N3O7S: 696.1105; found: 696.1123 [M + H]+. Pico STol NHTroc O N OPico p-Tolyl 4-[N-(methyl)-N-(picoloyl)-amino]-2,4-dideoxy-3-O-picoloyl-1-thio-2-(2,2,2-trichloroethyloxyc arbonylamino)-β-L-fucopyranoside (33) Compound 19 (157 mg, 0.343 mmol) was dissolved in DCM (5 mL), followed by addition of picolinic acid (127 mg, 1.03 mmol), EDC·HCl (237 mg, 1.24 mmol) and DMAP (8.4 mg, 0.069 85 mmol). The reaction was stirred at room temperature overnight before concentrated and washed with sat. NaHCO3 solution. The organic phase was dried over Na2SO4, concentrated and purified through column (DCM/MeOH = 8/1) to obtain compound 33 in a yield of 99%. 1HNMR (500 MHz, CDCl3): δ = 1.21&1.39&1.43 (d, 3H, J = 6.5 Hz, H-6), 2.29&2.32&2.35 (s, 3H, STol-Me), 3.08&3.12 (s, 3H, N-Me), 3.73-3.79&3.92-3.96&4.07-4.14 (m, 1H, H-5), 4.18-4.31&5.33-5.38 (m, 1H, H-2), 4.62&4.74&4.80 (d, 1H, J = 12.0 Hz, Troc-CH2), 4.61-4.67 (m, 1H, Troc-CH2), 4.68&3.91 (dd, 1H, J = 3.0, 6.5 Hz, H-4), 4.73&4.85 (d, 1H, J = 10.0 Hz, H-1), 5.42&5.75 (dd, 1H, J = 6.5, 11.0 Hz, H-3), 5.58-5.67&5.91-6.01 (m, 1H, Troc-NH), 7.04-7.19 (m, 3H), 7.26-7.53 (m, 4H), 7.65-8.01 (m, 2H), 8.20-8.77 (m, 2H). 13CNMR (125 MHz, CDCl3): δ = 16.99, 17.13, 17.41, 21.29, 21.35, 32.47, 36.32, 51.94, 52.41, 52.57, 57.4, 60.24, 72.59, 72.62, 74.07, 74.39, 74.93, 86.09, 86.8, 95.47, 95.56, 123.16, 123.84, 124.33, 124.43, 125.07, 125.32, 125.68, 126.13, 126.62, 127.19, 127.28, 127.44, 129.69, 129.74, 129.77, 134.56, 134.73, 135.25, 136.92, 137.06, 137.12, 137.44, 138.71, 138.8, 139.29, 147.09, 147.14, 147.16, 148.1, 148.61, 150.03, 150.27, 153.8, 153.94, 154.37, 154.59, 163.73, 164.12, 169.94, 171.18, 171.33. HRMS: m/z calc. for C29H30Cl3N4O6S: 667.0952; found: 667.0966 [M + H]+. Cbz STol NHTroc O N OLev p-Tolyl 4-[N-(methyl)-benzyloxycarbonylamino]-2,4-dideoxy-3-O-levuniloyl-1-thio-2-(2,2,2-trichloroet hyloxycarbonylamino)-β-L-fucopyranoside (34) 86 Compound 5 (1.81 g, 3.06 mmol) was dissolved in DCM (40 mL), followed by addition of levulinic acid (626 μL, 6.12 mmol), EDC·HCl (1.46 g, 7.64 mmol) and DMAP (38 mg, 0.31 mmol). The reaction was stirred at room temperature overnight before concentrated and washed with sat. NaHCO3 solution. The organic phase was dried over Na2SO4, concentrated and purified through column (Hexanes/DCM/EtOAc =1/1/1) to obtain compound 34 in a yield of 93%. 1HNMR (500 MHz, CDCl3): δ = 1.21&1.25 (d, 3H, J = 6.5 Hz, H-6), 2.06&2.08 (s, 3H, Lev-Me), 2.10-2.60 (m, 4H, Lev-CH2), 2.31 (s, 3H, STol-Me), 2.87&2.92 (s, 3H, N-Me), 3.87-3.99 (m, 2H, H-2, H-5), 4.50&4.57 (dd, 1H, J = 3.0, 6.0 Hz, H-4), 4.64&4.69 (d, 1H, J = 10.5 Hz, H-1), 4.68&4.87&4.90 (d, 2H, J = 12.0 Hz, Troc-CH2), 5.02&5.05&5.16 (d, 2H, J = 12.5 Hz, Cbz-CH2), 5.11 (dd, 1H, J = 6.0, 10.0 Hz, H-3), 5.51&5.61 (d, 1H, J = 9.5 Hz, Troc-NH), 7.07-7.12 (m, 2H), 7.27-7.37 (m, 5H), 7.38-7.45 (m, 2H). 13CNMR (125 MHz, CDCl3): δ = 17.04, 17.09, 21.27, 27.83, 29.75, 29.76, 32.81, 33.19, 37.76, 37.84, 51.63, 51.89, 54.61, 54.78, 67.44, 67.68, 71.88, 72.15, 74.43, 74.56, 86.27, 86.79, 95.73, 127.87, 128.09, 128.19, 128.23, 128.56, 128.58, 129.66, 129.69, 134.13, 134.53, 136.51, 136.87, 138.62, 138.87, 154.43, 157.27, 158.01, 171.86, 172.15, 206.52. HRMS: m/z calc. for C30H36Cl3N2O8S: 689.1258; found: 689.1236 [M + H]+. STol NHTroc O OTBS NH p-Tolyl 3-O-tert-butyldimethylsilyl-2,4-dideoxy-4-methylamino-1-thio-2-(2,2,2-trichloroethyloxycarbon 87 ylamino)-β-L-fucopyranoside (35) Compound 19 (1.2 g, 2.62 mmol) was dissolved in DCM (50 mL) followed by the addition of t-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf, 0.9 mL, 3.93 mmol) and 2, 6-lutidine (0.61 mL, 5.24 mmol) at -40℃. The mixture was allowed to warm up to r.t. and stirred overnight. Compound 35 was obtained through column chromatography (DCM/EtOAc = 3/1) as a white solid in a yield of 87%. 1HNMR (500 MHz, CDCl3): δ = 0.06 (s, 3H), 0.08 (s, 3H), 0.88 (s, 9H), 1.33 (d, 3H, J = 6.5 Hz), 2.31 (s, 3H), 2.51 (dd, 1H, J = 1.5, 4.5 Hz), 2.53 (s, 3H), 3.46 (q, 1H, J = 9.5 Hz), 3.59 (q, 1H, J = 6.5 Hz), 3.94 (dd, 1H, J = 3.5, 10.0 Hz), 4.64 (d, 1H, J = 11.5 Hz), 4.70 (d, 1H, J = 12.0 Hz), 4.82 (d, 1H, J = 10.5 Hz), 4.99 (d, 1H, J = 8.5 Hz), 7.07 (d, 2H, J = 8.0 Hz), 7.39 (d, 2H, J = 8.0 Hz). 13CNMR (125 MHz, CDCl3): δ = -4.83, -4.31, 18.01, 18.23, 21.19, 25.75, 39.01, 54.54, 64.93, 73.81, 74.75, 75.68, 86.87, 95.34, 129.62, 130.2, 132.48, 137.57, 153.78. HRMS: m/z calc. for C23H38Cl3N2O4SSi: 571.1387; found: 571.1378 [M + H]+. AcO AcO BnO BnO N Cbz LevO O TrocHN OBn O O O AcO O O NHTroc Bn N Cbz N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 4-[N-(methyl)-benzyloxycarbonylamino]-2,4-dideoxy-3-O-levuniloyl-2-(2,2,2-trichloroethyloxy carbonylamino)-α-L-fucopyranosyl-(1→6)-[6,7-di-O-acetyl-2,3,4-tri-O-benzyl-L-glycero-α-D-m 88 annoheptopyranosyl-(1→4)]-3-O-acetyl-2-deoxy-2-(2,2,2-trichloroethyloxycarbonylamino)-β-D- glucopyranoside (36α) A solution of compound 34 (51 mg, 0.0735 mmol), 25 (72 mg, 0.0588 mmol) and freshly activated 4 Å molecular sieves (100 mg) in DCM (3 mL) was stirred at r.t. for 20 min and then cooled to -78 ℃. To the above solution was added AgOTf (47 mg, 0.184 mmol) in Et2O/DCM (3/0.5 mL). The mixture was stirred for 10 min and p-TolSCl (9.7 μL, 0.0735 mmol) was added directly into it via microsyringe. The reaction was allowed to warm up to r.t. over a period of 2 h before quenched with Et3N. The mixture was then filtered through celite and concentrated. Column chromatography (Hexanes/DCM/EtOAc = 1/1/1) gave compound 36α (60 mg, 57%) and 36β (30 mg, 28%). 1HNMR (500 MHz, CDCl3): δ = 1.05&1.11 (d, 3H, J = 6.5 Hz), 1.64-1.78 (m, 2H), 1.90&1.91 (s, 3H), 2.02&2.04 (s, 3H), 2.09 (s, 3H), 2.10&2.11 (s, 3H), 2.11-2.49 (m, 4H), 2.59-2.68 (m, 1H), 2.84-2.91 (m, 1H), 3.22 (s, 3H), 3.25-3.45 (m, 3H), 3.50-3.98 (m, 9H), 4.14-4.37 (m, 5H), 4.39-4.88 (m, 12H), 5.00-5.30 (m, 8H), 5.38 (d, 1H, J = 9.5 Hz), 5.55-5.64 (m, 1H), 6.47-6.57 (m, 1H), 7.10-7.20 (m, 2H), 7.22-7.50 (m, 28H). 13CNMR (125 MHz, CDCl3): δ = 16.36, 16.45, 20.86, 20.89, 21.0, 21.03, 27.4, 27.82, 27.84, 29.78, 29.81, 29.87, 33.27, 33.66, 37.61, 37.78, 42.76, 50.0, 50.2, 50.33, 50.82, 54.8, 54.9, 56.37, 56.43, 63.4, 65.42, 65.55, 67.02, 67.37, 67.66, 68.75, 69.22, 72.14, 72.37, 72.71, 72.75, 73.78, 74.22, 74.35, 74.61, 74.77, 75.0, 79.34, 95.68, 95.78, 98.08, 98.28, 100.12, 100.2, 100.98, 127.27, 127.55, 127.57, 127.7, 127.78, 127.87, 128.08, 128.11, 128.25, 128.37, 128.42, 128.52, 128.55, 128.59, 128.73, 136.55, 136.76, 136.92, 137.44, 137.94, 138.01, 154.38, 154.45, 155.13, 156.68, 157.32, 157.99, 170.44, 170.46, 170.62, 89 170.65, 172.02, 172.22, 205.95, 206.32. Three C1-H1 coupling constants (175.0, 173.0, 162.0 Hz) confirmed the stereochemistry. Most peaks were split due to the secondary amides on the fucose and the linker. HRMS: m/z calc. for C84H97Cl6N4O26: 1787.4522; found: 1787.4478 [M + H]+. AcO AcO BnO BnO OBn O TrocHN O N Cbz LevO O O AcO O O NHTroc Bn N Cbz N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 4-[N-(methyl)-benzyloxycarbonylamino]-2,4-dideoxy-3-O-levuniloyl-2-(2,2,2-trichloroethyloxy carbonylamino)-β-L-fucopyranosyl-(1→6)-[6,7-di-O-acetyl-2,3,4-tri-O-benzyl-L-glycero-α-D-m annoheptopyranosyl-(1→4)]-3-O-acetyl-2-deoxy-2-(2,2,2-trichloroethyloxycarbonylamino)-β-D- glucopyranoside (36β) 1HNMR (500 MHz, CDCl3): δ = 1.14&1.20 (d, 3H, J = 6.5 Hz), 1.67-1.75 (m, 2H), 1.76&1.81 (s, 3H), 2.03&2.05 (s, 3H), 2.10 (s, 6H), 2.27-2.54 (m, 4H), 2.57-2.67 (m, 1H), 3.00-3.10 (m, 1H), 3.17&3.18 (s, 3H), 3.25-3.46 (m, 3H), 3.52-3.96 (m, 11H), 4.20-4.36 (m, 4H), 4.41-4.87 (m, 13H), 5.02-5.23 (m, 6H), 5.57-5.66 (m, 1H), 5.70-5.84 (m, 1H), 5.97-6.12 (m, 1H), 7.11-7.19 (m, 2H), 7.23-7.44 (m, 28H). 13CNMR obtained from HSQC: δ = 16.4, 16.5, 18.22, 19.32, 20.79, 20.87, 20.97, 21.27, 22.67, 27.58, 29.57, 29.71, 29.81, 31.42, 31.52, 33.2, 33.29, 37.67, 37.7, 49.99, 52.23, 54.1, 54.3, 63.47, 63.54, 65.72, 67.25, 67.46, 67.65, 68.58, 68.62, 70.16, 70.29, 71.11, 71.67, 71.99, 72.02, 72.09, 72.17, 72.52, 72.8, 73.65, 73.73, 73.76, 74.13, 74.27, 74.5, 90 74.55, 74.64, 75.18, 75.27, 75.31, 75.89, 77.94, 79.48, 99.93, 100.84, 102.61, 124.67, 125.58, 125.64, 125.66, 126.07, 126.3, 126.55, 127.13, 127.29, 127.48, 128.05, 128.33, 128.41, 130.07, 130.13, 131.08. Three C1-H1 coupling constants (172.0, 162.0, 160.0 Hz) confirmed the stereochemistry. Most peaks were split due to the secondary amides on the fucose and the linker. HRMS: m/z calc. for C84H97Cl6N4O26: 1787.4522; found: 1787.4562 [M + H]+. AcO AcO BnO BnO TrocHN OBn O O AcO TBSO N Cbz O O O O NHTroc Bn N Cbz N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 4-[N-(methyl)-benzyloxycarbonylamino]-3-O-tert-butyldimethylsilyl-2,4-dideoxy-2-(2,2,2-trichl oroethyloxycarbonylamino)-α-L-fucopyranosyl-(1→6)-[6,7-di-O-acetyl-2,3,4-tri-O-benzyl-L-gly cero-α-D-mannoheptopyranosyl-(1→4)]-3-O-acetyl-2-deoxy-2-(2,2,2-trichloroethyloxycarbonyl amino)-β-D-glucopyranoside (37α) A solution of compound 35 (0.89 g, 1.56 mmol), compound 25 (1.53 g, 1.25 mmol) and freshly activated 4 Å molecular sieves (1.6 g) in DCM (35 mL) was stirred at r.t. for 20 min and then cooled to -78 ℃. To the above solution was added AgOTf (1.0 g, 3.91 mmol) in DCM/MeCN (5/1 mL). The mixture was stirred for 10 min and p-TolSCl (207 μL, 1.56 mmol) was added directly into it via microsyringe. The reaction was allowed to warm up to r.t. over a period of 2 h. TLC indicated that acceptor was not completely consumed yet. The reaction was cooled to -78 ℃ 91 followed by addition of 35 (0.53 g, 0.94 mmol), AgOTf (0.60 g, 2.35 mmol) and p-TolSCl (124 μL, 0.94 mmol) in order. After warming up over another period of 2 h, the mixture was quenched with Et3N, filtered through celite and concentrated. Column chromatography gave inseparable α and β mixtures, which was dissolved in THF/water (40/10 mL) and treated with benzyl chloroformate (535 μL, 3.75 mmol) and sodium carbonate (0.66 g, 6.25 mmol). The reaction was stirred at r.t. for 4 h followed by concentration, dilution with EtOAc and wash with saturated NaHCO3 and brine. The organic layer was dried, concentrated and purified through column chromatography (Hexanses/DCM/EtOAc = 3/2/2) to give compound 37α (225 mg, 10%) and 37β (1.46 g, 73%) over 2 steps. 1HNMR (500 MHz, CDCl3): δ = -0.07-0.14 (m, 6H, TBS), 0.83 (s, 9H), 1.06&1.13 (d, 3H, J = 6.5Hz, Fuc-6-Me), 1.66-1.74 (m, 2H), 1.89 (s, 3H), 1.98 (s, 1H) + 2.05 (s, 2H, Ac), 2.10 (s, 3H), 2.85-2.91 (m, 1H), 3.15-3.29 (m, 5H), 3.40-3.57 (m, 2H), 3.62-3.74 (m, 3H), 3.76-3.92 (m, 6H), 4.00 (dd, 1H, J = 6.5, 11.0 Hz), 4.14-4.22 (m, 2H), 4.22-4.32 (m, 3H), 4.40-4.50 (m, 2H), 4.51-4.56 (m, 1H), 4.57-4.66 (m, 4H), 4.66-4.84 (m, 6H), 5.01-5.12 (m, 4H), 5.13-5.23 (m, 3H), 5.58-5.64 (m, 1H), 6.45-6.57 (m, 1H), 7.13-7.17 (m, 2H), 7.24-7.45 (m, 28H).. 13CNMR (125 MHz, CDCl3): δ = -4.93, -4.85, -4.83, -4.73, 16.5, 16.55, 17.77, 17.78, 17.8, 20.81, 20.91, 20.98, 21.05, 25.59, 25.6, 27.41, 33.1, 33.31, 33.71, 42.75, 50.02, 52.99, 53.04, 53.56, 56.25, 56.32, 57.44, 57.54, 63.34, 65.92, 66.78, 66.83, 67.42, 67.64, 67.68, 68.17, 68.28, 68.66, 68.75, 72.17, 72.21, 72.75, 73.86, 74.37, 74.75, 74.9, 75.02, 75.09, 75.19, 75.52, 95.47, 95.77, 98.63, 100.62, 100.91, 127.27, 127.58, 127.68, 127.87, 127.9, 127.92, 127.98, 128.01, 128.15, 128.42, 128.49, 128.54, 128.55, 128.58, 128.75, 136.53, 136.77, 136.87, 137.38, 137.83, 138.01, 138.05, 154.23, 92 154.27, 155.16, 156.67, 157.21, 157.72, 170.42, 170.57. Three C1-H1 coupling constants (175.5, 172.0, 160.0 Hz) confirmed the stereochemistry. Most peaks were split due to the secondary amides on the fucose and the linker. HRMS: m/z calc. for C85H105Cl6N4O24Si: 1803.5019; found: 1803.5009 [M + H]+. AcO AcO BnO BnO OBn O TrocHN O TBSO N Cbz O O AcO O O NHTroc Bn N Cbz N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 4-[N-(methyl)-benzyloxycarbonylamino]-3-O-tert-butyldimethylsilyl-2,4-dideoxy-2-(2,2,2-trichl oroethyloxycarbonylamino)-β-L-fucopyranosyl-(1→6)-[6,7-di-O-acetyl-2,3,4-tri-O-benzyl-L-gly cero-α-D-mannoheptopyranosyl-(1→4)]-3-O-acetyl-2-deoxy-2-(2,2,2-trichloroethyloxycarbonyl amino)-β-D-glucopyranoside (37β) 1HNMR (500 MHz, CDCl3): δ = -0.09-0.12 (m, 6H), 0.83 (s, 9H), 1.09-1.22 (m, 3H), 1.67-1.77 (m, 2H), 1.84 (s, 3H), 2.04 (s, 3H), 2.10-2.12(m, 3H), 2.97-3.07 (m, 1H), 3.14-3.18 (m, 3H), 3.24-3.42 (m, 3H), 3.56-3.77 (m, 5H), 3.77-3.90 (m, 5H), 3.92-4.05 (m, 2H), 4.16-4.25 (m, 1H), 4.26-4.40 (m, 3H), 4.42-4.51 (m, 2H), 4.53-4.80 (m, 10H), 4.91 (d, 1H, J = 12.0 Hz), 4.96-5.08 (m, 2H), 5.10-5.21 (m, 3H), 5.64-5.85 (m, 3H), 6.11-6.18 (m, 1H), 7.10-7.19 (m, 2H), 7.23-7.47 (m, 28H). 13CNMR (125 MHz, CDCl3): δ = -4.99, -4.93, -4.81, -4.77, 16.58, 17.8, 17.83, 20.94, 21.04, 25.62, 27.51, 29.79, 33.29, 33.72, 43.28, 50.15, 56.2, 56.92, 57.23, 57.39, 64.06, 65.31, 66.94, 67.41, 67.57, 67.63, 68.47, 68.54, 70.15, 70.42, 70.96, 71.93, 72.02, 72.12, 73.65, 74.34, 93 74.68, 74.89, 75.31, 76.17, 79.74, 95.76, 99.82, 100.9, 102.16, 127.07, 127.27, 127.56, 127.61, 127.66, 127.78, 127.93, 127.97, 128.1, 128.21, 128.47, 128.51, 128.53, 128.57, 128.61, 128.74, 136.53, 136.75, 136.87, 137.59, 138.02, 138.13, 141.07, 154.14, 154.25, 154.84, 156.54, 157.16, 157.78, 170.38, 170.52, 171.25, 171.39. Three C1-H1 coupling constants (173.0, 160.5, 160.5 Hz) confirmed the stereochemistry. Most peaks were split due to the secondary amides on the fucose and the linker. HRMS: m/z calc. for C85H105Cl6N4O24Si: 1803.5019; found: 1803.4955 [M + H]+. AcO AcO BnO BnO OBn O TrocHN O N Cbz HO O O AcO O O NHTroc Bn N Cbz N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 4-[N-(methyl)-benzyloxycarbonylamino]- 2,4-dideoxy-2-(2,2,2-trichloroethyloxycarbonylamino)-β-L-fucopyranosyl-(1→6)-[6,7-di-O-acet yl-2,3,4-tri-O-benzyl-L-glycero-α-D-mannoheptopyranosyl-(1→4)]-3-O-acetyl-2-deoxy-2-(2,2,2 -trichloroethyloxycarbonylamino)-β-D-glucopyranoside (38) Compound 37β (1.80 g, 1.0 mmol) was dissolved with pyridine (10 mL) in a plastic centrifuge tube. After cooling to 0 ℃, HF pyridine complex (5 mL) was added and the reaction was allowed to warm up to r.t. and continued to stir for 3 days. The reaction was then diluted with DCM, washed with saturated CuSO4 solution, 1 M HCl and saturated NaHCO3 solution successively. The organic layer was dried, concentrated and purified through column chromatography (Hexanes/DCM/EtOAc = 1/1/2) to give 38 as white foam in a yield of 85%. 94 1HNMR (500 MHz, CDCl3): δ = 1.19&1.24 (d, 3H, J = 6.5 Hz, Fuc-6-Me), 1.70-1.76 (m, 2H), 1.85 (s, 3H), 2.03&2.05 (s, 3H), 2.10&2.11 (s, 3H), 3.00-3.09 (m, 1H), 3.16 (s, 3H), 3.29 (d, 1H, J = 9.5 Hz), 3.34-3.48 (m, 2H), 3.57-3.93 (m, 11 Hz), 3.94-4.13 (m, 2H), 4.29-4.37 (m, 4H), 4.40-4.53 (m, 3H), 4.56-4.75 (m, 7H), 4.76-4.88 (m, 3H), 5.06-5.27 (m, 6H), 5.60-5.69 (m, 1H), 5.93-6.04 (m, 1H), 6.06-6.20 (m, 1H), 7.13-7.20 (m, 2H), 7.24-7.44 (m, 28H). 13CNMR (125 MHz, CDCl3): δ = 16.75, 16.76, 20.95, 21.02, 27.63, 29.78, 33.27, 33.7, 43.31, 50.15, 56.26, 56.44, 56.55, 56.81, 63.51, 67.06, 67.4, 67.58, 68.64, 70.58, 70.81, 71.87, 71.95, 72.07, 72.25, 72.42, 73.69, 74.32, 74.45, 74.59, 75.21, 75.35, 75.6, 79.66, 95.72, 95.82, 99.78, 100.94, 102.84, 127.23, 127.57, 127.68, 127.78, 127.86, 127.95, 128.0, 128.08, 128.48, 128.52, 128.56, 128.59, 128.76, 136.73, 136.87, 137.49, 137.9, 138.07, 138.13, 154.7, 156.58, 157.85, 158.97, 170.35, 170.57, 170.84, 170.94. Most peaks were split due to the secondary amides on the fucose and the linker. HRMS: m/z calc. for C79H90Cl6FeN4O24: 872.1713; found: 872.1685 [M + Fe]2+. Ph BnO O AcO AcO BnO OBn N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl Bn N Cbz O O O N3 TrocHN O OLev O O N Cbz O AcO O O NHTroc 3-azido-4, 6-O-benzylidene-3-deoxy-2-levulinoyl-β-D-glucopyranosyl-(1→3)-4-[N-(methyl)-benzyloxycar bonylamino]-2,4-dideoxy-2-(2,2,2-trichloroethyloxycarbonylamino)-β-L-fucopyranosyl-(1→6)-[ 95 6,7-di-O-acetyl-2,3,4-tri-O-benzyl-L-glycero-α-D-mannoheptopyranosyl-(1→4)]-3-O-acetyl-2-d eoxy-2-(2,2,2-trichloroethyloxycarbonylamino)-β-D-glucopyranoside (39) A solution of compound 6 (756 mg, 1.52 mmol), 38 (1.54 g, 0.91 mmol) and freshly activated 4 Å molecular sieves (1.2 g) in DCM (20 mL) was stirred at r.t. for 20 min and then cooled to -78 ℃. To the above solution was added AgOTf (974 mg, 3.79 mmol) in DCM/MeCN (5/0.5 mL). The mixture was stirred for 10 min and p-TolSCl (201 μL, 1.52 mmol) was added directly into it via microsyringe. The reaction was allowed to warm up to r.t. over a period of 2 h before quenched with Et3N. The mixture was then filtered through celite and concentrated. Column chromatography (Hexanes/DCM/EtOAc = 2/2/3) gave compound 39 in a yield of 65%. 1HNMR (500 MHz, CDCl3): δ = 1.18 (d, 3H, J = 6.0 Hz), 1.68-1.75 (m, 2H), 1.86 (s, 3H), 2.04&2.05 (s, 3H), 2.09-2.13 (m, 4H), 2.17-2.22 (m, 2H), 2.40-2.58 (m, 2H), 2.58-2.76 (3H), 2.76-2.87 (m, 1H), 2.95-3.05 (m, 1H), 3.08 + 3.11 (s, 3H), 3.21-3.44 (m, 4H), 3.47-3.58 (m, 2H), 3.58-3.73 (m, 5H), 3.74-3.90 (m, 6H), 3.92-4.03 (m, 1H), 4.16-4.23 (m, 1H), 4.24-4.39 (m, 4H), 4.40-4.52 (m, 2H), 4.53-4.63 (m, 3H), 4.63-4.90 (m, 9H), 5.01 (d, 1H, J = 11.5 Hz), 5.12-5.30 (m, 4H), 5.38 (d, 1H, J = 12.0 Hz), 5.45-5.57 (m, 1H), 5.58-5.73 (m, 2H), 6.08-6.19 (m, 1H), 7.12-7.20 (m, 2H), 7.22-7.55 (m, 33H).. 13CNMR (125 MHz, CDCl3): δ = 16.33, 16.58, 20.9, 20.95, 20.99, 21.02, 27.48, 27.69, 29.74, 29.83, 29.89, 33.02, 33.32, 37.73, 37.91, 43.21, 50.09, 50.87, 52.86, 53.02, 54.71, 54.91, 56.06, 62.91, 63.16, 63.39, 63.56, 66.7, 67.03, 67.14, 67.35, 67.54, 67.63, 67.71, 68.49, 68.56, 70.48, 70.63, 70.7, 71.35, 71.72, 71.8, 71.85, 72.07, 72.22, 73.64, 74.25, 74.31, 74.79, 75.31, 77.36, 79.12, 79.33, 79.72, 89.09, 95.79, 96.08, 96.9, 97.2, 99.53, 100.28, 100.9, 101.41, 101.51, 126.01, 126.08, 127.24, 127.53, 127.58, 127.74, 127.89, 96 128.02, 128.07, 128.21, 128.36, 128.4, 128.44, 128.45, 128.49, 128.53, 128.59, 128.65, 128.7, 128.71, 128.99, 129.22, 129.26, 136.55, 136.59, 136.67, 136.7, 136.76, 137.52, 137.95, 138.11, 153.93, 154.79, 156.52, 157.68, 170.39, 170.45, 170.48, 170.8, 170.92, 171.24, 171.55, 205.95, 206.37. Most peaks were split due to the secondary amides on the fucose and the linker. HRMS: m/z calc. for C97H117Cl6N9O30: 1048.8018; found: 1048.7975 [M + Na + NH4]2+. Ph BnO O AcO AcO BnO OBn N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl Bn N Cbz O O N3 TrocHN O O OH O N Cbz O O AcO O O NHTroc 3-azido-4,6-O-benzylidene-3-deoxy -β-D-glucopyranosyl-(1→3)-4-[N-(methyl)-benzyloxycarbonylamino]-2,4-dideoxy-2-(2,2,2-trich loroethyloxycarbonylamino)-β-L-fucopyranosyl-(1→6)-[6,7-di-O-acetyl-2,3,4-tri-O-benzyl-L-gl ycero-α-D-mannoheptopyranosyl-(1→4)]-3-O-acetyl-2-deoxy-2-(2,2,2-trichloroethyloxycarbony lamino)-β-D-glucopyranoside (40) Compound 39 (1.22 g, 0.59 mmol) was dissolved in DCM/AcOH/Pyridine (15/1/1.5 mL) followed by addition of N2H4·H2O (200 μL, 64%). The mixture was stirred at r.t. for 4 h before it was diluted with DCM and washed with brine. The organic layer was dried and concentrated. Column chromatography (Hexanes/DCM/EtOAc = 2/2/3) gave 40 as white foam in a yield of 84%. 1HNMR (500 MHz, CDCl3): δ = 1.26 (d, 3H, J = 5.5 Hz), 1.68-1.76 (m, 2H), 1.84 (s, 3H), 2.05 97 (s, 3H), 2.11 (s, 3H), 3.00-3.14 (m, 2H), 3.20 (s, 3H), 3.24-3.32 (d, 1H, J = 9.5 Hz), 3.34-3.51 (m, 4H), 3.53-3.72 (m, 5H), 3.72-3.91 (m, 8H), 3.94-4.09 (m, 2H), 4.18-4.26 (m, 1H), 4.26-4.44 (m, 5H), 4.44-4.50 (d, 1H, J = 10.0 Hz), 4.50-4.80 (m, 10H), 4.95-5.08 (m, 2H), 5.10-5.28 (m, 6H), 5.53 (s, 1H), 5.65 (t, 1H, J = 6.5 Hz), 5.79 (d, 1H, J = 8.5 Hz), 6.10 (d, 1H, J = 8.5 Hz), 7.14-7.21 (m, 2H), 7.23-7.47 (m, 31 H), 7.48-7.54 (m, 2H). 13CNMR (125 MHz, CDCl3): δ = 16.85, 20.93, 21.01, 21.03, 27.58, 29.78, 33.45, 43.36, 50.22, 54.78, 55.9, 56.16, 60.49, 63.65, 64.52, 66.83, 67.26, 67.47, 67.56, 68.14, 68.56, 68.65, 69.83, 71.84, 72.1, 73.28, 73.65, 74.18, 74.32, 74.44, 74.75, 75.12, 75.3, 76.01, 77.36, 79.04, 79.71, 95.77, 95.95, 99.8, 100.88, 101.6, 102.24, 106.29, 126.15, 127.28, 127.58, 127.77, 127.81, 127.92, 128.05, 128.1, 128.35, 128.39, 128.47, 128.49, 128.52, 128.53, 128.62, 128.7, 128.75, 129.24, 136.25, 136.71, 136.77, 137.56, 137.97, 138.06, 138.12, 154.44, 154.81, 156.54, 159.85, 170.36, 170.46, 170.99. Four C1-H1 coupling constants (174.5, 163.0, 163.0, 160.0 Hz) confirmed the stereochemistry. Most peaks were split due to the secondary amides on the fucose and the linker. HRMS: m/z calc. for C92H103Cl6FeN7O28: 1009.7166; found: 1009.7142 [M + Fe]2+. Ph BnO O AcO AcO BnO OBn N3 O O O N3 TrocHN O O N Cbz O O AcO O O NHTroc Bn N Cbz N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 2,3-diazido-4,6-O-benzylidene-2,3-dideoxy-β-D-mannopyranosyl-(1→3)-4-[N-(methyl)-benzylo 98 xycarbonylamino]-2,4-dideoxy-2-(2,2,2-trichloroethyloxycarbonylamino)-β-L-fucopyranosyl-(1 →6)-[6,7-di-O-acetyl-2,3,4-tri-O-benzyl-L-glycero-α-D-mannoheptopyranosyl-(1→4)]-3-O-acet yl-2-deoxy-2-(2,2,2-trichloroethyloxycarbonylamino)-β-D-glucopyranoside (41) Compound 40 (974 mg, 0.50 mmol) was dissolved in anhydrous DCM (10 mL) and cooled to -30 ℃. Pyridine (400 μL, 4.95 mmol) and Tf2O (333 μL, 1.98 mmol) were added and the mixture was allowed to warm up to r.t. over a period over 4 h. It was then quenched with MeOH, diluted with DCM and washed with brine. The organic layer was dried, concentrated and dissolved with DMF (10 mL). NaN3 (200 mg, 3.1 mmol) was added and the mixture was heated at 50 ℃ overnight. After diluting with EtOAc and washing with brine, compound 41 was purified through column chromatography in a yield of 86% over 2 steps. 1HNMR (500 MHz, CDCl3): δ = 1.25 (d, 3H, J = 6.5 Hz), 1.68-1.77 (m, 2H), 1.82 (s, 3H), 2.04 (s, 3H), 2.11 (s, 3H), 2.98-3.08 (m, 1H), 3,19 (s, 3H), 3.24-3.48 (m 6H), 3.57-3.89 (m, 12H), 3.98-4.08 (m, 1H), 4.20 (d, 1H, J = 8.0 Hz), 4.27 (dd, 1H, J = 5.5, 10.5 Hz), 4.29-4.37 (m, 3H), 4.46 (d, 1H, J = 9.5 Hz), 4.51 (dd, 1H, J = 3.0, 6.0 Hz), 4.53-4.78 (m, 10H), 4.80-4.92 (m, 2H), 4.95 (d, 1H, J = 12.0 Hz), 5.00-5.07 (m, 1H), 5.08 (d, 1H, J = 12.5 Hz), 5.14-5.21 (m, 3H), 5.23 (d, 1H, J = 12.5 Hz), 5.44-5.52 (m, 1H), 5.55 (s, 1H), 5.64 (t, 1H, J = 6.5 Hz), 6.09 (d, 1H, J = 9.0 Hz), 7.17 (d, 2H, J = 7.5 Hz), 7.25-7.46 (m, 31H), 7.46-7.50 (m, 2H). 13CNMR (125 MHz, CDCl3): δ = 16.79, 16.84, 20.94, 20.99, 21.06, 21.08, 27.54, 29.8, 33.19, 43.36, 50.22, 53.86, 54.29, 56.13, 59.89, 60.51, 62.36, 63.58, 66.89, 67.11, 67.57, 67.62, 67.9, 68.46, 68.63, 69.98, 71.83, 72.0, 72.1, 73.68, 74.21, 74.34, 74.47, 74.92, 75.2, 75.35, 76.1, 76.57, 76.69, 77.36, 79.75, 95.81, 96.07, 98.45, 99.78, 100.97, 101.67, 102.28, 125.92, 125.98, 127.29, 127.58, 127.61, 99 127.67, 127.77, 127.97, 128.12, 128.39, 128.49, 128.53, 128.59, 128.63, 128.69, 128.76, 128.98, 129.19, 129.23, 136.51, 136.7, 136.77, 137.59, 138.01, 138.15, 138.18, 154.4, 154.81, 156.55, 156.84, 158.46, 170.43, 170.53, 170.88. Most peaks were split due to the secondary amides on the fucose and the linker. HRMS: m/z calc. for C92H102Cl6FeN10O27: 1022.2198; found: 1022.2167 [M + Fe]2+. BnO O AcO AcO BnO OBn N3 O HO HO N3 TrocHN O O N Cbz O O AcO O O NHTroc Bn N Cbz N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 2,3-diazido-2,3-dideoxy -β-D-mannopyranosyl-(1→3)-4-[N-(methyl)-benzyloxycarbonylamino]-2,4-dideoxy-2-(2,2,2-tric hloroethyloxycarbonylamino)-β-L-fucopyranosyl-(1→6)-[6,7-di-O-acetyl-2,3,4-tri-O-benzyl-L-g lycero-α-D-mannoheptopyranosyl-(1→4)]-3-O-acetyl-2-deoxy-2-(2,2,2-trichloroethyloxycarbon ylamino)-β-D-glucopyranoside (42) Compound 41 (850 mg, 0.43 mmol) was dissolved in DCM/TFA/water (15/1.5/0.5 mL) and stirred at r.t. for 30 min. The reaction was then washed with brine, dried and concentrated. Column chromatography gave compound 42 as white foam in a yield of 86%. 1HNMR (500 MHz, CDCl3): δ = 1.24 (d, 3H, J = 6.5 Hz), 1.70-1.77 (m, 2H), 1.79-1.93 (m, 3H, Ac), 2.05 (s, 3H), 2.10 (s, 3H), 3.00-3.50 (m, 11H), 3.51-3.93 (m, 14H), 3.93-4.09 (m, 2H), 4.17-4.37 (m, 4H), 4.43-4.52 (m, 2H), 4.54-4.71 (m, 7H), 4.71-4.77 (m, 2H), 4.79-4.93 (m, 2H), 100 4.93-5.30 (m, 7H), 5.64 (t, 1H, J = 6.5 Hz), 6.01-6.27 (m, 2H), 7.13-7.22 (m, 2H), 7.24-7.47 (m, 28H). 13CNMR (125 MHz, CDCl3): δ = 16.78, 20.93, 21.0, 27.44, 33.26, 43.38, 50.17, 50.86, 54.14, 54.38, 56.05, 62.04, 62.23, 63.23, 63.69, 66.92, 67.55, 68.57, 70.02, 71.82, 72.06, 73.64, 74.32, 74.97, 75.34, 77.36, 79.71, 95.74, 95.92, 97.9, 99.61, 100.95, 102.12, 127.26, 127.56, 127.58, 127.73, 127.91, 128.09, 128.46, 128.5, 128.58, 128.65, 128.73, 136.5, 136.7, 137.53, 137.96, 138.12, 138.15, 154.63, 154.84, 156.58, 158.58, 170.41, 170.52, 171.17. Most peaks were split due to the secondary amides on the fucose and the linker. HRMS: m/z calc. for C85H98Cl6FeN10O27: 978.2041; found: 978.2010 [M + Fe]2+. BnO O AcO AcO BnO OBn MeOOC HO N3 O N3 TrocHN O O N Cbz O O AcO O O NHTroc Bn N Cbz N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl methyl 2,3-diazido-2,3-dideoxy -β-D-mannopyranosyluronate-(1→3)-4-[N-(methyl)-benzyloxycarbonylamino]-2,4-dideoxy-2-(2, 2,2-trichloroethyloxycarbonylamino)-β-L-fucopyranosyl-(1→6)-[6,7-di-O-acetyl-2,3,4-tri-O-ben zyl-L-glycero-α-D-mannoheptopyranosyl-(1→4)]-3-O-acetyl-2-deoxy-2-(2,2,2-trichloroethyloxy carbonylamino)-β-D-glucopyranoside (43) Compound 42 (700 mg, 0.37 mmol) was dissolved in DCM/t-BuOH/water (4/4/1 mL) followed by addition of BAIB (473 mg, 1.47 mmol) and TEMPO (23 mg, 0.15 mmol). The reaction was stirred at r.t. overnight. It was then diluted with DCM, washed with brine, dried and concentrated. 101 The crude product was dissolved in DMF (10 mL) and treated with MeI (228 μL, 3.67 mmol) and K2CO3 (507 mg, 3.67 mmol). Upon completion by TLC, the mixture was diluted with EtOAc and washed with brine. The organic layer was dried, concentrated and purified through column chromatography to afford compound 43 in a yield of 66% over 2 steps. 1HNMR (500 MHz, CDCl3): δ = 1.27 (d, 3H, J = 6.5 Hz), 1.62-1.78 (m, 2H), 1.71&1.91 (s, 3H, Ac), 2.04 (s, 3H), 2.12 (s, 3H), 2.93-3.04 (m, 1H), 3.10-3.17 (m, 1H), 3.20 (s, 3H), 3.23-3.27 (dd, 1H, J = 3.5, 9.5 Hz), 3.28-3.48 (m, 3H), 3.52-3.68 (m, 3H), 3.68-3.78 (m, 4H), 3.81 (s, 3H), 3.83-3.88 (m, 3H), 3.89-4.07 (m, 3H), 4.07-4.19 (m, 2H), 4.21-4.42 (m, 4H), 4.42-4.58 (m, 6H), 4.60-4.80 (m, 6H), 4.86 (d, 1H, J = 10.0 Hz), 4.92-5.02 (m, 1H), 5.05 (d, 1H, J = 12.0 Hz), 5.17 (d, 1H, J = 7.0 Hz), 5.22 (d, 1H, J = 12.0 Hz), 5.24-5.30 (m, 1H), 5.59-5.75 (m, 2H), 6.16 (d, 1H, J = 7.5 Hz), 7.10-7.20 (m, 2H), 7.22-7.51 (m, 28H), . 13CNMR (125 MHz, CDCl3): δ = 16.84, 20.76, 20.99, 21.11, 27.59, 29.81, 33.25, 43.29, 50.24, 53.41, 54.1, 56.14, 60.98, 62.1, 63.31, 66.99, 67.13, 67.61, 67.64, 68.67, 69.90, 71.49, 71.7, 72.11, 73.63, 74.22, 75.13, 75.38, 76.11, 77.36, 79.80, 95.92, 96.27, 98.32, 99.59, 101.17, 103.82, 127.25, 127.61, 127.63, 127.71, 127.91, 128.02, 128.05, 128.15, 128.48, 128.50, 128.57, 128.63, 128.68, 128.78, 136.74, 137.57, 138.06, 138.21, 138.26, 154.75, 154.95, 156.58, 158.67, 169.83, 170.32, 170.58, 170.70. Most peaks were split due to the secondary amides on the fucose and the linker. HRMS: m/z calc. for C86H98Cl6FeN10O28: 992.2016; found: 992.1984 [M + Fe]2+. 102 BnO BnO OAc O N3 O BnO O AcO AcO BnO OBn N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl Bn N Cbz MeOOC N3 O N3 TrocHN O O N Cbz O O AcO O O NHTroc 6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl 2,3-diazido-2,3-dideoxy-β-D-mannopyranosyluronate-(1→3)-4-[N-(methyl)-benzyloxycarbonyla mino]-2,4-dideoxy-2-(2,2,2-trichloroethyloxycarbonylamino)-β-L-fucopyranosyl-(1→6)-[6,7-di- O-acetyl-2,3,4-tri-O-benzyl-L-glycero-α-D-mannoheptopyranosyl-(1→4)]-3-O-acetyl-2-deoxy-2 -(2,2,2-trichloroethyloxycarbonylamino)-β-D-glucopyranoside (44) A solution of compound 7 (258 mg, 0.48 mmol), 43 (467 mg, 0.24 mmol) and freshly activated 4 Å molecular sieves (400 mg) in DCM (5 mL) was stirred at r.t. for 20 min and then cooled to -78 ℃. To the above solution was added AgOTf (310 mg, 1.21 mmol) in Et2O/DCM (6/1 mL). The mixture was stirred for 10 min and p-TolSCl (64 μL, 0.48 mmol) was added directly into it via microsyringe. The reaction was allowed to warm up to r.t. over a period of 3 h before quenched with Et3N. The mixture was then filtered through celite and concentrated. Column chromatography (Hexanes/DCM/EtOAc = 2/2/3) gave compound 44 in a yield of 63%. 1HNMR (500 MHz, CDCl3): δ = 1.26 (d, 3H, J = 6.5 Hz), 1.69-1.77 (m, 2H), 1.82 (s, 3H), 2.04 (s, 3H), 2.05 (s, 3H), 2.11 (s, 3H), 2.98-3.08 (m, 1H), 3.22 (s, 3H), 3.26-3.44 (m, 4 H), 3.44-3.50 (m, 1H), 3.53-3.62 (m, 2H), 3.63-3.93 (m, 15H), 3.95-4.10 (m, 3H), 4.18-4.25 (m, 2H), 4.25-4.38 103 (m, 4H), 4.40-4.52 (m, 3H), 4.52-4.65 (m, 5H), 4.65-4.81 (m, 7H), 4.82-4.89 (m, 3H), 4.90-4.94 (m, 1H), 4.99-5.06 (m, 1H), 5.09 (d, 1H, J = 13.0 Hz), 5.14-5.23 (m, 3H), 5.26 (d, 1H, J = 12.5 Hz), 5.36-5.52 (m, 2H), 5.60-5.68 (m, 1H), 6.06-6.16 (m, 1H), 7.15-7.22 (m, 2H), 7.26-7.48 (m, 38H). 13CNMR (125 MHz, CDCl3): δ = 16.75, 20.86, 20.95, 20.99, 27.47, 29.71, 33.2, 43.27, 50.16, 53.03, 53.65, 54.04, 56.14, 61.41, 62.06, 62.71, 63.32, 63.82, 66.85, 67.09, 67.42, 67.47, 68.57, 69.89, 70.26, 71.67, 71.81, 72.0, 73.59, 74.04, 74.22, 74.28, 74.39, 74.77, 75.08, 75.16, 75.22, 75.65, 75.68, 75.76, 75.93, 76.05, 77.19, 77.3, 79.63, 80.19, 80.23, 95.75, 95.98, 97.67, 98.89, 99.59, 100.91, 102.18, 127.22, 127.47, 127.53, 127.63, 127.68, 127.82, 127.9, 127.98, 128.05, 128.08, 128.19, 128.4, 128.43, 128.5, 128.52, 128.56, 128.62, 128.66, 129.17, 136.73, 136.76, 137.48, 137.56, 137.97, 138.07, 138.13, 154.35, 154.65, 156.46, 156.68, 158.33, 167.28, 170.3, 170.44, 170.58, 170.74. Most peaks were split due to the secondary amides on the fucose and the linker. HRMS: m/z calc. for C108H129Cl6N15O33: 1186.8504; found: 1186.8445 [M + 2NH4]2+. BnO BnO OAc O AcHN MeOOC O AcHN NHAc O O N Cbz AcHN O O O O NHAc Bn N Cbz BnO O AcO AcO BnO OBn O AcO N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 2-acetamido-6-O-acetyl-3, 4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl 2,3-diacetamido-2,3-dideoxy 104 -β-D-mannopyranosyluronate-(1→3)-2-acetamido-4-[N-(methyl)-benzyloxycarbonylamino]-2,4- dideoxy-β-L-fucopyranosyl-(1→6)-[6,7-di-O-acetyl-2,3,4-tri-O-benzyl-L-glycero-α-D-mannohep topyranosyl-(1→4)]-2-acetamido-3-O-acetyl-2-deoxy-β-D-glucopyranoside (45) Compound 44 (357 mg, 0.15 mmol) was dissolved in THF (10 mL) followed by addition of Ac2O (1.73 mL, 18.3 mmol), AcOH (1.04 mL, 18.3 mmol) and Zn (1.48 g, 22.9 mmol). The reaction was stirred at r.t. overnight before it was quenched with MeOH and filtered through celite to remove the insoluble impurities. The filtrate was concentrated, diluted with EtOAc and washed with saturated NaHCO3 solution. The organic layer was dried, concentrated and purified through column chromatography (DCM/MeOH = 10/1) to give compound 45 in a yield of 65%. 1HNMR (500 MHz, CDCl3): δ = 1.08&1.16 (d, 2H, J = 6.5 Hz, Fuc-6-Me), 1.64-1.72 (m, 2H), 1.81 (s, 3H), 1.89 (s, 3H), 1.98-2.01 (m, 6H), 2.03 (s, 9H), 2.04 (s, 3H), 2.08 (s, 3H), 2.90-2.98 (m, 1H), 3.03-3.10 (m, 1H), 3.15 + 3.18 (s, 3H, Fuc-4-N-Me), 3.19-3.33 (m, 3H), 3.60-3.73 (m, 6H), 3.76-3.92 (m, 8H), 4.01-4.12 (m, 2H), 4.20-4.35 (m, 6H), 4.38 (dd, 1H, J = 5.5, 12.0 Hz), 4.40-4.47 (m, 3H), 4.47-4.60 (m, 5H), 4.60-4.65 (m, 1H), 4.65-4.74 (m, 4H), 4.75-4.80 (m, 1H), 4.80-4.88 (m, 4H), 4.94 (d, 1H, J = 12.0 Hz), 4.97 (dd, 1H, J = 3.5, 7.0 Hz), 5.01 (d, 1H, J = 3.5 Hz), 5.04 (d, 1H, J = 3.5 Hz), 5.10-5.22 (m, 4H), 5.29 (d, 1H, J = 12.5 Hz), 5.57-5.63 (m, 1H), 6.42 (d, 1H, J = 9.5 Hz), 6.85 (m, 2H), 7.13-7.21 (m, 2H), 7.21-7.42 (m, 38H) . 13CNMR (125 MHz, CDCl3): δ = 16.36, 16.43, 20.81, 20.88, 20.91, 20.94, 20.97, 22.73, 22.8, 22.86, 22.92, 23.0, 23.06, 23.17, 24.03, 24.06, 26.97, 33.0, 33.42, 42.66, 49.74, 52.49, 52.71, 52.8, 53.81, 54.1, 62.14, 62.42, 62.66, 63.4, 63.67, 64.35, 66.37, 67.43, 67.62, 68.09, 68.29, 68.68, 69.98, 70.17, 71.67, 71.9, 71.97, 72.16, 73.54, 73.73, 74.24, 74.42, 75.1, 75.3, 75.33, 75.38, 75.49, 75.98, 105 76.35, 77.36, 79.72, 80.49, 99.55, 99.76, 99.85, 100.8, 102.39, 103.05, 127.3, 127.47, 127.51, 127.54, 127.57, 127.61, 127.65, 127.69, 127.71, 127.74, 127.78, 127.8, 127.85, 127.88, 127.92, 127.98, 128.01, 128.09, 128.15, 128.26, 128.37, 128.41, 128.46, 128.48, 128.5, 128.57, 128.62, 128.72, 136.0, 136.08, 136.7, 137.28, 137.67, 137.72, 137.91, 138.04, 138.1, 138.18, 138.28, 156.59, 158.27, 170.45, 170.59, 170.69, 170.79, 170.8, 170.82, 170.84, 171.02, 171.22, 171.44, 171.53, 171.61, 171.67, 172.0, 172.24, 175.36. Five C1-H1 coupling constants (174.5, 171.0, 163.0, 164.0, 160.5 Hz) confirmed the stereochemistry. Most peaks were split due to the secondary amides on the fucose and the linker. HRMS: m/z calc. for C112H137N7O34: 1061.9603; found: 1061.9554 [M + 2H]2+. HO HO OH O AcHN HOOC O AcHN NHAc O O NH AcHN O O O O NHAc O HO HO O HO HO HO OH NH2 3-Aminopropyl 2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→4)-2,3-diacetamido-2,3-dideoxy -β-D-mannopyranosyluronate-(1→3)-2-acetamido-2,4-dideoxy-4-methylamino-β-L-fucopyranos yl-(1→6)-[L-glycero-α-D-mannoheptopyranosyl-(1→4)]-2-acetamido-2-deoxy-β-D-glucopyrano side (2) Compound 45 (210 mg, 0.099 mmol) was dissolved in THF/water (20/5 mL) followed by addition of 1M LiOH solution (2.5 mL) at 0 ℃. The reaction was allowed to warm up to r.t. and 106 stirred overnight. H+ resin was added to neutralize the solution and filtered off through sintered glass funnel. The filtrate was concentrated and purified through column chromatography (DCM/MeOH = 4/1). To a solution of the product in THF/water/AcOH (2/2/2 mL) was added Pd(OH)2/C (100 mg) and it was stirred at r.t. overnight under H2 atmosphere. Pd(OH)2/C was filtered off and the filtrate was concentrated and purified through a G10 column followed by Na+ ion exchange column. The final aqueous solution was lyophilized to afford compound 2 in a yield of 68%. 1HNMR (500 MHz, D2O): δ = 1.25 (d, 3H, J = 6.5 Hz), 1.74 (s, 3H), 1.75 (s, 3H), 1.76-1.80 (m, 2H), 1.83 (s, 3H), 1.88 (s, 3H), 1.89 (s, 3H), 1.92 (s, 3H), 2.61 (s, 3H), 2.90 (t, 2H, J = 7.5 Hz), 3.32 (t, 1H, J = 10.0 Hz), 3.36-3.43 (m, 4H), 3.45-3.60 (m, 8H), 3.60-3.69 (m, 5H), 3.69-3.75 (m, 2H), 3.76-3.92 (m, 6H), 4.06 (dd, 1H, J = 4.0, 11.0 Hz), 4.11 (dd, 1H, J = 4.0, 10.5 Hz), 4.18 (d, 1H, J = 4.0 Hz), 4.26 (m, 1H), 4.43 (d, 1H, J = 8.0 Hz), 4.82 (s, 1H), 4.95 (d, 1H, J = 4.0 Hz), 5.11 (s, 1H). 13CNMR (125 MHz, D2O): δ = 15.98, 21.65, 21.73, 21.81, 21.97, 22.42, 23.19, 26.57, 36.21, 37.3, 50.89, 51.47, 53.22, 53.37, 55.66, 59.67, 60.97, 63.05, 65.75, 67.67, 68.29, 68.46, 68.89, 69.28, 70.06, 70.19, 70.24, 71.57, 72.48, 73.48, 73.78, 75.15, 75.75, 78.71, 96.5, 96.66, 101.07, 101.1, 101.88, 173.62, 173.93, 174.17, 174.63, 174.81, 175.07, 181.34. HRMS: m/z calc. for C45H79N7O26: 566.7537; found: 566.7537 [M + 2H]2+. 107 HO HO OH O AcHN HO O HO HO HO OH HOOC O AcHN NHAc O O NH AcHN O O O O NHAc O HO O N H O N O O O N-(5-(succinimidyloxycarbonyl)pentanoyl)-3-aminopropyl 2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→4)-2,3-diacetamido-2,3-dideoxy-β-D-mannopyra nosyluronate-(1→3)-2-acetamido-2,4-dideoxy-4-methylamino-β-L-fucopyranosyl-(1→6)-[L-gly cero-α-D-mannoheptopyranosyl-(1→4)]-2-acetamido-2-deoxy-β-D-glucopyranoside (47) Compound 2 (15 mg, 0.01324 mmol) together with compound 46 (22.5 mg, 0.066 mmol) was dissolved in dry DMF (2.5 mL) followed by addition of DIPEA (2 μL). The reaction was stirred at r.t. for 3 h and then DMF was removed by vacuum. The residue was washed with DCM and 47 was used for Qβ coupling without further purification. 1HNMR (500 MHz, CD3OD): δ = 1.35 (d, 3H, J = 6.5 Hz), 1.63-1.79 (m, 5H), 1.89 (s, 3H), 1.97 (s, 3H), 1.98 (s, 3H), 2.03 (s, 3H), 2.09 (s, 3H), 2.20-2.29 (m, 2H), 2.50-2.72 (m, 6H), 2.84 (s, 3H), 3.02-3.22 (m, 3H), 3.35 (s, 4H), 3.45-4.10 (m, 24H), 4.27 (dd, 1H, J = 4.0, 9.5 Hz), 4.32-4.38 (m, 2H), 4.40 (d, 1H, J = 8.0 Hz), 5.11 (d, 1H, J = 3.5 Hz), 5.41 (s, 1H). HRMS: m/z calc. for C55H89N8O31: 1357.5633; found: 1357.5582 [M + H]+. 108 APPENDIX 109 Product Characterization Spectra AcO N3 OAc O OAc STol 9 9 8 . 1 6 9 . 1 1 0 . 1 9 9 . 0 7 0 . 1 4 9 . 1 1 1 . 2 8 0 . 3 7 9 . 2 9 9 . 2 0 0 . 3 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1 Figure 2.8. 1H-NMR of 9 (500 MHz CDCl3) 1 8 . 0 7 1 7 3 . 9 6 1 5 2 . 9 6 1 3 9 . 8 3 1 9 7 . 3 3 1 6 8 . 9 2 1 2 0 . 8 2 1 9 5 . 6 8 2 5 . 6 7 9 1 . 0 7 4 4 . 8 6 9 9 . 5 6 7 3 . 2 6 8 3 . 1 2 6 0 . 1 2 6 9 . 0 2 5 8 . 0 2 AcO N3 OAc O OAc STol 9 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.9. 13C-NMR of 9 (125 MHz CDCl3) 110 AcO N3 OAc O OAc STol 9 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Figure 2.10. 1H-1H gCOSY of 9 (500 MHz CDCl3) AcO N3 OAc O OAc STol 9 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.11. 1H-13C gHSQCAD of 9 (500 MHz CDCl3) 111 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f Ph O O N3 O OH STol 10 9 9 . 0 0 0 . 1 3 0 . 1 0 8 . 1 9 7 . 1 7 7 . 2 1 8 . 1 3 0 . 2 5 0 . 1 3 0 . 1 7 9 . 0 8 9 . 0 3 9 . 2 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1 Figure 2.12. 1H-NMR of 10 (500 MHz CDCl3) 1 4 . 9 3 1 8 7 . 6 3 1 2 1 . 4 3 1 0 2 . 0 3 1 7 3 . 9 2 1 2 5 . 8 2 1 1 7 . 6 2 1 7 1 . 6 2 1 1 7 . 1 0 1 4 3 . 9 8 3 3 . 9 7 0 7 . 1 7 4 6 . 1 7 4 7 . 8 6 7 9 . 5 6 1 4 . 1 2 Ph O O N3 O OH STol 10 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.13. 13C-NMR of 10 (125 MHz CDCl3) 112 Ph O O N3 O OH STol 10 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Figure 2.14. 1H-1H gCOSY of 10 (500 MHz CDCl3) Ph O O N3 O OH STol 10 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Figure 2.15. 1H-13C gHSQCAD of 10 (500 MHz CDCl3) 113 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 ) m p p ( 1 f 10 20 30 40 50 60 70 80 90 100 110 120 130 140 ) m p p ( 1 f Ph O O N3 6 0 0 . 1 0 9 . 1 4 5 . 4 5 8 . 1 O OLev STol 0 0 . 1 2 0 . 1 6 0 . 1 6 0 . 2 4 0 . 1 6 0 . 1 7 1 . 2 1 1 . 2 8 8 . 2 9 6 . 2 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1 Figure 2.16. 1H-NMR of 6 (500 MHz CDCl3) 2 1 . 6 0 2 0 3 . 1 7 1 2 9 . 8 3 1 3 6 . 6 3 1 9 7 . 3 3 1 0 9 . 9 2 1 0 3 . 9 2 1 4 4 . 8 2 1 1 9 . 7 2 1 7 0 . 6 2 1 Ph O O N3 6 4 5 . 1 0 1 O 0 3 . 7 8 7 1 . 9 7 4 3 . 1 7 9 6 . 0 7 8 5 . 8 6 4 7 . 4 6 4 9 . 7 3 0 0 . 0 3 9 0 . 8 2 3 3 . 1 2 STol OLev 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.17. 13C-NMR of 6 (125 MHz CDCl3) 114 Ph O O N3 6 O OLev STol 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.18. 1H-1H gCOSY of 6 (500 MHz CDCl3) Ph O O N3 6 O OLev STol 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.19. 1H-13C gHSQCAD of 6 (500 MHz CDCl3) 115 0 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f STol HO O AcO 11 OH 1 9 . 1 2 1 . 2 4 9 . 0 0 0 . 1 6 9 . 0 1 0 . 1 4 1 . 1 0 9 . 1 0 8 . 2 3 8 . 2 4 1 . 3 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 2.20. 1H-NMR of 11 (500 MHz CDCl3) 2 0 . 5 7 9 6 . 1 7 1 2 . 1 7 0 0 . 0 7 7 2 . 1 2 5 2 . 1 2 8 5 . 7 1 0 6 . 1 7 1 4 0 . 8 3 1 3 3 . 2 3 1 4 0 . 0 3 1 4 9 . 9 2 1 1 1 . 8 8 STol HO O AcO 11 OH 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.21. 13C-NMR of 11 (125 MHz CDCl3) 116 STol HO O AcO 11 OH 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.22. 1H-1H gCOSY of 11 (500 MHz CDCl3) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 ) m p p ( 1 f STol HO O AcO 11 OH 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.23. 1H-13C gHSQCAD of 11 (500 MHz CDCl3) 117 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f HO O STol HO OH 12 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 2 9 . 1 5 0 . 2 9 0 . 1 5.0 f1 (ppm) Figure 2.24. 1H-NMR of 12 (500 MHz CD3OD) 6 9 . 0 6 0 . 1 9 3 . 1 2 1 . 1 0 0 . 3 6 6 . 3 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 0 2 . 8 3 1 0 3 . 3 3 1 7 8 . 1 3 1 2 6 . 0 3 1 4 0 . 9 8 8 8 . 7 7 1 9 . 5 7 9 1 . 4 7 4 6 . 3 7 5 0 . 1 2 4 2 . 8 1 HO O STol HO OH 12 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.25. 13C-NMR of 12 (125 MHz CD3OD) 118 HO O STol HO OH 12 ) m p p ( 1 f 1 2 3 4 5 6 7 8 9 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.26. 1H-1H gCOSY of 12 (500 MHz CD3OD) HO O STol HO OH 12 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.27. 1H-13C gHSQCAD of 12 (500 MHz CD3OD) 119 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 ) m p p ( 1 f HO BzO O STol OH 13 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 8 8 . 1 5 0 . 1 0 8 . 3 6 8 . 1 4 0 . 1 0 0 . 1 2 0 . 1 8 0 . 1 5.0 f1 (ppm) 4.5 4.0 8 0 . 1 3.5 9 8 . 0 4 9 . 2 0 8 . 0 3.0 2.5 2.0 Figure 2.28. 1H-NMR of 13 (500 MHz CDCl3) 3 8 . 6 6 1 0 3 . 8 3 1 7 7 . 3 3 1 0 6 . 2 3 1 4 0 . 0 3 1 2 0 . 0 3 1 4 8 . 9 2 1 4 4 . 9 2 1 9 6 . 8 2 1 6 4 . 7 8 0 8 . 7 7 3 1 . 7 7 8 9 . 0 7 3 8 . 0 7 0 0 . 3 1.5 1.0 0.5 0.0 -0.5 -1.0 9 2 . 1 2 8 0 . 8 1 HO BzO O STol OH 13 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.29. 13C-NMR of 13 (125 MHz CDCl3) 120 HO BzO O STol OH 13 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.30. 1H-1H gCOSY of 13 (500 MHz CDCl3) HO BzO O STol OH 13 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.31. 1H-13C gHSQCAD of 13 (500 MHz CDCl3) 121 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 ) m p p ( 1 f ) m p p ( 1 f O N3 OBz PhthN 14 8 0 . 1 7 0 . 1 2 9 . 0 2 0 . 1 8 1 . 2 8 6 . 2 8 1 . 2 3 0 . 2 8 0 . 2 6 1 . 2 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 Figure 2.32. 1H-NMR of 14 (500 MHz CDCl3) 9 1 . 5 6 1 0 4 . 8 3 1 4 2 . 4 3 1 6 6 . 3 3 1 2 1 . 3 3 1 7 0 . 0 3 1 3 0 . 0 3 1 1 9 . 9 2 1 4 8 . 9 2 1 7 7 . 9 2 1 0 8 . 8 2 1 2 7 . 8 2 1 8 5 . 8 2 1 8 7 . 3 2 1 STol 9 1 . 1 4.0 1 2 . 3 0 0 . 3 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 5 4 . 9 8 9 5 . 3 7 9 1 . 3 7 5 1 . 2 6 0 7 . 1 5 7 3 . 1 2 7 0 . 7 1 O N3 STol OBz PhthN 14 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.33. 13C-NMR of 14 (125 MHz CDCl3) 122 O N3 STol OBz PhthN 14 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Figure 2.34. 1H-1H gCOSY of 14 (500 MHz CDCl3) O N3 STol OBz PhthN 14 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.35. 1H-13C gHSQCAD of 14 (500 MHz CDCl3) 123 1 2 3 4 5 6 7 8 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 ) m p p ( 1 f ) m p p ( 1 f HO HN O STol OH CCl3 O 16 4 8 . 1 9 8 . 1 9 8 . 1 6 9 . 0 7 9 . 0 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 Figure 2.36. 1H-NMR of 16 (500 MHz CDCl3) 0 0 . 1 3.5 9 2 . 2 1 1 . 3 8 6 . 0 3.0 2.5 2.0 0 0 . 3 1.5 1.0 0.5 0.0 -0.5 1 5 . 4 8 5 5 . 2 8 4 0 . 0 8 3 1 . 6 7 6 7 . 1 7 3 1 . 1 2 4 7 . 8 1 6 8 . 7 3 1 5 7 . 1 3 1 0 8 . 0 3 1 7 7 . 9 2 1 8 9 . 6 1 1 1 5 . 3 0 1 HO HN O STol OH CCl3 O 16 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.37. 13C-NMR of 16 (125 MHz CDCl3) 124 HO HN O STol OH CCl3 O 16 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Figure 2.38. 1H-1H gCOSY of 16 (500 MHz CDCl3) HO HN O STol OH CCl3 O 16 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.39. 1H-13C gHSQCAD of 16 (500 MHz CDCl3) 125 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 ) m p p ( 1 f -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f STol N3 O N O CCl3 17 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 2 8 . 1 1 9 . 1 2 0 . 1 5.0 f1 (ppm) Figure 2.40. 1H-NMR of 17 (500 MHz CDCl3) 4 6 . 2 6 1 0 9 . 8 3 1 2 1 . 4 3 1 3 8 . 9 2 1 1 7 . 6 2 1 0 0 . 1 3 0 . 1 9 0 . 1 2 9 . 0 2 1 . 3 1 2 . 3 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1. l l l 3 c d c 8 2 . 7 7 3 c d c 2 0 . 7 7 3 c d c 7 7 . 6 7 1 3 . 6 8 7 3 . 5 8 6 8 . 4 8 3 0 . 3 7 8 7 . 8 6 1 3 . 2 6 6 1 . 1 2 6 1 . 8 1 STol N3 O N O CCl3 17 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.41. 13C-NMR of 17 (125 MHz CDCl3) 126 STol N3 O N O CCl3 17 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Figure 2.42. 1H-1H gCOSY of 17 (500 MHz CDCl3) STol N3 O N O CCl3 17 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.43. 1H-13C gHSQCAD of 17 (500 MHz CDCl3) 127 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 ) m p p ( 1 f -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f O N3 STol OH H2N 15 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 8 8 . 1 6 9 . 1 0 0 . 1 0 1 . 1 7 0 . 1 3 0 . 1 7 0 . 1 7 1 . 3 4.5 4.0 3.5 3.0 2.5 7 7 . 2 2.0 5.0 f1 (ppm) 8 2 . 3 1.5 1.0 0.5 0.0 -0.5 -1. Figure 2.44. 1H-NMR of 15 (500 MHz CDCl3) 2 7 . 8 3 1 8 9 . 3 3 1 7 7 . 9 2 1 6 5 . 7 2 1 4 0 . 6 8 7 0 . 5 7 1 6 . 3 7 5 3 . 3 6 1 3 . 4 5 1 2 . 1 2 6 1 . 7 1 O N3 STol OH H2N 15 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.45. 13C-NMR of 15 (125 MHz CDCl3) 128 O N3 STol OH H2N 15 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f2 (ppm) 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.46. 1H-1H gCOSY of 15 (500 MHz CDCl3) O N3 STol OH H2N 15 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.47. 1H-13C gHSQCAD of 15 (500 MHz CDCl3) 129 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f STol NH2 O OH 18 NH 6 8 . 1 3 9 . 1 0 0 . 1 7 0 . 1 5 0 . 1 9 9 . 0 0 0 . 1 4 8 . 2 0 0 . 3 3 0 . 3 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 2.48. 1H-NMR of 18 (500 MHz CD3OD) 8 6 . 7 3 1 5 1 . 2 3 1 5 4 . 9 2 1 6 2 . 9 2 1 O OH 18 NH 5 1 . 9 8 0 2 . 5 7 0 1 . 4 7 0 5 . 3 6 6 3 . 2 5 9 5 . 7 3 3 7 . 9 1 5 8 . 6 1 STol NH2 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.49. 13C-NMR of 18 (125 MHz CD3OD) 130 STol NH2 O OH 18 NH 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.50. 1H-1H gCOSY of 18 (500 MHz CD3OD) STol NH2 O OH 18 NH 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.51. 1H-13C gHSQCAD of 18 (500 MHz CD3OD) 131 1 2 3 4 5 6 7 8 9 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f STol NHTroc O OH NH 19 5 7 . 1 2 8 . 1 0 0 . 1 7 9 . 0 9 9 . 0 9 9 . 0 4 0 . 1 1 0 . 1 0 1 . 1 9 0 . 1 9 8 . 2 2 9 . 2 9 8 . 2 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 2.52. 1H-NMR of 19 (500 MHz CDCl3) 8 6 . 5 7 5 5 . 4 7 2 5 . 1 7 7 0 . 3 6 3 4 . 5 5 9 3 . 8 3 3 1 . 1 2 3 9 . 7 1 1 5 . 4 5 1 5 9 . 7 3 1 5 5 . 2 3 1 7 6 . 9 2 1 3 6 . 9 2 1 4 5 . 5 9 0 3 . 7 8 STol NHTroc O OH NH 19 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.53. 13C-NMR of 19 (125 MHz CDCl3) 132 STol NHTroc O OH NH 19 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Figure 2.54. 1H-1H gCOSY of 19 (500 MHz CDCl3) STol NHTroc O OH NH 19 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.55. 1H-13C gHSQCAD of 19 (500 MHz CDCl3) 133 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 ) m p p ( 1 f -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f STol NHTroc O N OH 5 Cbz 1 0 . 1 6 9 . 6 8 0 . 2 8 4 . 0 2 6 . 0 7 6 . 1 7 4 . 0 5 0 . 1 3 2 . 1 9 0 . 1 5 6 . 0 5 4 . 0 9 2 . 2 3 1 . 1 3 7 . 1 0 1 . 1 2 1 . 1 0 8 . 1 5 8 . 1 4 2 . 1 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 2.56. 1H-NMR of 5 (500 MHz d6-DMSO) STol NHTroc O N OH 5 Cbz 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 2.57. 1H-NMR of 5 (500 MHz d6-DMSO, VT at 90 ℃) 134 9 1 . 7 5 1 7 9 . 6 5 1 6 4 . 4 5 1 0 2 . 7 3 1 3 1 . 7 3 1 9 9 . 6 3 1 0 9 . 6 3 1 0 8 . 1 3 1 3 6 . 1 3 1 9 5 . 9 2 1 2 5 . 9 2 1 9 4 . 9 2 1 6 4 . 9 2 1 0 4 . 8 2 1 1 3 . 8 2 1 8 6 . 7 2 1 1 6 . 7 2 1 7 2 . 7 2 1 6 1 . 7 2 1 6 2 . 6 9 2 3 . 6 8 4 0 . 4 7 0 9 . 3 7 1 5 . 3 7 9 4 . 3 7 4 7 . 9 6 6 6 . 9 6 9 2 . 6 6 7 1 . 6 6 2 9 . 6 5 7 8 . 6 5 1 1 . 4 5 1 0 . 4 5 8 9 . 2 3 6 5 . 2 3 4 6 . 0 2 5 9 . 6 1 7 8 . 6 1 STol NHTroc O N OH 5 Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.58. 13C-NMR of 5 (125 MHz d6-DMSO) STol NHTroc O N OH 5 Cbz 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.59. 1H-1H gCOSY of 5 (500 MHz d6-DMSO) 135 1 2 3 4 5 6 7 8 ) m p p ( 1 f -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f STol NHTroc O N OH 5 Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.60. 1H-13C gHSQCAD of 5 (500 MHz d6-DMSO) 136 OBn O BnO BnO STol 21 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 8 0 . 6 1 4 9 . 1 2 1 . 1 6.0 1 1 . 1 7 0 . 1 3 0 . 2 5.5 3 2 . 1 5.0 9 0 . 4 5 0 . 1 4.5 f1 (ppm) 0 1 . 1 0 0 . 1 5 0 . 1 4.0 9 9 . 2 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 2.61. 1H-NMR of 21 (500 MHz CDCl3) 4 5 . 8 3 1 8 3 . 8 3 1 4 0 . 8 3 1 7 7 . 7 3 1 3 1 . 5 3 1 9 2 . 2 3 1 2 7 . 0 3 1 3 9 . 9 2 1 3 5 . 8 2 1 1 5 . 8 2 1 6 4 . 8 2 1 9 1 . 8 2 1 1 1 . 8 2 1 4 9 . 7 2 1 7 8 . 7 2 1 1 8 . 7 2 1 0 8 . 7 2 1 3 4 . 8 1 1 6 2 . 6 8 6 8 . 9 7 7 9 . 8 7 2 4 . 7 7 6 1 . 7 7 1 9 . 6 7 7 5 . 6 7 0 4 . 5 7 7 8 . 3 7 7 4 . 2 7 9 1 . 2 7 5 2 . 1 2 OBn O BnO BnO STol 21 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.62. 13C-NMR of 21 (125 MHz CDCl3) 137 OBn O BnO BnO STol 21 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.63. 1H-1H gCOSY of 21 (500 MHz CDCl3) OBn O BnO BnO STol 21 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.64. 1H-13C gHSQCAD of 21 (500 MHz CDCl3) 138 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f HO HO BnOBnO OBn O STol 22 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 2 7 . 5 1 7 9 . 1 9 0 1 0 . . 1 1 5.5 4 0 . 3 3 9 . 0 5.0 8 1 . 1 8 0 . 1 3 1 . 1 4.5 7 9 . 0 8 8 . 0 4.0 f1 (ppm) 2 1 . 1 9 0 . 1 0 1 . 1 3.5 0 0 . 1 3.0 5 6 . 2 2.5 0 1 . 1 2.0 1.5 1.0 0.5 Figure 2.65. 1H-NMR of 22 (500 MHz CDCl3) 5 2 . 8 3 1 2 7 . 7 3 1 8 6 . 7 3 1 7 5 . 7 3 1 5 7 . 2 3 1 6 9 . 9 2 1 7 5 . 9 2 1 7 5 . 8 2 1 2 5 . 8 2 1 7 4 . 8 2 1 8 1 . 8 2 1 5 0 . 8 2 1 3 9 . 7 2 1 0 9 . 7 2 1 6 8 . 7 2 1 5 2 . 6 8 5 2 . 0 8 3 6 . 6 7 7 8 . 5 7 8 0 . 5 7 6 6 . 2 7 0 3 . 2 7 4 2 . 2 7 3 9 . 1 7 9 0 . 3 6 4 1 . 1 2 HO HO BnOBnO OBn O STol 22 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.66. 13C-NMR of 22 (125 MHz CDCl3) 139 HO HO BnOBnO OBn O STol 22 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Figure 2.67. 1H-1H gCOSY of 22 (500 MHz CDCl3) HO HO BnOBnO OBn O STol 22 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.68. 1H-13C gHSQCAD of 22 (500 MHz CDCl3) 140 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 ) m p p ( 1 f 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f HO HO BnOBnO OBn O 23 STol 10.5 10.0 9.5 9.0 8.5 8.0 5 7 . 3 1 7.5 4 9 . 1 2 9 . 1 7.0 0 0 . 1 5.5 6.5 6.0 8 0 . 1 5 0 . 1 5.0 2 8 . 1 8 0 . 1 7 1 . 1 3 1 . 1 4.5 0 0 . 2 2 1 . 1 4.0 9 1 . 2 3 1 . 1 3.5 f1 (ppm) 0 1 . 3 7 3 . 1 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 2.69. 1H-NMR of 23 (500 MHz CDCl3) 1 4 . 8 3 1 7 3 . 8 3 1 8 1 . 8 3 1 6 9 . 7 3 1 7 4 . 2 3 1 1 2 . 0 3 1 4 5 . 9 2 1 1 6 . 8 2 1 6 2 . 8 2 1 5 0 . 8 2 1 0 0 . 8 2 1 9 9 . 7 2 1 7 9 . 7 2 1 3 9 . 7 2 1 6 3 . 6 8 8 0 . 0 8 5 0 . 6 7 6 5 . 5 7 1 5 . 4 7 0 0 . 4 7 2 5 . 2 7 7 3 . 2 7 5 2 . 9 6 1 2 . 5 6 9 2 . 1 2 HO HO BnOBnO OBn O 23 STol 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.70. 13C-NMR of 23 (125 MHz CDCl3) 141 HO HO BnOBnO OBn O 23 STol 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.71. 1H-1H gCOSY of 23 (500 MHz CDCl3) HO HO BnOBnO OBn O 23 STol 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.72. 1H-13C gHSQCAD of 23 (500 MHz CDCl3) 142 0 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f AcO AcO BnO BnO OBn O STol 3 10.5 10.0 9.5 9.0 8.5 8.0 4 1 . 4 1 7.5 8 8 . 1 0 0 . 2 7.0 4 0 . 1 5 9 . 0 5.5 6.5 6.0 8 1 . 1 6 9 . 1 2 1 . 1 8 0 . 1 5.0 1 1 . 1 5 0 . 1 4.5 1 0 . 1 9 1 . 2 4.0 3 0 . 2 3.5 0 0 . 3 6 7 . 2 7 6 . 2 2.0 3.0 2.5 f1 (ppm) Figure 2.73. 1H-NMR of 3 (500 MHz CDCl3) 1 4 . 0 7 1 1 3 . 0 7 1 8 8 . 7 3 1 9 7 . 7 3 1 2 7 . 7 3 1 5 2 . 1 3 1 0 0 . 0 3 1 1 9 . 9 2 1 7 5 . 8 2 1 8 4 . 8 2 1 5 4 . 8 2 1 8 3 . 8 2 1 6 9 . 7 2 1 1 9 . 7 2 1 5 8 . 7 2 1 0 8 . 7 2 1 4 5 . 5 8 6 3 . 0 8 2 2 . 7 7 7 4 . 5 7 6 3 . 5 7 7 6 . 3 7 0 0 . 2 7 7 7 . 1 7 7 7 . 0 7 4 2 . 8 6 9 2 . 2 6 1.5 1.0 0.5 0.0 -0.5 -1 7 0 . 1 2 5 9 . 0 2 5 6 . 0 2 AcO AcO BnO BnO OBn O STol 3 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.74. 13C-NMR of 3 (125 MHz CDCl3) 143 AcO AcO BnO BnO OBn O STol 3 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Figure 2.75. 1H-1H gCOSY of 3 (500 MHz CDCl3) 1 2 3 4 5 6 7 8 9 ) m p p ( 1 f AcO AcO BnO BnO OBn O STol 3 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.76. 1H-13C gHSQCAD of 3 (500 MHz CDCl3) 144 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f AcO AcO AcO O O NHTroc S3 Bn N Cbz 5 0 . 7 8 2 . 1 3 7 . 0 0 0 . 3 4 2 . 1 9 4 . 3 4 4 . 0 2 5 . 1 2 3 . 1 1 1 . 1 3 8 . 0 9 9 . 0 4 8 . 0 0 1 . 1 5 7 . 0 1 6 . 1 5 8 . 0 0 0 . 8 6 0 . 2 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 2.77. 1H-NMR of S3 (500 MHz CDCl3) 5 7 . 0 7 1 4 6 . 0 7 1 6 4 . 9 6 1 0 5 . 6 5 1 4 4 . 7 3 1 6 7 . 6 3 1 4 6 . 8 2 1 3 5 . 8 2 1 3 0 . 8 2 1 5 9 . 7 2 1 7 4 . 7 2 1 0 2 . 7 2 1 9 1 . 1 0 1 1 3 . 4 7 9 0 . 3 7 4 7 . 1 7 4 6 . 8 6 9 4 . 7 6 7 0 . 7 6 9 9 . 1 6 3 1 . 6 5 8 0 . 0 5 3 0 . 3 4 1 3 . 7 2 7 7 . 0 2 6 6 . 0 2 AcO AcO AcO O O NHTroc S3 Bn N Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.78. 13C-NMR of S3 (125 MHz CDCl3) 145 AcO AcO AcO O O NHTroc S3 Bn N Cbz 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Figure 2.79. 1H-1H gCOSY of S3 (500 MHz CDCl3) 1 2 3 4 5 6 7 8 ) m p p ( 1 f AcO AcO AcO O O NHTroc S3 Bn N Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.80. 1H-13C gHSQCAD of S3 (500 MHz CDCl3) 146 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f Ph O O HO O O NHTroc S4 Bn N Cbz 1 0 . 2 1 9 . 9 9 4 . 1 6 7 . 0 3 1 . 1 7 1 . 2 7 8 . 0 0 9 . 0 4 5 . 1 6 4 . 0 1 0 . 1 0 8 . 1 1 4 . 2 5 1 . 2 0 0 . 2 4 3 . 2 7 8 . 0 9 3 . 2 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1 Figure 2.81. 1H-NMR of S4 (500 MHz CDCl3) 5 6 . 6 5 1 8 3 . 6 5 1 7 3 . 7 3 1 0 1 . 7 3 1 5 6 . 6 3 1 5 2 . 9 2 1 7 6 . 8 2 1 2 6 . 8 2 1 3 5 . 8 2 1 7 3 . 8 2 1 4 3 . 8 2 1 3 2 . 8 2 1 5 1 . 8 2 1 9 8 . 7 2 1 0 5 . 7 2 1 0 2 . 7 2 1 8 3 . 6 2 1 9 8 . 1 0 1 2 3 . 1 0 1 0 6 . 5 9 3 4 . 1 8 1 6 . 4 7 8 9 . 2 7 0 6 . 8 6 6 5 . 7 6 8 3 . 7 6 1 9 . 6 6 3 1 . 6 6 3 0 . 6 6 4 0 . 9 5 2 0 . 9 5 6 7 . 0 5 5 0 . 0 5 4 3 . 3 4 2 9 . 2 4 5 4 . 7 2 Ph O O HO O O NHTroc S4 Bn N Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.82. 13C-NMR of S4 (125 MHz CDCl3) 147 Ph O O HO O O NHTroc S4 Bn N Cbz 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.83. 1H-1H gCOSY of S4 (500 MHz CDCl3) Ph O O HO O O NHTroc S4 Bn N Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.84. 1H-13C gHSQCAD of S4 (500 MHz CDCl3) 148 0 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f HO HO AcO O O NHTroc S5 Bn N Cbz 8 3 . 7 8 3 . 1 9 6 . 0 7 2 . 2 4 4 . 1 6 4 . 1 1 2 . 2 0 9 . 1 6 3 . 3 7 5 . 2 1 2 . 1 9 9 . 1 6 8 . 1 2 9 . 0 0 0 . 3 0 9 . 2 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 2.85. 1H-NMR of S5 (500 MHz CDCl3) 4 2 . 2 7 1 5 6 . 6 5 1 9 9 . 4 5 1 9 5 . 7 3 1 0 7 . 6 3 1 8 7 . 8 2 1 6 6 . 8 2 1 1 2 . 8 2 1 4 0 . 8 2 1 0 6 . 7 2 1 4 3 . 7 2 1 1 4 . 1 0 1 3 8 . 5 9 7 2 . 6 7 3 7 . 5 7 4 4 . 4 7 1 7 . 9 6 5 6 . 7 6 2 4 . 7 6 6 2 . 2 6 2 1 . 6 5 1 3 . 0 5 3 6 . 3 4 8 3 . 3 4 6 1 . 8 2 5 6 . 7 2 4 1 . 1 2 HO HO AcO O O NHTroc S5 Bn N Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.86. 13C-NMR of S5 (125 MHz CDCl3) 149 HO HO AcO O O NHTroc S5 Bn N Cbz 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Figure 2.87. 1H-1H gCOSY of S5 (500 MHz CDCl3) HO HO AcO O O NHTroc S5 Bn N Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.88. 1H-13C gHSQCAD of S5 (500 MHz CDCl3) 150 1 2 3 4 5 6 7 8 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f TBDPSO HO AcO O O NHTroc 4 Bn N Cbz 1.0 10.5 10.0 9.5 9.0 8.5 8.0 9 8 . 3 0 4 . 2 1 7.5 3 4 . 1 1 7 . 0 7.0 6.5 6.0 5.5 9 2 . 2 3 3 . 1 5.0 2 4 . 2 4 4 . 1 4.5 2 4 . 1 7 0 . 2 1 7 . 1 1 5 . 3 4.0 9 6 . 0 5 6 . 2 3.5 9 7 . 0 3.0 9 8 . 2 5 5 . 2 9 7 . 8 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1. f1 (ppm) Figure 2.89. 1H-NMR of 4 (500 MHz CDCl3) 2 9 . 1 7 1 7 7 . 1 7 1 1 5 . 6 5 1 1 0 . 5 5 1 8 2 . 4 5 1 1 9 . 7 3 1 0 7 . 7 3 1 3 8 . 6 3 1 6 7 . 5 3 1 7 6 . 5 3 1 3 9 . 2 3 1 4 7 . 2 3 1 4 0 . 0 3 1 3 7 . 8 2 1 3 6 . 8 2 1 6 1 . 8 2 1 6 0 . 8 2 1 0 0 . 8 2 1 5 9 . 7 2 1 1 9 . 7 2 1 3 5 . 7 2 1 3 3 . 7 2 1 4 1 . 1 0 1 4 6 . 0 0 1 7 8 . 5 9 8 0 . 6 7 8 9 . 4 7 6 8 . 4 7 1 6 . 4 7 0 4 . 4 7 8 2 . 1 7 4 5 . 7 6 0 4 . 7 6 6 6 . 6 6 9 7 . 4 6 4 5 . 0 6 9 9 . 5 5 7 9 . 0 5 2 3 . 0 5 6 4 . 3 4 9 7 . 4 3 1 7 . 1 3 3 3 . 8 2 9 4 . 7 2 3 0 . 7 2 1 9 . 6 2 0 4 . 5 2 8 7 . 2 2 6 1 . 1 2 4 8 . 0 2 2 3 . 9 1 3 3 . 4 1 7 2 . 4 1 8 5 . 1 1 TBDPSO HO AcO O O NHTroc 4 Bn N Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.90. 13C-NMR of 4 (125 MHz CDCl3) 151 TBDPSO HO AcO O O NHTroc 4 Bn N Cbz 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.91. 1H-1H gCOSY of 4 (500 MHz CDCl3) TBDPSO HO AcO O O NHTroc 4 Bn N Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.92. 1H-13C gHSQCAD of 4 (500 MHz CDCl3) 152 0 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f AcO AcO BnO BnO OBn O O AcO 24 OTBDPS O O NHTroc Bn N Cbz 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7 3 . 4 5 7 . 9 2 7.5 3 6 . 1 8 0 . 1 3 4 . 1 2 6 . 3 0 5 . 1 5.5 1 4 . 1 7 0 . 2 2 8 . 4 5.0 7 1 . 1 4.5 3 4 . 1 5 0 . 1 5 3 . 1 0 9 . 0 4.0 9 7 . 4 3 4 . 3 3.5 7 2 2 3 . . 3 3 3.0 7 1 . 1 2.5 5 8 . 2 2 3 . 2 1 8 . 2 9 8 . 2 2.0 1.5 0 0 . 9 1.0 0.5 0.0 -0.5 -1. 7.0 6.5 6.0 f1 (ppm) Figure 2.93. 1H-NMR of 24 (500 MHz CDCl3) 4 5 . 0 7 1 8 4 . 0 7 1 8 2 . 0 7 1 5 5 . 6 5 1 2 2 . 8 3 1 3 1 . 8 3 1 1 7 . 7 3 1 9 8 . 5 3 1 8 6 . 5 3 1 2 4 . 3 3 1 2 0 . 3 3 1 1 9 . 9 2 1 0 8 . 9 2 1 7 7 . 8 2 1 5 6 . 8 2 1 1 6 . 8 2 1 7 5 . 8 2 1 1 5 . 8 2 1 4 4 . 8 2 1 4 1 . 8 2 1 8 9 . 7 2 1 7 8 . 7 2 1 5 8 . 7 2 1 9 7 . 7 2 1 4 6 . 7 2 1 8 5 . 7 2 1 4 3 . 7 2 1 5 7 . 0 0 1 1 2 . 9 9 0 8 . 9 7 6 8 . 5 7 8 5 . 5 7 0 5 . 5 7 9 1 . 5 7 2 7 . 4 7 2 4 . 4 7 2 8 . 3 7 3 4 . 2 7 9 2 . 2 7 2 0 . 2 7 0 7 . 8 6 2 6 . 7 6 3 5 . 6 6 9 2 . 3 6 1 1 . 3 6 8 6 . 6 5 1 3 . 0 5 4 3 . 3 4 6 4 . 7 2 4 0 . 7 2 3 9 . 6 2 7 0 . 1 2 6 8 . 0 2 2 4 . 9 1 AcO AcO BnO BnO OBn O O AcO 24 OTBDPS O O NHTroc Bn N Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.94. 13C-NMR of 24 (125 MHz CDCl3) 153 AcO AcO BnO BnO OBn O O AcO 24 OTBDPS O O NHTroc Bn N Cbz 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.95. 1H-1H gCOSY of 24 (500 MHz CDCl3) AcO AcO BnO BnO OBn O O AcO 24 OTBDPS O O NHTroc Bn N Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.96. 1H-13C gHSQCAD of 24 (500 MHz CDCl3) 154 0 1 2 3 4 5 6 7 8 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f AcO AcO BnO BnO OBn O O AcO OH O O NHTroc 25 Bn N Cbz 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7.0 6.5 6.0 5.5 f1 (ppm) 3 7 . 1 7 7 . 0 2 7.5 2 0 . 1 3 1 . 1 2 4 . 4 1 6 . 1 3 6 . 1 4 5 . 6 5.0 3 0 . 3 9 2 . 1 4.5 2 1 . 4 6 1 . 6 4.0 3 9 . 1 0 6 . 1 3.5 8 9 . 0 3.0 2 8 . 2 0 1 . 3 4 0 . 1 2.5 5 4 . 2 0 0 . 3 2.0 1.5 1.0 0.5 0.0 -0.5 -1. Figure 2.97. 1H-NMR of 25 (500 MHz CDCl3) 6 7 . 0 7 1 9 5 . 0 7 1 9 4 . 0 7 1 6 5 . 6 5 1 7 8 . 4 5 1 4 1 . 8 3 1 9 0 . 8 3 1 1 9 . 7 3 1 7 5 . 7 3 1 3 7 . 6 3 1 1 7 . 8 2 1 7 5 . 8 2 1 2 5 . 8 2 1 9 4 . 8 2 1 5 4 . 8 2 1 9 0 . 8 2 1 5 9 . 7 2 1 9 7 . 7 2 1 4 6 . 7 2 1 7 5 . 7 2 1 2 5 . 7 2 1 6 2 . 7 2 1 7 1 . 1 0 1 2 2 . 9 9 3 7 . 5 9 6 5 . 9 7 4 6 . 5 7 1 1 . 5 7 9 9 . 4 7 8 8 . 4 7 6 3 . 4 7 9 0 . 4 7 5 7 . 3 7 8 7 . 2 7 0 2 . 2 7 0 0 . 2 7 5 8 . 8 6 8 5 . 7 6 2 3 . 7 6 6 0 . 3 6 3 3 . 1 6 9 5 . 6 5 1 2 . 0 5 1 4 . 3 4 7 7 . 9 2 8 5 . 7 2 5 1 . 1 2 2 0 . 1 2 2 9 . 0 2 AcO AcO BnO BnO OBn O O AcO OH O O NHTroc 25 Bn N Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.98. 13C-NMR of 25 (125 MHz CDCl3) 155 AcO AcO BnO BnO OBn O O AcO OH O O NHTroc 25 Bn N Cbz 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.99. 1H-1H gCOSY of 25 (500 MHz CDCl3) AcO AcO BnO BnO OBn O O AcO OH O O NHTroc 25 Bn N Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.100. 1H-13C gHSQCAD of 25 (500 MHz CDCl3) 156 1 2 3 4 5 6 7 8 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f Cbz O N Ph O O O N3 TrocHN TolS 26 OLev O 7 0 . 2 9 8 . 8 3 8 . 1 6 5 . 0 7 4 . 0 5 5 . 0 6 9 . 0 1 6 . 0 3 3 . 1 5 8 . 2 5 5 . 0 1 6 . 0 6 5 . 0 1 6 . 0 0 1 . 1 8 0 . 1 1 6 . 0 4 4 . 1 4 7 . 4 1 0 . 1 6 4 . 1 8 1 . 1 9 7 . 4 7 5 . 2 1 1 . 1 7 2 . 1 4 6 . 2 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 2.101. 1H-NMR of 26 (500 MHz CDCl3) 0 6 . 7 5 1 9 4 . 6 5 1 9 6 . 8 3 1 9 4 . 8 3 1 4 4 . 6 3 1 6 1 . 4 3 1 5 8 . 3 3 1 0 7 . 9 2 1 7 6 . 9 2 1 6 2 . 9 2 1 3 2 . 9 2 1 5 9 . 8 2 1 0 9 . 8 2 1 5 5 . 8 2 1 8 3 . 8 2 1 9 1 . 8 2 1 2 9 . 7 2 1 1 0 . 6 2 1 5 9 . 5 2 1 7 5 . 1 0 1 4 9 . 9 9 4 8 . 6 9 8 4 . 5 8 6 5 . 1 7 5 2 . 1 7 8 6 . 7 6 2 2 . 7 6 6 0 . 3 6 8 7 . 2 6 8 1 . 3 5 5 5 . 1 5 2 8 . 7 3 9 5 . 7 3 3 7 . 2 3 2 8 . 9 2 1 6 . 7 2 9 1 . 1 2 Cbz O N Ph O O O N3 TrocHN TolS 26 OLev O 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.102. 13C-NMR of 26 (125 MHz CDCl3) 157 Cbz O N Ph O O O N3 TrocHN TolS 26 OLev O 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.103. 1H-1H gCOSY of 26 (500 MHz CDCl3) Cbz O N Ph O O O N3 TrocHN TolS 26 OLev O 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.104. 1H-13C gHSQCAD of 26 (500 MHz CDCl3) 158 0 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 ) m p p ( 1 f ) m p p ( 1 f Ph O OH O O N3 TrocHN TolS 27 Cbz O N O 5 1 . 2 7 7 . 9 6 9 . 1 6 0 . 1 8 9 . 0 6 1 . 1 1 2 . 1 7 0 . 1 0 9 . 0 6 9 . 0 4 0 . 1 6 1 . 1 1 1 . 1 6 0 . 1 1 0 . 1 2 0 . 1 0 1 . 1 1 2 . 1 3 9 . 1 5 9 . 0 4 8 . 2 1 0 . 3 7 8 . 2 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1 Figure 2.105. 1H-NMR of 27 (500 MHz CDCl3) 7 8 . 9 5 1 2 2 . 4 5 1 2 9 . 8 3 1 7 6 . 6 3 1 3 2 . 6 3 1 2 4 . 4 3 1 0 8 . 9 2 1 7 3 . 9 2 1 6 1 . 9 2 1 8 7 . 8 2 1 9 4 . 8 2 1 6 4 . 8 2 1 5 3 . 8 2 1 5 1 . 8 2 1 5 7 . 7 2 1 8 1 . 6 2 1 2 4 . 5 2 1 3 5 . 6 0 1 3 7 . 1 0 1 2 6 . 5 9 7 2 . 6 8 0 7 . 1 8 2 0 . 9 7 3 6 . 4 7 5 3 . 4 7 2 3 . 3 7 8 5 . 8 6 7 2 . 8 6 8 5 . 7 6 9 4 . 4 6 1 2 . 6 5 0 4 . 3 5 0 2 . 3 3 3 3 . 1 2 4 3 . 7 1 Ph O OH O O N3 TrocHN TolS 27 Cbz O N O 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.106. 13C-NMR of 27 (125 MHz CDCl3) 159 Ph O OH O O N3 TrocHN TolS 27 Cbz O N O 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.107. 1H-1H gCOSY of 27 (500 MHz CDCl3) Ph O OH O O N3 TrocHN TolS 27 Cbz O N O 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.108. 1H-13C gHSQCAD of 27 (500 MHz CDCl3) 160 1 2 3 4 5 6 7 8 9 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f Cbz O N Ph N3 O O O N3 TrocHN TolS 28 O 10.5 10.0 9.5 9.0 8.5 8.0 5 2 . 1 1 7.5 1 9 . 1 6 8 . 0 8 0 . 1 9 8 . 0 4 9 . 0 0 0 . 1 0 8 . 0 5.5 3 8 . 0 5.0 0 0 . 1 8 7 . 1 4.5 6 0 . 1 4.0 9 0 . 3 3 0 . 1 3.5 7 0 . 1 2 0 . 1 6 8 . 2 9 7 . 0 3.0 0 0 . 3 5 0 . 3 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 7.0 6.5 6.0 f1 (ppm) Figure 2.109. 1H-NMR of 28 (500 MHz CDCl3) 8 4 . 8 5 1 2 2 . 4 5 1 4 7 . 8 3 1 7 6 . 6 3 1 3 6 . 6 3 1 3 5 . 4 3 1 9 1 . 4 3 1 7 7 . 9 2 1 4 7 . 9 2 1 0 3 . 9 2 1 6 2 . 9 2 1 8 9 . 8 2 1 2 7 . 8 2 1 1 6 . 8 2 1 3 4 . 8 2 1 5 0 . 8 2 1 9 9 . 5 2 1 2 9 . 5 2 1 6 7 . 1 0 1 1 6 . 1 0 1 0 6 . 8 9 9 6 . 5 9 5 7 . 6 8 3 7 . 6 7 7 0 . 6 7 4 6 . 4 7 9 3 . 4 7 7 3 . 8 6 4 9 . 7 6 4 7 . 7 6 8 3 . 2 6 6 9 . 9 5 3 1 . 4 5 3 8 . 2 5 8 8 . 2 3 2 3 . 1 2 0 3 . 7 1 Cbz O N Ph N3 O O O N3 TrocHN TolS 28 O 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.110. 13C-NMR of 28 (125 MHz CDCl3) 161 Cbz O N Ph N3 O O O N3 TrocHN TolS 28 O 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.111. 1H-1H gCOSY of 28 (500 MHz CDCl3) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 ) m p p ( 1 f Cbz O N Ph N3 O O O N3 TrocHN TolS 28 O 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.112. 1H-13C gHSQCAD of 28 (500 MHz CDCl3) 162 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f Ph OBn O AcO AcO BnOBnO N3 O O O N3 TrocHN O O AcO 29 Cbz O N O O O NHTroc Bn N Cbz 1.0 10.5 10.0 9.5 9.0 8.5 8.0 4 5 . 8 2 7.5 2 6 . 1 4 2 . 1 9 7 . 0 7.0 7 9 . 0 6.5 5 1 . 1 3 0 . 1 2 2 . 1 2 0 . 3 6.0 7 1 . 2 5.5 1 3 . 3 6 8 . 3 5.0 3 8 . 4 3 8 . 1 0 5 . 1 4.5 9 2 . 5 6 6 . 1 4.0 1 2 . 1 4 5 . 8 3.5 1 6 . 2 3 4 . 1 8 3 . 1 4 9 . 3 3.0 4 8 . 2 2.5 8 9 . 0 5 7 . 2 2.0 1 7 . 2 1 9 . 2 6 8 . 2 2 3 . 2 1.5 f1 (ppm) 1.0 0.5 0.0 -0.5 -1. Figure 2.113. 1H-NMR of 29 (500 MHz CDCl3) 5 5 . 0 7 1 2 4 . 0 7 1 9 4 . 8 5 1 1 4 . 4 5 1 4 0 . 8 3 1 8 9 . 7 3 1 6 7 . 6 3 1 2 6 . 6 3 1 1 2 . 9 2 1 6 1 . 9 2 1 6 7 . 8 2 1 8 6 . 8 2 1 0 6 . 8 2 1 8 5 . 8 2 1 7 4 . 8 2 1 9 3 . 8 2 1 2 1 . 8 2 1 0 1 . 8 2 1 2 0 . 8 2 1 6 9 . 7 2 1 9 8 . 7 2 1 6 6 . 7 2 1 1 6 . 7 2 1 8 5 . 7 2 1 6 2 . 7 2 1 3 0 . 6 2 1 5 9 . 5 2 1 5 7 . 1 0 1 8 0 . 9 9 6 3 . 7 7 9 6 . 6 7 2 5 . 5 7 4 4 . 4 7 9 3 . 4 7 2 0 . 4 7 3 2 . 2 7 8 0 . 2 7 4 6 . 8 6 2 4 . 8 6 8 8 . 7 6 9 6 . 7 6 6 8 . 6 6 0 0 . 5 6 6 5 . 2 6 2 9 . 9 5 3 3 . 6 5 4 5 . 4 5 2 1 . 1 5 1 2 . 3 3 1 8 . 9 2 6 0 . 1 2 0 0 . 1 2 5 9 . 0 2 4 7 . 6 1 Ph OBn O AcO AcO BnOBnO N3 O O O N3 TrocHN O O AcO 29 Cbz O N O O O NHTroc Bn N Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.114. 13C-NMR of 29 (125 MHz CDCl3) 163 Ph OBn O AcO AcO BnOBnO N3 O O O N3 TrocHN O O AcO 29 Cbz O N O O O NHTroc Bn N Cbz 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.115. 1H-1H gCOSY of 29 (500 MHz CDCl3) Ph OBn O AcO AcO BnOBnO N3 O O O N3 TrocHN O O AcO 29 Cbz O N O O O NHTroc Bn N Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.116. 1H-13C gHSQCAD of 29 (500 MHz CDCl3) 164 0 1 2 3 4 5 6 7 8 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f STol NHTroc O N OAc 31 Cbz 7 9 . 1 9 6 . 4 8 9 . 1 7 0 . 1 0 8 . 0 2 9 . 0 9 4 . 0 8 3 . 0 7 7 . 0 8 9 . 0 1 0 . 1 9 4 . 0 7 3 . 1 2 4 . 0 7 9 . 0 2 2 . 1 7 7 . 2 0 0 . 3 3 2 . 3 5 5 . 3 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 2.117. 1H-NMR of 31 (500 MHz CDCl3) 1 5 . 0 7 1 7 0 . 8 5 1 1 3 . 7 5 1 5 3 . 4 5 1 4 1 . 4 5 1 1 8 . 8 3 1 2 8 . 6 3 1 7 3 . 6 3 1 4 7 . 4 3 1 0 4 . 4 3 1 4 7 . 9 2 1 0 7 . 9 2 1 4 6 . 8 2 1 0 6 . 8 2 1 5 3 . 8 2 1 3 3 . 8 2 1 3 1 . 8 2 1 0 9 . 7 2 1 5 5 . 7 2 1 5 6 . 5 9 5 9 . 6 8 8 4 . 6 8 4 6 . 4 7 9 5 . 4 7 3 8 . 1 7 1 7 . 1 7 3 7 . 7 6 7 4 . 7 6 8 7 . 4 5 4 6 . 4 5 6 8 . 1 5 4 7 . 1 5 7 1 . 3 3 8 7 . 2 3 0 3 . 1 2 5 5 . 0 2 5 4 . 0 2 8 0 . 7 1 7 0 . 7 1 STol NHTroc O N OAc 31 Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.118. 13C-NMR of 31 (125 MHz CDCl3) 165 STol NHTroc O N OAc 31 Cbz 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Figure 2.119. 1H-1H gCOSY of 31 (500 MHz CDCl3) 1 2 3 4 5 6 7 8 9 ) m p p ( 1 f STol NHTroc O N OAc 31 Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.120. 1H-13C gHSQCAD of 31 (500 MHz CDCl3) 166 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f STol NHTroc O N OPico 32 Cbz 1 1 . 1 7 4 . 5 1 7 . 1 7 7 . 4 0 5 . 0 7 6 . 0 1 5 . 0 5 5 . 0 3 5 . 0 6 5 . 1 0 5 . 0 7 5 . 0 0 5 . 0 5 6 . 0 3 6 . 0 2 5 . 0 3 0 . 1 9 5 . 0 1 5 . 0 7 6 . 2 7 8 . 2 0 0 . 3 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 2.121. 1H-NMR of 32 (500 MHz CDCl3) 5 8 . 3 6 1 3 6 . 3 6 1 8 9 . 7 5 1 9 3 . 7 5 1 8 6 . 4 5 1 6 3 . 0 5 1 7 1 . 0 5 1 1 1 . 7 4 1 1 8 . 6 4 1 7 7 . 8 3 1 1 5 . 7 3 1 4 1 . 7 3 1 5 6 . 6 3 1 6 9 . 5 3 1 0 0 . 5 3 1 3 8 . 4 3 1 1 7 . 9 2 1 0 6 . 9 2 1 9 4 . 8 2 1 9 3 . 8 2 1 3 1 . 8 2 1 9 0 . 8 2 1 6 9 . 7 2 1 0 7 . 7 2 1 2 3 . 7 2 1 1 2 . 7 2 1 9 9 . 6 2 1 6 4 . 5 2 1 7 1 . 5 2 1 5 6 . 5 9 4 5 . 5 9 4 1 . 6 8 0 0 . 6 8 4 5 . 4 7 1 5 . 4 7 4 3 . 4 7 0 8 . 3 7 4 4 . 3 7 2 7 . 7 6 8 2 . 7 6 2 0 . 5 5 7 9 . 4 5 4 7 . 1 5 7 6 . 1 5 8 2 . 3 3 4 9 . 2 3 0 3 . 1 2 5 1 . 7 1 2 1 . 7 1 STol NHTroc O N OPico 32 Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.122. 13C-NMR of 32 (125 MHz CDCl3) 167 STol NHTroc O N OPico 32 Cbz 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f2 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Figure 2.123. 1H-1H gCOSY of 32 (500 MHz CDCl3) 0 1 2 3 4 5 6 7 8 9 ) m p p ( 1 f STol NHTroc O N OPico 32 Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.124. 1H-13C gHSQCAD of 32 (500 MHz CDCl3) 168 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 ) m p p ( 1 f STol NHTroc O N OPico 33 Pico 4 5 . 0 9 3 . 0 8 3 . 0 6 1 . 0 0 5 . 0 0 4 . 0 1 5 . 0 2 7 . 1 4 7 . 3 3 4 . 0 8 8 . 2 2 5 . 0 9 4 . 0 0 6 . 0 7 6 . 0 3 4 . 0 1 5 . 0 1 2 . 0 9 3 . 0 9 4 . 0 6 6 . 0 1 4 . 1 8 9 . 0 2 5 . 0 0 4 . 0 4 2 . 0 3 2 . 0 9 4 . 2 9 9 . 2 3 5 . 2 6 3 . 0 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1 Figure 2.125. 1H-NMR of 33 (500 MHz CDCl3) 3 3 . 1 7 1 8 1 . 1 7 1 2 1 . 4 6 1 3 7 . 3 6 1 9 5 . 4 5 1 7 3 . 4 5 1 0 8 . 3 5 1 7 2 . 0 5 1 3 0 . 0 5 1 1 6 . 8 4 1 0 1 . 8 4 1 6 1 . 7 4 1 4 1 . 7 4 1 9 2 . 9 3 1 0 8 . 8 3 1 1 7 . 8 3 1 4 4 . 7 3 1 2 1 . 7 3 1 6 0 . 7 3 1 2 9 . 6 3 1 5 2 . 5 3 1 6 5 . 4 3 1 7 7 . 9 2 1 4 7 . 9 2 1 9 6 . 9 2 1 8 2 . 7 2 1 9 1 . 7 2 1 2 6 . 6 2 1 3 1 . 6 2 1 8 6 . 5 2 1 2 3 . 5 2 1 7 0 . 5 2 1 3 4 . 4 2 1 3 3 . 4 2 1 4 8 . 3 2 1 6 1 . 3 2 1 0 8 . 6 8 9 0 . 6 8 3 9 . 4 7 9 3 . 4 7 7 0 . 4 7 2 6 . 2 7 9 5 . 2 7 0 4 . 7 5 7 5 . 2 5 1 4 . 2 5 4 9 . 1 5 2 3 . 6 3 7 4 . 2 3 5 3 . 1 2 9 2 . 1 2 1 4 . 7 1 3 1 . 7 1 9 9 . 6 1 STol NHTroc O N OPico 33 Pico 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.126. 13C-NMR of 33 (125 MHz CDCl3) 169 STol NHTroc O N OPico 33 Pico 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f2 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Figure 2.127. 1H-1H gCOSY of 33 (500 MHz CDCl3) STol NHTroc O N OPico 33 Pico 0 1 2 3 4 5 6 7 8 9 ) m p p ( 1 f Figure 2.128. 1H-13C gHSQCAD of 33 (500 MHz CDCl3) 170 STol NHTroc O N OLev 34 Cbz 1 9 . 1 8 4 . 4 0 9 . 1 8 0 . 1 1 0 . 1 9 0 . 1 1 4 . 0 4 7 . 0 4 7 . 0 6 3 . 0 3 2 . 1 0 7 . 0 3 8 . 0 7 3 . 0 4 0 . 2 3 6 . 2 5 3 . 2 9 4 . 3 4 2 . 1 3 9 . 2 0 0 . 3 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 2.129. 1H-NMR of 34 (500 MHz CDCl3) 2 5 . 6 0 2 5 1 . 2 7 1 6 8 . 1 7 1 1 0 . 8 5 1 7 2 . 7 5 1 3 4 . 4 5 1 2 6 . 8 3 1 7 8 . 6 3 1 1 5 . 6 3 1 3 5 . 4 3 1 3 1 . 4 3 1 9 6 . 9 2 1 6 6 . 9 2 1 8 5 . 8 2 1 6 5 . 8 2 1 3 2 . 8 2 1 9 1 . 8 2 1 9 0 . 8 2 1 7 8 . 7 2 1 3 7 . 5 9 9 7 . 6 8 7 2 . 6 8 6 5 . 4 7 3 4 . 4 7 5 1 . 2 7 8 8 . 1 7 8 6 . 7 6 4 4 . 7 6 8 7 . 4 5 1 6 . 4 5 9 8 . 1 5 3 6 . 1 5 4 8 . 7 3 6 7 . 7 3 9 1 . 3 3 1 8 . 2 3 6 7 . 9 2 5 7 . 9 2 3 8 . 7 2 7 2 . 1 2 9 0 . 7 1 4 0 . 7 1 STol NHTroc O N OLev 34 Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.130. 13C-NMR of 34 (125 MHz CDCl3) 171 STol NHTroc O N OLev 34 Cbz 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.131. 1H-1H gCOSY of 34 (500 MHz CDCl3) STol NHTroc O N OLev 34 Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.132. 1H-13C gHSQCAD of 34 (500 MHz CDCl3) 172 0 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f STol NHTroc O OTBS NH 35 4 9 . 1 0 0 . 2 8 9 . 0 5 9 . 0 2 0 . 1 3 1 . 1 9 9 . 0 6 0 . 1 7 0 . 1 3 9 . 2 5 0 . 1 0 0 . 3 6 0 . 1 9 1 . 3 8 1 . 9 7 9 . 5 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 f1 (ppm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 2.133. 1H-NMR of 35 (500 MHz CDCl3) 8 7 . 3 5 1 7 5 . 7 3 1 8 4 . 2 3 1 0 2 . 0 3 1 2 6 . 9 2 1 O OTBS NH 35 8 6 . 5 7 5 7 . 4 7 1 8 . 3 7 3 9 . 4 6 4 5 . 4 5 1 0 . 9 3 5 7 . 5 2 9 1 . 1 2 3 2 . 8 1 1 0 . 8 1 1 3 . 4 - 3 8 . 4 - 4 3 . 5 9 7 8 . 6 8 STol NHTroc 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.134. 13C-NMR of 35 (125 MHz CDCl3) 173 STol NHTroc O OTBS NH 35 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f2 (ppm) 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 2.135. 1H-1H gCOSY of 35 (500 MHz CDCl3) STol NHTroc O OTBS NH 35 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.136. 1H-13C gHSQCAD of 35 (500 MHz CDCl3) 174 0 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f AcO AcO BnO BnO N Cbz LevO O TrocHN OBn O O O O NHTroc O AcO 36a Bn N Cbz 1.0 10.5 10.0 9.5 9.0 8.5 8.0 6 8 . 1 7 5 . 2 2 7.5 7.0 7 9 . 0 6.5 6.0 5 0 . 8 2 1 . 1 6 9 . 0 5.5 8 1 . 2 1 4 5 . 5 5.0 f1 (ppm) 4.5 4.0 8 7 . 9 7 1 . 3 9 6 . 2 3.5 5 8 . 0 0 0 . 1 3.0 5 0 . 3 1 6 . 1 2.5 6 6 . 4 6 6 . 2 7 3 . 2 7 5 . 2 2.0 6 7 . 2 1.5 1.0 0.5 0.0 -0.5 -1 Figure 2.137. 1H-NMR of 36α (500 MHz CDCl3) 2 3 . 6 0 2 2 6 . 0 7 1 6 4 . 0 7 1 4 4 . 0 7 1 9 9 . 7 5 1 5 4 . 4 5 1 1 0 . 8 3 1 4 9 . 7 3 1 2 9 . 6 3 1 3 7 . 8 2 1 9 5 . 8 2 1 5 5 . 8 2 1 2 5 . 8 2 1 2 4 . 8 2 1 7 3 . 8 2 1 5 2 . 8 2 1 1 1 . 8 2 1 8 0 . 8 2 1 7 8 . 7 2 1 8 7 . 7 2 1 0 7 . 7 2 1 7 5 . 7 2 1 5 5 . 7 2 1 7 2 . 7 2 1 2 1 . 0 0 1 8 6 . 5 9 7 7 . 4 7 1 6 . 4 7 5 3 . 4 7 2 2 . 4 7 5 7 . 2 7 7 3 . 2 7 4 1 . 2 7 5 7 . 8 6 6 6 . 7 6 7 3 . 7 6 5 5 . 5 6 0 4 . 3 6 0 8 . 4 5 0 2 . 0 5 8 7 . 7 3 1 6 . 7 3 6 6 . 3 3 7 2 . 3 3 7 8 . 9 2 1 8 . 9 2 4 8 . 7 2 2 8 . 7 2 3 0 . 1 2 0 0 . 1 2 9 8 . 0 2 6 8 . 0 2 5 4 . 6 1 6 3 . 6 1 AcO AcO BnO BnO N Cbz LevO O TrocHN OBn O O O O NHTroc O AcO 36a Bn N Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.138. 13C-NMR of 36α (125 MHz CDCl3) 175 AcO AcO BnO BnO N Cbz LevO O TrocHN OBn O O O O NHTroc O AcO 36a Bn N Cbz 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Figure 2.139. 1H-1H gCOSY of 36α (500 MHz CDCl3) 1 2 3 4 5 6 7 8 9 ) m p p ( 1 f AcO AcO BnO BnO N Cbz LevO O TrocHN OBn O O O O NHTroc O AcO 36a Bn N Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.140. 1H-13C gHSQCAD of 36α (500 MHz CDCl3) 176 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 ) m p p ( 1 f AcO AcO BnO BnO OBn O TrocHN O N Cbz LevO O O O NHTroc O AcO 36β Bn N Cbz 1.0 10.5 10.0 9.5 9.0 8.5 8.0 6 8 . 1 4 3 . 3 2 7.5 7.0 6.5 6 1 . 1 9 2 . 1 5 1 . 1 6.0 5 5 . 6 1 4 . 3 1 5 8 . 3 5.5 5.0 f1 (ppm) 4.5 4.0 2 1 . 1 1 0 9 . 2 6 0 . 1 3 0 . 3 3.5 0 2 . 1 2 1 . 5 0 3 . 4 2.5 8 2 . 2 6 3 2 9 . . 3 2 2.0 3.0 0 0 . 3 1.5 1.0 0.5 0.0 -0.5 -1 Figure 2.141. 1H-NMR of 36β (500 MHz CDCl3) AcO AcO BnO BnO OBn O TrocHN O N Cbz LevO O O O NHTroc O AcO 36β Bn N Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.142. 1H-13C gHSQCAD of 36β (500 MHz CDCl3) 177 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 ) m p p ( 1 f AcO AcO BnO BnO TBSO N Cbz O O O O NHTroc TrocHN OBn O O AcO 37a Bn N Cbz 10.5 10.0 9.5 9.0 8.5 8.0 0 7 . 1 0 9 . 4 2 7.5 7.0 0 5 . 1 6.5 6.0 7 8 . 3 8 4 . 1 5 6 . 3 5.5 1 1 . 4 3 9 . 5 5.0 5 0 2 1 . . 1 2 4.5 3 4 . 3 3 5 . 2 4.0 4 4 . 1 8 9 . 5 3.5 0 4 . 3 5 8 . 2 7 2 . 1 9 6 . 4 3.0 2 7 . 2 2.5 5 0 . 2 4 0 . 1 2.0 8 8 . 2 0 5 . 2 1.5 f1 (ppm) 0 2 . 2 0 0 . 9 3 0 . 1 1.0 9 4 . 5 1 0 . 1 0.0 0.5 -0.5 -1. Figure 2.143. 1H-NMR of 37α (500 MHz CDCl3) 7 5 . 0 7 1 2 4 . 0 7 1 2 7 . 7 5 1 7 2 . 4 5 1 5 0 . 8 3 1 1 0 . 8 3 1 7 8 . 6 3 1 5 7 . 8 2 1 8 5 . 8 2 1 5 5 . 8 2 1 4 5 . 8 2 1 9 4 . 8 2 1 2 4 . 8 2 1 5 1 . 8 2 1 1 0 . 8 2 1 8 9 . 7 2 1 2 9 . 7 2 1 0 9 . 7 2 1 7 8 . 7 2 1 8 6 . 7 2 1 8 5 . 7 2 1 7 2 . 7 2 1 3 6 . 8 9 2 0 . 5 7 0 9 . 4 7 7 3 . 4 7 5 7 . 2 7 1 2 . 2 7 7 1 . 2 7 5 7 . 8 6 8 2 . 8 6 8 6 . 7 6 4 6 . 7 6 2 4 . 7 6 3 8 . 6 6 4 4 . 7 5 6 5 . 3 5 4 0 . 3 5 1 7 . 3 3 1 3 . 3 3 0 6 . 5 2 9 5 . 5 2 5 0 . 1 2 8 9 . 0 2 1 9 . 0 2 1 8 . 0 2 8 7 . 7 1 7 7 . 7 1 5 5 . 6 1 0 5 . 6 1 3 7 . 4 - 3 8 . 4 - 5 8 . 4 - 3 9 . 4 - AcO AcO BnO BnO TBSO N Cbz O O O O NHTroc TrocHN OBn O O AcO 37a Bn N Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.144. 13C-NMR of 37α (125 MHz CDCl3) 178 AcO AcO BnO BnO TBSO N Cbz O O O O NHTroc TrocHN OBn O O AcO 37a Bn N Cbz 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f2 (ppm) 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 2.145. 1H-1H gCOSY of 37α (500 MHz CDCl3) AcO AcO BnO BnO TBSO N Cbz O O O O NHTroc TrocHN OBn O O AcO 37a Bn N Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.146. 1H-13C gHSQCAD of 37α (500 MHz CDCl3) 179 0 1 2 3 4 5 6 7 8 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f AcO AcO BnO BnO OBn O TrocHN O TBSO N Cbz O O O NHTroc O AcO 37β Bn N Cbz 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7.0 6.5 6.0 7 5 . 1 8 1 . 3 2 7.5 1 2 . 1 8 8 . 2 3 0 . 1 1 4 0 . 2 0 4 . 3 5.5 4 7 . 0 5.0 f1 (ppm) 1 1 . 3 7 5 . 1 1 6 . 2 4.5 5 1 . 5 9 4 . 2 4.0 0 3 . 3 2 4 . 5 3.5 6 0 . 1 6 2 . 3 3.0 3 0 . 3 8 0 . 2 9 5 . 2 7 7 . 2 2.0 1.5 2.5 7 2 . 3 7 0 . 8 1.0 8 7 . 4 5 0 . 1 0.0 0.5 -0.5 -1. Figure 2.147. 1H-NMR of 37β (500 MHz CDCl3) 2 5 . 0 7 1 8 7 . 7 5 1 4 5 . 6 5 1 5 2 . 4 5 1 3 1 . 8 3 1 2 0 . 8 3 1 7 8 . 6 3 1 4 7 . 8 2 1 1 6 . 8 2 1 7 5 . 8 2 1 3 5 . 8 2 1 1 5 . 8 2 1 7 4 . 8 2 1 1 2 . 8 2 1 0 1 . 8 2 1 7 9 . 7 2 1 3 9 . 7 2 1 8 7 . 7 2 1 6 6 . 7 2 1 1 6 . 7 2 1 6 5 . 7 2 1 7 2 . 7 2 1 7 0 . 7 2 1 6 1 . 2 0 1 6 7 . 5 9 4 7 . 9 7 1 3 . 5 7 9 8 . 4 7 8 6 . 4 7 4 3 . 4 7 5 6 . 3 7 2 1 . 2 7 3 9 . 1 7 6 9 . 0 7 2 4 . 0 7 4 5 . 8 6 3 6 . 7 6 7 5 . 7 6 1 4 . 7 6 4 9 . 6 6 1 3 . 5 6 3 2 . 7 5 0 2 . 6 5 5 1 . 0 5 9 2 . 3 3 2 6 . 5 2 4 0 . 1 2 4 9 . 0 2 3 8 . 7 1 0 8 . 7 1 8 5 . 6 1 1 8 . 4 - 3 9 . 4 - 9 9 . 4 - AcO AcO BnO BnO OBn O TrocHN O TBSO N Cbz O O O NHTroc O AcO 37β Bn N Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.148. 13C-NMR of 37β (125 MHz CDCl3) 180 AcO AcO BnO BnO OBn O TrocHN O TBSO N Cbz O O O NHTroc O AcO 37β Bn N Cbz 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f2 (ppm) 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0 Figure 2.149. 1H-1H gCOSY of 37β (500 MHz CDCl3) 0 1 2 3 4 5 6 7 8 9 ) m p p ( 1 f AcO AcO BnO BnO OBn O TrocHN O TBSO N Cbz O O O NHTroc O AcO 37β Bn N Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.150. 1H-13C gHSQCAD of 37β (500 MHz CDCl3) 181 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f AcO AcO BnO BnO OBn O TrocHN O N Cbz HO O O O NHTroc O AcO 38 Bn N Cbz 1.0 10.5 10.0 9.5 9.0 8.5 8.0 0 6 . 1 1 6 . 2 2 7.5 7.0 6.5 6 4 . 1 5 4 . 1 0 3 . 1 6.0 5.5 4 3 . 6 4 0 . 7 2 3 . 3 5.0 5 5 . 3 4.5 5 4 . 4 5 4 . 2 0 2 . 1 1 4.0 7 4 . 2 1 2 . 3 2 4 . 1 3.5 1 1 . 1 3.0 f1 (ppm) 9 8 . 2 3 3 . 2 7 0 . 2 8 0 . 1 7 7 . 2 6 1 . 3 2.0 2.5 1.5 1.0 0.5 0.0 -0.5 Figure 2.151. 1H-NMR of 38 (500 MHz CDCl3) 4 8 . 0 7 1 7 5 . 0 7 1 5 3 . 0 7 1 7 9 . 8 5 1 8 5 . 6 5 1 0 7 . 4 5 1 3 1 . 8 3 1 7 0 . 8 3 1 0 9 . 7 3 1 9 4 . 7 3 1 3 7 . 6 3 1 6 7 . 8 2 1 9 5 . 8 2 1 6 5 . 8 2 1 2 5 . 8 2 1 8 4 . 8 2 1 8 0 . 8 2 1 0 0 . 8 2 1 5 9 . 7 2 1 6 8 . 7 2 1 8 7 . 7 2 1 8 6 . 7 2 1 7 5 . 7 2 1 3 2 . 7 2 1 4 9 . 0 0 1 8 7 . 9 9 2 7 . 5 9 5 3 . 5 7 9 5 . 4 7 5 4 . 4 7 2 3 . 4 7 9 6 . 3 7 5 2 . 2 7 7 0 . 2 7 5 9 . 1 7 7 8 . 1 7 8 5 . 0 7 4 6 . 8 6 8 5 . 7 6 0 4 . 7 6 6 0 . 7 6 1 5 . 3 6 5 5 . 6 5 4 4 . 6 5 6 2 . 6 5 5 1 . 0 5 1 3 . 3 4 0 7 . 3 3 7 2 . 3 3 3 6 . 7 2 2 0 . 1 2 5 9 . 0 2 6 7 . 6 1 5 7 . 6 1 AcO AcO BnO BnO OBn O TrocHN O N Cbz HO O O O NHTroc O AcO 38 Bn N Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.152. 13C-NMR of 38 (125 MHz CDCl3) 182 AcO AcO BnO BnO OBn O TrocHN O N Cbz HO O O O NHTroc O AcO 38 Bn N Cbz 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.153. 1H-1H gCOSY of 38 (500 MHz CDCl3) AcO AcO BnO BnO OBn O TrocHN O N Cbz HO O O O NHTroc O AcO 38 Bn N Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.154. 1H-13C gHSQCAD of 38 (500 MHz CDCl3) 183 0 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f Ph BnO O AcO AcO BnO OBn O O O N3 TrocHN O OLev O O N Cbz O O NHTroc O AcO 39 Bn N Cbz 10.5 10.0 9.5 9.0 8.5 8.0 2 6 7 1 . 4 . 2 2 7.5 1 7 . 1 7.0 6.5 7 5 . 1 4 2 . 1 8 2 . 2 6.0 5 8 . 0 5.5 9 6 . 0 8 7 . 3 1 3 . 1 5.0 0 5 . 9 7 6 . 2 7 0 . 3 4.5 1 3 . 4 4.0 9 4 . 1 5 6 . 1 9 8 . 4 2 1 . 6 3.5 4 5 . 2 5 2 . 2 3.0 7 3 5 8 . . 1 2 2.5 5 8 . 1 6 6 . 1 2.0 8 7 . 2 0 1 . 2 1.5 4 9 . 1 1 0 . 4 0 9 . 2 0 9 . 2 1 7 . 2 1.0 9 3 . 2 0.5 f1 (ppm) 0.0 -0.5 Figure 2.155. 1H-NMR of 39 (500 MHz CDCl3) 4 2 . 1 7 1 8 6 . 7 5 1 2 5 . 6 5 1 1 1 . 8 3 1 2 2 . 9 2 1 9 9 . 8 2 1 1 7 . 8 2 1 0 7 . 8 2 1 5 6 . 8 2 1 9 5 . 8 2 1 3 5 . 8 2 1 9 4 . 8 2 1 5 4 . 8 2 1 4 4 . 8 2 1 0 4 . 8 2 1 6 3 . 8 2 1 1 2 . 8 2 1 7 0 . 8 2 1 2 0 . 8 2 1 9 8 . 7 2 1 4 7 . 7 2 1 8 5 . 7 2 1 3 5 . 7 2 1 4 2 . 7 2 1 8 0 . 6 2 1 1 0 . 6 2 1 1 5 . 1 0 1 6 3 . 7 7 1 3 . 5 7 1 3 . 4 7 5 2 . 4 7 4 6 . 3 7 7 0 . 2 7 0 8 . 1 7 2 7 . 1 7 6 5 . 8 6 4 5 . 7 6 4 1 . 7 6 3 0 . 7 6 6 1 . 3 6 1 9 . 7 3 3 7 . 7 3 2 3 . 3 3 9 8 . 9 2 3 8 . 9 2 4 7 . 9 2 9 6 . 7 2 8 4 . 7 2 2 0 . 1 2 9 9 . 0 2 5 9 . 0 2 0 9 . 0 2 8 5 . 6 1 3 3 . 6 1 Ph BnO O AcO AcO BnO OBn O O O N3 TrocHN O OLev O O N Cbz O O NHTroc O AcO 39 Bn N Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.156. 13C-NMR of 39 (125 MHz CDCl3) 184 Ph BnO O AcO AcO BnO OBn O O O N3 TrocHN O OLev O O N Cbz O O NHTroc O AcO 39 Bn N Cbz 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.157. 1H-1H gCOSY of 39 (500 MHz CDCl3) Ph BnO O AcO AcO BnO OBn O O O N3 TrocHN O OLev O O N Cbz O O NHTroc O AcO 39 Bn N Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.158. 1H-13C gHSQCAD of 39 (500 MHz CDCl3) 185 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f Ph BnO O AcO AcO BnO OBn O O N3 TrocHN O OH O O N Cbz O O O NHTroc O AcO 40 Bn N Cbz 10.5 10.0 9.5 9.0 8.5 8.0 2 0 4 0 . 5 . 2 2 7.5 0 7 . 1 7.0 3 1 . 1 6.5 3 3 . 1 6 4 . 1 8 4 . 1 6.0 2 5 . 5 5.5 5 5 . 1 6 2 . 3 0 5 . 1 8 2 . 1 4 9 . 4 4.5 3 9 4 9 . . 2 7 4.0 9 4 . 4 9 0 . 4 3.5 6 3 . 1 2 1 . 2 6 9 . 2 3.0 6 2 . 0 1 5.0 f1 (ppm) 4 0 . 3 8 0 . 2 1 0 . 4 2 6 . 2 1 3 . 3 2.0 2.5 1.5 1.0 0.5 0.0 -0.5 Figure 2.159. 1H-NMR of 40 (500 MHz CDCl3) 9 9 . 0 7 1 6 4 . 0 7 1 6 3 . 0 7 1 5 8 . 9 5 1 2 1 . 8 3 1 6 0 . 8 3 1 7 9 . 7 3 1 7 7 . 6 3 1 1 7 . 6 3 1 4 2 . 9 2 1 5 7 . 8 2 1 0 7 . 8 2 1 2 6 . 8 2 1 3 5 . 8 2 1 2 5 . 8 2 1 9 4 . 8 2 1 7 4 . 8 2 1 9 3 . 8 2 1 5 3 . 8 2 1 0 1 . 8 2 1 5 0 . 8 2 1 2 9 . 7 2 1 1 8 . 7 2 1 7 7 . 7 2 1 8 5 . 7 2 1 8 2 . 7 2 1 5 1 . 6 2 1 0 6 . 1 0 1 4 0 . 9 7 6 3 . 7 7 0 3 . 5 7 5 7 . 4 7 4 4 . 4 7 2 3 . 4 7 5 6 . 3 7 8 2 . 3 7 0 1 . 2 7 4 8 . 1 7 3 8 . 9 6 5 6 . 8 6 6 5 . 8 6 4 1 . 8 6 6 5 . 7 6 7 4 . 7 6 2 5 . 4 6 5 6 . 3 6 6 1 . 6 5 2 2 . 0 5 6 3 . 3 4 5 4 . 3 3 3 0 . 1 2 1 0 . 1 2 3 9 . 0 2 5 8 . 6 1 Ph BnO O AcO AcO BnO OBn O O N3 TrocHN O OH O O N Cbz O O O NHTroc O AcO 40 Bn N Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.160. 13C-NMR of 40 (125 MHz CDCl3) 186 Ph BnO O AcO AcO BnO OBn O O N3 TrocHN O OH O O N Cbz O O O NHTroc O AcO 40 Bn N Cbz 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Figure 2.161. 1H-1H gCOSY of 40 (500 MHz CDCl3) 1 2 3 4 5 6 7 8 ) m p p ( 1 f Ph BnO O AcO AcO BnO OBn O O N3 TrocHN O OH O O N Cbz O O O NHTroc O AcO 40 Bn N Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.162. 1H-13C gHSQCAD of 40 (500 MHz CDCl3) 187 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f Ph BnO O AcO AcO BnO OBn N3 O O O N3 TrocHN O O N Cbz O O O NHTroc O AcO 41 Bn N Cbz 10.5 10.0 9.5 9.0 8.5 8.0 9 5 . 7 2 8 3 . 1 9 8 . 1 7.5 7.0 8 4 . 1 9 8 . 0 6 3 . 1 6.5 5 9 . 0 6.0 2 4 . 1 5 7 . 2 3 1 . 1 5.5 5 9 . 0 1 1 . 2 9 8 . 0 5.0 0 5 9 2 . . 9 1 4.5 2 8 . 1 6 6 . 2 4.0 3 5 . 1 5 2 . 1 3.5 f1 (ppm) 6 5 . 2 1 9 6 . 1 9 6 . 2 4 1 . 6 3.0 7 0 . 1 2.5 6 6 . 2 6 8 . 2 2.0 0 9 . 2 7 9 . 1 9 8 . 3 1.5 1.0 0.5 0.0 -0.5 Figure 2.163. 1H-NMR of 41 (500 MHz CDCl3) 8 8 . 0 7 1 3 5 . 0 7 1 6 4 . 8 5 1 0 4 . 4 5 1 8 1 . 8 3 1 5 1 . 8 3 1 1 0 . 8 3 1 7 7 . 6 3 1 0 7 . 6 3 1 3 2 . 9 2 1 9 1 . 9 2 1 8 9 . 8 2 1 6 7 . 8 2 1 9 6 . 8 2 1 3 6 . 8 2 1 9 5 . 8 2 1 3 5 . 8 2 1 9 4 . 8 2 1 9 3 . 8 2 1 2 1 . 8 2 1 7 9 . 7 2 1 7 7 . 7 2 1 7 6 . 7 2 1 1 6 . 7 2 1 8 5 . 7 2 1 9 2 . 7 2 1 8 9 . 5 2 1 2 9 . 5 2 1 7 6 . 1 0 1 6 3 . 7 7 9 6 . 6 7 5 3 . 5 7 2 9 . 4 7 7 4 . 4 7 4 3 . 4 7 8 6 . 3 7 0 1 . 2 7 3 8 . 1 7 8 9 . 9 6 3 6 . 8 6 6 4 . 8 6 0 9 . 7 6 2 6 . 7 6 7 5 . 7 6 9 8 . 9 5 3 1 . 6 5 2 2 . 0 5 6 3 . 3 4 9 1 . 3 3 8 0 . 1 2 6 0 . 1 2 9 9 . 0 2 4 9 . 0 2 4 8 . 6 1 Ph BnO O AcO AcO BnO OBn N3 O O O N3 TrocHN O O N Cbz O O O NHTroc O AcO 41 Bn N Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.164. 13C-NMR of 41 (125 MHz CDCl3) 188 Ph BnO O AcO AcO BnO OBn N3 O O O N3 TrocHN O O N Cbz O O O NHTroc O AcO 41 Bn N Cbz 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.165. 1H-1H gCOSY of 41 (500 MHz CDCl3) Ph BnO O AcO AcO BnO OBn N3 O O O N3 TrocHN O O N Cbz O O O NHTroc O AcO 41 Bn N Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.166. 1H-13C gHSQCAD of 41 (500 MHz CDCl3) 189 0 1 2 3 4 5 6 7 8 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f N3 O HO HO N3 TrocHN O O N Cbz O O O NHTroc O AcO 42 Bn N Cbz BnO O AcO AcO BnO OBn 2 3 . 2 5 3 . 1 10.5 10.0 9.5 9.0 8.5 8.0 7.0 6.5 6.0 5.5 3.5 3.0 2.5 7 9 . 2 7 1 . 2 2 8 . 2 7 5 . 2 0 8 . 2 2.0 1.5 1.0 0.5 0.0 -0.5 -1. 9 5 . 1 5 8 . 3 2 7.5 0 1 . 7 6 3 . 2 5.0 2 7 . 1 9 1 . 7 4.5 6 3 . 2 1 2 . 4 7 9 . 3 1 8 0 . 2 4.0 f1 (ppm) 0 2 . 1 1 Figure 2.167. 1H-NMR of 42 (500 MHz CDCl3) 7 1 . 1 7 1 2 5 . 0 7 1 1 4 . 0 7 1 8 5 . 8 5 1 8 5 . 6 5 1 4 8 . 4 5 1 3 6 . 4 5 1 5 1 . 8 3 1 2 1 . 8 3 1 6 9 . 7 3 1 3 5 . 7 3 1 0 7 . 6 3 1 3 7 . 8 2 1 5 6 . 8 2 1 8 5 . 8 2 1 0 5 . 8 2 1 6 4 . 8 2 1 9 0 . 8 2 1 1 9 . 7 2 1 3 7 . 7 2 1 8 5 . 7 2 1 6 5 . 7 2 1 6 2 . 7 2 1 2 1 . 2 0 1 1 6 . 9 9 0 9 . 7 9 2 9 . 5 9 4 7 . 5 9 1 7 . 9 7 6 3 . 7 7 4 3 . 5 7 7 9 . 4 7 2 3 . 4 7 4 6 . 3 7 6 0 . 2 7 2 8 . 1 7 2 0 . 0 7 7 5 . 8 6 5 5 . 7 6 2 9 . 6 6 9 6 . 3 6 3 2 . 3 6 3 2 . 2 6 4 0 . 2 6 5 0 . 6 5 8 3 . 4 5 4 1 . 4 5 7 1 . 0 5 8 3 . 3 4 6 2 . 3 3 4 4 . 7 2 0 0 . 1 2 3 9 . 0 2 8 7 . 6 1 N3 O HO HO N3 TrocHN O O N Cbz O O O NHTroc O AcO 42 Bn N Cbz BnO O AcO AcO BnO OBn 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.168. 13C-NMR of 42 (125 MHz CDCl3) 190 N3 O HO HO N3 TrocHN O O N Cbz O O O NHTroc O AcO 42 Bn N Cbz BnO O AcO AcO BnO OBn 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f2 (ppm) 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.169. 1H-1H gCOSY of 42 (500 MHz CDCl3) N3 O HO HO N3 TrocHN O O N Cbz O O O NHTroc O AcO 42 Bn N Cbz BnO O AcO AcO BnO OBn 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.170. 1H-13C gHSQCAD of 42 (500 MHz CDCl3) 191 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 ) m p p ( 1 f 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f MeOOC HO N3 O N3 TrocHN O O N Cbz O O O NHTroc O AcO 43 Bn N Cbz BnO O AcO AcO BnO OBn 1.0 10.5 10.0 9.5 9.0 8.5 8.0 0 0 . 4 2 7.5 6 9 . 1 0 4 . 2 4 0 . 1 7.0 8 1 . 1 7 9 . 0 6.5 8 3 . 1 6.0 0 5 . 1 3 2 . 1 9 6 . 1 5.5 6 9 . 5 6 8 . 3 8 5 . 5 5.0 0 5 0 3 . . 2 3 4.5 0 1 . 3 7 9 . 3 4.0 5 7 . 3 2 1 . 3 3.5 4 6 . 2 8 4 . 1 3 9 . 2 3.0 5 4 . 1 1 7 . 2 4 1 . 1 2.5 7 7 . 2 2.0 7 1 . 1 4 5 . 4 5 1 . 4 f1 (ppm) 1.5 1.0 0.5 0.0 -0.5 Figure 2.171. 1H-NMR of 43 (500 MHz CDCl3) 0 7 . 0 7 1 8 5 . 0 7 1 2 3 . 0 7 1 8 5 . 6 5 1 5 7 . 4 5 1 6 2 . 8 3 1 1 2 . 8 3 1 6 0 . 8 3 1 7 5 . 7 3 1 4 7 . 6 3 1 8 7 . 8 2 1 8 6 . 8 2 1 3 6 . 8 2 1 7 5 . 8 2 1 0 5 . 8 2 1 8 4 . 8 2 1 5 1 . 8 2 1 5 0 . 8 2 1 2 0 . 8 2 1 1 9 . 7 2 1 1 7 . 7 2 1 3 6 . 7 2 1 1 6 . 7 2 1 5 2 . 7 2 1 9 5 . 9 9 0 8 . 9 7 6 3 . 7 7 1 1 . 6 7 8 3 . 5 7 3 1 . 5 7 2 2 . 4 7 3 6 . 3 7 1 1 . 2 7 0 7 . 1 7 9 4 . 1 7 0 9 . 9 6 7 6 . 8 6 4 6 . 7 6 1 6 . 7 6 9 9 . 6 6 1 3 . 3 6 0 1 . 2 6 4 1 . 6 5 0 1 . 4 5 1 4 . 3 5 4 2 . 0 5 9 2 . 3 4 5 2 . 3 3 1 8 . 9 2 9 5 . 7 2 1 1 . 1 2 9 9 . 0 2 6 7 . 0 2 4 8 . 6 1 MeOOC HO N3 O N3 TrocHN O O N Cbz O O O NHTroc O AcO 43 Bn N Cbz BnO O AcO AcO BnO OBn 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.172. 13C-NMR of 43 (125 MHz CDCl3) 192 MeOOC HO N3 O N3 TrocHN O O N Cbz O O O NHTroc O AcO 43 Bn N Cbz BnO O AcO AcO BnO OBn 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.173. 1H-1H gCOSY of 43 (500 MHz CDCl3) MeOOC HO N3 O N3 TrocHN O O N Cbz O O O NHTroc O AcO 43 Bn N Cbz BnO O AcO AcO BnO OBn 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.174. 1H-13C gHSQCAD of 43 (500 MHz CDCl3) 193 1 2 3 4 5 6 7 8 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f BnO BnO OAc O N3 O BnO O AcO AcO BnO OBn MeOOC N3 O N3 TrocHN O O N Cbz O O O NHTroc O AcO 44 Bn N Cbz 10.5 10.0 9.5 9.0 8.5 8.0 6 7 . 1 7 3 . 2 3 7.5 7.0 9 1 . 1 9 4 . 1 6.5 4 8 . 1 2 2 . 1 6.0 4 3 . 3 0 3 . 1 5 3 . 1 4 2 . 1 5.5 2 2 . 3 5.0 9 8 . 6 3 4 . 5 4 5 . 4 2 3 . 3 4.5 8 8 . 1 4.0 1 9 . 2 0 0 . 5 1 3.5 7 9 . 1 9 2 . 1 4 5 . 4 3.0 0 3 . 3 1 3 . 1 2.5 3 6 . 2 6 5 . 5 2.0 6 0 . 3 6 0 . 2 6 3 . 3 f1 (ppm) 1.5 1.0 0.5 0.0 -0.5 Figure 2.175. 1H-NMR of 44 (500 MHz CDCl3) 4 7 . 0 7 1 8 5 . 0 7 1 4 4 . 0 7 1 3 3 . 8 5 1 3 1 . 8 3 1 7 0 . 8 3 1 6 5 . 7 3 1 8 4 . 7 3 1 3 7 . 6 3 1 6 6 . 8 2 1 2 6 . 8 2 1 6 5 . 8 2 1 2 5 . 8 2 1 0 5 . 8 2 1 3 4 . 8 2 1 0 4 . 8 2 1 9 1 . 8 2 1 8 0 . 8 2 1 5 0 . 8 2 1 8 9 . 7 2 1 0 9 . 7 2 1 2 8 . 7 2 1 8 6 . 7 2 1 3 6 . 7 2 1 3 5 . 7 2 1 7 4 . 7 2 1 2 2 . 7 2 1 9 8 . 8 9 3 2 . 0 8 0 3 . 7 7 9 1 . 7 7 6 7 . 5 7 8 6 . 5 7 2 2 . 5 7 8 0 . 5 7 9 3 . 4 7 8 2 . 4 7 2 2 . 4 7 9 5 . 3 7 0 0 . 2 7 7 6 . 1 7 6 2 . 0 7 9 8 . 9 6 7 5 . 8 6 7 4 . 7 6 2 4 . 7 6 2 8 . 3 6 6 0 . 2 6 4 1 . 6 5 3 0 . 3 5 0 2 . 3 3 5 9 . 0 2 6 8 . 0 2 5 7 . 6 1 BnO BnO OAc O N3 O BnO O AcO AcO BnO OBn MeOOC N3 O N3 TrocHN O O N Cbz O O O NHTroc O AcO 44 Bn N Cbz 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.176. 13C-NMR of 44 (125 MHz CDCl3) 194 BnO BnO OAc O N3 O BnO O AcO AcO BnO OBn MeOOC N3 O N3 TrocHN O O N Cbz O O O NHTroc O AcO 44 Bn N Cbz 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Figure 2.177. 1H-1H gCOSY of 44 (500 MHz CDCl3) 1 2 3 4 5 6 7 8 ) m p p ( 1 f BnO BnO OAc O N3 O BnO O AcO AcO BnO OBn MeOOC N3 O N3 TrocHN O O N Cbz O O O NHTroc O AcO 44 Bn N Cbz 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.178. 1H-13C gHSQCAD of 44 (500 MHz CDCl3) 195 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f BnO BnO OAc O AcHN MeOOC O AcHN NHAc O O N Cbz BnO O AcO AcO BnO OBn AcHN O O O O NHAc Bn N Cbz O AcO 45 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 5 6 . 3 3 4 3 . 2 6 6 . 1 7.0 4 3 . 1 7 0 . 1 6.5 0 8 . 0 6.0 3 8 . 3 9 7 . 0 6 7 . 0 5.5 7 9 . 0 7 2 . 1 5.0 0 8 . 3 9 3 . 1 4.5 7 9 . 3 2 2 . 1 7 0 . 1 8 6 . 3 4.0 6 3 . 3 3.5 4 2 . 1 8 2 . 6 3.0 5 7 . 2 5 3 . 8 5 1 . 2 2.5 1 3 . 4 5 9 . 2 2.0 2 6 . 2 8 2 . 1 9 7 . 2 9 9 . 0 1.5 1 0 . 3 1.0 f1 (ppm) 3 0 . 0 1 4 6 . 4 1 4 . 6 0.5 0 0 7 3 . . 2 2 0.0 6 6 . 1 0 7 . 0 -0.5 -1. Figure 2.179. 1H-NMR of 45 (500 MHz CDCl3) 6 3 . 5 7 1 4 2 . 2 7 1 7 6 . 1 7 1 4 8 . 0 7 1 0 8 . 0 7 1 9 7 . 0 7 1 8 2 . 8 3 1 4 0 . 8 3 1 2 7 . 8 2 1 2 6 . 8 2 1 7 5 . 8 2 1 0 5 . 8 2 1 8 4 . 8 2 1 6 4 . 8 2 1 1 4 . 8 2 1 7 3 . 8 2 1 6 2 . 8 2 1 5 1 . 8 2 1 9 0 . 8 2 1 1 0 . 8 2 1 8 9 . 7 2 1 2 9 . 7 2 1 8 8 . 7 2 1 5 8 . 7 2 1 0 8 . 7 2 1 8 7 . 7 2 1 4 7 . 7 2 1 1 7 . 7 2 1 9 6 . 7 2 1 5 6 . 7 2 1 1 6 . 7 2 1 7 5 . 7 2 1 4 5 . 7 2 1 1 5 . 7 2 1 7 4 . 7 2 1 0 3 . 7 2 1 9 4 . 5 7 0 1 . 5 7 2 4 . 4 7 0 9 . 1 7 7 1 . 0 7 0 8 . 2 5 9 4 . 2 5 0 0 . 3 3 6 0 . 3 2 2 9 . 2 2 6 8 . 2 2 3 7 . 2 2 7 9 . 0 2 4 9 . 0 2 1 9 . 0 2 8 8 . 0 2 1 8 . 0 2 6 3 . 6 1 BnO BnO OAc O AcHN MeOOC O AcHN NHAc O O N Cbz BnO O AcO AcO BnO OBn AcHN O O O O NHAc Bn N Cbz O AcO 45 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.180. 13C-NMR of 45 (125 MHz CDCl3) 196 BnO BnO OAc O AcHN MeOOC O AcHN NHAc O O N Cbz BnO O AcO AcO BnO OBn AcHN O O O O NHAc Bn N Cbz O AcO 45 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Figure 2.181. 1H-1H gCOSY of 45 (500 MHz CDCl3) 1 2 3 4 5 6 7 8 9 ) m p p ( 1 f BnO BnO OAc O AcHN MeOOC O AcHN NHAc O O N Cbz BnO O AcO AcO BnO OBn AcHN O O O O NHAc Bn N Cbz O AcO 45 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.182. 1H-13C gHSQCAD of 45 (500 MHz CDCl3) 197 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f HO HO OH O AcHN HO O HO HO HO OH HOOC O AcHN NHAc O O NH AcHN O O O O NHAc O HO 2 NH2 0 9 . 0 0 8 . 0 8 7 . 0 9 0 . 1 1 1 . 1 4 0 . 1 8 9 . 0 6 0 . 1 4 4 . 6 8 6 . 1 7 2 . 5 1 1 . 8 6 8 . 3 7 1 . 1 2 2 . 2 6 0 . 3 8 3 . 2 2 6 . 5 4 6 . 2 7 2 . 2 2 7 . 3 1 4 . 2 9 2 . 3 6.5 6.0 5.5 5.0 4.5 4.0 3.5 f1 (ppm) 3.0 2.5 2.0 1.5 1.0 0.5 Figure 2.183. 1H-NMR of 2 (500 MHz D2O, PRESAT) 4 3 . 1 8 1 7 0 . 5 7 1 1 8 . 4 7 1 3 6 . 4 7 1 7 1 . 4 7 1 3 9 . 3 7 1 2 6 . 3 7 1 8 8 . 1 0 1 0 1 . 1 0 1 7 0 . 1 0 1 6 6 . 6 9 0 5 . 6 9 1 7 . 8 7 5 7 . 5 7 5 1 . 5 7 8 7 . 3 7 8 4 . 3 7 8 4 . 2 7 7 5 . 1 7 4 2 . 0 7 9 1 . 0 7 6 0 . 0 7 8 2 . 9 6 9 8 . 8 6 6 4 . 8 6 9 2 . 8 6 7 6 . 7 6 5 7 . 5 6 5 0 . 3 6 7 9 . 0 6 7 6 . 9 5 6 6 . 5 5 7 3 . 3 5 2 2 . 3 5 7 4 . 1 5 9 8 . 0 5 0 3 . 7 3 1 2 . 6 3 7 5 . 6 2 9 1 . 3 2 2 4 . 2 2 7 9 . 1 2 1 8 . 1 2 3 7 . 1 2 5 6 . 1 2 8 9 . 5 1 HO HO OH O AcHN HO O HO HO HO OH HOOC O AcHN NHAc O O NH AcHN O O O O NHAc O HO 2 NH2 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.184. 13C-NMR of 2 (125 MHz D2O) 198 HO HO OH O AcHN HO O HO HO HO OH HOOC O AcHN NHAc O O NH AcHN O O O O NHAc O HO 2 NH2 6.5 6.0 5.5 5.0 4.5 4.0 3.5 f2 (ppm) 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Figure 2.185. 1H-1H gCOSY of 2 (500 MHz D2O) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 ) m p p ( 1 f HO HO OH O AcHN HO O HO HO HO OH HOOC O AcHN NHAc O O NH AcHN O O O O NHAc O HO 2 NH2 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 2.186. 1H-13C gHSQCAD of 2 (500 MHz D2O) 199 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f HO HO OH O AcHN HO O HO HO HO OH HOOC O AcHN NHAc O O NH AcHN O O O O NHAc O HO 2 NH2 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 f2 (ppm) 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 Figure 2.187. 1H-1H TOCSY of 2 (500 MHz D2O) HO HO OH O AcHN HO O HO HO HO OH HOOC O AcHN NHAc O O NH AcHN O O O O NHAc O HO 2 NH2 Figure 2.188. ESI-MS of 2 200 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 ) m p p ( 1 f HO HO OH O AcHN HOOC O AcHN NHAc O O NH HO O HO HO HO OH O HO AcHN O O O O NHAc 47 O N H O O N O O 6.5 6.0 4 1 . 1 5.5 8 9 . 0 5.0 4 9 . 1 8 1 . 1 5 9 . 0 4.5 4 9 . 3 2 4.0 3.5 f1 (ppm) 7 7 . 3 1 7 . 1 0 6 . 2 4 1 . 6 6 0 . 1 3.0 2 1 . 2 9 7 . 2 7 7 . 2 5 1 . 3 7 4 . 5 2 3 . 5 2.0 2.5 5 1 . 3 1.5 1.0 0.5 Figure 2.189. 1H-NMR of 47 (500 MHz CD3OD) HO HO OH O AcHN HOOC O AcHN NHAc O O NH HO O HO HO HO OH O HO AcHN O O O O NHAc 47 O N H O O N O O Figure 2.190. ESI-MS of 47 201 REFERENCES 202 REFERENCES 1. Niedziela, T.; Letowska, I.; Lukasiewicz, J.; Kaszowska, M.; Czarnecka, A.; Kenne, L.; Lugowski, C., Epitope of the vaccine-type Bordetella pertussis strain 186 lipooligosaccharide and antiendotoxin activity of antibodies directed against the terminal pentasaccharide-tetanus toxoid conjugate. Infect. Immun. 2005, 73 (11), 7381-7389. 2. Guo, J.; Frost, J. W., Kanosamine biosynthesis:  a likely source of the aminoshikimate pathway's nitrogen atom. J. Am. Chem. Soc. 2002, 124 (36), 10642-10643. 3. Mo, K.-F.; Li, X.; Li, H.; Low, L. Y.; Quinn, C. P.; Boons, G.-J., Endolysins of Bacillus anthracis bacteriophages recognize unique carbohydrate epitopes of vegetative cell wall polysaccharides with high affinity and selectivity. J. Am. Chem. Soc. 2012, 134 (37), 15556-15562. 4. Visansirikul, S.; Yasomanee, J. P.; Pornsuriyasak, P.; Kamat, M. N.; Podvalnyy, N. M.; Gobble, C. P.; Thompson, M.; Kolodziej, S. A.; Demchenko, A. V., A concise synthesis of the repeating unit of capsular polysaccharide Staphylococcus aureus type 8. Org. Lett. 2015, 17 (10), 2382-2384. 5. Emmadi, M.; Kulkarni, S. S., Expeditious synthesis of bacterial, rare sugar building blocks to access the prokaryotic glycome. Org. Biomol. Chem. 2013, 11 (19), 3098-3102. 6. Emmadi, M.; Kulkarni, S. S., Synthesis of orthogonally protected bacterial, rare-sugar and D-glycosamine building blocks. Nat. Protoc. 2013, 8 (10), 1870-1889. 7. Van den Bos, L. J.; Codée, J. D.; Van Boom, J. H.; Overkleeft, H. S.; Van der Marel, G. A., A novel strategy towards the synthesis of orthogonally functionalised 4-aminoglycosides. Org. Biomol. Chem. 2003, 1 (23), 4160-4165. 8. Van den Bos, L. J.; Boltje, T. J.; Provoost, T.; Mazurek, J.; Overkleeft, H. S.; Van der Marel, G. A., A synthetic study towards the PSA1 tetrasaccharide repeating unit. Tetrahedron Lett. 2007, 48 (15), 2697-2700. 9. Crich, D.; Banerjee, A., Synthesis and stereoselective glycosylation of D- and L-glycero-β-D-manno-heptopyranoses. Org. Lett. 2005, 7 (7), 1395-1398. 10. Ohara, T.; Adibekian, A.; Esposito, D.; Stallforth, P.; Seeberger, P. H., Towards the synthesis of a Yersinia pestis cell wall polysaccharide: enantioselective synthesis of an L-glycero-D-manno-heptose building block. Chem. Commun. 2010, 46 (23), 4106-4108. 11. Durka, M.; Tikad, A.; Périon, R.; Bosco, M.; Andaloussi, M.; Floquet, S.; Malacain, E.; Moreau, F.; Oxoby, M.; Gerusz, V.; Vincent, S. P., Systematic synthesis of inhibitors of the two 203 first enzymes of the bacterial heptose biosynthetic pathway: Towards antivirulence molecules targeting lipopolysaccharide biosynthesis. Chem. Eur. J. 2011, 17 (40), 11305-11313. 12. Mulani, S. K.; Cheng, K.-C.; Mong, K.-K. T., General homologation strategy for synthesis of L-glycero-and D-glycero-heptopyranoses. Org. Lett. 2015, 17 (22), 5536-5539. C.; I. R., 13. Stanetty, crystalline 1,2,3,4,6,7-hexa-O-acetyl-L-glycero-α-D-manno-heptopyranose. Eur. J. Org. Chem. 2015, 2718-2726. Large-Scale Baxendale, synthesis of 14. Yasomanee, J. P.; Demchenko, A. V., Effect of remote picolinyl and picoloyl substituents on the stereoselectivity of chemical glycosylation. J. Am. Chem. Soc. 2012, 134 (49), 20097-20102. 15. Medgyes, A.; Farkas, E.; Lipták, A.; Pozsgay, V., Synthesis of the monosaccharide units of the O-specific polysaccharide of Shigella sonnei. Tetrahedron 1997, 53 (12), 4159-4178. 16. Medgyes, A.; Bajza, I.; Farkas, E.; Pozsgay, V.; Lipták, A., Synthetic studies towards the O-specific polysaccharide of Shigella Sonnei. J. Carbohydr. Chem. 2000, 19 (3), 285-310. 17. Pfister, H. B.; Mulard, L. A., Synthesis of the zwitterionic repeating unit of the O-antigen from Shigella sonnei and chain elongation at both ends. Org. Lett. 2014, 16 (18), 4892-4895. 18. Podilapu, A. R.; Kulkarni, S. S., Total synthesis of repeating unit of O-polysaccharide of Providencia alcalifaciens O22 via one-pot glycosylation. Org. Lett. 2017, 19 (19), 5466-5469. 19. Leyva, A.; Quintana, A.; Sánchez, M.; Rodríguez, E. N.; Cremata, J.; Sánchez, J. C., Rapid and sensitive anthrone–sulfuric acid assay in microplate format to quantify carbohydrate in biopharmaceutical products: method development and validation. Biologicals 2008, 36 (2), 134-141. 20. Zlamy, M., Rediscovering pertussis. Front. Pediatr. 2016, 4:52. 21. Marzouqi, I.; Richmond, P.; Fry, S.; Wetherall, J.; Mukkur, T., Development of improved vaccines against whooping cough: current status. Hum. Vaccin. 2010, 6 (7), 543-553. 22. Friedman, R. L.; Nordensson, K.; Wilson, L.; Akporiaye, E.; Yocum, D., Uptake and intracellular survival of Bordetella pertussis in human macrophages. Infect. Immun. 1992, 60 (11), 4578-4585. 23. Lamberti, Y.; Perez Vidakovics, M. L.; van der Pol, L.-W.; Rodríguez, M. E., Cholesterol-rich domains are involved in Bordetella pertussis phagocytosis and intracellular survival in neutrophils. Microb. Pathog. 2008, 44 (6), 501-511. 24. Le Blay, K.; Caroff, M.; Blanchard, F.; Perry, M. B.; Chaby, R., Epitopes of Bordetella 204 pertussis lipopolysaccharides as potential markers for typing of isolates with monoclonal antibodies. Microbiology 1996, 142 (4), 971-978. 25. Demizu, Y.; Kubo, Y.; Miyoshi, H.; Maki, T.; Matsumura, Y.; Moriyama, N.; Onomura, O., Regioselective protection of sugars catalyzed by dimethyltin dichloride. Org. Lett. 2008, 10 (21), 5075-5077. 26. Crich, D.; Picione, J., Direct synthesis of the β-l-rhamnopyranosides. Org. Lett. 2003, 5 (5), 781-784. 205 Chapter 3. Heparin Nanoparticles for β Amyloid Binding and Mitigation of β Amyloid Associated Cytotoxicity 3.1. Introduction Alzheimer’s disease (AD) has become the most common form of dementia, which is affecting about 5.2 million Americans.1 The number of AD patients is predicted to increase significantly and expected to triple by 2050.2 One of the main pathological hallmarks of AD is the senile plaques formed by Aβ. Aβ is derived from amyloid precursor protein processing by β- and γ-secretases and proposed to be a causative agent of AD.3 Aβ can aggregate into highly toxic oligomers and deposit as plaques on the cerebral cortex damaging the nervous system.4-6 Glycosaminoglycans (GAGs) are believed to play a central role in the amyloidosis pathway with many GAG bearing proteoglycans (PGs) found in both diffuse and neuritic amyloid plaques.7-8 GAGs on surface of neuronal cells can serve as nucleating sites for Aβ aggregation, contribute to the formation of neurotoxic Aβ deposits on cells.9-14 Heparin is a member of the GAG family, which is known to be able to bind with Aβ 12, 15-16. We envision that nanoparticles coated with heparin can be utilized to mimic PG bearing neuronal cells and potentially compete for their interactions with Aβ. Although heparin nanoparticles have been utilized for cancer targeting, anti-coagulation, tissue engineering and drug delivery,17-22 their interactions with Aβ have not been studied before. Herein we report the synthesis of heparin-functionalized magnetic glyconanoparticles. These nanoparticles can bind with Aβ, induce the formation of fibril, and protect neuronal cells from Aβ induced cell death. 206 3.2. Results and Discussion 3.2.1. Preparation and Characterization of Hep-SPION. We selected magnetic nanoparticles as the core of our heparin nanoparticles since magnetic nanoparticles are a powerful platform for biological applications due to their high surface area, biocompatibility and magnetic relaxivity.23-25 Two methods were explored to immobilize heparin polysaccharides onto magnetic nanoparticles. In the first approach, we adapted our previous synthesis of colloidal hyaluronan nanoparticles.26 Magnetite nanoparticles were first produced through the thermal decomposition method, resulting in hydrophobic magnetic nanoparticles mainly coated with oleic acid (Scheme 3.1A). Exchanging the oleic acid ligand with heparin was performed in a water/toluene biphasic system. However, although the resulting nanoparticles could be dispersed in water or phosphate buffered saline (PBS), they quickly precipitated out of aqueous solutions. This was most likely due to the lower efficiency of ligand displacement from the hydrophobic nanoparticles by heparin as compared to hyaluronan, as heparin is more charged and presumably less soluble in the organic solvent for ligand exchange. An entirely aqueous solution based synthetic route was tested next. The magnetite core was constructed by the co-precipitation method by mixing ferric chloride and ferrous chloride with ammonium hydroxide in water (Scheme 3.1B).22 The resulting superparamagnetic iron oxide nanoparticles (SPION) were collected with a magnet and resuspended in water. Heparin sodium salt was then added, which could chelate with the SPIONs to form a stable colloid suspension. Removal of excess heparin by ultrafiltration produced the heparin-coated SPIONs 207 (Hep-SPION). A) Fe(acac)3 Oleic acid + + 1,2-hexadecanediol Benzyl ether 200 oC 2h then 300 oC 1h Fe3O4 Toluene : H2O Heparin reflux Fe3O4 Oleylamine + B) FeCl3•6H2O FeCl2•4H2O + conc. NH3•H2O H2O, N2 Fe3O4 SPION Heparin Fe3O4 Hep-SPION Scheme 3.1. Synthesis of heparin coated magnetic nanoparticles by A) the thermal decomposition and ligand exchange method, and B) the co-precipitation method. Hep-SPION was characterized by a series of techniques including transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential and thermogravimetric analysis (TGA). TEM images showed that the magnetite core had an average diameter around 10 nm (Figure 3.1A), with the hydrodynamic diameters of 68 nm in water and 59 nm in PBS buffer respectively. The successful attachment of heparin was supported by TGA analysis. While the amount of organic compounds only accounted for 3% of the gross weight of SPION by TGA analysis, heating the Hep-SPION to above 700 °C led to 63% weight loss suggesting that about 60% of the Hep-SPION mass was due to heparin attachment (Figure 3.1B). Furthermore, the zeta potential of the nanoparticles changed from +12.3 mV (SPION) to -53.3 mV (Hep-SPION), consistent with the high negative charge of heparin on Hep-SPION. 208 (A) (B) 100 ——— SPION Hep-SPION ) % ( t h g i e W d e i f i d o M 90 80 70 60 50 40 30 100 200 300 400 500 600 Temperature (°C) 700 800 900 Figure 3.1. (A) TEM characterization of Hep-SPION; (B) TGA of SPION and Hep-SPION. 3.2.2. Assessment of Binding between Aβ and Hep-SPION by ELISA. With the Hep-SPION in hand, its interaction with Aβ was investigated. Naturally isolated Aβ peptides exist in variable lengths ranging from 36 to 42 amino acid residues. We chose Aβ1-42 for our study, as it is the more amyloidogenic Aβ form.27-28 Furthermore, it is prone to aggregation6, 29 and is the major species found in the senile plaques of AD brains30. Aβ1-42 monomers were dissolved in 10 mM NaOH, neutralized with HCl and were incubated at 37℃ for 2 days. The resulting fibrils were added to a 96-well plate, which could adhere to the surface of the wells. Upon removal of the unbound peptide, the bound Aβ was detected by an anti-Aβ IgG monoclonal antibody 6E10 (mAb) through an enzyme linked immunosorbent assay (ELISA) using a horseradish peroxidase (HRP) conjugated anti-IgG secondary antibody and 3, 3’, 5, 5’-tetramethylbenzidine (TMB) substrate. The relative amounts of Aβ bound could be determined from the absorbance at 450 nm. If heparin could bind with Aβ, the heparin should coat the surface of the fibril and shield the Aβ from adhesion to 209 the plate. To test heparin binding, Aβ fibrils were pre-mixed with varying concentrations of Hep-SPION and incubated in each well overnight. Upon washing off unbound material, the amounts of Aβ remaining in the well were semi-quantified by ELISA. A concentration dependent decrease in absorbance at 450 nm was observed with increasing amounts of Hep-SPION (Figure 3.2A). Incubation of Aβ with uncoated SPION showed no effect on the absorbance, which revealed the crucial role of heparin on the surface of nanoparticles (Figure 3.2B). Hep-SPION did not bind to the surface of wells (data not shown), thus precluding the possibility that the decrease in absorbance was due to Hep-SPION passivating the wells. (A) m n 0 5 4 t a e c n a b r o s b A 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 (B) m n 0 5 4 t a e c n a b r o s b A 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0.01 0.1 [SPION] in µg/mL 0.01 0.1 [Hep-SPION] in µg/mL 1 Figure 3.2. (A) Aβ binding to plate decreased with increasing concentrations of Hep-SPION. The bound Aβ was detected by an anti-Aβ IgG mAb 6E10, followed by addition of HRP-conjugated anti-IgG secondary antibody and the TMB substrate. (B) ELISA curve for Aβ incubated with increasing concentrations of SPION. SPIONs without heparin coating showed little effect on Aβ binding to the plate. 3.2.3. Effect of Hep-SPION on Aβ Aggregation. As Hep-SPION can bind with Aβ, we analyzed its effects on Aβ aggregation by native polyacrylamide gel electrophoresis (PAGE). Aβ monomers (25 μM) were incubated with 210 Hep-SPION at 37℃ for 2 days followed by analysis via native PAGE. Without any Hep-SPION, Aβ existed as a mixture of low molecular weight oligomers and high molecular weight fibril (Figure 3.3A). With increasing concentrations of Hep-SPION (0.0078, 0.0156, 0.0312, 0.125 mg/mL), the relative amounts of the low-molecular-weight Aβ oligomers decreased (Figure 3.3A). At the concentration of 0.125 mg/mL Hep-SPION, almost all Aβ (>90%) had formed large fibrils appearing at higher molecular weight region on the gel (Figure 3.3A, B). This suggested that Hep-SPION can eliminate low-molecular-weight oligomers by direct binding or facilitating the conversion of Aβ oligomers to fibrils. Figure 3.3. (A) PAGE gel of Aβ only (lane 1) or Aβ (25 μM) incubated with 0.0078 mg/mL (lane 2), 0.0156 mg/mL (lane 3), 0.0312mg/mL (lane 4) and 0.125 mg/mL (lane 5) of Hep-SPION. (B) Percentage of low-molecular-weight Aβ oligomer in total Aβ in presence of various concentrations of Hep-SPION. The percentage was calculated by dividing the intensity of the low molecular weight oligomer band by the sum of the intensities of all bands in the specific lane. To further confirm the effect of Hep-SPION on Aβ aggregation, a thioflavin T (ThT) binding 211 assay was performed. ThT is a cationic benzothiazole dye31 that displays enhanced fluorescence when interacting with β-sheet structures (Figure 3.4A, 1st vs 2nd column). When incubated with Aβ, Hep-SPION accelerated β-sheet formation and gave rise to markedly enhanced ThT fluorescence (Figure 3.4A, 3rd – 5th column). Hep-SPION itself did not result in any fluorescence enhancement of ThT even at the highest nanoparticle concentration tested (Figure 3.4A, 6th column), which excluded the direct effect of Hep-SPION on ThT fluorescence. Uncoated SPION without any heparin did not impact the fluorescence of ThT (Figure 3.4B, 2nd column), which further confirmed the imperative role of heparin. It has been proposed that heparin can function as a structural template and facilitate the nucleation step of Aβ aggregation.13 Our results that Hep-SPION induced extensive aggregation of Aβ were consistent with the reported effects of heparin on Aβ1-40.11 (A) (B) ) U A ( e c n e c s e r o u F T h T l 100 80 60 40 20 0 1 2 3 4 5 6 ) U A ( e c n e c s e r o u F T h T l 120 100 80 60 40 20 0 1 2 3 4 5 Figure 3.4. The intensities of ThT fluorescence at 489 nm (λex = 440 nm). (A) Incubation of Aβ in the presence of Hep-SPION significantly enhanced ThT fluorescence. From left to right: ThT fluorescence in the presence of 1) H2O; 2) Aβ (25 μM); 3) Aβ (25 μM) + Hep-SPION (0.0020 mg/mL); 4) Aβ (25 μM) + Hep-SPION (0.0078 mg/mL); 5) Aβ (25 μM) + Hep-SPION (0.0312 mg/mL); 6) Hep-SPION (0.0312 mg/mL). (B) ThT fluorescence in the presence of 1) Aβ (25 μM) + Hep-SPION (0.0625 mg/mL); 2) Aβ (25 μM) + SPION (0.0625 mg/mL); 3) Aβ (25 μM); 4) SPION (0.0625 mg/mL); and 5) H2O. 212 3.2.4. Effect of Hep-SPION on Aβ-Induced Cytotoxicity Although it remains debatable whether Aβ peptides cause AD, the toxicity of Aβ on neuronal cells is an important contributing factor to the pathology of the disease. While Aβ can exist in monomer, oligomer and fibril forms, the soluble Aβ oligomers have been proven to be the most toxic among all Aβ species.4 Shifting the equilibrium between the oligomers and fibril towards the more benign fibrils can potentially reduce the adverse effects of Aβ.32-33 As Hep-SPION can convert Aβ oligomers into the fibrillar forms (Figure 3.3A), we hypothesized that Hep-SPION could protect neuronal cells from Aβ induced toxicity. To test the effects of Aβ and Hep-SPION on cells, cell viability assays were performed with SH-SY5Y neuroblastoma cells, a common model utilized in Aβ toxicity studies.33-34 Various concentrations of Aβ were incubated with SH-SY5Y cells in a 96-well cell culture plate. Cells in each well were collected and then mixed with 7-aminoactinomycin D (7-AAD), a fluorescent stain specific to dead cells. The numbers of live and dead cells were counted via fluorescence-activated cell sorting (FACS), with the percentage of live cells without any treatment set as 100%. Aβ peptide exhibited dose dependent cytotoxicities (Figure 3.5A) with about 75% cell viability when treated with 5 μM Aβ. The SH-SY5Y cells were then incubated with Aβ (5 μM) in the presence of increasing concentrations of Hep-SPION. As shown in Figure 3.5B, Hep-SPION could protect the cells from Aβ induced toxicity with 0.01 mg/mL of Hep-SPION enough to fully mitigate the effect of Aβ on the cells. Hep-SPION by itself did not have a significant impact on the viability of the cells, demonstrating the biocompatibility of the nanoparticles. The protective effect of Hep-SPION can potentially be due to two factors: 1) the 213 nanoparticles can induce the transformation of Aβ into the more benign fibril form; and 2) by binding with Aβ, the Hep-SPION can serve as a sink to reduce the Aβ available for interactions with the neuronal cells. (A) (B) y t i l i b a i V l l e C % 100 90 80 70 0.01 0.1 1 [Aβ] in µM 10 100 No treatment Aβ Aβ + Hep-SPION 0.001 mg/mL 0.01 mg/mL 0.5 mg/mL Hep-SPION (0.5 mg/mL) Figure 3.5. Cell viability assay of SH-SY5Y cells. (A) Increasing concentrations of Aβ induced higher cytotoxicity against SH-SY5Y cells. (B) Addition of Hep-SPION protected SH-SY5Y cells from Aβ induced cytotoxicity. Incubation of cells with Hep-SPION (0.5 mg/mL) did not exhibit any cytotoxicity indicating the high biocompatibility of the nanoparticles. Aβ peptides exist in a dynamic equilibrium among monomers, oligomers and fibrils, while heparin can perturb the equilibrium and therefore affect the aggregation process. It has been reported that heparin bind to fibrillar Aβ in an analysis via affinity co-electrophoresis,15 which is also proved in our study. Hep-SPION could eliminate the most toxic oligomer form and protect SH-SY5Y cells. However, more details about how heparin is involved in the process remain concealed. Further study on the effect of Hep-SPION on different forms of Aβ will help with better understanding of the role of heparin in the aggregation of Aβ. 214 3.3. Conclusions We demonstrated that Hep-SPION could bind Aβ and heparin was essential for the interaction. Furthermore, Hep-SPION promoted the transition of Aβ into the more benign fibrils. This in turn could protect neuronal cells from Aβ induced cytotoxicity. As iron oxide nanoparticles have been widely applied as MRI contrast agents and drug carriers,23-25 Hep-SPION can potentially be a useful platform for future imaging and drug delivery studies targeting Aβ. 3.4. Experimental Section 3.4.1. Materials and Instrumentation Unless otherwise indicated, all starting materials, reagents and solvents were obtained from commercial suppliers and used as supplied without further purifications. Ferric chloride hexahydrate (FeCl3·6H2O) was purchased from Honeywell Riedel-de Haen. Ferrous chloride tetrahydrate (FeCl2·4H2O), ferric acetylacetonate [Fe(acac)3], oleic acid, 1, 2-hexadecanediol, 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol, Cameo syringe filter (0.22 micron) and 7-aminoactinomycin D (7-AAD) were purchased from Sigma-Aldrich. Ammonium hydroxide (NH4OH, 28−30%) and hydrogen peroxide (H2O2, 30%) were purchased from CCI. Benzyl ether and 3, 3’, 5, 5’-tetramethylbenzidine were purchased from Acros Organics. Oleyl amine was purchased from Fluka. Heparin sodium was purchased from Celsus Laboratories, Inc. Thioflavin T (ThT), UltraPure Grade was purchased from AnaSpec. Aβ1-42 was purchased from GL Biochem. (Shanghai) Ltd. (No. 52487). Anti Aβ1-16 IgG (6E10) monoclonal antibody was purchased 215 from Covance. Goat anti-mouse HRP-conjugated secondary antibody was purchased from Jackson ImmunoResearch Laboratory. SH-SY5Y cells were purchased from American Type Culture Collection (ATCC). Ultrathin-carbon type A, 400 mesh copper grids for TEM were purchased form Ted Pella, Inc. Ultrafiltration membranes and centrifugal filters were purchased from Millipore. All cell culture media was supplemented with 10% heat inactivated FBS, 1% Pen-Strep mixture, glutamine (2 mM), and sodium pyruvate (1 mM). Dynamic light scattering (DLS) and zeta potential measurements were performed on a Zetasizer Nano zs apparatus (Malvern, U.K.). Transmission electron microscopy (TEM) images were collected on a JEM-2200FS operating at 200 kV using Gatan multiscan CCD camera with Digital Micrograph imaging software. Thermogravimetric analysis (TGA) was carried on a Thermal Advantage (TA-Instruments-Waters LLC) TGA-Q500 series and the samples were burned under nitrogen. Native-PAGE gel analysis was performed via ImageJ 1.42q (NIH). FACS experiments were conducted on a BD Vantage SE flow cytometer. 3.4.2. Synthesis of Hep-SPION A. Thermal decomposition approach Fe(acac)3 (0.71 g, 2 mmol), 1, 2-hexadecanediol (2.58 g, 10 mmol), oleic acid (1.69 g, 6 mmol), oleyl amine (1.61 g, 6 mmol), and benzyl ether (40 mL) were mixed and stirred under a nitrogen atmosphere. The mixture was heated to 200 ℃ for 2 h followed by refluxing for 1 h. The black mixture was cooled down to room temperature and ethanol (50 mL) was added. The iron oxide nanoparticles were collected by an external magnet and washed three times with 216 ethanol to remove excess starting materials. The nanoparticles were then dispersed in hexane (50 mL) and the mixture was placed on an external magnet to remove undispersed magnetic material. The supernatant containing nanoparticles was centrifuged to remove large particulates and give the OA-SPION (6 mg/mL). OA-SPION (25 mg) was dried from hexane and re-dissolved in toluene (15 mL). Heparin sodium salt (50 mg) was dissolved in MilliQ water (30 mL) and pH of the solution was adjusted to 8.5 with NaOH solution. The heparin solution was mixed with OA-SPION in toluene and the two phase system was refluxed for 24 h under rapid stirring. The aqueous layer containing the Hep-SPION was collected using a separatory funnel, centrifuged to remove large particulates and purified by ultrafiltration (MWCO 100,000) to remove excess heparin and NaOH. The purified Hep-SPION was diluted with MillQ water to a final volume of 30 mL (0.8 mg/mL). B. Co-precipitation approach FeCl3·6H2O (500 mg, 1.85 mmol) and FeCl2·4H2O (185 mg, 0.93 mmol) were dissolved in MilliQ water (30 mL) that had been deoxygenated by bubbling with nitrogen for 20 min. The solution was filtered through 0.22 μm syringe filter to remove any undissolved solid. To this solution, NH4OH (30%, 2 mL) was added under a nitrogen atmosphere while stirring vigorously for 1 h at room temperature. An external magnet was used to collect iron oxide nanoparticles and the supernatant solution was discarded. The nanoparticles were washed three times and re-suspended in MilliQ water (30 mL). Heparin sodium salt (0.5 g) in 10 mL MilliQ water was added and the mixture was stirred for 2 h followed by sonication for 1 h. The solution was centrifuged to remove any aggregation and then heated at 80 ℃ for 1 h to achieve stabilization. 217 Excess heparin was removed by ultrafiltration and the final Hep-SPION solution (2.5 mg/mL) was kept at 4 °C for further use. 3.4.3. Transmission Electron Microscopy (TEM) Procedure 10 μL of the Hep-SPION solution was deposited on ultrathin-carbon type A, 400 mesh copper grids and let to evaporate under the hood. Once dry, 1% solution of uranyl acetate was added for 10 seconds and the solution was wicked away with filter paper. The grids were then washed with water and dried for 15 min at room temperature. 3.4.4. Preparation of Aβ Aβ peptide (0.5 mg) was dissolved in spectroscopy grade 99.9% 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (1.5 mL), sonicated for 15 min, and lyophilized for 72 h. The thin film was then dissolved in 0.22 μm filtered solution of 10 mM NaOH solution (0.25 mL). The pH of the solution was adjusted to 6 with 10 mM HCl solution and diluted with deionized water to a total volume of 1.0 mL (the concentration of Aβ stock solution was 100 μM). For experiments that needed Aβ fibrils, the stock solution was incubated at 37℃ for 48 h. 3.4.5. Native-PAGE Gel Electrophoresis Aβ monomers (25 μM) were incubated either without Hep-SPION or with different concentration of Hep-SPION (0.125, 0.0312, 0.0156, 0.0078 mg/mL) at 37℃ for 2 days. After incubation, 20 μL of the mixture was added to 5 μL of non-SDS sample buffer and was subjected to electrophoresis (200 V) on a 15% native-PAGE gel. The gels were then stained with silver staining. 218 3.4.6. Thioflavin T Assay ThT fluorescence measurements were performed in a clear bottom black 96-well fluorescence plate (COSTAR 3695-96) on a FLUOstar OPTIMA (BMG Labtechnologies). The control solution was 220 μL water and 200 μL ThT (25 μM) + 20 μL water. 20 μL Aβ (25 μM) solutions incubated with different concentration of Hep-SPION were added to 200 μL ThT (25 μM) solutions. ThT fluorescence measurements were performed with λex = 440 nm and λem = 489 nm. 3.4.7. ELISA Assay Aβ fibrils (100 nM) along with Hep-SPION at different concentrations (0.008, 0.016, 0.031, 0.062, 0.125, 0.25, 0.50, 1.0 μg/mL) were added into a 96-well plate (100 μL/well) and incubated IgG (6E10) monoclonal antibody (100 μL/well, 0.82 nM, 1 : 4000 in 1% BSA containing PBS) at 22℃ overnight. All wells were washed with 300 μL PBST three times and blocked with 1% BSA (300 μL/well) at 22℃ for 1 h. After washing with 300 μL PBST three times, anti Aβ1-16 was added and then incubated at 37℃ for 1 h. The solutions were then discarded and washed incubated at 37℃ for 1 h followed by washing with 300 μL PBST three times. To a freshly prepared 3, 3’, 5, 5’-tetramethylbenzidine (TMB) solution (5 mg of TMB was dissolved in 2 mL again with 300 μL PBST three times. The goat anti-mouse HRP-conjugated secondary antibody (100 μL/well, 5.1 nM, 1: 6000 in 1% BSA containing PBS) was added into each well and of DMSO and then diluted to 20 mL with citrate phosphate buffer), 20 μL of H2O2 was added. This mixture (150 μL/well) was immediately added to the plate and a blue color was allowed to develop for 20 min. The reaction was then quenched by 0.5 M H2SO4 (50 μL/well) and the 219 absorbance was measured at 450 nm on an iMark microplate reader. 3.4.8. Cell Viability Assay. Different concentrations of Hep-SPION solutions (0.002, 0.02, 0.2, 1 mg/mL) were pre-incubated with or without Aβ fibrils for 24h and then added into 96-well plate (50 μL/well). In each well, 2*104 cells were added in 4% serum solution. The final solutions in those wells are Aβ (5 μM), Hep-SPION (0.001, 0.01, 0.1, 0.5 mg/mL) in 2% serum (100 μL/well). The plate was incubated for 24 h at 37℃. All media were collected in separate eppendorf tubes. Trypsin (50 μL) was then added into each well to digest cells and 4% culture media (200 μL*2) was used to wash wells and combined with original media in the eppendorf tubes. Cells were pelleted by centrifugation and resuspended in FACS buffer (300 μL) in FACS tubes. 7-AAD (3 μL) was added into each tube, followed by incubation at 0℃ for 10 min. All solutions were then analyzed by a flow cytometer to evaluate the cell viability. 220 REFERENCES 221 REFERENCES 1. Alzhermer's disease facts and figures. http://www.alz.org/downloads/facts_figures_2013.pdf, 2013. 2. Hebert, L. E.; Scherr, P. A.; Bienias, J. L.; Bennett, D. A.; Evans, D. A., Alzheimer disease in the US population: prevalence estimates using the 2000 census. Arch. Neurol. 2003, 60 (8), 1119-1122. 3. Hardy, J. A.; Higgins, G. A., Alzheimer's disease: the amyloid cascade hypothesis. Science 1992, 256, 184-185. 4. Dahlgren, K. N.; Manelli, A. M.; Stine, W. B.; Baker, L. K.; Krafft, G. A.; LaDu, M. J., Oligomeric and fibrillar species of amyloid-β peptides differentially affect neuronal viability. J. Biol. Chem. 2002, 277 (35), 32046-32053. 5. Bieschke, J.; Herbst, M.; Wiglenda, T.; Friedrich, R. P.; Boeddrich, A.; Schiele, F.; Kleckers, D.; del Amo, J. M. L.; Grüning, B. A.; Wang, Q.; Schmidt, M. R.; Lurz, R.; Anwyl, R.; Schnoegl, S.; Fändrich, M.; Frank, R. F.; Reif, B.; Günther, S.; Walsh, D. M.; Wanker, E. E., Small-molecule conversion of toxic oligomers to nontoxic β-sheet–rich amyloid fibrils. Nat. Chem. Biol. 2012, 8 (1), 93-101. 6. Masters, C. L.; Selkoe, D. J., Biochemistry of amyloid β-protein and amyloid deposit in Alzheimer disease. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, 2012. 7. Snow, A. D.; Mar, H.; Nochlin, D.; Kimata, K.; Kato, M.; Suzuki, S.; Hassell, J.; Wight, T., The presence of heparan sulfate proteoglycans in the neuritic plaques and congophilic angiopathy in Alzheimer's disease. Am. J. Pathol. 1988, 133 (3), 456-463. 8. Snow, A. D.; Sekiguchi, R. T.; Nochlin, D.; Fraser, P.; Kimata, K.; Mizutani, A.; Arai, M.; Schreier, W. A.; Morgan, D. G., An imporant role of heparan sulfate proteoglycan (perlecan) in a model system for the deposition and persistence of fibrillar Aβ-amyloid in rat brain. Neuron 1994, 12, 219-234. 9. Gupta-Bansal, R.; Frederickson, R. C.; Brunden, K. R., Proteoglycan-mediated inhibition of Aβ proteolysis. A potential cause of senile plaque accumulation. J. Biol. Chem. 1995, 270 (31), 18666-18671. 10. Bame, K. J.; Danda, J.; Hassall, A.; Tumova, S., Aβ (1–40) Prevents heparanase-catalyzed degradation of heparan sulfate glycosaminoglycans and proteoglycans in vitro. A role for heparan sulfate proteoglycan turnover in Alzheimer's disease. J. Biol. Chem. 1997, 272 (27), 17005-17011. 222 11. Castillo, G. M.; Lukito, W.; Wight, T. N.; Snow, A. D., The sulfate moieties of glycosaminoglycans are critical for the enhancement of β-Amyloid protein fibril formation. J. Neurochem. 1999, 72 (4), 1681-1687. 12. Walzer, M.; Lorens, S.; Hejna, M.; Fareed, J.; Hanin, I.; Cornelli, U.; Lee, J. M., Low molecular weight glycosaminoglycan blockade of β-amyloid induced neuropathology. Eur. J. Pharmacol. 2002, 445, 211-220. 13. McLaurin, J.; Franklin, T.; Zhang, X.; Deng, J.; Fraser, P. E., Interactions of Alzheimer amyloid‐β peptides with glycosaminoglycans. Eur. J. Biochem. 1999, 266 (3), 1101-1110. 14. Fraser, P. E.; Darabie, A. A.; McLaurin, J., Amyloid-β interactions with chondroitin sulfate-derived monosaccharides and disaccharides: implications for drug development. J. Biol. Chem. 2001, 276 (9), 6412-6419. 15. Watson, D. J.; Lander, A. D.; Selkoe, D. J., Heparin-binding properties of the amyloidogenic peptides Aβ and amylin dependence on aggregation state and inhibition by congo red. J. Biol. Chem. 1997, 272 (50), 31617-31624. 16. Lindahl, B.; Westling, C.; Giménez-Gallego, G.; Lindahl, U.; Salmivirta, M., Common binding sites for β-amyloid fibrils and fibroblast growth factor-2 in heparan sulfate from human cerebral cortex. J. Biol. Chem. 1999, 274, 30631–30635. 17. Kemp, M. M.; Linhardt, R. J., Heparin-based nanoparticles. WIREs Nanomed. Nanobiotechnol. 2010, 2, 77-87 and references cited therein. 18. Tan, Q.; Tang, H.; Hu, J.; Hu, Y.; Zhou, X.; Tao, Y.; Wu, Z., Controlled release of chitosan/heparin nanoparticledelivered VEGF enhances regeneration of decellularized tissue-engineered scaffolds. Int. J. Nanomed. 2011, 6, 929-942. 19. Nurunnabi, M.; Khatun, Z.; Moon, W.-C.; Lee, G.; Lee, Y.-K., Heparin based nanoparticles for cancer targeting and noninvasive imaging. Quant. Imaging Med. Surg. 2012, 2, 219-226. 20. Shahbazi, M.-A.; Hamidi, M.; Mohammadi-Samani, S., Preparation, optimization, and in-vitro/in-vivo/ex-vivo characterization of chitosan-heparin nanoparticles: drug-induced gelation. J. Pharm. Pharmacol. 2013, 65, 1118-1133. 21. Zhang, J.; Shin, M. C.; David, A. E.; Zhou, J.; Lee, K.; He, H.; Yang, V. C., Long-circulating heparin-functionalized magnetic nanoparticles for potential application as a protein drug delivery platform. Mol. Pharm. 2013, 10, 3892-3902. 22. Lee, J.-h.; Jung, M. J.; Hwang, Y. H.; Lee, Y. J.; Lee, S.; Lee, D. Y.; Shin, H., Heparin-coated superparamagnetic iron oxide for in vivo MR imaging of human MSCs. Biomaterials 2012, 33 (19), 4861-4871. 223 23. Corot, C.; Robert, P.; Idée, J.-M.; Port, M., Recent advances in iron oxide nanocrystal technology for medical imaging. Adv. Drug Del. Rev. 2006, 58, 1471-1504. 24. Colombo, M.; Carregal-Romero, S.; Casula, M. F.; Gutiérrez, L.; Morales, M. P.; Böhm, I. B.; Heverhagen, J. T.; Prosperi, D.; Parak, W. J., Biological applications of magnetic nanoparticles. Chem. Soc. Rev. 2012, 41, 4306-4334. 25. He, H.; David, A.; Chertok, B.; Cole, A.; Lee, K.; Zhang, J.; Wang, J.; Huang, Y.; Yang, V. C., Magnetic nanoparticles for tumor imaging and therapy: a so-called theranostic system. Pharm. Res. 2013, 30, 2445-2458. 26. El-Dakdouki, M. H.; El-Boubbou, K.; Zhu, D. C.; Huang, X., A simple method for the synthesis of hyaluronic acid coated magnetic nanoparticles for highly efficient cell labelling and in vivo imaging. RSC Adv. 2011, 1, 1449-1452. 27. García-Matas, S.; de Vera, N.; Aznar, A. O.; Marimon, J. M.; Adell, A.; Planas, A. M.; Cristòfol, R.; Sanfeliu, C., In vitro and in vivo activation of astrocytes by amyloid-beta is potentiated by pro-oxidant agents. J. Alzheimers Dis. 2010, 20, 229-245. 28. Allaman, I.; Gavillet, M.; Bélanger, M.; Laroche, T.; Viertl, D.; Lashuel, H. A.; Magistretti, P. J., Amyloid-beta aggregates cause alterations of astrocytic metabolic phenotype: impact on neuronal viability. J. Neurosci. 2010, 30, 3326-3338. 29. Bitan, G.; Vollers, S. S.; Teplow, D. B., Elucidation of primary structure elements controlling early amyloid β-protein oligomerization. J. Biol. Chem. 2003, 278 (37), 34882-34889. 30. Roher, A. E.; Lowenson, J. D.; Clarke, S.; Woods, A. S.; Cotter, R. J.; Gowing, E.; Ball, M. J., Beta-amyloid-(1−42) is a major component of cerebrovascular amyloid deposits: Implications for the pathology of Alzheimer disease. Proc. Natl. Acad. Sci., U.S.A. 1993, 90, 10836−10840. 31. LeVine, H., Quantification of β-sheet amyloid fibril structures with Thioflavin T. Meth. Enzymol. 1999, 309, 274-284. 32. Vilasi, S.; Sarcina, R.; Maritato, R.; De Simone, A.; Irace, G.; Sirangelo, I., Heparin induces harmless fibril formation in amyloidogenic W7FW14F apomyoglobin and amyloid aggregation in wild-type protein in vitro. PloS One 2011, 6 (7), e22076. 33. Kouyoumdjian, H.; Zhu, D. C.; El-Dakdouki, M. H.; Lorenz, K.; Chen, J.; Li, W.; Huang, X., Glyconanoparticle Aided detection of β-amyloid by magnetic resonance imaging and attenuation of β-amyloid induced cytotoxicity. ACS Chem. Neurosci. 2013, 4, 575-584. 34. Luo, J.; Mohammed, I.; Wärmländer, S. K. T. S.; Hiruma, Y.; Gräslund, A.; Abrahams, J. P., Endogenous polyamines reduce the toxicity of soluble Aβ peptide aggregates associated with Alzheimer’s disease. Biomacromolecules 2014, 15 (6), 1985-1991. 224 Chapter 4. Mitigation of Neurotoxicities of Toxic Tau Oligomers by Heparin Like Oligosaccharides 4.1. Introduction Alzheimer’s disease (AD) is a progressive degenerative brain disease, which is estimated to affect 5.5 million Americans in 2017.1 Although the causes for most AD cases have not been firmly established, the pathology of tau protein is believed to play important roles.2 Tau protein in its native state exists as a soluble monomer, which is critical in stabilizing microtubules. However, tau can misfold and aggregate leading to the formation of oligomers and hyperphosphorilated tau aggregates known as neurofibrillary tangles (NFTs), a hallmark of AD.3 While NFTs are abundant in the brains of late stage AD patients, some patients show neuronal loss and cognitive deficits prior to the formation of histologically identifiable NFTs.4 In animal studies, NFTs have been found not to be associated with neuronal death, suggesting that these large insoluble aggregates may not be the key toxic species in AD.5-7 In order to explain tau pathology, tau oligomer hypothesis has been proposed recently with strong evidence supporting that the soluble, oligomeric tau rather than the NFTs are likely the most toxic species producing disease pathology.8-9 Tau oligomers (TauO) are found in human AD patients and are able to propagate extracellularly through different brain regions, contributing to neuronal cell death in addition to learning and memory deficits.10-12 Injection of tau oligomers isolated from the cerebral cortex of AD brains initiated tau pathology in cognitively normal mice, and cause synaptic and mitochondrial dysfunction associated with memory loss in their brains.11-13 Even brief exposure to human tau oligomers could produce an immediate impairment 225 of long-term potentiation and memory.14 The tau oligomer hypothesis was further strengthened by the observations that lowering tau oligomer levels protected against behavioral deficits and tau pathology in multiple mouse models without affecting NFTs levels.13, 15 Therefore, strategies that can reduce the oligomer associated neurotoxicity are highly desirable. Heparan sulfate (HS) and its more sulfated analog heparin are a class of highly negatively charged polysaccharides present on mammalian cells including neuronal cells.16-17 HS and heparin are composed of repeating disaccharide subunits with D-glucosamine (GlcN) α-1,4 linked with a uronic acid (either L-iduronic acid (IdoA) or D-glucuronic acid (GlcA)).18-19 The amine moiety, 3-OH and 6-OH of GlcN and 2-OH of the uronic acid of heparin can be sulfated. Heparin is known to bind with tau.20-24 Yi Liang and coworkers characterized a tight 1:1 complex between tau fragment Tau244-372 and heparin (average molecular mass = 7 kDa).20 They also proposed a model for tau filament formation where the formation of the complex with heparin initiated nucleation and promoted elongation of tau fibrils. NMR analysis mapped the binding region to the paired helical filament (PHF) core region, which was rich in positive charges.22 A study using enzyme-cleaved fragments of heparin with different sulfation patterns and lengths revealed a key 6-O-sulfate residue in the tau-binding affinity, as reported by Fuming Zhang and coworkers.24 Although HSPG on cell surface has been found important in the uptake of tau aggregates,21 it is not known whether heparin can interact with tau oligomers. Herein, using structurally well-defined synthetic oligosaccharides, we report for the first time that heparin-like oligosaccharides as small as tetrasaccharides can bind and interact with the toxic tau oligomers. Furthermore, treatment of human neuroblastoma cell line with heparin like oligosaccharides can 226 protect the cells from tau oligomers-induced toxicity providing an exciting new direction in addressing tauopathy. 4.2. Results and Discussion 4.2.1. Preparation of Heparin Oligosaccharide Backbones In order to obtain heparin like oligosaccharides, we based our synthetic design on disaccharide modules 1, 2 and 3. Disaccharides 1 and 2 were synthesized starting from disaccharide 4 following literature procedures.25 For the non-reducing end disaccharide module, while TBS bearing disaccharide 4 could be used, we found it was impossible to remove the TBS group from sulfated oligosaccharides during late stage deprotection of sulfated glycans.25 This consideration prompted us to prepare the 4’-O-Bn protected disaccharide 3. Pre-activation of benzyl (Bn) protected glucosamine donor 5 with p-TolSCl and AgOTf26 followed by the addition of acceptor 6, gave the α-linked disaccharide 7 in 87% yield (JH1-C1 = 166.5, 171.0 Hz) (Scheme 4.1). Protective group manipulation of 7 yielded disaccharide module 3. 227 OAc O OLev OBn O STol HO BnO N3 O 1 BzO OAc O OLev OBn O STol BnO BnO N3 O 3 BzO p-TolSCl, AgOTf, 4Å MS, -78°C OAc O OLev HO BnO N3 O 2 BzO OAc O OBn O O OLev Bn N Cbz TBSO BnO N3 O 4 BzO OBn O STol BnO BnO OAc O N3 5 BnO BnO STol STol then HO 6 OPMB OBn O BzO 87% OAc O OPMB OBn O STol N3 O 7 BzO OAc O BnO BnO OLev OBn O STol N3 O 3 BzO 1) DDQ 2) LevOH, EDC·HCl, DMAP 89% Scheme 4.1. Synthesis of non-reducing end disaccharide module 3. With the necessary disaccharide building blocks in hand, we performed glycosylation to elongate the backbones. Reaction of the donor 3 with acceptor 1 generated tetrasaccharide 8 in 85% yield (Scheme 4.2). Alternatively, 3 glycosylated the reducing end disaccharide module 2 giving tetrasaccharide 9 (Scheme 4.2b). In a similar manner, TBS bearing tetrasaccharide donor 10 was formed (Scheme 4.2c). 228 a) 3 b) 3 b) 4 p-TolSCl, AgOTf, 4Å MS, -78°C then 1 85% p-TolSCl, AgOTf, 4Å MS, -78°C 8 9 then 2 88% p-TolSCl, AgOTf, 4Å MS, -78°C then 1 72% TBS 10 O BnO Bn O BnO OAc O N3 O OLev R OBn O BzO 2 8: R = STol 9: R = O Cbz N Bn OAc O N3 O 10 OLev STol OBn O BzO 2 Scheme 4.2. Construction of heparin tetrasaccharide backbones. To produce the hexasaccharide backbone, a 4+2 glycosylation was carried out between the tetrasaccharide donor 8 and acceptor 2 producing hexasaccharide 11 (Scheme 4.3a). The 4-O-TBS protected tetrasaccharide donor 10 also reacted well with disaccharide 2. Removal of the TBS group from the glycosylation product led to the hexasaccharide acceptor 12 (Scheme 4.3b), which was subsequently glycosylated by tetrasaccharide donor 8, forming decasaccharide 13 in 84% yield (Scheme 4.3c). 229 a) 8 b) 10 c) 8 p-TolSCl, AgOTf, 4Å MS, -78°C then 2 58% 1) p-TolSCl, AgOTf, 4Å MS, -78°C then 2 2) HF.pyridine, pyridine 81% p-TolSCl, AgOTf, 4Å MS, -78°C then 12 84% Bn O BnO OAc O N3 O 11 OAc O H O BnO N3 O 12 OLev O OBn O BzO 3 OLev O OBn O BzO 3 Bn O BnO OAc O N3 O 13 OLev O OBn O BzO 5 Cbz N Bn Cbz N Bn Cbz N Bn Scheme 4.3. Constructions of heparin hexa- and deca-saccharide backbones. 4.2.2. Deprotection and Sulfation The deprotections and modifications of the backbones were carried out first by removal of 6-O-Lev from fully protected tetra-, hexa- and decasaccharide 9, 11 and 13 respectively with hydrazine exposing the 6-OH (Scheme 4.4). The conversion of these primary hydroxyl groups to carboxylic acids was mediated by bis(acetoxy)iodobenzene (BAIB) assisted 2, 2, 6, 6-tetramethyl-1-piperidinyloxyl (TEMPO) oxidation.27 Since free carboxylic acids were found to lead to low yields in subsequent sulfation reactions,25 they were protected as either methyl (83% for tetrasaccharide 17 in 2 steps) or benzyl (77% for hexasaccharide 18 and 81% for decasaccharide 19 in 2 steps) esters. Removal of the acyl protecting groups was accomplished by treating oligosaccharides 17-19 with sodium methoxide, which gave 20, 21 and 22 respectively. 230 Bn O BnO OLev R OBn O BzO n OAc O N3 O 9: n = 2 11: n = 3 13: n = 5 N2H4 ·H2O, AcOH, Py, 0°C Bn O BnO OAc O N3 O OH R OBn O BzO n 1. BAIB, TEMPO, t-BuOH/DCM/H2O 2. MeI, Na2CO3, DMF or PhCH2N2, DCM 14: n = 2, 86% 15: n = 3, 93% 16: n = 5, 83% OH O MeO2C N3 O R OBn O R = O Cbz N Bn HO n OAc O RO2C N3 O Bn O BnO BzO n R OBn O NaOMe, DCM/MeOH Bn O BnO 17: n = 2, R = Me, 83% 18: n = 3, R = Bn, 77% 19: n = 5, R = Bn, 81% 20: n = 2, 79% 21: n = 3, 97% 22: n = 5, 90% Scheme 4.4. Deprotection of heparin oligosaccharides. The two azido groups in tetrasaccharide 20 were reduced by zinc powder in acetic acid and acetic anhydride leading to N-acetylated tetrasaccharide 23 in 99% yield, while performing the reaction in the absence of acetic anhydride provided 24 with two free amine groups (Scheme 4.5). Sulfations of free hydroxyls and amines of the tetrasaccharide 24 were performed stepwise. Firstly, 24 was dissolved in methanol with aqueous NaOH solution adjusting the pH to 9.5 in order to deprotonate amine groups and N-sulfation was performed by adding excess SO3·Et3N complex to the mixture to give 25 in 78% yield. Hydrogenolysis and saponification of 25 gave the N-sulfated heparin like tetrasaccharide 27. Alternatively, 25 was subjected to O-sulfation with SO3·pyridine complex in pyridine overnight at 55 °C. Subsequent hydrogenolysis and saponification produced N, O-sulfated tetrasaccharide 29. In a similar manner, from the N-acetylated tetrasaccharide 23, tetrasaccharides 26 and 28 were generated. 231 Zn, AcOH, Ac2O, THF 20 99% Bn O BnO Zn, AcOH, THF 88% Bn O BnO OH O MeO2C H2N O 24 OBn O HO 2 R SO3 ·Et3N, NaOH 78% Bn AcHN O 23 OH O MeO2C R OBn O HO 2 O BnO - O3SHN O 25 OH O MeO2C R OBn O HO 2 1. P d( O 2. Li O 2 2 H )2/C, H 2 O H , H 6 5 % 83% 3 1. SO · 2. Pd(O Py, Py 3. LiO H)2/C, H H, H 2O 2 2 OH O O HO AcHN - OSO3 O O HO AcHN H H O 26 HO2C O - 28 HO2C O OH O NH2 HO 2 O OH O NH2 O3SO 2 1. P d( O 2. Li O 2 2 H )2/C, H 2 O H , H 3 % 8 81% 1. SO · 2. Pd(O Py, Py 3. LiO H)2/C, H H, H 3 2O 2 2 H H OH O O HO - O3SHN O 27 HO2C O OH O NH2 HO 2 - OSO3 O O HO - O3SHN HO2C O OH O NH2 O - 29 O3SO 2 Scheme 4.5. Sulfation and deprotection of tetrasaccharides. For the heparin hexasaccharide 21, the reduction was performed with 1, 3-propanedithiol and triethylamine over 3 days25 in a yield of 76% (Scheme 4.6a). Similar stepwise sulfation as in synthesis of tetrasaccharide 25 was attempted on hexasaccharide 30, which only led to decomposition of the starting materials. Analysis of the reaction mixture showed the formation of side products due to β-elimination with the oligosaccharide backbone cleaved. Instead, treatment of the hexasaccharide 30 with 600 mM SO3·py complex in pyridine at 55 ℃ successfully installed both N- and O-sulfation in one step, which was followed by catalytic hydrogenation and methyl ester hydrolysis, giving the final heparin like hexasaccharide 31 at 64% yield over 3 steps (Scheme 4.6a). Analogously, the heparin like decasaccharide 32 was synthesized with an overall yield of 42% from 22 (Scheme 4.6b). 232 a) 21 b) 22 1,3-propanedithiol, Et3N 76% OH O H2N Bn O BnO · 1. 1,3-propanedithiol, Et3N Py, Py 2. SO3 3. Pd(OH)2, H2 4. LiOH, H2O2 42% H MeO2C R OBn O HO 3 O 30 - OSO3 O 1. SO3·Py, Py 2. Pd(OH)2/C, H2 3. LiOH, H2O2 H 64% - OSO3 O O HO - O3SHN HO2C O OH O NH2 O - 31 O3SO 3 HO2C O OH O NH2 O HO - O3SHN O - 32 O3SO 5 Scheme 4.6. Sulfation and deprotection of hexa- and deca-saccharide. 4.2.3. Binding Assay with Tau Oligomers The majority of heparin – tau studies to date have been performed using polysaccharides isolated from nature, which are heterogeneous mixtures of many sequences with various backbone length and sulfation patterns. Structurally well-defined heparin oligosaccharides can provide useful information on structure-activity relationship. With the synthetic oligosaccharides 26-29, 31, 32 in hand, their binding with tau oligomers were analyzed. The sensorgrams showed that oligosaccharide as short as a tetrasaccharide (29) could exhibit significant binding to Tau oligomers with a KD value of 2.79 × 10-7 M. Comparison within the tetrasaccharide series 26-29 indicated that tetrasaccharides with higher degree of sulfation are associated with stronger binding to tau oligomers (Figure 4.1a-d). Increasing the backbone length of the oligosaccharide to hexa- and deca-saccharides led to enhancement in tau oligomer binding, with KD values of 1.41 × 10-7 M and 3.49 × 10-8 M for oligosaccharides 31 and 32 respectively. These results suggest that electrostatic interactions may play an important role in heparin – tau oligomer binding. 233 a) ) m n ( g n i d n i B 2.5 2.0 1.5 1.0 0.5 0.0 d) ) m n ( g n i d n i B 2.5 2.0 1.5 1.0 0.5 0.0 0 0 4.36 μM 2.18 μM 1.09 μM 0.545 μM 50 100 150 200 250 300 350 Time (sec) 4.36 μM 2.18 μM 1.09 μM 0.545 μM 0.272 μM 0.136 μM b) ) m n ( g n i d n i B 2.5 2.0 1.5 1.0 0.5 0.0 e) ) m n ( g n i d n i B 2.5 2.0 1.5 1.0 0.5 0.0 4.36 μM 2.18 μM 1.09 μM 0.545 μM 0 50 100 150 200 250 300 350 Time (sec) 4.36 μM 2.18 μM 1.09 μM 0.545 μM 0.272 μM 0.136 μM 50 100 150 200 250 300 350 Time (sec) 0 50 100 150 200 250 300 350 Time (sec) c) ) m n ( g n i d n i B 2.5 2.0 1.5 1.0 0.5 0.0 f) ) m n ( g n i d n i B 2.5 2.0 1.5 1.0 0.5 0.0 0 0 4.36 μM 2.18 μM 1.09 μM 0.545 μM 0.272 μM 0.136 μM 50 100 150 200 250 300 350 Time (sec) 4.36 μM 2.18 μM 1.09 μM 0.545 μM 0.272 μM 0.136 μM 0.0681 μM 50 100 150 200 250 300 350 Time (sec) Figure 4.1. Sensograms of heparin like oligosaccharide binding with tau oligomers. Fit curves of interactions between BLI sensors loaded with a)26, b)27, c)28, d)29, e)31 and f)32 and tau oligomers at various concentrations were obtained using models from Octet Data Analysis 9.0.0.12. Higher sulfation degree or longer backbone length led to stronger binding with Tau oligomers. 4.2.4. Heparin Oligosaccharides Mitigates Cytotoxicity of Tau Oligomers (Done by Dr. Rakez Kayed lab) In this study, we pursued an alternative approach to evaluate heparin like oligosaccharides’ ability to modulate the aggregation state and toxicity of preformed tau oligomers.28 Therefore, highly purified oligomeric tau species were incubated with and without oligosaccharides (5X) at room temperature on an orbital shaker, without stirring, for 16 hours under oligomerization conditions.28 Atomic force microscopy (AFM) was performed to visualize and characterize the morphology and aggregation state of the end product of each reaction. AFM images of TauO displayed a homogeneous spherical morphology (Figure 4.2a) while, in the presence of 234 heparin-like oligosaccharides (Figure 4.2b-e), we observed the tendency of tau oligomers to aggregate leading to the formation of fibrils and protofibrils (26-28) and compound with higher sulfatation degree (29) or longer backbone length (31-32) convert them into larger non-toxic tau aggregates. Figure 4.2. Biophysical characterization of Tau oligomers alone and in the presence of heparin-like oligosaccharides. Atomic Force Microscopy images of TauO without (a) and after incubation with heparin-like oligosaccharides (5X) b)26, c)27, d)28, e)29, f)31 and g)32. AFM images show the ability of the compounds to modulate tau aggregation state forming larger tau aggregates. Scale bars = 100 nm. Next, we evaluated the toxicity of tau aggregated species, resulting from the co-incubation of TauO alone and with heparin-like oligosaccharides, on human neuroblastoma cell line SH-SY5Y. Therefore, cells were treated with tau oligomers alone and in the presence of the compounds (Figure 4.3). SH-SY5Y cytotoxicity significantly increased after treatment with TauO alone while, in the presence of oligosaccharides (final concentration 10 μM), we observed 235 decreased LDH release as compared to TauO (Figure 4.3a). Furthermore, SH-SY5Y viability significantly decreased after treatment with TauO alone while cells exposed to TauO in the presence of heparin-like oligosaccharides reduced TauO-induced toxicity as shown by the higher level of cell viability using a resazurin based assay (Figure 4.3b). Moreover, cells exposed to each condition were evaluated for morphological differences, showing cells shrinkage and loss of their processes once they were exposed to TauO as compared to either the untreated control or to cells treated with tau oligomers in the presence of heparin-like oligosaccharides (Figure 4.3c). Taken together these results suggest that heparin-like oligosaccharides interact and remodel toxic tau oligomers converting them into less toxic high molecular weight aggregates. 236 Figure 4.3. Viability and Cytotoxicity assays of Tau oligomers alone and in the presence of heparin-like oligosaccharides on human SH-SY5Y neuroblastoma cell line. (A) SH-SY5Y cells cytotoxicity after exposure to 2µM tau oligomers, or 2µM tau oligomers with 10µM of each heparin-like oligosaccharide (26-29, 31, 32) and untreated control (Ctrl). Treatment of SH-SY5Y cells with TauO had significantly higher LDH release compared to untreated control and cells exposed to TauO in the presence of heparin-like oligosaccharides. (B) SH-SY5Y cells cytotoxicity after exposure to 2µM tau oligomers, or 2µM tau oligomers with 10µM of each heparin-like oligosaccharide (26-29, 31, 32) and Ctrl. Cells treated with TauO had significantly lower viability compared to untreated control while cells exposed to TauO in the presence of heparin-like oligosaccharides show to rescue TauO-induced toxicity. Each experiment was performed in triplicate (n = 3). Data were compared by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test: Ctrl vs TauO, 26-29, 31, 32, Fibrils: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05; TauO vs 26-29, 31, 32: §§§§p<0.0001, §§§p<0.001, §§p<0.01. Bars and error bars represent means and standard deviations performed. (C) Untreated SH-SY5Y cells were compared to cells treated for 24 hours with TauO alone or TauO in the presence of heparin-like oligosaccharides (26-29, 31, 32) and evaluated for morphological changes. Scale bar =20 µm. 237 4.3. Conclusions The binding of heparin with tau aggregates and HSPG mediated endocytosis suggested that heparin may play an important role in the propagation of tau pathology. However, the structural heterogeneity of naturally extracted heparin has been the obstacle in the study of the interactions. Also, no prior research has been performed on the interaction between heparin and tau oligomers, which is considered as the main culprit in tau pathology. We synthesized a series of heparin oligosaccharides, including different sulfation patterns and backbone lengths up to fully-sulfated decasaccharide. Direct binding between heparin oligosaccharides and tau oligomers were observed through BLI study, in which higher sulfation degree or longer backbone length both contributed to stronger binding the tau oligomers. AFM images displayed two different ways of heparin oligosaccharide promoting the aggregation of tau oligomers. Partially sulfated tetrasaccharides led to the formation of fibrils and protofibrils, while longer backbone length of heparin resulted in the formation of amorphous larger aggregates. Both effects of heparin binding were proven to be protective in the SH-SY5Y cytotoxicity or viability assay. This study may help understanding the structural-activity relationship of heparin in the binding the tau oligomers for the first time and elucidating the role of heparin in tauopathy. 4.4. Experimental Section 4.4.1. General Procedure for Preactivation Based Glycosylation. A solution of donor (1.0 equiv) and freshly activated 4 Å molecular sieves (1 g per 20 mL of final solvent) in CH2Cl2 was stirred at room temperature for 10 min and then cooled to −78 °C. 238 AgOTf (2.5 equiv) dissolved in Et2O/CH2Cl2 (10/1) was added directly to the solution. After 10 min, the orange-colored promoter p-TolSCl (1.0 equiv) was added with a microsyringe directly to the flask to avoid freezing the promoter on the walls of the flask. The color of p-TolSCl disappeared rapidly, indicating the consumption of p-TolSCl. After TLC indicated that the donor was fully activated (about 5 min at −78 °C), a solution of acceptor (0.8-1.0 equiv) in CH2Cl2 along with TTBP (1.0 equiv) was slowly added along the walls of the flask. This was done to allow the acceptor solution to cool before mixing with the activated donor. The final ratio of Et2O/CH2Cl2 was 1/1 after all reagents were added. The reaction mixture was slowly warmed to 0 ℃ over 2 h. The mixture was quenched with Et3N, diluted with CH2Cl2 and filtered through Celite. After washing the Celite with CH2Cl2 until all organic compounds were removed, as verified by TLC, the CH2Cl2 fractions were combined and washed with sat. NaHCO3 solution and brine. The organic layer was collected and dried over Na2SO4. After removal of the solvent, the product was purified by silica gel chromatography unless noted. 4.4.2. General Procedure for TBS Removal The TBS-containing oligosaccharide was dissolved in pyridine (5 mL per 1 g oligosaccharide) and transferred to a 50 mL plastic centrifuge tube. The pyridine solution was cooled to 0 °C, followed by dropwise addition of HF·pyridine (2.5 mL per 1 g oligosaccharide) while stirring. The reaction was then allowed to warm to room temperature and kept overnight or 3 days. The reaction was diluted with CH2Cl2 and washed sequentially with sat. CuSO4, sat. NaHCO3, and 1M HCl. The organic layer was dried over Na2SO4, concentrated, and purified by silica gel chromatography. 239 4.4.3. General Procedure for Removal of Levulinoyl Esters A solution of the oligosaccharide containing Lev esters (1 equiv) in pyridine/AcOH (3/2) was cooled to 0 °C. To this was added hydrazine hydrate (5 equiv per Lev ester). The reaction was stirred at 0 °C for 3 h or until TLC showed that the reaction was complete. To quench the reaction, excess acetone was added and the reaction was stirred at room temperature for 30 min. The reaction mixture was then diluted with ethyl acetate and washed with 1 M HCl, sat. NaHCO3 and brine. The resulting organic layer was then dried over Na2SO4, concentrated, and purified by silica gel chromatography. 4.4.4. General Procedure for Oxidation of 6-OH The desired compound to be oxidized (1 equiv) was dissolved in a solution of DCM/t-BuOH/H2O (4/4/1). To this solution was added TEMPO (0.3 equiv per 6-OH), followed by BAIB (3 equiv per 6-OH). The reaction was then stirred at room temperature overnight. After ensuring that the reaction was complete by TLC, the reaction was quenched by addition of excess Na2S2O3 solution and allowed to stir at room temperature for 15 min. The mixture was then diluted with DCM and washed with brine. The organic layers were combined, dried over Na2SO4, and concentrated. The crude product could then be protected as a methyl or benzyl ester. 4.4.5. General Procedure for Methyl Ester Formation after Oxidation The crude product from oxidation was dissolved in DMF. To this solution was added K2CO3 (5 equiv per COOH), followed by CH3I (2.5 equiv per COOH), and the reaction was allowed to stir overnight at room temperature. After verifying that the reaction was complete by TLC, the reaction was diluted with ethyl acetate and water. The mixture was then washed with 1 M HCl 240 and sat. NaHCO3, dried over Na2SO4, concentrated, and purified by silica gel chromatography. 4.4.6. General Procedure for Benzyl Ester Formation after Oxidation The crude product from oxidation was dissolved in DCM. To this was added phenyl diazomethane until a deep red color persisted. The reaction was allowed to stir overnight. After TLC indicated that the reaction was complete, the mixture was concentrated and purified by silica gel chromatography. 4.4.7. General Procedure for Transesterification The ester containing oligosaccharide was dissolved in a mixture of DC/MeOH (1/1). NaOMe solution was added to the oligosaccharide solution until the pH reached 10. The reaction was maintained at pH 10 and stirred at room temperature. After the reaction was confirmed complete by TLC, it was quenched by adding H+ resin. The quenched reaction was filtered, concentrated and purified by silica gel chromatography. 4.4.8. General Procedure for 1, 3-Propanedithiol Mediated Azide Reduction The starting oligosaccharide was dissolved in anhydrous MeOH (dried over 4 Å molecular sieves) and protected from light. To this solution were added triethylamine (30 equiv per N3) and 1, 3-propanedithiol (30 equiv per N3), and the reaction was stirred at room temperature for 72 h. The reaction was concentrated and purified by silica gel chromatography. 4.4.9. General Procedure for Selective N-Sulfation To a solution of NH2-containing compound (1 equiv) in MeOH was added 1 M aqueous NaOH solution at 0 °C until the pH reaches 10. SO3·pyridine (10 equiv) was added to the solution at the same temperature followed by NaOH to adjust the pH back to 10. The solution 241 was allowed to warm up to room temperature and stirred overnight. The reaction was concentrated and purified by silica gel chromatography. 4.4.10. General Procedure for Simultaneous O, N-Sulfation A compound (1 equiv) containing both free OH and NH2 groups was dissolved in dry pyridine (1 mL per 5 mg compound, dried over 4 Å molecular sieves). To this mixture was added SO3·pyridine (100 mg per 1mL pyridine), which had been previously washed with H2O, MeOH, and DCM and dried under vacuum. The reaction was protected from light and stirred for 24 h at 55 °C. The reaction was diluted with 1:1 DCM:MeOH and eluted from a Sephadex LH-20 column, ensuring that all pyridine was removed. The fractions containing sugar were concentrated and further purified by prep TLC (EtOAc/MeOH/H2O = 3/1/1). 4.4.11. General Procedure for Global Debenzylation A mixture of the Bn-containing compound (for 6 mg of compound, 1 equiv), MeOH/H2O (4 mL/2 mL), and Pd(OH)2/C (100 mg) was stirred under H2 at room temperature overnight and then filtered. The filtrate was concentrated to dryness under vacuum and then diluted with H2O (15 mL). The aqueous phase was further washed with CH2Cl2 (3 × 5 mL) and EtOAc (3 × 5 mL), and then the aqueous phase was dried under vacuum. The crude product was further purified by a Sephadex G-15 column. 4.4.12. General Procedure for Methyl Ester Saponification The solution of compound (1 equiv) in THF was cooled to 0 °C and 1 M LiOH (15 equiv per COOMe) was added dropwise, followed by addition of H2O2 (150 equiv per COOMe, 30%). Additional LiOH was added to adjust the pH to 9. The reaction was warmed up to room 242 temperature and stirred overnight. Then the mixture was eluted from a Sephadex G-15 column with H2O. To simplify mass spectrometry analysis, the product was then eluted from a column of Dowex 50WX4-Na+ to convert the compound into the sodium salt form. 4.4.13. Preparation of Tau Oligomers Recombinant tau protein (tau-441 (2N4R) MW 45.9 kDa) was expressed and purified as described.29-30 Tau pellet was treated with 8M urea followed by overnight dialysis against 1X phosphate-buffered saline (PBS), pH 7.4. Tau concentration was measured using bicinchoninic acid protein assay (Micro BCA kit, Pierce) and diluted to 1 mg/ml using 1X PBS. Aliquots of tau monomer in PBS were stored at -20°C. Each 300 µl of tau stock (0.3 mg) was added to 700 µl of 1X PBS and incubated for 1 hour on an orbital shaker at room temperature. After shaking, the resulting tau oligomers were purified by fast protein liquid chromatography (FPLC, Superdex 200HR 10/30 column, Amersham Biosciences). 4.4.14. BLI Binding Assay of Heparin and Tau Oligomers The heparin oligosaccharides were biotinylated by reaction with sulfo-N-hydroxysuccinimide long-chain biotin (ApexBio Tech LLC) following a previously reported method.31 The binding assay was performed on the Octet K2 System (Pall ForteBio). The biotinylated heparin oligosaccharides were absorbed to streptavidin (SA) sensor at a concentration of 50 μM for 2 min. The sensor was then balanced in the assay buffer (PBS containing 0.005% P20) and dipped into tau oligomer solution in assay buffer at different concentration (4.36, 2.18, 1.09, 0.545, 0.272, 0.136, 0.0681 μM). After 2 min of association, the 243 sensor was brought back to the previous assay buffer for a 3-min dissociation step. At the end of the assay, the sensor was regenerated in 1 M NaCl to remove the bound tau oligomers. Each measurement was repeated 3 times on the same sensor. The control assay was done with another sensor loaded with saturated biotin solution. 4.4.15. Preparation of Tau Oligomers in the Presence of Heparin-like Oligosaccharides 100 μl of tau oligomers (1µg/μl) were incubated with heparin-like oligosaccharides (1:5). Oligosaccharides were dissolved in ddH2O at a final concentration of 50 mM and diluted in 1X PBS or cells medium for incubation or toxicity assays. Tau oligomers in the presence of oligosaccharides and controls were incubated on an orbital shaker, without stirring, for 16 hours under oligomerization conditions. 4.4.16. Atomic Force Microscopy (AFM) Tau oligomers were characterized by AFM as previously described.28 Briefly, samples were prepared by adding 10 µl tau oligomers in the absence or presence of AC on freshly-cleaved mica and were allowed to adsorb to the surface. Mica were then washed three times with distilled water to remove unbound protein and impurities followed by air-drying. Samples were then imaged with Multimode 8 AFM machine (Veeco, CA) using a non-contact tapping method (ScanAsyst-Air). 4.4.17. Cell Toxicity Assays Human neuroblastoma SH-SY5Y cells were cultured and treated for measuring cytotoxicity using either lactate dehydrogenase (LDH) release assay (Cytotoxicity Detection KitPLUS -LDH, 244 Roche) or a resazurin-based assay (PrestoBlueTM, Invitrogen) following manufacturers’ instructions as previously described.28, 32 Briefly, cells were maintained in Dulbecco`s modified Eagle’s medium (DMEM) and grown to confluency in 96-well plates. Cells (≈10,000 cells /well) were treated for 24 hours with 2.0 µM tau oligomers and 2.0 µM tau oligomers incubated with 10 µM of heparin-like oligosaccharides (26-29, 31, 32) followed by assaying with LDH or PrestoBlue. Optical density (OD) was measured at 490 nm and 570 nm, for LDH and PrestoBlue, respectively, with POLARstar OMEGA microplate reader (BMG Labtech). All measurements were performed in triplicate and corrected by the vehicle background. Statistical analysis was based on one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test performed using GraphPad Prism 6.01. 4.4.18. Product Preparation and Characterization Data OAc O BnO BnO N3 O OLev OBn O STol BzO p-Tolyl 6-O-acetyl-2-azido-3, 4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl- 3-O-benzyl-6-O-levulinoyl-1-thio-α-L-idopyranoside (3) Compound 3 was prepared from compound 7 in 2 steps. Firstly, compound 7 (1.04 g, 1.03 mmol) was dissolved in DCM/H2O (45/5 mL), cooled to 0 °C and DDQ (467 mg, 2.06 mmol) was added. The reaction was allowed to warm up to room temperature and stirred overnight. Upon completion, the reaction was quenched with sat. NaHCO3, diluted with DCM and washed sequentially with water and sat. NaHCO3. The organic phase was dried over Na2SO4, 245 concentrated, and purified through silica gel (Hexanes/EtOAc = 2/1). The product was then diluted in DCM (25 mL). To this solution was added EDC·HCl (522 mg, 2.77 mmol), DMAP (10 mg, 0.08 mmol) and levulinic acid (257 μL, 2.52 mmol), and the reaction was stirred at room temperature overnight. The mixture was then diluted with DCM, washed with sat. NaHCO3, dried over Na2SO4, concentrated, and purified by silica gel column to afford compound 3 in 89% yield over 2 steps. 1HNMR (500 MHz, CDCl3): δ = 2.02 (s, 3H), 2.16 (s, 3H), 2.35 (s, 3H), 2.56-2.62 (m, 2H), 2.69-2.75 (m, 2H), 3.29 (dd, 1H, J = 3.5, 10.0 Hz), 3.38 (t, 1H, J = 9.5 Hz), 3.56 (t, 1H, J = 9.5 Hz), 3.64 (s, 1H), 3.91 (d, 1H, J = 10.5 Hz), 3.93-3.97 (m, 1H), 4.17 (s, 1H), 4.20-4.26 (m, 2H), 4.26-4.31 (m, 1H), 4.33 (dd, 1H, J = 4.0, 11.5 Hz), 4.42 (dd, 1H, J = 8.0, 12.0 Hz), 4.50 (d, 1H, J = 10.5 Hz), 4.56 (d, 1H, J = 4.0 Hz), 4.73 (d, 1H, J = 10.5 Hz), 4.78 (d, 1H, J = 11.5 Hz), 4.94-4.98 (m, 1H), 5.00 (d, 1H, J = 12.0 Hz), 5.39 (s, 1H), 5.58 (s, 1H), 7.10-7.18 (m, 4H), 7.21-7.44 (m, 14H), 7.46-7.53 (m, 4H), 8.13-8.18 (m, 2H). 13CNMR (125 MHz, CDCl3): δ = 20.85, 21.22, 27.86, 29.91, 37.93, 62.79, 63.88, 63.94, 66.06, 69.55, 70.41, 71.34, 72.64, 75.04, 75.13, 76.08, 77.64, 80.82, 86.5, 99.26, 127.95, 128.06, 128.14, 128.16, 128.38, 128.46, 128.54, 128.58, 128.71, 129.8, 129.89, 130.05, 131.97, 132.23, 133.29, 137.29, 137.45, 137.47, 137.8, 165.73, 170.74, 172.4, 206.45. HRMS: m/z calc. for C54H61N4O13S: 1005.3956; found: 1005.3941 [M + NH4]+. 246 OAc O BnO BnO N3 O OPMB OBn O STol BzO p-Tolyl 6-O-acetyl-2-azido-3, 4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl- 3-O-benzyl-6-O-p-methoxybenzyl-1-thio-α-L-idopyranoside (7) Compound 7 was prepared from compound 5 (533 mg, 1.0 mmol) and 6 (600 mg, 1.0 mmol) by following the general procedure for preactivation based glycosylation. The reaction was performed at -78 °C until quenched by Et3N at the same temperature to avoid decomposition under acidic conditions. Purification through silica gel column (Hexanes/EtOAc = 3/1) provided compound 7 in 87% yield. 1HNMR (500 MHz, CDCl3): δ = 2.02(s, 3H), 2.34 (s, 3H), 3.31 (dd, 1H, J = 3.5, 10.0 Hz), 3.43 (dd, 1H, J = 8.5, 10.0 Hz), 3.64 (dd, 1H, J = 9.0, 10.5 Hz), 3.74 (t, 1H, J = 3.0 Hz), 3.79 (d, 2H, J = 6.0 Hz), 3.82 (s, 3H), 3.98 (dt, 1H, J = 3.5, 10.0 Hz), 4.14 (d, 1H, J = 10.5 Hz), 4.17-4.21 (m, 3H), 4.32 (d, 1H, J = 10.5 Hz), 4.50-4.55 (m, 3H), 4.72 (d, 1H, J = 3.5 Hz), 4.76 (d, 1H, J = 3.5 Hz), 4.78 (d, 1H, J = 5.0 Hz), 4.94 (dd. 1H, J = 2.5, 6.5 Hz), 4.97 (d, 1H, J = 12.0 Hz), 5.42 (s, 1H), 5.57 (s, 1H), 6.88 (d, 2H, J = 9.0 Hz), 7.07 (d, 2H, J = 7.5 Hz), 7.17-7.21 (m, 2H), 7.25-7.43 (m, 16H), 7.48 (d, 4H, J = 8.0 Hz), 8.13-8.16 (m, 2H). 13CNMR (125 MHz, CDCl3): δ = 20.9, 21.24, 55.35, 62.82, 64.06, 67.18, 69.13, 70.06, 70.12, 72.08, 72.67, 73.08, 75.12, 75.17, 77.72, 80.8, 86.52, 98.64, 113.84, 127.99, 128.02, 128.08, 128.17, 128.21, 128.5, 128.52, 128.52, 128.62, 128.69, 129.44, 129.74, 129.95, 130.01, 130.26, 131.92, 132.52, 133.27, 137.52, 137.56, 137.59, 137.66, 159.28, 165.76, 170.65. HRMS: m/z calc. for C57H63N4O12S:1027.4163; found: 247 1027.4120 [M + NH4]+. Bn O BnO OAc O N3 O OLev STol OBn O BzO 2 p-Tolyl 6-O-acetyl-2-azido-3, 4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl -3-O-benzyl-6-O-levulinoyl-α-L-idopyranosyl-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α -D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-1-thio-α-L-idopyranoside (8) Compound 8 was prepared from compound 3 (412 mg, 0.42 mmol) and 1 (374 mg, 0.42 mmol) by following the general procedure for preactivation based glycosylation. Purification through silica gel column (Hexanes/EtOAc = 1/1) provided compound 8 in 85% yield. 1HNMR (500 MHz, CDCl3): δ = 1.99 (s, 3H), 2.04 (s, 3H), 2.10 (s, 3H), 2.15 (s, 3H), 2.35 (s, 3H), 2.43-2.51 (m, 2H), 2.51-2.58 (m, 2H), 2.58-2.63 (m, 2H), 2.65-2.75 (m, 2H), 3.27 (ddd, 2H, J = 2.5, 4.0, 10.0 Hz), 3.45 (t, 1H, J = 9.5 Hz), 3.48 (t, 1H, J = 9.5 Hz), 3.62 (t, 1H, J = 2.5 Hz), 3.66-3.73 (m, 4H), 3.83-3.88 (m, 2H), 4.02-4.07 (m, 2H), 4.14-4.17 (m, 1H), 4.20-4.40 (m, 9H), 4.47-4.56 (m, 4H), 4.71 (d, 1H, J = 4.0 Hz), 4.74-4.80 (m, 3H), 4.83 (d, 1H, J = 11.5 Hz), 4.94 (ddd, 1H, J = 1.5, 4.0, 7.0 Hz), 4.98 (d, 1H, J = 12.0 Hz), 5.08 (d, 1H, J = 3.5 Hz), 5.12 (t, 1H, J = 4.0 Hz), 5.38 (t, 1H, J = 2.0 Hz), 5.59 (s, 1H), 7.14 (d, 4H, J = 8.0 Hz), 7.20-7.53 (m, 31H), 8.10 (d, 2H, J = 7.0 Hz), 8.16 (d, 2H, J = 6.5 Hz). 13CNMR (125 MHz, CDCl3): δ = 20.7, 20.8, 21.18, 27.76, 27.79, 29.83, 29.84, 37.76, 37.89, 62.18, 62.34, 62.56, 63.6, 63.67, 63.9, 65.94, 67.61, 69.53, 70.11, 70.15, 70.23, 71.19, 72.6, 73.43, 74.14, 74.73, 74.88, 75.17, 75.22, 75.29, 248 75.76, 77.62, 79.17, 80.46, 86.42, 97.8, 98.81, 98.91, 127.61, 127.99, 128.03, 128.07, 128.11, 128.13, 128.16, 128.25, 128.28, 128.33, 128.44, 128.49, 128.5, 128.62, 128.68, 129.61, 129.76, 129.84, 129.86, 129.92, 131.92, 132.15, 133.35, 133.42, 137.24, 137.31, 137.34, 137.5, 137.72, 137.74, 165.44, 165.73, 170.62, 170.74, 172.25, 172.26, 206.36, 206.39. HRMS: m/z calc. for C94H104N7O26S:1778.6752; found:1778.6780 [M + NH4]+. Bn O BnO OAc O N3 O OLev O OBn O BzO 2 Cbz N Bn N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-acetyl-2-azido-3, 4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-α-L -idopyranosyl-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4) -2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-α-L-idopyranoside (9) Compound 9 was prepared from compound 3 (153 mg, 0.16 mmol) and 2 (150 mg, 0.14 mmol) by following the general procedure for preactivation based glycosylation. Purification through silica gel column (Hexanes/EtOAc = 1/1) provided compound 9 in 88% yield. 1HNMR (500 MHz, CDCl3): δ = 1.80-1.91 (m, 2H), 1.98 (s, 3H), 2.04 (s, 3H), 2.10 (s, 3H), 2.14 (s, 3H), 2.41-2.51 (m, 4H), 2.60 (t, 2H, J = 7.0 Hz), 2.64-2.73 (m, 2H), 3.23-3.28 (m, 2H), 3.31-3.40 (m, 2H), 3.41-3.52 (m, 2H), 3.54-3.60 (m, 1H), 3.60-3.77 (m, 5H), 3.80-3.87 (m, 2H), 3.93 (d, 1H, J = 10.0 Hz), 4.00-4.07 (m, 3H), 4.15-4.22 (m, 1H), 4.22-4.30 (m, 4H), 4.30-4.40 (m, 5H), 4.43-4.54 (m, 4H), 4.59-4.66 (m, 2H), 4.68 (d, 1H, J = 3.5 Hz), 4.69-4.74 (m, 1H), 249 4.74-4.86 (m, 4H), 4.88-4.98 (m, 1H), 5.06-5.13 (m, 3H), 5.16 (d, 2H, J = 10.0 Hz), 7.10-7.20 (m, 3H), 7.20-7.50 (m, 38H), 8.07-8.11 (m, 2H), 8.12-8.15 (m, 2H). 13CNMR (125 MHz, CDCl3): δ = 20.85, 20.87, 27.82, 29.9, 37.83, 62.33, 62.62, 63.75, 63.9, 65.41, 67.24, 67.49, 68.75, 70.01, 70.19, 70.21, 72.31, 73.43, 73.96, 74.93, 74.95, 75.18, 75.26, 75.34, 77.68, 79.19, 80.54, 97.91, 98.39, 98.87, 127.36, 127.68, 127.94, 128.03, 128.08, 128.15, 128.22, 128.29, 128.33, 128.41, 128.48, 128.51, 128.55, 128.58, 128.6, 128.67, 128.72, 129.69, 129.87, 129.9, 133.4, 137.36, 137.38, 137.54, 137.79, 165.52, 165.76, 170.67, 170.77, 172.29, 172.35, 206.4. HRMS: m/z calc. for C105H117N8O29: 1953.7926; found: 1953.7886 [M + NH4]+. TBS OAc O O BnO N3 O OLev STol OBn O BzO 2 p-Tolyl 6-O-acetyl-2-azido-3-O-benzyl-4-O-tert-butyldimethylsilyl-2-deoxy-α-D-glucopyranosyl-(1→4) -2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-α-L-idopyranosyl-(1→4)-6-O-acetyl-2-azido-3-O-benz yl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-1-thio-α-L-idop yranoside (10) Compound 10 was prepared from compound 4 (93 mg, 0.092 mmol) and 1 (83 mg, 0.092 mmol) by following the general procedure for preactivation based glycosylation. Purification through silica gel column (Hexanes/EtOAc = 2/1) provided compound 10 in 72% yield. 1HNMR (500 MHz, CDCl3): δ = -0.07 (s, 3H), 0.00 (s, 3H), 0.88 (s, 9H), 1.99 (s, 3H), 2.01 (s, 250 3H), 2.11 (s, 3H), 2.13 (s, 3H), 2.33 (s, 3H), 2.45-2.55 (m, 4H), 2.60-2.75 (m, 4H), 3.24 (ddd, 2H, J = 3.5, 9.0, 10.0 Hz), 3.42-3.50 (m, 2H), 3.56 (dd, 1H, J = 8.5, 9.5 Hz), 3.60 (t, 1H, J = 2.5 Hz), 3.65 (dd, 1H, J = 8.5, 10.0 Hz), 3.69 – 3.76 (m, 3H), 3.82 (ddd, 1H, J = 2.0, 4.5, 10.0 Hz), 4.00-4.08 (m, 3H), 4.09-4.14 (m, 2H), 4.20 (dd, 1H, J = 2.0, 12.0 Hz), 4.24 (dd, 1H, J = 5.0, 11.5 Hz), 4.27-4.34 (m, 4H), 4.35-4.42 (m, 2H), 4.49 (t, 2H, J = 10.5 Hz), 4.54 (d, 1H, J = 4.0 Hz), 4.72 – 4.78 (m, 3H), 4.80 (d, 1H, J = 3.5 Hz), 4.90 (ddd, 1H, J = 2.0, 4.5, 7.5 Hz), 4.95 (d, 1H, J = 12.0 Hz), 5.05 (d, 1H, J = 4.0 Hz), 5.12 (t, 1H, J = 4.5 Hz), 5.35-5.37 (m, 1H), 5.55 (s, 1H), 7.10-7.14 (m, 4H), 7.20-7.26 (m, 6H), 7.25-7.33 (m, 8H), 7.35-7.51 (m, 12H), 8.05-8.09 (m, 2H), 8.11-8.14 (m, 2H). Comparison with literature data confirmed its identity.25 Bn O BnO OAc O N3 O OLev O OBn O BzO 3 Cbz N Bn N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-acetyl-2-azido-3, 4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-α-L -idopyranosyl-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O- benzoyl-3-O-benzyl-6-O-levulinoyl-α-L-idopyranosyl-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2- deoxy-α-D-glucopyranosyl-(1→4) -2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-α-L-idopyranoside (11) Compound 11 was prepared from compound 8 (110 mg, 0.062 mmol) and 2 (67 mg, 0.062 mmol) by following the general procedure for preactivation based glycosylation. Purification through 251 silica gel column (Hexanes/DCM/EtOAc = 1/1/1) provided compound 11 in 58% yield. 1HNMR (500 MHz, CDCl3): δ = 1.78-1.92 (m, 2H), 1.98 (s, 3H), 2.02 (s, 6H), 2.10 (s, 6H), 2.14 (s, 3H), 2.39-2.74 (m, 12H), 3.21-3.28 (m, 3H), 3.29-3.40 (m, 3H), 3.43 (t, 2H, J = 9.5 Hz), 3.55 (t, 1H, J = 9.5 Hz), 3.58-3.72 (m, 6H), 3.73-3.78 (m, 2H), 3.91 (d, 1H, J = 10.5 Hz), 3.97-4.09 (m, 5H), 4.14-4.30 (m, 8H), 4.30-4.39 (m, 6H), 4.41-4.55 (m, 5H), 4.57-4.63 (m, 2H), 4.63-4.85 (m, 10H), 4.86-4.97 (m, 2H), 5.03-5.10 (m, 3H), 5.10-5.19 (m, 4H), 7.10-7.53 (m, 54H), 8.01-8.18 (m, 6H). 13CNMR (125 MHz, CDCl3): δ = 20.8, 20.85, 27.8, 27.82, 29.89, 37.8, 37.86, 43.93, 44.95, 50.83, 51.05, 62.04, 62.26, 62.32, 62.62, 62.96, 63.25, 63.43, 63.64, 63.74, 63.84, 65.4, 65.54, 65.74, 67.23, 67.44, 67.86, 68.73, 69.98, 70.15, 70.22, 70.43, 70.67, 71.31, 72.31, 72.37, 73.42, 73.58, 73.86, 74.36, 74.69, 74.9, 74.94, 75.11, 75.27, 75.35, 75.43, 77.67, 78.92, 79.07, 80.08, 80.56, 97.81, 97.97, 98.35, 98.47, 98.87, 127.36, 127.64, 127.73, 127.85, 127.9, 127.94, 127.96, 128.01, 128.04, 128.08, 128.11, 128.14, 128.19, 128.21, 128.26, 128.28, 128.3, 128.33, 128.41, 128.43, 128.46, 128.48, 128.53, 128.56, 128.58, 128.61, 128.68, 128.71, 128.75, 129.58, 129.68, 129.89, 129.92, 133.45, 133.5, 136.81, 136.91, 137.32, 137.35, 137.38, 137.53, 137.73, 137.78, 137.85, 137.98, 138.05, 156.22, 156.77, 165.52, 165.56, 165.75, 170.68, 170.72, 170.78, 171.95, 172.26, 172.31, 172.34, 206.42, 206.46. HRMS: m/z calc. for C145H164N12O42: 1372.5533; found: 1372.5518 [M + 2NH4]2+. H OAc O O BnO N3 O OLev O OBn O BzO 3 Cbz N Bn 252 N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6- O-levulinoyl-α-L-idopyranosyl-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyran osyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-α-L-idopyranosyl-(1→4)-6-O-acetyl-2-azid o-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-α-L- idopyranoside (12) Compound 12 was prepared from compound 10 (200 mg, 0.11 mmol) and 2 (96 mg, 0.09 mmol) by following the general procedure for preactivation based glycosylation and TBS removal. Purification through silica gel column (Hexanes/DCM/EtOAc = 1/1/2) provided compound 12 in 84% yield over 2 steps. 1HNMR (500 MHz, CDCl3): δ = 1.80-1.90 (m, 2H), 2.01 (s, 3H), 2.02 (s, 3H), 2.04 (s, 3H), 2.10 (s, 3H), 2.12 (s, 3H), 2.13 (s, 3H), 2.36-2.56 (m, 6H), 2.57-2.73 (m, 6H), 3.21 (dd, 1H, J = 3.5, 10.0 Hz), 3.23-3.27 (m, 2H), 3.30-3.50 (m, 5H), 3.54 (t, 1H, J = 9.5 Hz), 3.57-3.65 (m, 3H), 3.65-3.71(m, 3H), 3.72-3.85 (m, 5H), 3.91 (d, 1H, J = 10.0 Hz), 3.98-4.07 (m, 5H), 4.10-4.24 (m, 5H), 4.25-4.39 (m, 8H), 4.41 (d, 1H, J = 11.0 Hz), 4.43-4.47 (m, 1H), 4.49 (d, 1H, J = 4.0 Hz), 4.51 (d, 1H, J = 4.0 Hz), 4.59 (d, 2H, J = 10.0 Hz), 4.61 (d, 1H, J = 7.5 Hz), 4.66-4.84 (m, 9H), 4.87-4.95 (m, 1H), 5.03-5.10 (m, 3H), 5.10-5.18 (m, 4H), 7.10-7.38 (m, 40H), 7.39-7.54 (m, 9H), 8.06-8.15 (m, 6H). 13CNMR (125 MHz, CDCl3): δ = 20.85, 20.86, 27.82, 28.41, 29.89, 29.92, 37.86, 37.9, 43.93, 44.95, 50.83, 51.05, 62.08, 62.21, 62.28, 62.4, 62.84, 63.1, 63.44, 63.72, 63.85, 65.41, 65.54, 65.75, 67.23, 67.63, 67.81, 68.73, 70.15, 70.21, 70.31, 70.38, 70.72, 71.26, 72.32, 72.45, 73.44, 73.56, 73.79, 74.16, 74.3, 74.7, 74.93, 75.07, 75.18, 75.3, 75.41, 78.84, 253 79.07, 79.81, 97.8, 97.86, 98.35, 98.44, 98.47, 98.52, 127.35, 127.63, 127.67, 127.84, 127.88, 127.93, 128.0, 128.1, 128.13, 128.16, 128.17, 128.24, 128.4, 128.46, 128.48, 128.6, 128.61, 128.68, 128.7, 128.73, 129.57, 129.88, 129.91, 133.42, 133.49, 133.54, 136.79, 136.89, 137.3, 137.37, 137.72, 137.78, 137.84, 137.89, 137.96, 137.98, 138.03, 156.2, 156.76, 165.51, 165.58, 165.75, 170.77, 170.8, 171.84, 172.25, 172.34, 172.43, 206.42, 206.48, 206.93. HRMS: m/z calc. for C138H154N11O42: 2637.0253; found: 2637.0212 [M + NH4]+. Bn O BnO OAc O N3 O OLev O OBn O BzO 5 Cbz N Bn N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-acetyl-2-azido-3, 4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-α-L -idopyranosyl-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O- benzoyl-3-O-benzyl-6-O-levulinoyl-α-L-idopyranosyl-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2- deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-α-L-idopyranosyl-(1 →4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-ben zyl-6-O-levulinoyl-α-L-idopyranosyl-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-gluco pyranosyl-(1→4) -2-O-benzoyl-3-O-benzyl-6-O-levulinoyl-α-L-idopyranoside (13) Compound 13 was prepared from compound 8 (808 mg, 0.46 mmol) and 12 (841 mg, 0.32 mmol) by following the general procedure for preactivation based glycosylation. Purification through silica gel column (Hexanes/DCM/EtOAc = 1/1/1) provided compound 13 in 84% yield. 254 1HNMR (500 MHz, CDCl3): δ = 1.82-1.93 (m, 2H), 1.99 (s, 3H), 2.02 (s, 6H), 2.04 (s, 6H), 2.11 (s, 12H), 2.14 (s, 3H), 2.40-2.73 (m, 20H), 3.24-3.30 (m, 5H), 3.32-3.41 (m, 3H), 3.45 (t, 2H, J = 9.5 Hz), 3.56 (t, 1H, J = 9.5 Hz), 3.61-3.73 (m, 10H), 3.73-3.81 (m, 6H), 3.81-3.88 (m, 3H), 3.92 (d, 1H, J = 10.5 Hz), 3.99-4.09 (m, 9H), 4.14-4.26 (m, 10H), 4.26-4.39 (m, 14H), 4.43-4.49 (m, 2H), 4.49-4.55 (dd, 3H, J = 7.0, 10.5 Hz), 4.58-4.64 (m, 2H), 4.68 (d, 1H, J = 3.5 Hz), 4.71-4.85 (m, 15H), 4.88-4.97 (m, 2H), 5.05-5.12 (m, 5H), 5.12-5.20 (m, 6H), 7.11-7.55 (m, 80H), 8.07-8.17 (m, 10H). 13CNMR (125 MHz, CDCl3): δ = 20.7, 20.78, 27.74, 29.81, 37.72, 37.77, 37.79, 43.86, 44.88, 50.76, 50.98, 61.96, 62.18, 62.26, 62.31, 62.55, 63.36, 63.61, 63.68, 63.76, 65.31, 65.45, 65.67, 67.15, 67.39, 67.75, 68.64, 69.93, 70.09, 70.12, 70.15, 70.34, 72.23, 72.33, 73.35, 73.49, 73.52, 73.81, 74.18, 74.22, 74.34, 74.56, 74.61, 74.81, 74.85, 75.02, 75.06, 75.19, 75.28, 75.32, 75.38, 77.6, 78.72, 78.75, 78.87, 78.99, 80.48, 97.72, 97.8, 97.9, 98.29, 98.39, 98.79, 127.29, 127.57, 127.62, 127.67, 127.78, 127.82, 127.87, 127.94, 127.97, 128.0, 128.03, 128.07, 128.1, 128.12, 128.15, 128.24, 128.26, 128.34, 128.38, 128.41, 128.48, 128.51, 128.54, 128.61, 128.66, 128.68, 129.48, 129.49, 129.6, 129.82, 133.38, 133.43, 133.49, 136.75, 136.84, 137.25, 137.28, 137.32, 137.47, 137.67, 137.78, 137.83, 137.95, 156.14, 156.68, 165.43, 165.46, 165.48, 165.68, 170.59, 170.64, 170.7, 172.17, 172.2, 172.24, 172.27, 206.34, 206.38. HRMS: m/z calc. for C225H246N17O68: 4273.6314; found: 4273.6372 [M + NH4]+. Bn O BnO OAc O N3 O OH O OBn O BzO 2 Cbz N Bn 255 N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-acetyl-2-azido-3, 4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-α-L-idopyranosyl-( 1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-be nzyl-α-L-idopyranoside (14) Compound 14 was prepared from compound 9 (1.0 g, 0.52 mmol) by following the general procedure for removal of levulinoyl esters. Purification through silica gel column (Hexanes/DCM/EtOAc = 1/1/1) provided compound 14 in 86% yield. 1HNMR (500 MHz, CDCl3): δ = 1.80-1.90 (m, 2H), 2.01 (s, 3H), 2.04 (s, 3H), 2.60 (br, 1H), 3.17-3.37 (m, 5H), 3.38-3.44 (m, 1H), 3.50-3.61 (m, 3H), 3.61-3.80 (m, 5H), 3.81-4.00 (m, 5H), 4.03-4.14 (m, 3H), 4.14-4.29 (m, 4H), 4.29-4.43 (m, 3H), 4.43-4.60 (m, 5H), 4.64-4.74 (m, 2H), 4.76 (d, 2H, J = 10.5 Hz), 4.81-4.97 (m, 3H), 4.98-5.05 (m, 1H), 5.05-5.21 (m, 4H), 7.10-7.50 (m, 41H), 8.08-8.17 (m, 4H). 13CNMR (125 MHz, CDCl3): δ = 20.89, 28.19, 28.35, 44.87, 50.61, 61.3, 62.72, 62.8, 63.79, 64.17, 66.33, 67.41, 68.9, 69.13, 70.01, 70.37, 72.14, 72.99, 74.03, 75.09, 75.24, 75.28, 77.74, 79.45, 80.7, 97.98, 98.22, 127.35, 127.42, 127.92, 127.95, 128.03, 128.07, 128.19, 128.22, 128.27, 128.39, 128.43, 128.5, 128.54, 128.57, 128.59, 128.65, 128.7, 129.73, 129.79, 129.92, 130.1, 133.26, 133.42, 137.4, 137.46, 137.52, 137.54, 137.87, 165.81, 170.58, 170.69. HRMS: m/z calc. for C95H102N7O25:1740.6925; found: 1740.6924 [M + H]+. Bn O BnO OAc O N3 O OH O OBn O BzO 3 Cbz N Bn 256 N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-acetyl-2-azido-3, 4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-α-L-idopyranosyl-( 1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-be nzyl-α-L-idopyranosyl-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→ 4) -2-O-benzoyl-3-O-benzyl-α-L-idopyranoside (15) Compound 15 was prepared from compound 11 (500 mg, 0.18 mmol) by following the general procedure for removal of levulinoyl esters. Purification through silica gel column (Hexanes/DCM/EtOAc = 1/1/1) provided compound 15 in 93% yield. 1HNMR (500 MHz, CDCl3): δ = 1.81-1.89 (m, 2H), 2.00 (s, 3H), 2.04 (s, 6H), 3.12-3.23 (m, 3H), 3.23-3.38 (m, 4H), 3.38-3.44 (m, 2H), 3.44-3.63 (m, 7H), 3.63-3.72 (m, 2H), 3.72-3.82 (m, 4H), 3.82-4.00 (m, 5H), 4.01-4.09 (m, 3H), 4.15-4.29 (m, 7H), 4.30-4.62 (m, 9H), 4.63-4.80 (m, 6H), 4.81-4.97 (m, 4H), 4.98-5.22 (m, 8H), 7.12-7.47 (m, 54H), 8.08-8.17 (m, 6H). 13CNMR (125 MHz, CDCl3): δ = 14.32, 20.88, 20.94, 21.18, 28.18, 50.59, 60.51, 61.24, 61.33, 62.41, 62.72, 62.79, 63.8, 64.02, 67.41, 67.67, 69.01, 70.0, 70.07, 70.42, 72.14, 72.91, 72.99, 73.14, 73.69, 74.03, 75.15, 75.18, 75.24, 75.27, 77.73, 79.35, 79.47, 80.73, 97.34, 97.89, 97.95, 98.22, 127.34, 127.43, 127.89, 127.93, 128.03, 128.07, 128.12, 128.23, 128.26, 128.29, 128.32, 128.4, 128.47, 128.54, 128.6, 128.66, 128.68, 129.66, 129.76, 129.88, 129.9, 130.1, 133.27, 133.41, 137.4, 137.48, 137.52, 137.86, 165.85, 165.91, 170.6, 170.7, 171.27. HRMS: m/z calc. for C130H139N10O36: 2415.9353; found: 2415.9319 [M + H]+. 257 Bn O BnO OAc O N3 O OH O OBn O BzO 5 Cbz N Bn N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-acetyl-2-azido-3, 4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-α-L-idopyranosyl-( 1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-be nzyl-α-L-idopyranosyl-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→ 4)-2-O-benzoyl-3-O-benzyl-α-L-idopyranosyl-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-α -D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-α-L-idopyranosyl-(1→4)-6-O-acetyl-2-azid o-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-α-L-idopyranoside (16) Compound 16 was prepared from compound 13 (120 mg, 0.028 mmol) by following the general procedure for removal of levulinoyl esters. Purification through silica gel column (Hexanes/DCM/EtOAc = 1/1/1) provided compound 16 in 83% yield 1HNMR (500 MHz, CDCl3): δ = 1.80-1.89 (m, 2H), 1.99 (s, 3H), 2.01 (s, 3H), 2.02 (s, 6H), 2.03 (s, 3H), 3.07-3.24 (m, 5H), 3.28 (dd, 1H, J = 3.5, 10.0 Hz), 3.28-3.37 (m, 5H), 3.37-3.49 (m, 6H), 3.50-3.62 (m, 8H), 3.62-3.70 (m, 3H), 3.70-3.99 (m, 13H), 3.99-4.08 (m, 5H), 4.08-4.37 (m, 18H), 4.37-4.60 (m, 9H), 4.62-4.80 (m, 10H), 4.80-4.96 (m, 6H), 4.97-5.06 (m, 4H), 5.06-5.21 (m, 7H), 7.10-7.48 (m, 80H), 8.06-8.17 (m, 10H). 13CNMR (125 MHz, CDCl3): δ = 20.9, 20.96, 29.83, 61.31, 62.43, 62.74, 62.81, 63.82, 64.07, 67.45, 67.53, 67.7, 69.09, 70.01, 70.13, 70.45, 72.17, 72.8, 72.94, 73.03, 73.12, 73.17, 73.59, 73.7, 74.04, 75.2, 75.26, 75.3, 77.75, 79.4, 80.74, 258 97.34, 97.89, 97.98, 98.23, 127.44, 127.91, 127.94, 128.05, 128.09, 128.22, 128.24, 128.29, 128.31, 128.36, 128.42, 128.49, 128.53, 128.56, 128.59, 128.62, 128.68, 128.71, 128.74, 129.7, 129.77, 129.81, 129.88, 129.92, 133.47, 137.41, 137.43, 137.49, 137.53, 137.88, 165.87, 165.94, 165.97, 170.61, 170.72. HRMS: m/z calc. for C200H213N16O58: 3766.4209; found: 3766.4148 [M + H]+. Bn O BnO OAc O MeO2C N3 O BzO O OBn O 2 Cbz N Bn N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-acetyl-2-azido-3, 4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl 2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deo xy-α-D-glucopyranosyl-(1→4)-methyl 2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate (17) Compound 17 was prepared from compound 14 (696 mg, 0.40 mmol) by following the general procedure for oxidation of 6-OH and formation of methyl esters after oxidation. Purification through silica gel column (Hexanes/DCM/EtOAc = 3/2/2) provided compound 14 in 83% yield over 2 steps. 1HNMR (500 MHz, CDCl3): δ = 1.77-1.88 (m, 2H), 1.99 (s, 3H), 2.11 (s, 3H), 3.18-3.26 (m, 2H), 3.27-3.46 (m, 3H), 3.47-3.57 (m, 3H), 3.60 (s, 3H), 3.65 (s, 3H), 3.73-3.84 (m, 3H), 3.87 (d, 1H, J = 10.0 Hz), 3.93-4.00 (m, 2H), 4.03-4.17 (m, 3H), 4.22-4.35 (m, 4H), 4.35-4.54 (m, 5H), 4.57 (d, 1H, J = 11.0 Hz), 4.65 (t, 2H, J = 3.5 Hz), 4.68-4.90 (m, 6H), 4.94 (d, 1H, J = 3.0 Hz), 259 5.02-5.20 (m, 5H), 5.48 (d, 1H, J = 4.0 Hz), 7.07-7.41 (m, 36H), 7.43-7.57 (m, 5H), 8.08-8.17 (m, 4H). 13CNMR (125 MHz, CDCl3): δ = 20.91, 20.95, 52.13, 52.27, 61.82, 62.29, 63.51, 63.61, 67.28, 67.45, 68.0, 69.71, 70.11, 70.25, 70.51, 72.28, 72.5, 74.09, 74.63, 74.86, 75.08, 75.11, 75.54, 75.71, 78.74, 80.03, 98.49, 99.1, 99.26, 127.69, 127.98, 128.03, 128.08, 128.1, 128.17, 128.19, 128.24, 128.43, 128.53, 128.57, 128.6, 128.62, 128.82, 128.85, 129.36, 129.98, 130.09, 133.53, 137.31, 137.57, 137.7, 137.78, 165.27, 169.51, 170.6, 170.79. HRMS: m/z calc. for C97H105N8O27: 1813.7089; found: 1813.7002 [M + NH4]+. Bn O BnO OAc O BnO2C N3 O BzO O OBn O 3 Cbz N Bn N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-acetyl-2-azido-3, 4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-benzyl 2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deo xy-α-D-glucopyranosyl-(1→4)-benzyl 2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deo xy-α-D-glucopyranosyl-(1→4)-benzyl 2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate (18) Compound 18 was prepared from compound 15 (290 mg, 0.18 mmol) by following the general procedure for oxidation of 6-OH and formation of benzyl esters after oxidation. Purification through silica gel column (Hexanes/DCM/EtOAc = 3/2/1) provided compound 18 in 77% yield over 2 steps. 260 1HNMR (500 MHz, CDCl3): δ = 1.75-1.83 (m, 2H), 1.98 (s, 3H), 2.04 (s, 3H), 2.09 (s, 3H), 3.13 (dd, 1H, J = 3.5, 10.0 Hz), 3.19-3.24 (m, 2H), 3.25-3.35 (m, 2H), 3.40 (t, 2H, J = 9.5 Hz), 3.47-3.54 (m, 3H), 3.61 (dd, 1H, J = 9.0, 10.0 Hz), 3.65-3.79 (m, 4H), 3.85-3.98 (m, 6H), 4.03-4.18 (m, 6H), 4.20-4.27 (m, 2H), 4.28-4.37 (m, 6H), 4.40-4.46 (m, 2H), 4.48 (d, 1H, J = 5.0 Hz), 4.54 (d, 1H, J = 11.0 Hz), 4.62 (d, 1H, J = 3.5 Hz), 4.64 (d, 1H, J = 10.5 Hz), 4.68 (d, 1H, J = 4.5 Hz), 4.72-4.81 (m, 6H), 4.86 (d, 1H, J = 4.0 Hz), 4.93 (d, 1H, J = 3.5 Hz), 4.99 (s, 2H), 5.02-5.10 (m, 4H), 5.10-5.16 (m, 4H), 5.17-5.22 (m, 2H), 5.49 (d, 1H, J = 4.5 Hz), 5.54 (d, 1H, J = 5.0 Hz), 7.08-7.18 (m, 7H), 7.19-7.41 (m, 54H), 7.44-7.52 (m, 6H), 7.52-7.59 (m, 2H), 8.08-8.17 (m, 6H). 13CNMR (125 MHz, CDCl3): δ = 20.8, 20.91, 50.77, 51.06, 61.7, 61.84, 62.31, 63.24, 63.48, 63.6, 64.12, 67.12, 67.29, 67.64, 67.77, 69.31, 69.75, 70.02, 70.14, 70.86, 71.2, 71.51, 72.42, 73.17, 74.3, 74.39, 74.64, 74.8, 75.03, 75.23, 75.7, 75.96, 76.07, 76.91, 77.16, 77.36, 77.41, 77.53, 78.35, 78.41, 80.04, 98.36, 98.55, 99.14, 99.21, 100.18, 127.32, 127.52, 127.74, 127.91, 128.04, 128.07, 128.09, 128.12, 128.24, 128.27, 128.33, 128.36, 128.41, 128.49, 128.51, 128.56, 128.58, 128.61, 128.63, 128.68, 128.73, 128.77, 128.94, 128.97, 129.24, 129.31, 129.78, 130.01, 130.07, 130.12, 133.11, 133.68, 133.74, 134.89, 135.18, 137.23, 137.32, 137.65, 137.67, 137.84, 137.96, 165.25, 165.32, 169.0, 169.11, 170.58, 170.71, 170.75. HRMS: m/z calc. for C151H155N11O39: 1373.0242; found: 1373.0178 [M + H + NH4]2+. Bn O BnO OAc O BnO2C N3 O BzO O OBn O 5 Cbz N Bn 261 N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 6-O-acetyl-2-azido-3, 4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-benzyl 2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deo xy-α-D-glucopyranosyl-(1→4)-benzyl 2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deo xy-α-D-glucopyranosyl-(1→4)-benzyl 2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deo xy-α-D-glucopyranosyl-(1→4)-benzyl 2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate-(1→4)-6-O-acetyl-2-azido-3-O-benzyl-2-deo xy-α-D-glucopyranosyl-(1→4)-benzyl 2-O-benzoyl-3-O-benzyl-α-L-idopyranosyluronate (19) Compound 19 was prepared from compound 16 (80 mg, 0.021 mmol) by following the general procedure for oxidation of 6-OH and formation of benzyl esters after oxidation. Purification through silica gel column (Hexanes/DCM/EtOAc = 3/2/1) provided compound 19 in 81% yield over 2 steps. 1HNMR (500 MHz, CDCl3): δ = 1.75-1.85 (m, 2H), 1.98 (s, 3H), 2.01 (s, 6H), 2.03 (s, 3H), 2.09 (s, 3H), 3.10-3.24 (m, 6H), 3.37-3.54 (m, 8H), 3.60 (d, 1H, J = 10.0 Hz), 3.65-3.72 (m, 2H), 3.72-3.90 (m, 9H), 3.91-3.99 (m, 5H), 4.05-4.47 (m, 24H), 4.47-4.63 (m, 7H), 4.63-4.80 (m, 13H), 4.80-4.88 (m, 4H), 4.92-5.01 (m, 7H), 5.01-5.10 (m, 4H), 5.10-5.22 (m, 8H), 5.45-5.49 (m, 3H), 5.54 (d, 1H, J = 10.0 Hz), 7.05-7.42 (m, 90H), 7.42-7.59 (m, 15H), 8.07-8.17 (m, 10H). 13CNMR (125 MHz, CDCl3): δ = 20.8, 20.89, 26.6, 50.79, 51.05, 53.56, 61.67, 61.75, 61.86, 62.31, 63.11, 63.24, 63.48, 63.58, 65.46, 66.42, 67.12, 67.16, 67.29, 67.64, 67.78, 69.8, 70.03, 262 70.15, 70.88, 71.24, 71.6, 72.42, 73.17, 74.28, 74.33, 74.43, 74.62, 74.83, 75.06, 75.23, 75.72, 75.99, 76.09, 76.25, 76.91, 77.16, 77.36, 77.42, 77.53, 78.07, 78.14, 78.3, 78.44, 80.04, 84.05, 98.31, 98.36, 98.55, 99.12, 99.2, 100.16, 127.08, 127.65, 127.7, 127.74, 127.77, 127.8, 127.91, 127.94, 128.02, 128.04, 128.07, 128.11, 128.18, 128.2, 128.22, 128.24, 128.25, 128.33, 128.36, 128.41, 128.45, 128.47, 128.49, 128.51, 128.56, 128.58, 128.61, 128.68, 128.73, 128.77, 128.88, 128.95, 129.18, 129.24, 130.02, 130.07, 130.13, 133.7, 133.84, 134.88, 135.13, 135.18, 137.23, 137.28, 137.3, 137.33, 137.65, 137.67, 137.83, 137.88, 137.95, 165.23, 165.26, 165.29, 165.53, 169.0, 169.09, 169.11, 169.13, 170.58, 170.69, 170.71, 170.74. HRMS: m/z calc. for C235H234N16O63: 2143.7799; found: 2143.7791 [M + 2H]2+. OH O Bn O BnO MeO2C N3 O O OBn O HO 2 Cbz N Bn N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl 3-O-benzyl-α-L-idopyranosyluronate-(1→4)-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-( 1→4)-methyl 3-O-benzyl-α-L-idopyranosyluronate (20) Compound 20 was prepared from compound 17 (400 mg, 0.22 mmol) by following the general procedure for transesterification. Purification through silica gel column (DCM/MeOH = 20/1) provided compound 20 in 79% yield. 1HNMR (500 MHz, CDCl3): δ = 1.67 (br, 1H), 1.78-1.91 (m, 2H), 2.22(br, 1H), 3.35-3.44 (m, 263 2H), 3.46(s, 3H), 3.47-3.65 (m, 7H), 3.65-3.72 (m, 3H), 3.76 (s, 3H), 3.77-3.84 (m, 4H), 3.84-3.90 (m, 3H), 3.94 (t, 1H, J = 9.5 Hz), 4.04 (t, 1H, J = 3.5 Hz), 4.13-4.18 (m, 1H), 4.43-4.54 (m, 3H), 4.58 (d, 1H, J = 11.5 Hz), 4.63 (d, 1H, J = 6.5 Hz), 4.65 (d, 1H, J = 6.5 Hz), 4.70 (d, 1H, J = 4.5 Hz), 4.75 (d, 2H, J = 11.0 Hz), 4.80-4.86 (m, 4H), 4.95-5.05 (m, 3H), 5.17 (s, 2H), 5.28-5.30 (m, 1H), 7.11-7.15 (m, 1H), 7.17-7.45 (m, 34H). 13CNMR (125 MHz, CDCl3): δ = 27.9, 28.36, 43.81, 44.53, 50.53, 50.86, 52.29, 52.52, 61.21, 61.26, 63.67, 64.16, 65.34, 65.36, 66.26, 66.62, 66.94, 67.34, 67.63, 68.05, 71.87, 71.95, 72.02, 72.22, 72.37, 72.64, 72.84, 73.06, 74.18, 74.81, 75.17, 75.86, 77.3, 79.35, 80.78, 95.55, 100.97, 101.84, 127.04, 127.23, 127.32, 127.39, 127.75, 127.94, 128.03, 128.06, 128.21, 128.24, 128.34, 128.46, 128.56, 128.57, 128.59, 128.64, 128.8, 136.71, 136.82, 137.14, 137.53, 137.69, 137.86, 141.04, 156.31, 156.78, 169.56, 170.24. HRMS: m/z calc. for C79FeH89N7O23: 779.7679; found: 779.7664 [M + Fe]2+. OH O Bn O BnO MeO2C N3 O O OBn O HO 3 Cbz N Bn N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl 3-O-benzyl-α-L-idopyranosyluronate-(1→4)-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-( 1→4)-methyl 3-O-benzyl-α-L-idopyranosyluronate-(1→4)-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-( 1→4)-methyl 3-O-benzyl-α-L-idopyranosyluronate (21) 264 Compound 21 was prepared from compound 18 (89 mg, 0.033 mmol) by following the general procedure for transesterification. Purification through silica gel column (DCM/MeOH = 20/1) provided compound 21 in 97% yield. 1HNMR (500 MHz, CDCl3): δ = 1.71-1.90 (m, 2H), 2.00-2.35 (br, 5H), 3.27-3.38 (m, 2H), 3.40 (s, 3H), 3.44 (s, 3H), 3.46-3.58 (m, 6H), 3.58-3.64 (m, 3H), 3.64-3.74 (m, 5H), 3.75 (s, 3H), 3.77-3.83 (m, 4H), 3.83-3.97 (m, 6H), 4.00-4.05 (m, 2H), 4.12-4.17 (m, 1H), 4.44-4.59 (m, 5H), 4.60-4.66 (m, 3H), 4.67-4.77 (m, 7H), 4.77-4.90 (m, 5H), 4.93-5.04 (m, 4H), 5.16 (s, 2H), 5.26 (s, 1H), 5.29 (s, 1H), 7.10-7.15 (m, 1H), 7.15-7.46 (m, 44H). 13CNMR (125 MHz, CDCl3): δ = 27.9, 28.36, 43.81, 44.54, 50.54, 50.86, 52.18, 52.29, 52.52, 61.21, 63.64, 64.05, 64.2, 65.35, 65.38, 66.26, 67.35, 67.64, 67.82, 68.08, 71.98, 72.15, 72.22, 72.39, 72.61, 72.67, 72.79, 72.87, 73.11, 73.94, 74.09, 74.82, 75.07, 75.85, 77.31, 79.16, 79.35, 80.75, 95.59, 100.89, 100.95, 101.84, 127.06, 127.28, 127.39, 127.7, 127.74, 127.76, 127.77, 127.94, 128.03, 128.07, 128.2, 128.21, 128.24, 128.32, 128.37, 128.39, 128.47, 128.5, 128.55, 128.57, 128.6, 128.63, 128.8, 128.82, 137.08, 137.12, 137.52, 137.7, 137.84, 140.99, 156.32, 169.55, 169.57. HRMS: m/z calc. for C106H120N10Na2O33: 1053.3907; found: 1053.3929 [M + 2Na]2+. OH O Bn O BnO MeO2C N3 O O OBn O HO 5 Cbz N Bn N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl 265 3-O-benzyl-α-L-idopyranosyluronate-(1→4)-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-( 1→4)-methyl 3-O-benzyl-α-L-idopyranosyluronate-(1→4)-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-( 1→4)-methyl 3-O-benzyl-α-L-idopyranosyluronate-(1→4)-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-( 1→4)-methyl 3-O-benzyl-α-L-idopyranosyluronate-(1→4)-2-azido-3-O-benzyl-2-deoxy-α-D-glucopyranosyl-( 1→4)-methyl 3-O-benzyl-α-L-idopyranosyluronate (22) Compound 22 was prepared from compound 19 (25 mg, 0.0058 mmol) by following the general procedure for transesterification. Purification through silica gel column (DCM/MeOH = 8/1) provided compound 22 in 90% yield. 1HNMR (500 MHz, CDCl3): δ = 1.75-1.88 (m, 2H), 2.25 (br, 10H), 3.28-3.35 (m, 2H), 3.38 (s, 3H), 3.39 (s, 3H), 3.43 (s, 3H), 3.45-3.53 (m, 7H), 3.53-3.63 (m, 6H), 3.63-3.68 (m, 4H), 3.69-3.81 (m, 10H), 3.74 (s, 3H), 3.81-3.97 (m, 9H), 3.98-4.04 (m, 3H), 4.14 (s, 1H), 4.42-4.57 (m, 7H), 4.57-4.65 (m, 5H), 4.65-4.75 (m, 14H), 4.75-4.85 (m, 6H), 4.90-5.02 (m, 5H), 5.15 (s, 2H), 5.23 (s, 2H), 5.27 (s, 1H), 7.09-7.13 (m, 1H), 7.14-7.45 (m, 64H). 13CNMR (125 MHz, CDCl3): δ = 17.1, 26.59, 39.83, 52.21, 52.32, 63.68, 64.06, 64.14, 64.24, 65.44, 66.29, 67.38, 67.69, 67.87, 68.13, 72.0, 72.21, 72.25, 72.66, 72.84, 72.91, 73.15, 73.95, 74.16, 74.85, 75.11, 75.88, 79.17, 79.37, 80.78, 83.98, 95.61, 95.68, 100.9, 100.97, 101.86, 127.1, 127.28, 127.31, 127.43, 127.76, 127.8, 127.98, 128.07, 128.1, 128.23, 128.26, 128.41, 128.44, 128.49, 128.53, 128.58, 128.6, 128.62, 128.65, 128.67, 128.82, 128.84, 137.08, 137.1, 137.14, 137.54, 137.72, 266 137.85, 140.97, 156.34, 169.58, 169.61. HRMS: m/z calc. for C160H182N16Na2O53: 1610.5917; found: 1610.5923 [M + 2Na]2+. Bn OH O MeO2C O BnO AcHN O O OBn O HO 2 Cbz N Bn N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl 3-O-benzyl-α-L-idopyranosyluronate-(1→4)-2-acetamido-3-O-benzyl-2-deoxy-α-D-glucopyrano syl-(1→4)-methyl 3-O-benzyl-α-L-idopyranosyluronate (23) Compound 20 (60 mg, 0.04 mmol) was dissolved in THF (4 mL), followed by the addition of Zn (104 mg, 1.6 mmol), AcOH (70 μL, 1.2 mmol) and Ac2O (116 μL, 1.2 mmol) The reaction was stirred at room temperature overnight. Upon completion, the mixture was filtered, concentrated and purification through silica gel column (DCM/MeOH = 8/1) provided compound 23 in 99% yield. 1HNMR (500 MHz, CDCl3): δ = 1.60 (s, 3H), 1.74 (s, 3H), 1.76-1.84 (m, 2H), 3.20-3.39 (m, 4H), 3.44 (s, 3H), 3.48-3.61 (m, 4H), 3.65 (s, 3H), 3.67-3.87 (m, 8H), 3.90-4.19 (m, 7H), 4.34-4.56 (m, 6H), 4.57-4.70 (m, 5H), 4.72 (d, 1H, J = 11.0 Hz), 4.78 (d, 1H, J = 11.0 Hz), 4.82-5.02 (m, 5H), 5.08-5.20 (m, 2H), 5.31 (s, 1H), 5.96-6.18 (m, 2H), 6.73 (br, 1H), 7.05-7.45 (m, 35H). 13CNMR (125 MHz, CDCl3): δ =22.9, 22.99, 27.71, 29.73, 31.53, 36.63, 43.71, 44.26, 50.39, 50.77, 51.91, 52.17, 52.31, 52.87, 52.94, 60.95, 61.5, 66.2, 67.33, 67.35, 67.52, 67.88, 68.03, 71.59, 71.87, 267 72.02, 72.18, 72.33, 72.73, 72.89, 73.44, 74.36, 74.57, 74.81, 74.98, 77.41, 77.69, 78.38, 80.0, 96.18, 96.7, 100.75, 101.4, 127.2, 127.4, 127.54, 127.6, 127.69, 127.71, 127.78, 127.86, 127.88, 127.91, 127.97, 128.02, 128.05, 128.11, 128.19, 128.35, 128.44, 128.47, 128.52, 128.55, 128.6, 128.62, 136.52, 136.74, 137.39, 137.55, 137.57, 137.69, 137.71, 137.75, 138.02, 138.17, 138.24, 138.43, 156.37, 156.79, 162.97, 169.84, 170.06, 170.59, 170.82, 171.06. HRMS: m/z calc. for C83FeH97N3O25: 795.7880; found: 795.7868 [M + Fe]2+. Bn OH O MeO2C O BnO H2N O O OBn O HO 2 Cbz N Bn N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 2-amino-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl 3-O-benzyl-α-L-idopyranosyluronate-(1→4)-2-amino-3-O-benzyl-2-deoxy-α-D-glucopyranosyl- (1→4)-methyl 3-O-benzyl-α-L-idopyranosyluronate (24) Compound 20 (60 mg, 0.04 mmol) was dissolved in THF (4 mL), followed by the addition of Zn (104 mg, 1.6 mmol) and AcOH (70 μL, 1.2 mmol). The reaction was stirred at room temperature for 3h. Upon completion, the mixture was filtered, concentrated and purification through silica gel column (DCM/MeOH = 6/1) provided compound 24 in 88% yield. 1HNMR (500 MHz, CDCl3): δ = 1.75-1.87 (m, 2H), 3.11 (d, 1H, J = 10.5 Hz), 3.20 (d, 1H, J = 10.5 Hz), 3.25-3.44 (m, 3H), 3.38 (s, 3H), 3.48 (d, 2H, J = 9.5 Hz), 3.57 (t, 2H, J = 9.5 Hz), 3.62-3.73 (m, 3H), 3.65 (s, 3H), 3.79 (d, 2H, J = 11.5 Hz), 3.85 (d, 3H, J = 11.5 Hz), 3.93-4.05 268 (m, 4H), 4.14 (s, 1H), 4.35-4.51 (m, 4H), 4.54 (d, 1H, J = 11.5 Hz), 4.58-4.75 (m, 4H), 4.75-4.99 (m, 6H), 5.14 (s, 3H), 5.27 (s, 2H), 7.07-7.15 (m, 4H), 7.15-7.40 (m, 31H). 13CNMR (125 MHz, CDCl3): δ = 22.1, 29.76, 43.88, 44.76, 50.65, 50.93, 52.12, 52.28, 53.98, 54.33, 60.37, 60.83, 65.38, 65.53, 66.04, 66.23, 66.9, 67.02, 67.29, 67.79, 70.09, 71.04, 71.74, 72.04, 72.3, 72.53, 72.98, 74.63, 74.79, 75.17, 77.84, 78.75, 93.09, 94.4, 100.88, 101.33, 127.22, 127.28, 127.54, 127.61, 127.71, 127.83, 127.89, 127.95, 128.01, 128.12, 128.14, 128.4, 128.44, 128.47, 128.52, 128.59, 128.64, 136.68, 136.8, 137.33, 137.72, 137.83, 137.88, 156.25, 156.76, 170.19. HRMS: m/z calc. for C79H95N3O23: 726.8178; found: 726.8185 [M + 2H]2+. Bn OH O MeO2C O BnO HO3SHN O O OBn O HO 2 Cbz N Bn N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 3,4-di-O-benzyl-2-deoxy-2-sulfoamino-α-D-glucopyranosyl-(1→4)-methyl 3-O-benzyl-α-L-idopyranosyluronate-(1→4)-3-O-benzyl-2-deoxy-2-sulfoamino-α-D-glucopyran osyl-(1→4)-methyl 3-O-benzyl-α-L-idopyranosyluronate (25) Compound 25 was prepared from compound 24 (15 mg, 0.010 mmol) by following the general procedure for selective N-sulfation. Purification through silica gel column (DCM/MeOH = 8/1) provided compound 25 in 78% yield. 1HNMR (500 MHz, CD3OD): δ = 1.74-1.85 (m, 2H), 3.23 (s, 2H), 3.32-3.34 (m, 1H), 3.35 (s, 3H), 3.42-3.52 (m, 5H), 3.53 (t, 1H, J = 10.0 Hz), 3.63 (t, 1H, J = (9.0 Hz), 3.69 (s, 3H), 269 3.70-3.71 (m, 2H), 3.73-3.79 (m, 1H), 3.81 (s, 2H), 3.83-3.90 (m, 2H), 3.98 (s, 1H), 4.07 (s, 1H), 4.14 (s, 2H), 4.20 (s, 1H), 4.37-4.44 (m, 3H), 4.59 (d, 2H, J = 11.5 Hz), 4.66 (t, 3H, J = 10.0 Hz), 4.74 (d, 1H, J = 10.0 Hz), 4.77 (d, 2H, J = 10.5 Hz), 4.87-4.94 (m, 2H), 5.03 (d, 1H, J = 1.5 Hz), 5.06 (d, 1H, J = 10.5 Hz), 5.09-5.14 (m, 2H), 5.18 (s, 1H), 5.40-5.44 (m, 2H), 7.06-7.12 (m, 1H), 7.14-7.36 (m, 28H), 7.37-7.45 (m, 4H), 7.46-7.50 (m, 2H). 13CNMR (125 MHz, CD3OD): δ = 24.7, 45.2, 46.05, 48.49, 48.66, 48.83, 49.0, 49.17, 49.34, 49.51, 49.85, 51.58, 52.73, 52.97, 59.42, 59.65, 61.42, 61.74, 66.9, 67.21, 68.33, 68.54, 72.75, 72.86, 73.02, 73.16, 73.38, 73.48, 74.08, 74.4, 74.63, 75.68, 76.12, 76.25, 78.48, 79.36, 81.47, 97.4, 97.54, 101.6, 102.69, 128.13, 128.25, 128.38, 128.51, 128.58, 128.66, 128.87, 128.9, 129.07, 129.13, 129.21, 129.27, 129.39, 129.54, 129.57, 137.97, 137.99, 139.07, 139.25, 139.41, 139.6, 139.76, 139.9, 140.29, 157.91, 158.36, 171.89, 172.2. HRMS: m/z calc. for C79H91N3O29S2: 804.7590; found: 804.7593 [M - 2H]2-. H OH O HO2C O HO AcHN O HO OH O O 2 NH2 3-Aminopropyl 2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→4)-α-L-idopyranosyluronate-(1→4)-2-acetamido- 2-deoxy-α-D-glucopyranosyl-(1→4)-α-L-idopyranosyluronate (26) Compound 26 was prepared from compound 23 (5 mg, 0.003 mmol) by following the general procedure for global debenzylation and methyl ester saponification, providing the final product 270 in 65% yield over 2 steps. 1HNMR (500 MHz, D2O): δ =1.80-1.89 (m, 2H), 1.84 (s, 3H), 1.86 (s, 3H), 2.98 (dt, 2H, J = 2.5, 6.5 Hz), 3.31 (t, 1H, J = 9.5 Hz), 3.45-3.50 (m, 1H), 3.50-3.61 (m, 6H), 3.61-3.74 (m, 10H), 3.75 (d, 1H, J = 4.0 Hz), 3.76-3.83 (m, 3H), 3.87 (t, 1H, J = 3.0 Hz), 3.92 (t, 1H, J = 3.5 Hz), 4.33 (d, 1H, J = 3.0 Hz), 4.58 (d, 1H, J = 3.0 Hz), 4.73 (d, 1H, J = 3.0 Hz), 4.75 (d, 1H, J = 4.0 Hz), 4.97 (d, 1H, J = 4.0 Hz), 5.03 (d, 1H, J = 3.5 Hz), . 13CNMR (125 MHz, D2O): δ = 21.7, 21.79, 26.2, 38.05, 53.41, 53.57, 59.46, 60.02, 66.3, 67.21, 68.12, 68.48, 69.55, 69.83, 70.95, 71.01, 71.83, 73.25, 74.19, 76.43, 94.23, 94.31, 100.29, 101.56, 174.2, 174.28, 174.81, 174.99. HRMS: m/z calc. for C31H50N3O23: 832.2835; found: 832.2836 [M - H]-. H OH O HO2C O HO HO3SHN O HO OH O O 2 NH2 3-Aminopropyl 2-deoxy-2-sulfoamino-α-D-glucopyranosyl-(1→4)-α-L-idopyranosyluronate-(1→4)-2-deoxy-2-s ulfoamino-α-D-glucopyranosyl-(1→4)-α-L-idopyranosyluronate (27) Compound 27 was prepared from compound 25 (5 mg, 0.003 mmol) by following the general procedure for global debenzylation and methyl ester saponification, providing the final product in 83% yield over 2 steps. 1HNMR (500 MHz, D2O): δ = 1.76-1.91 (m, 2H), 2.96-3.01 (m, 1H), 3.02-3.10 (m, 2H), 3.30 (t, 1H, J = 9.5 Hz), 3.47 (t, 1H, J = 10.0 Hz), 3.50-3.60 (m, 5H), 3.60-3.76 (m, 8H), 3.85-3.89 (m, 271 1H), 3.90-3.96 (m, 2H), 4.01 (s, 1H), 4.31 (d, 1H, J = 2.0 Hz), 4.62 (d, 1H, J = 2.5 Hz), 4.74 (s, 1H), 4.79 (d, 1H, J = 3.0 Hz), 5.16 (d, 1H, J = 4.0 Hz), 5.23 (d, 1H, J = 3.5 Hz). 13CNMR (125 MHz, D2O): δ = 26.1, 38.24, 57.78, 57.89, 59.61, 60.11, 66.36, 67.02, 67.73, 68.1, 68.74, 69.29, 69.64, 69.66, 70.85, 71.17, 71.57, 74.41, 74.72, 76.85, 95.29, 95.72, 100.27, 101.48, 175.24, 175.29. HRMS: m/z calc. for C31H50N3O35S4: 908.1760; found: 908.1729 [M - H]-. H OSO3H O AcHN O HO HO2C O HO3SO OH O O 2 NH2 3-Aminopropyl 2-acetamido-2-deoxy-6-O-sulfonato-α-D-glucopyranosyl-(1→4)-2-O-sulfonato-α-L-idopyranosy luronate-(1→4)-2-acetamido-2-deoxy-6-O-sulfonato-α-D-glucopyranosyl-(1→4)-2-O-sulfonato- α-L-idopyranosyluronate (28) Compound 28 was prepared from compound 23 (5 mg, 0.003 mmol) by following the general procedure for simultaneous O, N-sulfation, global debenzylation and methyl ester saponification, providing the final product in 83% yield over 3 steps. 1HNMR (500 MHz, D2O): δ = 1.83-1.89 (m, 2H), 1.90 (s, 3H), 1.92 (s, 3H), 3.01 (t, 2H, J = 6.5 Hz), 3.41 (t, 1H, J = 9.5 Hz), 3.49 (t, 1H, J = 9.0 Hz), 3.53-3.64 (m, 5H), 3.76-3.82 (m, 2H), 3.86 (dd, 2H, J = 3.5, 10.0 Hz), 3.91 (dd, 1H, J = 3.5, 10.5 Hz), 3.96 (t, 1H, J = 2.5 Hz), 3.98 (t, 1H, J = 2.5 Hz), 4.07-4.11 (m, 1H), 4.11-4.20 (m, 7H), 4.76 (d, 1H, J = 2.5 Hz), 4.99 (d, 2H, J = 3.5 Hz), 5.01 (s, 1H), 5.06-5.08 (m, 2H). 13CNMR (125 MHz, D2O): δ = 22.0, 22.1, 25.96, 38.19, 272 53.06, 53.21, 63.01, 64.61, 66.03, 66.41, 66.5, 66.72, 66.8, 69.03, 69.63, 69.78, 70.51, 70.79, 72.08, 73.0, 73.58, 76.6, 93.98, 95.11, 98.28, 99.08, 172.54, 172.65, 174.8. HRMS: m/z calc. for C31H46N3Na3O35S4: 608.5245; found: 608.5258 [M + 3Na - 5H]2-. H OSO3H O HO2C O HO HO3SHN O HO3SO OH O O 2 NH2 3-Aminopropyl 2-deoxy-2-sulfoamino-6-O-sulfonato-α-D-glucopyranosyl-(1→4)-2-O-sulfonato-α-L-idopyranos yluronate-(1→4)-2-deoxy-2-sulfoamino-6-O-sulfonato-α-D-glucopyranosyl-(1→4)-2-O-sulfonat o-α-L-idopyranosyluronate (29) Compound 29 was prepared from compound 25 (5 mg, 0.003 mmol) by following the general procedure for simultaneous O, N-sulfation, global debenzylation and methyl ester saponification, providing the final product in 81% yield over 3 steps. 1HNMR (500 MHz, D2O): δ = 1.80-1.90 (m, 2H), 3.02 (t, 2H, J = 1.5 Hz), 3.10 (dd, 1H, J = 3.5, 10.0 Hz), 3.13 (dd, 1H, J = 3.5, 10.5 Hz), 3.42 (t, 1H, J = 10.0 Hz), 3.50 (t, 1H, J = 10.0 Hz), 3.52-3.59 (m, 2H), 3.62 (t, 1H, J = 9.0 Hz), 3.80 (d, 2H, J = 7.5 Hz), 3.87 (d, 1H, J = 10.0 Hz), 3.93-3.99 (m, 2H), 4.02-4.15 (m, 5H), 4.18-4.27 (m, 3H), 4.40 (d, 1H, J = 3.0 Hz), 4.78 (d, 1H, J = 2.5 Hz), 4.97 (d, 1H, J = 3.5 Hz), 5.08 (s, 1H), 5.25 (d, 1H, J = 3.5 Hz), 5.27 (d, 1H, J = 3.5 Hz). 13CNMR (125 MHz, D2O): δ = 26.0, 38.31, 46.57, 57.84, 66.3, 66.57, 68.47, 68.58, 68.82, 273 68.87, 69.05, 69.18, 69.43, 69.93, 70.82, 75.25, 75.79, 75.92, 76.01, 76.4, 96.48, 97.34, 98.78, 99.07, 174.04, 174.26. HRMS: m/z calc. for C27H42N3Na3O39S6: 646.4708; found: 646.4703 [M + 3Na - 5H]2-. Bn OH O MeO2C O BnO H2N O O OBn O HO 3 Cbz N Bn N-(Benzyl)-benzyloxycarbonyl-3-aminopropyl 2-amino-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl-(1→4)-methyl 3-O-benzyl-α-L-idopyranosyluronate-(1→4)-2-amino-3-O-benzyl-2-deoxy-α-D-glucopyranosyl- (1→4)-methyl 3-O-benzyl-α-L-idopyranosyluronate-(1→4)-2-amino-3-O-benzyl-2-deoxy-α-D-glucopyranosyl- (1→4)-methyl 3-O-benzyl-α-L-idopyranosyluronate (30) Compound 30 was prepared from compound 21 (15 mg, 0.007 mmol) by following the general procedure for 1, 3-propanedithiol mediated azide reduction. Purification through silica gel column (DCM/MeOH = 6/1 with 2% Et3N) provided compound 27 in 76% yield. 1HNMR (500 MHz, CDCl3): δ = 1.78-1.88 (m, 2H), 2.83-2.91 (m, 2H), 3.23-3.32 (m, 2H), 3.34-3.51 (m, 10H), 3.53 (s, 3H), 3.55 (s, 3H), 3.65-3.68 (m, 2H), 3.70-3.73 (m, 2H), 3.74 (s, 3H), 3.75-3.82 (m, 5H), 3.82-3.91 (m, 4H), 3.94 (s, 3H), 3.96-4.02 (m, 2H), 4.14−4.22 (m, 3H), 4.41 (d, 2H, J = 11.5 Hz), 4.43−4.54 (m, 4H), 4.58 (d, 2H, J = 11.5 Hz), 4.64 (d, 2H, J = 11.0 Hz), 4.66 (s, 4H), 4.72 (dd, 2H, J = 3.5, 11.5 Hz), 4.81 (d, 2H, J = 11.5 Hz), 4.86 (d, 1H, J = 3.0 274 Hz), 4.87−4.91 (m, 3H), 4.92−4.97 (m, 5H), 4.99 (d, 1H, J = 12.0 Hz), 5.15 (s, 2H), 5.29 (dd, 2H, J = 3.5, 8.0 Hz), 7.10− 7.13 (m, 1H), 7.17−7.38 (m, 44H). Comparison with literature data confirmed its identity.25 H OSO3H O HO2C O HO HO3SHN O HO3SO OH O O 3 NH2 3-Aminopropyl 2-deoxy-2-sulfoamino-6-O-sulfonato-α-D-glucopyranosyl-(1→4)-2-O-sulfonato-α-L-idopyranos yluronate-(1→4)-2-deoxy-2-sulfoamino-6-O-sulfonato-α-D-glucopyranosyl-(1→4)-2-O-sulfonat o-α-L-idopyranosyluronate-(1→4)-2-deoxy-2-sulfoamino-6-O-sulfonato-α-D-glucopyranosyl-(1 →4)-2-O-sulfonato-α-L-idopyranosyluronate (31) Compound 31 was prepared from compound 30 (5 mg, 0.0025 mmol) by following the general procedure for simultaneous O, N-sulfation, global debenzylation and methyl ester saponification, providing the final product in 64% yield over 3 steps. 1HNMR (500 MHz, D2O): δ = 1.92−2.01 (m, 2H), 3.11−3.15 (m, 1H), 3.21 (dd, 1H, J = 3.5, 10.5 Hz), 3.25 (dd, 2H, J = 3.0, 10.5 Hz), 3.54 (t, 2H, J = 10.0 Hz), 3.59-3.67 (m, 4H), 3.70-3.79 (m, 3H), 3.90-3.94 (m, 1H), 3.95-3.98 (m, 1H), 3.98-4.03 (m, 2H), 4.06-4.10 (m, 3H), 4.14-4.20 (m, 4H), 4.22-4.27 (m, 3H), 4.29-4.41 (m, 5H), 4.47 (d, 1H, J = 3.0 Hz), 4.81 (d, 1H, J = 3.0 Hz), 5.06 (d, 1H, J = 3.5 Hz), 5.19 (d, 2H, J = 3.0 Hz), 5.39 (d, 2H, J = 3.5 Hz), 5.42 (d, 1H, J = 3.5 Hz). Comparison with literature data confirmed its identity.25 275 H OSO3H O HO2C O HO HO3SHN O HO3SO OH O O 5 3-Aminopropyl NH2 2-deoxy-2-sulfoamino-6-O-sulfonato-α-D-glucopyranosyl-(1→4)-2-O-sulfonato-α-L-idopyranos yluronate-(1→4)-2-deoxy-2-sulfoamino-6-O-sulfonato-α-D-glucopyranosyl-(1→4)-2-O-sulfonat o-α-L-idopyranosyluronate-(1→4)-2-deoxy-2-sulfoamino-6-O-sulfonato-α-D-glucopyranosyl-(1 →4)-2-O-sulfonato-α-L-idopyranosyluronate-(1→4)-2-deoxy-2-sulfoamino-6-O-sulfonato-α-D-g lucopyranosyl-(1→4)-2-O-sulfonato-α-L-idopyranosyluronate-(1→4)-2-deoxy-2-sulfoamino-6- O-sulfonato-α-D-glucopyranosyl-(1→4)-2-O-sulfonato-α-L-idopyranosyluronate (32) Compound 32 was prepared from compound 22 (5 mg, 0.0016 mmol) by following the general procedure for 1, 3-propanedithiol mediated azide reduction, simultaneous O, N-sulfation, global debenzylation and methyl ester saponification, providing the final product in 42% yield over 4 steps. 1HNMR (500 MHz, D2O): δ = 1.92-1.99 (m, 2H), 3.09-3.15 (m, 2H), 3.20 (dd, 2H, J = 3.0, 10.0 Hz), 3.25 (dd, 4H, J = 3.5, 10.5 Hz), 3.54 (t, 3H, J = 10.0 Hz), 3.58-3.66 (m, 5H), 3.68-3.79 (m, 6H), 3.92-4.02 (m, 7H), 4.08 (t, 4H, J = 3.5 Hz), 4.14-4.19 (m, 6H), 4.20-4.27 (m, 6H), 4.29-4.34 (m, 5H), 4.34-4.42 (m, 5H), 4.79-4.83 (m, 2H), 5.18 (d, 5H, J = 3.0 Hz), 5.39 (d, 5H, J = 3.0 Hz). δC (values obtained from F1 dimension of HSQC spectrum) = 26.0, 38.24, 57.81, 57.82, 61.11, 65.54, 66.22, 66.23, 66.26, 66.29, 66.51, 66.52, 68.60, 69.12, 69.17, 69.24, 69.28, 69.40, 69.42, 69.66, 69.73, 70.87, 70.88, 75.66, 75.70, 75.77, 75.80, 75.81, 96.57, 99.18. HRMS: m/z calc. for 276 C63H87N6Na13O96S15: 810.4149; found: 810.4116 [M + 13Na - 17H]4-. 277 APPENDIX 278 Product Characterization Spectra OAc O BnO BnO OLev OBn O STol N3 O 3 BzO Figure 4.4. 1H-NMR of 3 (500 MHz CDCl3) OAc O BnO BnO OLev OBn O STol N3 O 3 BzO Figure 4.5. 13C-NMR of 3 (125 MHz CDCl3) 279 OAc O BnO BnO OLev OBn O STol N3 O 3 BzO Figure 4.6. 1H-1H gCOSY of 3 (500 MHz CDCl3) OAc O BnO BnO OLev OBn O STol N3 O 3 BzO Figure 4.7. 1H-13C gHSQCAD of 3 (500 MHz CDCl3) 280 OAc O BnO BnO OPMB OBn O STol N3 O 7 BzO Figure 4.8. 1H-NMR of 7 (500 MHz CDCl3) OAc O BnO BnO OPMB OBn O STol N3 O 7 BzO Figure 4.9. 13C-NMR of 7 (125 MHz CDCl3) 281 OAc O BnO BnO OPMB OBn O STol N3 O 7 BzO Figure 4.10. 1H-1H gCOSY of 7 (500 MHz CDCl3) OAc O BnO BnO OPMB OBn O STol N3 O 7 BzO Figure 4.11. 1H-13C gHSQCAD of 7 (500 MHz CDCl3) 282 Bn O BnO OLev STol OBn O BzO 2 OAc O N3 O 8 Figure 4.12. 1H-NMR of 8 (500 MHz CDCl3) Bn O BnO OLev STol OBn O BzO 2 OAc O N3 O 8 Figure 4.13. 13C-NMR of 8 (125 MHz CDCl3) 283 Bn O BnO OLev STol OBn O BzO 2 OAc O N3 O 8 Figure 4.14. 1H-1H gCOSY of 8 (500 MHz CDCl3) Bn O BnO OLev STol OBn O BzO 2 OAc O N3 O 8 Figure 4.15. 1H-13C gHSQCAD of 8 (500 MHz CDCl3) 284 Bn O BnO OLev O OBn O BzO 2 OAc O N3 O 9 Cbz N Bn Figure 4.16. 1H-NMR of 9 (500 MHz CDCl3) Bn O BnO OLev O OBn O BzO 2 OAc O N3 O 9 Cbz N Bn Figure 4.17. 13C-NMR of 9 (125 MHz CDCl3) 285 Bn O BnO OLev O OBn O BzO 2 OAc O N3 O 9 Cbz N Bn Figure 4.18. 1H-1H gCOSY of 9 (500 MHz CDCl3) Bn O BnO OLev O OBn O BzO 2 OAc O N3 O 9 Cbz N Bn Figure 4.19. 1H-13C gHSQCAD of 9 (500 MHz CDCl3) 286 TBS O BnO OLev STol OBn O BzO 2 OAc O N3 O 10 Figure 4.20. 1H-NMR of 10 (500 MHz CDCl3) 287 Bn O BnO OAc O N3 O 11 OLev O OBn O BzO 3 Cbz N Bn Figure 4.21. 1H-NMR of 11 (500 MHz CDCl3) Bn O BnO OAc O N3 O 11 OLev O OBn O BzO 3 Cbz N Bn Figure 4.22. 13C-NMR of 11 (125 MHz CDCl3) 288 Bn O BnO OAc O N3 O 11 OLev O OBn O BzO 3 Cbz N Bn Figure 4.23. 1H-1H gCOSY of 11 (500 MHz CDCl3) Bn O BnO OAc O N3 O 11 OLev O OBn O BzO 3 Cbz N Bn Figure 4.24. 1H-13C gHSQCAD of 11 (500 MHz CDCl3) 289 OAc O H O BnO OLev O OBn O BzO 3 N3 O 12 Cbz N Bn Figure 4.25. 1H-NMR of 12 (500 MHz CDCl3) OAc O H O BnO OLev O OBn O BzO 3 N3 O 12 Cbz N Bn Figure 4.26. 13C-NMR of 12 (125 MHz CDCl3) 290 OAc O H O BnO OLev O OBn O BzO 3 N3 O 12 Cbz N Bn Figure 4.27. 1H-1H gCOSY of 12 (500 MHz CDCl3) OAc O H O BnO OLev O OBn O BzO 3 N3 O 12 Cbz N Bn Figure 4.28. 1H-13C gHSQCAD of 12 (500 MHz CDCl3) 291 Bn O BnO OAc O N3 O 13 OLev O OBn O BzO 5 Cbz N Bn Figure 4.29. 1H-NMR of 13 (500 MHz CDCl3) Bn O BnO OAc O N3 O 13 OLev O OBn O BzO 5 Cbz N Bn Figure 4.30. 13C-NMR of 13 (125 MHz CDCl3) 292 Bn O BnO OAc O N3 O 13 OLev O OBn O BzO 5 Cbz N Bn Figure 4.31. 1H-1H gCOSY of 13 (500 MHz CDCl3) Bn O BnO OAc O N3 O 13 OLev O OBn O BzO 5 Cbz N Bn Figure 4.32. 1H-13C gHSQCAD of 13 (500 MHz CDCl3) 293 Bn O BnO OAc O N3 O 14 OH O OBn O BzO 2 Cbz N Bn 1.0 10.5 10.0 9.5 9.0 8.5 8.0 7 7 . 3 6 9 . 2 2 4 . 4 3 7.5 7.0 6.5 6.0 6 4 . 4 1 6 . 1 5.5 2 0 . 3 6 0 . 2 5.0 0 3 . 2 7 1 . 5 4.5 2 3 . 3 2 9 . 4 f1 (ppm) 0 2 . 5 2 3 . 3 4.0 4 2 . 5 3.5 2 4 . 3 2 4 . 1 3.0 1 1 . 5 9 7 . 0 2.5 8 6 . 1 0 0 . 3 0 8 . 2 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 4.33. 1H-NMR of 14 (500 MHz CDCl3) 9 6 . 0 7 1 8 5 . 0 7 1 1 8 . 5 6 1 7 8 . 7 3 1 4 5 . 7 3 1 2 5 . 7 3 1 6 4 . 7 3 1 0 4 . 7 3 1 2 4 . 3 3 1 6 2 . 3 3 1 0 1 . 0 3 1 2 9 . 9 2 1 9 7 . 9 2 1 3 7 . 9 2 1 0 7 . 8 2 1 5 6 . 8 2 1 9 5 . 8 2 1 7 5 . 8 2 1 4 5 . 8 2 1 0 5 . 8 2 1 3 4 . 8 2 1 9 3 . 8 2 1 7 2 . 8 2 1 2 2 . 8 2 1 9 1 . 8 2 1 7 0 . 8 2 1 3 0 . 8 2 1 5 9 . 7 2 1 2 9 . 7 2 1 2 4 . 7 2 1 5 3 . 7 2 1 2 2 . 8 9 8 9 . 7 9 0 7 . 0 8 5 4 . 9 7 4 7 . 7 7 8 2 . 5 7 4 2 . 5 7 9 0 . 5 7 3 0 . 4 7 9 9 . 2 7 4 1 . 2 7 7 3 . 0 7 1 0 . 0 7 3 1 . 9 6 0 9 . 8 6 1 4 . 7 6 3 3 . 6 6 7 1 . 4 6 9 7 . 3 6 0 8 . 2 6 2 7 . 2 6 0 3 . 1 6 9 8 . 0 2 Bn O BnO OAc O N3 O 14 OH O OBn O BzO 2 Cbz N Bn 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 4.34. 13C-NMR of 14 (125 MHz CDCl3) 294 Bn O BnO OAc O N3 O 14 OH O OBn O BzO 2 Cbz N Bn 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Figure 4.35. 1H-1H gCOSY of 14 (500 MHz CDCl3) 0 1 2 3 4 5 6 7 8 9 ) m p p ( 1 f Bn O BnO OAc O N3 O 14 OH O OBn O BzO 2 Cbz N Bn 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 4.36. 1H-13C gHSQCAD of 14 (500 MHz CDCl3) 295 Bn O BnO OAc O N3 O 15 OH O OBn O BzO 3 Cbz N Bn 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 1 7 . 5 8 4 . 8 4 1 3 . 4 8 5 4 8 . . 5 2 6.0 7 1 . 1 5.5 9 9 . 2 9 4 . 2 5.0 f1 (ppm) 5 7 . 7 6 5 . 2 1 8 . 7 4.5 7 2 . 3 0 6 . 4 6 6 . 5 4.0 1 7 . 2 3.5 4 3 . 5 5 5 . 2 8 7 . 1 3.0 9 8 . 4 6 6 . 2 2.5 0 8 . 1 1 9 . 4 2 5 . 3 2 7 . 2 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 4.37. 1H-NMR of 15 (500 MHz CDCl3) 0 7 . 0 7 1 0 6 . 0 7 1 1 9 . 5 6 1 5 8 . 5 6 1 2 5 . 7 3 1 8 4 . 7 3 1 0 4 . 7 3 1 1 4 . 3 3 1 0 9 . 9 2 1 8 8 . 9 2 1 6 7 . 9 2 1 8 6 . 8 2 1 6 6 . 8 2 1 0 6 . 8 2 1 4 5 . 8 2 1 7 4 . 8 2 1 0 4 . 8 2 1 2 3 . 8 2 1 9 2 . 8 2 1 6 2 . 8 2 1 3 2 . 8 2 1 2 1 . 8 2 1 7 0 . 8 2 1 3 0 . 8 2 1 3 9 . 7 2 1 9 8 . 7 2 1 2 2 . 8 9 5 9 . 7 9 9 8 . 7 9 4 3 . 7 9 3 7 . 0 8 5 3 . 9 7 3 7 . 7 7 7 2 . 5 7 4 2 . 5 7 8 1 . 5 7 5 1 . 5 7 3 0 . 4 7 4 1 . 3 7 9 9 . 2 7 1 9 . 2 7 4 1 . 2 7 7 0 . 0 7 0 0 . 0 7 7 6 . 7 6 2 0 . 4 6 0 8 . 3 6 9 7 . 2 6 3 3 . 1 6 1 5 . 0 6 8 1 . 1 2 4 9 . 0 2 8 8 . 0 2 2 3 . 4 1 Bn O BnO OAc O N3 O 15 OH O OBn O BzO 3 Cbz N Bn 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 4.38. 13C-NMR of 15 (125 MHz CDCl3) 296 Bn O BnO OAc O N3 O 15 OH O OBn O BzO 3 Cbz N Bn 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 f2 (ppm) 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Figure 4.39. 1H-1H gCOSY of 15 (500 MHz CDCl3) Bn O BnO OAc O N3 O 15 OH O OBn O BzO 3 Cbz N Bn 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 4.40. 1H-13C gHSQCAD of 15 (500 MHz CDCl3) 297 1 2 3 4 5 6 7 8 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 ) m p p ( 1 f ) m p p ( 1 f Bn O BnO OAc O N3 O 16 OH O OBn O BzO 5 Cbz N Bn 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 0 2 . 9 9 3 . 2 7 1 9 . 4 3 7 . 6 7 8 . 7 5.5 7 9 . 0 1 5.0 5 4 . 0 1 0 6 . 0 2 4.5 4 6 . 5 5 9 . 4 1 4.0 f1 (ppm) 0 0 . 3 1 2 . 9 1 9 . 4 9 3 . 6 3.5 7 5 . 5 0 6 . 1 3.0 0 9 . 2 9 4 . 4 2 5 . 2 2.0 5 9 . 2 6 7 . 4 1.5 2.5 1.0 0.5 0.0 -0.5 Figure 4.41. 1H-NMR of 16 (500 MHz CDCl3) 2 7 . 0 7 1 1 6 . 0 7 1 7 9 . 5 6 1 4 9 . 5 6 1 7 8 . 5 6 1 3 5 . 7 3 1 9 4 . 7 3 1 3 4 . 7 3 1 1 4 . 7 3 1 7 4 . 3 3 1 2 9 . 9 2 1 8 8 . 9 2 1 1 8 . 9 2 1 7 7 . 9 2 1 0 7 . 9 2 1 4 7 . 8 2 1 1 7 . 8 2 1 8 6 . 8 2 1 2 6 . 8 2 1 9 5 . 8 2 1 6 5 . 8 2 1 3 5 . 8 2 1 9 4 . 8 2 1 2 4 . 8 2 1 6 3 . 8 2 1 1 3 . 8 2 1 9 2 . 8 2 1 4 2 . 8 2 1 2 2 . 8 2 1 9 0 . 8 2 1 5 0 . 8 2 1 4 9 . 7 2 1 1 9 . 7 2 1 3 2 . 8 9 9 8 . 7 9 4 3 . 7 9 0 4 . 9 7 5 7 . 7 7 0 3 . 5 7 6 2 . 5 7 0 2 . 5 7 9 5 . 3 7 7 1 . 3 7 2 1 . 3 7 4 9 . 2 7 7 1 . 2 7 3 1 . 0 7 9 0 . 9 6 7 0 . 4 6 3 4 . 2 6 1 3 . 1 6 3 8 . 9 2 6 9 . 0 2 0 9 . 0 2 Bn O BnO OAc O N3 O 16 OH O OBn O BzO 5 Cbz N Bn 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 4.42. 13C-NMR of 16 (125 MHz CDCl3) 298 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 f2 (ppm) 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 Figure 4.43. 1H-1H gCOSY of 16 (500 MHz CDCl3) 0 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 14 13 12 11 10 9 8 7 6 f2 (ppm) 5 4 3 2 1 0 -1 -2 Figure 4.44. 1H-13C gHSQCAD of 16 (500 MHz CDCl3) 299 ) m p p ( 1 f ) m p p ( 1 f OAc O Bn O BnO O OBn O 2 MeO2C N3 O BzO 17 Cbz N Bn Figure 4.45. 1H-NMR of 17 (500 MHz CDCl3) OAc O Bn O BnO O OBn O 2 MeO2C N3 O BzO 17 Cbz N Bn Figure 4.46. 13C-NMR of 17 (125 MHz CDCl3) 300 Figure 4.47. 1H-1H gCOSY of 17 (500 MHz CDCl3) Figure 4.48. 1H-13C gHSQCAD of 17 (500 MHz CDCl3) 301 OAc O Bn O BnO BnO2C N3 O BzO 18 O OBn O 3 Cbz N Bn Figure 4.49. 1H-NMR of 18 (500 MHz CDCl3) OAc O Bn O BnO BnO2C N3 O BzO 18 O OBn O 3 Cbz N Bn Figure 4.50. 13C-NMR of 18 (125 MHz CDCl3) 302 OAc O Bn O BnO BnO2C N3 O BzO 18 O OBn O 3 Cbz N Bn Figure 4.51. 1H-1H gCOSY of 18 (500 MHz CDCl3) OAc O Bn O BnO BnO2C N3 O BzO 18 O OBn O 3 Cbz N Bn Figure 4.52. 1H-13C gHSQCAD of 18 (500 MHz CDCl3) 303 OAc O Bn O BnO BnO2C N3 O BzO 19 O OBn O 5 Cbz N Bn Figure 4.53. 1H-NMR of 19 (500 MHz CDCl3) OAc O Bn O BnO BnO2C N3 O BzO 19 O OBn O 5 Cbz N Bn Figure 4.54. 13C-NMR of 19 (125 MHz CDCl3) 304 Figure 4.55. 1H-1H gCOSY of 19 (500 MHz CDCl3) Figure 4.56. 1H-13C gHSQCAD of 19 (500 MHz CDCl3) 305 OH O Bn O BnO MeO2C N3 O O OBn O Cbz N Bn HO 2 20 Figure 4.57. 1H-NMR of 20 (500 MHz CDCl3) OH O Bn O BnO MeO2C N3 O O OBn O Cbz N Bn HO 2 20 Figure 4.58. 13C-NMR of 20 (125 MHz CDCl3) 306 Figure 4.59. 1H-1H gCOSY of 20 (500 MHz CDCl3) Figure 4.60. 1H-13C gHSQCAD of 20 (500 MHz CDCl3) 307 OH O Bn O BnO MeO2C N3 O O OBn O Cbz N Bn HO 3 21 Figure 4.61. 1H-NMR of 21 (500 MHz CDCl3) OH O Bn O BnO MeO2C N3 O O OBn O Cbz N Bn HO 3 21 Figure 4.62. 13C-NMR of 21 (125 MHz CDCl3) 308 OH O Bn O BnO MeO2C N3 O O OBn O Cbz N Bn HO 3 21 Figure 4.63. 1H-1H gCOSY of 21 (500 MHz CDCl3) Figure 4.64. 1H-13C gHSQCAD of 21 (500 MHz CDCl3) 309 OH O Bn O BnO MeO2C N3 O O OBn O Cbz N Bn HO 5 22 Figure 4.65. 1H-NMR of 22 (500 MHz CDCl3) OH O Bn O BnO MeO2C N3 O O OBn O Cbz N Bn HO 5 22 Figure 4.66. 13C-NMR of 22 (125 MHz CDCl3) 310 Figure 4.67. 1H-1H gCOSY of 22 (500 MHz CDCl3) Figure 4.68. 1H-13C gHSQCAD of 22 (500 MHz CDCl3) 311 OH O MeO2C O OBn O Cbz N Bn Bn O BnO AcHN O 23 HO 2 Figure 4.69. 1H-NMR of 23 (500 MHz CDCl3) OH O MeO2C O OBn O Cbz N Bn Bn O BnO AcHN O 23 HO 2 Figure 4.70. 13C-NMR of 23 (125 MHz CDCl3) 312 OH O MeO2C O OBn O Cbz N Bn Bn O BnO H2N O 24 HO 2 Figure 4.71. 1H-NMR of 24 (500 MHz CDCl3) OH O MeO2C O OBn O Cbz N Bn HO 2 Bn O BnO H2N O 24 Figure 4.72. 13C-NMR of 24 (125 MHz CDCl3) 313 OH O MeO2C O OBn O Cbz N Bn Bn O BnO HO3SHN O 25 HO 2 Figure 4.73. 1H-NMR of 25 (500 MHz CD3OD) OH O MeO2C O OBn O Cbz N Bn Bn O BnO HO3SHN O 25 HO 2 Figure 4.74. 13C-NMR of 25 (125 MHz CD3OD) 314 Figure 4.75. 1H-1H gCOSY of 25 (500 MHz CD3OD) Figure 4.76. 1H-13C gHSQCAD of 25 (500 MHz CD3OD) 315 H OH O HO2C O HO AcHN O HO OH O O 2 26 NH2 Figure 4.77. 1H-NMR of 26 (500 MHz D2O) H OH O HO2C O HO AcHN O HO OH O O 2 26 NH2 Figure 4.78. 13C-NMR of 26 (125 MHz D2O) 316 H OH O HO2C O HO AcHN O HO OH O O 2 26 NH2 Figure 4.79. 1H-1H gCOSY of 26 (500 MHz D2O) H OH O HO2C O HO AcHN O HO OH O O 2 26 NH2 Figure 4.80. 1H-13C gHSQCAD of 26 (500 MHz D2O) 317 H OH O HO2C O HO AcHN O HO OH O O 2 26 NH2 Figure 4.81. ESI-MS of 26 318 H OH O HO2C O HO HO3SHN O HO OH O O 2 27 NH2 Figure 4.82. 1H-NMR of 27 (500 MHz D2O) H OH O HO2C O HO HO3SHN O HO OH O O 2 27 NH2 Figure 4.83. 13C-NMR of 27 (125 MHz D2O) 319 H OH O HO2C O HO HO3SHN O HO OH O O 2 27 NH2 Figure 4.84. 1H-1H gCOSY of 27 (500 MHz D2O) H OH O HO2C O HO HO3SHN O HO OH O O 2 27 NH2 Figure 4.85. 1H-13C gHSQCAD of 27 (500 MHz D2O) 320 H OH O HO2C O HO HO3SHN O HO OH O O 2 27 NH2 Figure 4.86. ESI-MS of 27 321 OSO3H O AcHN H O HO OH O O 2 HO2C O HO3SO 28 NH2 Figure 4.87. 1H-NMR of 28 (500 MHz D2O) OSO3H O AcHN H O HO OH O O 2 HO2C O HO3SO 28 NH2 Figure 4.88. 13C-NMR of 28 (125 MHz D2O) 322 OSO3H O AcHN H O HO OH O O 2 HO2C O HO3SO 28 NH2 Figure 4.89. 1H-1H gCOSY of 28 (500 MHz D2O) OSO3H O AcHN H O HO OH O O 2 HO2C O HO3SO 28 NH2 Figure 4.90. 1H-13C gHSQCAD of 28 (500 MHz D2O) 323 OSO3H O AcHN H O HO OH O O 2 HO2C O HO3SO 28 NH2 Figure 4.91. ESI-MS of 28 324 H O HO OSO3H O HO2C HO3SHN O HO3SO 29 OH O O 2 NH2 Figure 4.92. 1H-NMR of 29 (500 MHz D2O) H O HO OSO3H O HO2C HO3SHN O HO3SO 29 OH O O 2 NH2 Figure 4.93. 13C-NMR of 29 (125 MHz D2O) 325 H O HO OSO3H O HO2C HO3SHN O HO3SO 29 OH O O 2 NH2 Figure 4.94. 1H-1H gCOSY of 29 (500 MHz D2O) H O HO OSO3H O HO2C HO3SHN O HO3SO 29 OH O O 2 NH2 Figure 4.95. 1H-13C gHSQCAD of 29 (500 MHz D2O) 326 H O HO OSO3H O HO2C HO3SHN O HO3SO 29 OH O O 2 NH2 Figure 4.96. ESI-MS of 29 327 Bn OH O MeO2C O BnO H2N O 30 O OBn O Cbz N Bn HO 3 Figure 4.97. 1H-NMR of 30 (500 MHz CDCl3) H OSO3H O HO2C O HO HO3SHN O HO3SO 31 OH O O 3 NH2 Figure 4.98. 1H-NMR of 31 (500 MHz D2O) 328 H O HO OSO3H O HO2C HO3SHN O HO3SO 32 OH O O 5 NH2 Figure 4.99. 1H-NMR of 32 (500 MHz D2O) H O HO OSO3H O HO2C HO3SHN O HO3SO 32 OH O O 5 NH2 Figure 4.100. 1H-13C gHSQCAD of 32 (900 MHz D2O) 329 H O HO OSO3H O HO2C HO3SHN O HO3SO 32 OH O O 5 NH2 Figure 4.101. ESI-MS of 32 330 REFERENCES 331 REFERENCES 1. “2017 https://www.alz.org/documents_custom/2017-facts-and-figures.pdf 2017. Alzheimer’s Disease facts and figures,”. 2. Šimić, G.; Babić Leko, M.; Wray, S.; Harrington, C.; Delalle, I.; Jovanov-Milošević, N.; Bažadona, D.; Buée, L.; De Silva, R.; Di Giovanni, G., Tau protein hyperphosphorylation and aggregation in Alzheimer’s disease and other tauopathies, and possible neuroprotective strategies. Biomolecules 2016, 6 (1), 6. 3. Arriagada, P. V.; Growdon, J. H.; Hedley-Whyte, E. T.; Hyman, B. T., Neurofibrillary tangles but not senile plaques parallel duration and severity of Alzheimer's disease. Neurology 1992, 42 (3), 631-631. 4. Haroutunian, V.; Davies, P.; Vianna, C.; Buxbaum, J.; Purohit, D., Tau protein abnormalities associated with the progression of alzheimer disease type dementia. Neurobiol. Aging 2007, 28 (1), 1-7. 5. Kim, Y.; Choi, H.; Lee, W.; Park, H.; Kam, T.-I.; Hong, S.-h.; Nah, J.; Jung, S.; Shin, B.; Lee, H., Caspase-cleaved tau exhibits rapid memory impairment associated with tau oligomers in a transgenic mouse model. Neurobiol. Dis. 2016, 87, 19-28. 6. Cook, C.; Kang, S. S.; Carlomagno, Y.; Lin, W.-L.; Yue, M.; Kurti, A.; Shinohara, M.; Jansen-West, K.; Perkerson, E.; Castanedes-Casey, M., Tau deposition drives neuropathological, inflammatory and behavioral abnormalities independently of neuronal loss in a novel mouse model. Hum. Mol. Genet. 2015, 24 (21), 6198-6212. 7. Cowan, C. M.; Quraishe, S.; Mudher, A., What is the pathological significance of tau oligomers? Portland Press Limited: 2012. 8. Gerson, J. E.; Mudher, A.; Kayed, R., Potential mechanisms and implications for the formation of tau oligomeric strains. Crit. Rev. Biochem. Mol. Biol. 2016, 51 (6), 482-496. 9. Guerrero-Muñoz, M. J.; Gerson, J.; Castillo-Carranza, D. L., Tau oligomers: the toxic player at synapses in Alzheimer’s disease. Front. Cell. Neurosci. 2015, 9, 464. 10. Kopeikina, K. J.; Carlson, G. A.; Pitstick, R.; Ludvigson, A. E.; Peters, A.; Luebke, J. I.; Koffie, R. M.; Frosch, M. P.; Hyman, B. T.; Spires-Jones, T. L., Tau accumulation causes mitochondrial distribution deficits in neurons in a mouse model of tauopathy and in human Alzheimer's disease brain. Am. J. Pathol. 2011, 179 (4), 2071-2082. 11. Lasagna-Reeves, C. A.; Castillo-Carranza, D. L.; Sengupta, U.; Sarmiento, J.; Troncoso, J.; Jackson, G. R.; Kayed, R., Identification of oligomers at early stages of tau aggregation in 332 Alzheimer's disease. FASEB J. 2012, 26 (5), 1946-1959. 12. Lasagna-Reeves, C. A.; Castillo-Carranza, D. L.; Sengupta, U.; Guerrero-Munoz, M. J.; Kiritoshi, T.; Neugebauer, V.; Jackson, G. R.; Kayed, R., Alzheimer brain-derived tau oligomers propagate pathology from endogenous tau. Sci. Rep. 2012, 2, 700. 13. Castillo-Carranza, D. L.; Gerson, J. E.; Sengupta, U.; Guerrero-Muñoz, M. J.; Lasagna-Reeves, C. A.; Kayed, R., Specific targeting of tau oligomers in Htau mice prevents cognitive impairment and tau toxicity following injection with brain-derived tau oligomeric seeds. J. Alzheimer's Dis. 2014, 40 (s1), S97-S111. 14. Fá, M.; Puzzo, D.; Piacentini, R.; Staniszewski, A.; Zhang, H.; Baltrons, M. A.; Puma, D. L.; Chatterjee, I.; Li, J.; Saeed, F., Extracellular tau oligomers produce an immediate impairment of LTP and memory. Sci. Rep. 2016, 6, 19393. 15. Castillo-Carranza, D. L.; Sengupta, U.; Guerrero-Muñoz, M. J.; Lasagna-Reeves, C. A.; Gerson, J. E.; Singh, G.; Estes, D. M.; Barrett, A. D.; Dineley, K. T.; Jackson, G. R., Passive immunization with Tau oligomer monoclonal antibody reverses tauopathy phenotypes without affecting hyperphosphorylated neurofibrillary tangles. J. Neurosci. 2014, 34 (12), 4260-4272. 16. Sarrazin, S.; Lamanna, W. C.; Esko, J. D., Heparan sulfate proteoglycans. Cold Spring Harb. Perspect. Biol. 2011, 3 (7), a004952. 17. Capila, I.; Linhardt, R. J., Heparin–protein interactions. Angew. Chem. Int. Ed. 2002, 41 (3), 390-412. 18. Linhardt, R. J.; Dordick, J. S.; Deangelis, P. L.; Liu, J. Enzymatic synthesis of glycosaminoglycan heparin. Semin. Thromb. Hemost. 2007, 33 (5), 453-465. 19. Dulaney, S. B.; Huang, X., Strategies oligosaccharides: 2000–present. Adv. Carbohydr. Chem. Biochem. 2012, 67, 95-136. in synthesis of heparin/heparan sulfate 20. Zhu, H.-L.; Fernández, C.; Fan, J.-B.; Shewmaker, F.; Chen, J.; Minton, A. P.; Liang, Y., Quantitative characterization of heparin binding to tau protein implication for inducer-mediated tau filament formation. J. Biol. Chem. 2010, 285 (6), 3592-3599. 21. Holmes, B. B.; DeVos, S. L.; Kfoury, N.; Li, M.; Jacks, R.; Yanamandra, K.; Ouidja, M. O.; Brodsky, F. M.; Marasa, J.; Bagchi, D. P., Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds. Proc. Natl. Acad. Sci., U. S. A. 2013, 110 (33), E3138-E3147. 22. Sibille, N.; Sillen, A.; Leroy, A.; Wieruszeski, J.-M.; Mulloy, B.; Landrieu, I.; Lippens, G., Structural impact of heparin binding to full-length Tau as studied by NMR spectroscopy. Biochemistry 2006, 45 (41), 12560-12572. 333 23. Jangholi, A.; Ashrafi-Kooshk, M. R.; Arab, S. S.; Riazi, G.; Mokhtari, F.; Poorebrahim, M.; Mahdiuni, H.; Kurganov, B. I.; Moosavi-Movahedi, A. A.; Khodarahmi, R., Appraisal of role of the polyanionic inducer length on amyloid formation by 412-residue 1N4R Tau protein: a comparative study. Arch. Biochem. Biophys. 2016, 609, 1-19. 24. Zhao, J.; Huvent, I.; Lippens, G.; Eliezer, D.; Zhang, A.; Li, Q.; Tessier, P.; Linhardt, R. J.; Zhang, F.; Wang, C., Glycan Determinants of Heparin-Tau Interaction. Biophys. J. 2017, 112 (5), 921-932. 25. Dulaney, S. B.; Xu, Y.; Wang, P.; Tiruchinapally, G.; Wang, Z.; Kathawa, J.; El-Dakdouki, M. H.; Yang, B.; Liu, J.; Huang, X., Divergent synthesis of heparan sulfate oligosaccharides. J. Org. Chem. 2015, 80 (24), 12265-12279. 26. Huang, X.; Huang, L.; Wang, H.; Ye, X. S., Iterative one-pot synthesis of oligosaccharides. Angew. Chem.Int. Ed. 2004, 116 (39), 5333-5336. 27. van den Bos, L. J.; Codée, J. D. C.; van der Toorn, J. C.; Boltje, T. J.; van Boom, J. H.; Overkleeft, H. S.; van der Marel, G. A., Thioglycuronides:  synthesis and application in the assembly of acidic oligosaccharides. Org. Lett. 2004, 6 (13), 2165-2168. 28. Lasagna-Reeves, C. A.; Castillo-Carranza, D. L.; Guerrero-Muñoz, M. J.; Jackson, G. R.; Kayed, R., Preparation and characterization of neurotoxic tau oligomers. Biochemistry 2010, 49 (47), 10039-10041. 29. Margittai, M.; Langen, R., Template-assisted filament growth by parallel stacking of tau. Proc. Natl. Acad. Sci., U. S. A. 2004, 101 (28), 10278-10283. 30. Margittai, M.; Langen, R., Side chain-dependent stacking modulates tau filament structure. J. Biol. Chem. 2006, 281 (49), 37820-37827. 31. Hernaiz, M.; Liu, J.; Rosenberg, R. D.; Linhardt, R. J., Enzymatic modification of heparan sulfate on a biochip promotes its interaction with antithrombin III. Biochem. Biophys. Res. Commun. 2000, 276 (1), 292-297. 32. Kayed, R.; Head, E.; Thompson, J. L.; McIntire, T. M.; Milton, S. C.; Cotman, C. W.; Glabe, C. G., Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 2003, 300 (5618), 486-489. 334