CHEMICAL SYNTHESIS OF HEPARIN LIKE HEAD TO TAIL MULTIMERS By Jicheng Zhang A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Chemistry—Doctor of Philosophy 2020 ABSTRACT CHEMICAL SYNTHESIS OF HEPARIN LIKE HEAD TO TAIL MULTIMERS By Jicheng Zhang Heparin, a heterogeneously sulfated glycosaminoglycan (GAG), consists of α-1,4- linked glucosamine (GlcN) and uronic acid [either D-glucuronic acid (GlcA) or L-iduronic acid (IdoA)] disaccharide repeating units. It has been used as an anticoagulant drug for over 80 years. In addition, it plays significant roles in various biological processes such as inflammation, growth factor regulation, bacterial and viral infection, cell adhesion, cell growth, tumor metastasis, lipid metabolism and diseases of the nervous system. However, the complex heterogeneity of natural heparin polysaccharides has hindered efforts to understand the relationship between their diverse structures and biological functions. While chemical and enzymatic syntheses of heparin oligosaccharides have seen tremendous advances in recent years, it is still challenging to prepare heparin analogs approaching the length of polysaccharides with distinct backbone structures and sulfation patterns. In this project, we have developed a new strategy, where (GlcN-IdoA) disaccharide modules with defined sulfation pattern are synthesized and conjugated through amide bond formation. Novel “head-to-tail” multimers are accessed to mimic the linear connections in natural GAG polysaccharides. The ligand requirements of fibroblast growth factor 2 (FGF-2) are studied using bio-layer interferometry (BLI) and surface plasmon resonance (SPR), and the results show the synthetic multimers could mimic the natural heparin mimetics. Heparanase inhibitory activities have also been examined through the colorimetric assay. Copyright by JICHENG ZHANG 2020 ACKNOWLEDGEMENTS First and foremost, I would like to show my deepest gratitude to my supervisor, Professor Xuefei Huang, who has provided me with consistent and outstanding support through every aspect of my graduate career. Aside from Prof. Huang, many faculty members at the chemistry department have been paragons of aid. Professor Wulff graciously agreed to be my second reader and write letters of recommendation for my post-doc application. I would like to thank Prof. Frost and Prof. Hong for reviewing this thesis and serving on my graduate committee. I would also like to thank all my coworkers who have helped me to develop the fundamental and essential academic competence. Postdocs Weizhun, Kedar, Sherif, Xuanjun, and Tianlu are knowledgeable and generous with their time and talents. Graduate students in the Huang group have also been extremely helpful for solving both synthetic and biological problems. Support staff has also played important roles in my research. Professor Dan Jones, Lijun Chen, and Tony Schilmiller in the Mass Spectrometry facility assisted me to figure out the optimized conditions for my sugar. Tom Dexheimer in drug discovery spent tremendous time and effort on SPR binding assays. My parents, Weiguo and Yun, and my wife, Jiexi, have supported me all the time. I would like to thank them for their loving considerations and great confidence in me all through these years. iv TABLE OF CONTENTS LIST OF FIGURES ...................................................................................................................... vii LIST OF SCHEMES..................................................................................................................... xv KEY TO ABBREVIATIONS ...................................................................................................... xvi Chapter 1. A Review of Heparin Mimetics as Therapeutic Agents ................................................ 1 1.1. Introduction .......................................................................................................................... 1 1.2. Heparin Mimetics as Anticoagulants.................................................................................... 3 1.3 Heparin Mimetics as Growth Factor Binders ...................................................................... 13 1.4. Heparin Mimetics as Heparanase Inhibitors ...................................................................... 20 1.5. Conclusions ........................................................................................................................ 27 Chapter 2. Chemical Synthesis of Heparin Like Head to Tail Multimers .................................... 30 2.1. Introduction ........................................................................................................................ 30 2.2. Synthetic Challenges in Preparation of Disaccharide Modules ......................................... 32 2.3. Monosaccharide Building Block Preparation .................................................................... 41 2.4. Synthesis of Disaccharide modules with Various Sulfation Patterns ................................ 43 2.5. Synthesis of a Library of 27 Pseudo-hexasaccharide Heparin Mimetics ........................... 46 2.6. Conclusions ........................................................................................................................ 48 2.7. Experimental Section ......................................................................................................... 49 2.7.1. General synthetic procedures....................................................................................... 49 2.7.2. General procedure for pre-activation Based Single-step Glycosylation. .................... 49 2.7.3. General procedure for deprotection of PMB. .............................................................. 50 2.7.4. General procedure for protection of 6-OH with Lev. .................................................. 50 2.7.5. General procedure for O-Acetylation. ......................................................................... 51 2.7.6. General procedure for N-Acetylation. ......................................................................... 51 2.7.7. General procedure for protection of OH with Fmoc. .................................................. 51 2.7.8. General procedure for protection of NH2 with Fmoc. ................................................. 52 2.7.9. General procedure for preparation of methyl ester. ..................................................... 52 2.7.10. General procedure for NH-Fmoc Deprotection. ........................................................ 52 2.7.11. General procedure for preparation of pseudo-tetrasaccharides/hexasaccharides. ..... 53 2.7.12. General procedure for hydrogenolysis. ..................................................................... 53 2.7.13. General procedure for saponification of methyl esters. ............................................. 53 APPENDIX ............................................................................................................................. 106 Chapter 3. Biological Studies of Pseudo-hexasaccharide Heparin Mimetics ............................. 182 3.1. Bio-Layer Interferometry (BLI) Competition Assay on FGF-2-heparin interaction ....... 182 3.2. Binding Studies of Pseudo-hexasaccharides and FGF-2 Using Surface Plasmon Resonance (SPR) ....................................................................................................................................... 184 3.3. Heparanase Inhibitory Activity Tests Through the Colorimetric Assay .......................... 187 3.4. Experimental Section ....................................................................................................... 189 3.4.1. Bio-Layer Interferometry (BLI). ............................................................................... 189 v 3.4.2. Surface Plasmon Resonance (SPR). .......................................................................... 190 3.4.3. Heparanase Colorimetric Assays. .............................................................................. 190 REFERENCES ........................................................................................................................ 193 vi LIST OF FIGURES Figure 1.1. Structure of the disaccharide repeating unit of heparin and HS. ................................. 1 Figure 1.2. “Non-glycosamino” glycan analogs ............................................................................ 5 Figure 1.3. Idrabiotaparinux........................................................................................................... 6 Figure 1.4. A series of “non-glycosamino” glycan heparin mimetics. .......................................... 8 Figure 1.5. Maltotrioside 21, antithrombin binding domain 22, sulfo-SIAB 23, and glycoconjugates 24, 25 and 26. ..................................................................................................... 11 Figure 1.6. Structures of poly(GEMA) 27 and poly(GEMA) sulfate 28. .................................... 12 Figure 1.7. Heparin-based glycopolymers 29 and 30 prepared through ROMP.......................... 13 Figure 1.8. Cyclitol-based heparin mimetics 31 and 32. ............................................................. 14 Figure 1.9. Compound 33 synthesized via the click reaction. ..................................................... 15 Figure 1.10. Representative heparin mimetic compounds synthesized via the Ugi reaction for growth factor binding. ................................................................................................................... 15 Figure 1.11. Structures of heparin oligosaccharides and heparin glycodendrimers. ................... 17 Figure 1.12. A library of neoPGs 45 - 50. .................................................................................... 19 Figure 1.13. Linear heparin mimetics with glyco-amino acid oligomers 51-57 .......................... 20 Figure 1.14. PI-88, a heparin mimetic with heparanase inhibitory activities. ............................. 21 Figure 1.15. Structure of PG545. ................................................................................................. 22 Figure 1.16. Structure of JG3. ...................................................................................................... 23 Figure 1.17. Structure of suramin. ............................................................................................... 24 Figure 1.18. Diantennary and monoantennary monomers 62, 63 and 64. ................................... 25 Figure 1.19. Disaccharide motifs 65-70 with various sulfation patterns used for glycopolymer construction. .................................................................................................................................. 26 Figure 1.20. Dendrimer glycomimetics 71-76 for heparanase inhibition. ................................... 27 vii Figure 2.1. Target tetrasaccharide modules. ................................................................................ 33 Figure 2.2. 1H-NMR of 30 (500 MHz CDCl3) ........................................................................... 107 Figure 2.3. 13C-NMR of 30 (125 MHz CDCl3).......................................................................... 107 Figure 2.4. 1H-1H gCOSY of 30 (500 MHz CDCl3) .................................................................. 108 Figure 2.5. 1H-NMR of 31 (500 MHz CDCl3) ........................................................................... 108 Figure 2.6. 13C-NMR of 31 (125 MHz CDCl3).......................................................................... 109 Figure 2.7. 1H-1H gCOSY of 31 (500 MHz CDCl3) .................................................................. 109 Figure 2.8. 1H-NMR of 33 (500 MHz CDCl3) ........................................................................... 110 Figure 2.9. 13C-NMR of 33 (125 MHz CDCl3).......................................................................... 110 Figure 2.10. 1H-1H gCOSY of 33 (500 MHz CDCl3) ................................................................ 111 Figure 2.11. 1H-13C gHSQCAD of 33 (500 MHz CDCl3) ......................................................... 111 Figure 2.12. 1H-NMR of 34 (500 MHz CDCl3) ......................................................................... 112 Figure 2.13. 13C-NMR of 34 (125 MHz CDCl3)........................................................................ 112 Figure 2.14. 1H-1H gCOSY of 34 (500 MHz CDCl3) ................................................................ 113 Figure 2.15. 1H-13C gHSQCAD of 34 (500 MHz CDCl3) ......................................................... 113 Figure 2.16. 1H-NMR of 35 (500 MHz CDCl3) ......................................................................... 114 Figure 2.17. 13C-NMR of 35 (125 MHz CDCl3)........................................................................ 114 Figure 2.18. 1H-1H gCOSY of 35 (500 MHz CDCl3) ................................................................ 115 Figure 2.19. 1H-13C gHSQCAD of 35 (500 MHz CDCl3) ......................................................... 115 Figure 2.20. 1H-13C gHMBCAD of 35 (500 MHz CDCl3) ........................................................ 116 Figure 2.21. 1H-NMR of 36 (500 MHz CDCl3) ......................................................................... 116 Figure 2.22. 13C-NMR of 36 (125 MHz CDCl3)........................................................................ 117 Figure 2.23. 1H-NMR of 37 (500 MHz CD3OD) ....................................................................... 117 Figure 2.24. 13C-NMR of 37 (125 MHz CD3OD)...................................................................... 118 viii Figure 2.25. 1H-1H gCOSY of 37 (500 MHz CD3OD) .............................................................. 118 Figure 2.26. 1H-NMR of 38 (500 MHz CD3OD) ....................................................................... 119 Figure 2.27. 1H-NMR of 39 (500 MHz CD3OD) ....................................................................... 119 Figure 2.28. 13C-NMR of 39 (125 MHz CD3OD)...................................................................... 120 Figure 2.29. 1H-1H gCOSY of 39 (500 MHz CD3OD) .............................................................. 120 Figure 2.30. 1H-NMR of 40 (500 MHz CD3OD) ....................................................................... 121 Figure 2.31. 13C-NMR of 40 (125 MHz CD3OD)...................................................................... 121 Figure 2.32. 1H-NMR of 41(500 MHz D2O) ............................................................................. 122 Figure 2.33. 13C-NMR of 41 (125 MHz D2O) ........................................................................... 122 Figure 2.34. 1H-NMR of 42 (500 MHz D2O) ............................................................................ 123 Figure 2.35. 13C-NMR of 42 (125 MHz D2O) ........................................................................... 123 Figure 2.36. 1H-13C gHSQCAD of 42 (500 MHz D2O)............................................................. 124 Figure 2.37. 1H-NMR of 43 (500 MHz CD3OD) ....................................................................... 124 Figure 2.38. 13C-NMR of 43 (125 MHz CD3OD)...................................................................... 125 Figure 2.39. 1H-NMR of 44 (500 MHz CD3OD) ....................................................................... 125 Figure 2.40. 13C-NMR of 44 (125 MHz CD3OD)...................................................................... 126 Figure 2.41. 1H-NMR of 45 (500 MHz CD3OD) ....................................................................... 126 Figure 2.42. 13C-NMR of 45 (125 MHz CD3OD)...................................................................... 127 Figure 2.43. 1H-NMR of 46 (500 MHz CD3OD) ....................................................................... 127 Figure 2.44. 13C-NMR of 46 (125 MHz CD3OD)...................................................................... 128 Figure 2.45. 1H-13C gHSQCAD of 46 (500 MHz CD3OD) ....................................................... 128 Figure 2.46. 1H-NMR of 47 (500 MHz D2O) ............................................................................ 129 Figure 2.47. 13C-NMR of 47 (125 MHz D2O) ........................................................................... 129 ix Figure 2.48. 1H-NMR of 48 (500 MHz CD3OD) ....................................................................... 130 Figure 2.49. 13C-NMR of 48 (125 MHz CD3OD)...................................................................... 130 Figure 2.50. 1H-1H gCOSY of 48 (500 MHz CD3OD) .............................................................. 131 Figure 2.51. 1H-13C gHSQCAD of 48 (500 MHz CD3OD) ....................................................... 131 Figure 2.52. 1H-NMR of 49a (500 MHz D2O) .......................................................................... 132 Figure 2.53. 13C-NMR of 49a (125 MHz D2O) ......................................................................... 132 Figure 2.54. 1H-NMR of 49b (500 MHz D2O) .......................................................................... 133 Figure 2.55. 13C-NMR of 49b (125 MHz D2O) ......................................................................... 133 Figure 2.56. 1H-1H gCOSY of 49b (500 MHz D2O) ................................................................. 134 Figure 2.57. 1H-NMR of 49c (500 MHz CD3OD) ..................................................................... 134 Figure 2.58. 13C-NMR of 49c (125 MHz CD3OD) .................................................................... 135 Figure 2.59. 1H-NMR of 49d (500 MHz CD3OD) .................................................................... 135 Figure 2.60. 13C-NMR of 49d (125 MHz CD3OD) ................................................................... 136 Figure 2.61. 1H-NMR of 49e (500 MHz CD3OD) ..................................................................... 136 Figure 2.62. 13C-NMR of 49e (125 MHz CD3OD) .................................................................... 137 Figure 2.63. 1H-NMR of 49f (500 MHz CD3OD) ..................................................................... 137 Figure 2.64. 13C-NMR of 49f (125 MHz CD3OD) .................................................................... 138 Figure 2.65. 1H-1H gCOSY of 49f (500 MHz CD3OD)............................................................. 138 Figure 2.66. 1H-13C gHSQCAD of 49f (500 MHz CD3OD) ...................................................... 139 Figure 2.67. 1H-13C gHMBCAD of 49f (500 MHz CD3OD) .................................................... 139 Figure 2.68. 1H-NMR of 49g (500 MHz CD3OD) ..................................................................... 140 Figure 2.69. 13C-NMR of 49g (125 MHz CD3OD).................................................................... 140 Figure 2.70. 1H-NMR of 49h (500 MHz CD3OD) .................................................................... 141 x Figure 2.71. 13C-NMR of 49h (125 MHz CD3OD) ................................................................... 141 Figure 2.72. 1H-1H gCOSY of 49h (500 MHz CD3OD) ............................................................ 142 Figure 2.73. 1H-13C gHSQCAD of 49h (500 MHz CD3OD) ..................................................... 142 Figure 2.74. 1H-13C gHMBCAD of 49h (500 MHz CD3OD) .................................................... 143 Figure 2.75. 1H-NMR of 49i (500 MHz CD3OD)...................................................................... 143 Figure 2.76. 13C-NMR of 49i (125 MHz CD3OD) .................................................................... 144 Figure 2.77. 1H-1H gCOSY of 49i (500 MHz CD3OD) ............................................................. 144 Figure 2.78. 1H-13C gHSQCAD of 49i (500 MHz CD3OD) ...................................................... 145 Figure 2.79. 1H-13C gHSQCAD of 49i (500 MHz CD3OD) ...................................................... 145 Figure 2.80. 1H-13C gHMBCAD of 49i (500 MHz CD3OD) ..................................................... 146 Figure 2.81. 1H-NMR of 50 (500 MHz D2O) ............................................................................ 146 Figure 2.82. 13C-NMR of 50 (125 MHz D2O) ........................................................................... 147 Figure 2.83. 1H-1H gCOSY of 50 (500 MHz D2O) ................................................................... 147 Figure 2.84. 1H-NMR of 50a (500 MHz D2O) .......................................................................... 148 Figure 2.85. 13C-NMR of 50a (125 MHz D2O) ......................................................................... 148 Figure 2.86. 1H-13C gHSQCAD of 50a (500 MHz D2O)........................................................... 149 Figure 2.87. 1H-NMR of 50b (500 MHz D2O) .......................................................................... 149 Figure 2.88. 13C-NMR of 50b (125 MHz D2O) ......................................................................... 150 Figure 2.89. 1H-1H gCOSY of 50b (500 MHz D2O) ................................................................. 150 Figure 2.90. 1H-NMR of 50c (500 MHz D2O) .......................................................................... 151 Figure 2.91. 13C-NMR of 50c (125 MHz D2O) ......................................................................... 151 Figure 2.92. 1H-13C gHSQCAD of 50c (500 MHz D2O) ........................................................... 152 Figure 2.93. 1H-NMR of 50d (500 MHz D2O) .......................................................................... 152 xi Figure 2.94. 13C-NMR of 50d (125 MHz D2O) ......................................................................... 153 Figure 2.95. 1H-13C gHSQCAD of 50d (500 MHz D2O) .......................................................... 153 Figure 2.96. 1H-NMR of 50e (500 MHz D2O) .......................................................................... 154 Figure 2.97. 13C-NMR of 50e (125 MHz D2O) ......................................................................... 154 Figure 2.98. 1H-NMR of 50f (500 MHz D2O) ........................................................................... 155 Figure 2.99. 13C-NMR of 50f (125 MHz D2O) .......................................................................... 155 Figure 2.100. 1H-13C gHSQCAD of 50f (500 MHz D2O) ......................................................... 156 Figure 2.101. 1H-NMR of 50g (500 MHz D2O) ........................................................................ 156 Figure 2.102. 13C-NMR of 50g (125 MHz D2O) ....................................................................... 157 Figure 2.103. 1H-13C gHSQCAD of 50g (500 MHz D2O)......................................................... 157 Figure 2.104. 1H-NMR of 50h (500 MHz D2O) ........................................................................ 158 Figure 2.105. 13C-NMR of 50h (125 MHz D2O) ....................................................................... 158 Figure 2.106. 1H-13C gHSQCAD of 50h (500 MHz D2O) ........................................................ 159 Figure 2.107. 1H-NMR of 50i (500 MHz D2O) ......................................................................... 159 Figure 2.108. 1H-NMR of 50j (500 MHz D2O) ......................................................................... 160 Figure 2.109. 13C-NMR of 50j (125 MHz D2O) ........................................................................ 160 Figure 2.110. 1H-13C gHSQCAD of 50j (500 MHz D2O) ......................................................... 161 Figure 2.111. 1H-NMR of 50k (500 MHz D2O) ........................................................................ 161 Figure 2.112. 13C-NMR of 50k (125 MHz D2O) ....................................................................... 162 Figure 2.113. 1H-13C gHSQCAD of 50k (500 MHz D2O) ........................................................ 162 Figure 2.114. 1H-NMR of 50l (500 MHz D2O) ......................................................................... 163 Figure 2.115. 1H-1H gCOSY of 50l (500 MHz D2O) ................................................................ 163 Figure 2.116. 1H-NMR of 50m (500 MHz D2O) ....................................................................... 164 xii Figure 2.117. 1H-NMR of 50n (500 MHz D2O) ........................................................................ 164 Figure 2.118. 13C-NMR of 50n (125 MHz D2O) ....................................................................... 165 Figure 2.119. 1H-13C gHSQCAD of 50n (500 MHz D2O) ........................................................ 165 Figure 2.120. 1H-NMR of 50o (500 MHz D2O) ........................................................................ 166 Figure 2.121. 1H-13C gHSQCAD of 50o (500 MHz D2O)......................................................... 166 Figure 2.122. 1H-NMR of 50p (500 MHz D2O) ........................................................................ 167 Figure 2.123. 13C-NMR of 50p (125 MHz D2O) ....................................................................... 167 Figure 2.124. 1H-13C gHSQCAD of 50p (500 MHz D2O) ........................................................ 168 Figure 2.125. 1H-NMR of 50q (500 MHz D2O) ........................................................................ 168 Figure 2.126. 13C-NMR of 50q (125 MHz D2O) ....................................................................... 169 Figure 2.127. 1H-NMR of 50r (500 MHz D2O) ........................................................................ 169 Figure 2.128. 13C-NMR of 50r (125 MHz D2O) ....................................................................... 170 Figure 2.129. 1H-13C gHSQCAD of 50r (500 MHz D2O) ......................................................... 170 Figure 2.130. 1H-NMR of 50s (500 MHz D2O) ......................................................................... 171 Figure 2.131. 13C-NMR of 50s (125 MHz D2O)........................................................................ 171 Figure 2.132. 1H-13C gHSQCAD of 50s (500 MHz D2O) ......................................................... 172 Figure 2.133. 1H-NMR of 50t (500 MHz D2O) ......................................................................... 172 Figure 2.134. 13C-NMR of 50t (125 MHz D2O) ........................................................................ 173 Figure 2.135. 1H-13C gHSQCAD of 50t (500 MHz D2O) ......................................................... 173 Figure 2.136. 1H-NMR of 50u (500 MHz D2O) ........................................................................ 174 Figure 2.137. 13C-NMR of 50u (125 MHz D2O) ....................................................................... 174 Figure 2.138. 1H-13C gHSQCAD of 50u (500 MHz D2O) ........................................................ 175 Figure 2.139. 1H-NMR of 50v (500 MHz D2O) ........................................................................ 175 xiii Figure 2.140. 13C-NMR of 50v (125 MHz D2O) ....................................................................... 176 Figure 2.141. 1H-13C gHSQCAD of 50v (500 MHz D2O)......................................................... 176 Figure 2.142. 1H-NMR of 50w (500 MHz D2O) ....................................................................... 177 Figure 2.143. 13C-NMR of 50w (125 MHz D2O) ...................................................................... 177 Figure 2.144. 1H-13C gHSQCAD of 50w (500 MHz D2O) ........................................................ 178 Figure 2.145. 1H-NMR of 50x (500 MHz D2O) ........................................................................ 178 Figure 2.146. 1H-NMR of 50y (500 MHz D2O) ........................................................................ 179 Figure 2.147. 13C-NMR of 50y (125 MHz D2O) ....................................................................... 179 Figure 2.148. 1H-13C gHSQCAD of 50y (500 MHz D2O)......................................................... 180 Figure 2.149. 1H-NMR of 50z (500 MHz D2O) ........................................................................ 180 Figure 2.150. 13C-NMR of 50z (125 MHz D2O) ....................................................................... 181 Figure 3.1. The sensorgram of heparin-FGF-2 interaction. ....................................................... 183 Figure 3.2. The sensorgrams of competition BLI. A: heparin as the inhibitor. Concentrations from top to bottom: 0, 3.2, 6.5, 13.1, 26.1, 51.7, 77.8 nM. B: compound 50k. Concentrations from top to bottom: 0, 12.5, 25, 50, 100, 500, 1000 uM. C: fondaparinux. Concentrations from top to bottom: 0, 1.0, 10.0, 25, 50, 1000, 2000 uM. ................................................................... 183 Figure 3.3. The SPR sensorgram of FGF-2 and compound 50 interaction at different concentrations ............................................................................................................................. 184 Figure 3.4. Heparin hexa-, tetra- and disaccharides. .................................................................. 185 Figure 3.5. Measurement of inhibition of 27 pseudo-hexasaccharides on FGF-2-heparin interaction using competition SPR.............................................................................................. 186 Figure 3.6. Competition SPR for IC50 measurements for pseudo-hexasaccharides 50 (A), 50a (B), 50b (C), and 50r (D) ................................................................................................................... 187 Figure 3.7. Inhibition of heparanase by heparin ........................................................................ 189 Figure 3.8. Inhibition of heparanase by A: compound 50a (10, 50, 100, 1000, and 1000 nM). B: compound 50f (0.1, and 10 uM). ................................................................................................ 189 xiv LIST OF SCHEMES Scheme 2.1. “Head-to-tail” strategy via chemoselective oxime ligation. ................................................. 34 Scheme 2.2. Failed glycosylation with idose acceptor 2. .................................................................................. 35 Scheme 2.3. Glycosylation with various donors.................................................................................................... 35 Scheme 2.4. Preparation of disaccharide 9 ............................................................................................................... 36 Scheme 2.5. Hydroxylamine bond cleavage under hydrogenation conditions ....................................... 37 Scheme 2.6. Disaccharide module deprotection sequence. .............................................................................. 37 Scheme 2.7. Hydroxylamine group installation..................................................................................................... 38 Scheme 2.8. Synthesis of Fmoc protected hydroxylamine disaccharide 17. ........................................... 38 Scheme 2.9. Hydrolysis of ketoxime under pseudo-hexasaccharide formation condition. .............. 39 Scheme 2.10. Failure of reductive amination reaction to link the amine bearing disaccharide module and a ketone bearing disaccharide. .............................................................................................................. 40 Scheme 2.11. Lactone formation from carboxylic acid 26 during HTAU mediated amide formation reaction ................................................................................................................................................................. 41 Scheme 2.12. Glucosamine donor synthesis. .......................................................................................................... 42 Scheme 2.13. Idopyranosyluronate acceptor 34 preparation. .......................................................................... 43 Scheme 2.14. Disaccharide synthesis and modification. ................................................................................... 44 Scheme 2.15. Preparation of disaccharides bearing various sulfation patterns. ..................................... 45 Scheme 2.16. Reducing end sequence disaccharides 46-48. ........................................................................... 46 Scheme 2.17. Pseudo tetrasaccharide formation. .................................................................................................. 47 Scheme 2.18. A library of 27 pseudo-hexasaccharides. ..................................................................................... 48 Scheme. 3.1. Heparanase colorimetric assay. ....................................................................................................... 188 xv KEY TO ABBREVIATIONS Ac2O AcOH acetic anhydride acetic acid AF488 Alex Fluor 488 AgOTf silver trifluoromethanesulfonate APS ammonium peroxodisulfate ATM Ataxia-telangiectasia-mutated kinase AT-III antithrombin III BAIB BF3·Et2O bis(acetoxy)iodobenzene boron trifluoride etherate BLI bio-layer interferometry Bn benzyl BnBr benzyl bromide BSA Bz Cbz bovine serum albumin benzoyl benzyloxycarbonyl CH3CN acetonitrile CSA camphorsulfonic acid CVDs cardiovascular diseases DBU 1, 8-diazabicyclo[5.4.0]undec-7-ene DCM dichloromethane DDQ 2, 3-dichloro-5, 6-dicyanobenzoquinone DIPEA diisopropylethylamine DMAP 4-dimethylaminopyridine xvi DMF dimethylformamide DMSO dimethyl sulfoxide DTT dithiothreitol ECM extracellular matrix EDC·HCl 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride ELISA enzyme-linked immunosorbent assay ESI-MS electrospray-ionization mass spectrometry EtOAc ethyl acetate Et3N trimethylamine FDA Food and Drug Administration FGFs fibroblast growth factors FGFRs FGF receptors Fmoc 9-fluorenylmethoxycarbonyl FmocCl Fmoc chloride Fmoc-Osu Fmoc N-hydroxysuccinimide ester FXa activated factor X GAGs glycosaminoglycans GEMA glucosyloxyethyl methacrylate GlcA glucuronic acid GlcN glucosamine GlcNAc 2-acetamido-2-deoxy-glucose GlcNS N-sulfated glucosamine HCl hydrochloric acid xvii HIT heparin-induced thrombocytopenia H2O water H2O2 hydrogen peroxide HMBC heteronuclear multiple bond correlation HOBT hydroxybenzotriazole HPLC high performance liquid chromatography HRMS high resolution mass spectrometry HS heparan sulfate HSPGs heparan sulfate proteoglycans HSQC heteronuclear single quantum correlation IdoA iduronic acid IV intravenous K2CO3 potassium carbonate kDa kilo-dalton KPB potassium phosphate buffer LevOH levulinic acid LiAlH4 lithium aluminum hydride LiOH lithium hydroxide LMWH low-molecular-weight heparin MALDI-TOF matrix assisted laser desorption ionization-time of flight MeI methyl iodide MeOH methanol mESCs mouse embryonic stem cells MS mass spectrometry xviii NaBH4 sodium borohydride NaCNBH3 sodium cyanoborohydride NaH sodium hydride NaHCO3 sodium bicarbonate NaN3 sodium azide Na2SO4 sodium sulfate neoPGs neoproteoglycans NF-KB nuclear factor kappa B NH4OH ammonium hydroxide NHS N-hydroxysuccinimide NIS N-iodoxuccinimide NMO 4-methylmorpholine N-oxide NMR nuclear magnetic resonance spectroscopy OD Optical density OsO4 osmium tetraoxide PBS phosphate buffered saline PBST PBS/0.5% Tween-20 Pd(OH)2 palladium hydroxide PF4 platelet factor 4 Ph phenyl PMB p-methoxybenzyl p-TolSCl p-toluenesulfenyl chloride p-TolSH p-toluenethiol Py/Pyr pyridine xix ROMP ring-opening metathesis polymerization SA streptavidin sat. saturated SC subcutaneous SEC size exclusion chromatography SPR surface plasmon resonance TAMRA tetramethylrhodamine TBAI TBDPS tetrabutylammonium iodide t-butyldiphenylsilyl TBS t-butyldimethylsilyl t-Bu t-butyl TCT tracheal cytotoxin TEMPO 2, 2, 6, 6-tetramethyl-1-piperidinyloxyl TFA trifluoroacetic acid Tf2O trifluoromethanesulfonic anhydride THF tetrahydrofuran TLC thin layer chromatography TR-FRET Time-resolved fluorescence energy transfer TrocCl trichloroethyl chloroformate TTBP 2,4,6-tri-t-butylpyrimidine UFH unfractionated heparin ULMW ultralow molecular weight VEGF vascular endothelial growth factor WHO World Health Organization xx Chapter 1. A Review of Heparin Mimetics as Therapeutic Agents 1.1. Introduction Glycosaminoglycans (GAGs) are highly negatively charged, linear polysaccharides. As members of the GAG family, heparin and heparan sulfate (HS) have highest densities of negative charges among all biological macromolecules. The structures of heparin and HS are remarkably heterogeneous, which contain complex linear disaccharide repeating units of D-glucosamine (GlcN) α-(1-4)-linked to a uronic acid (90% L-iduronic acid (IdoA) and 10% D-glucuronic acid (GlcA)). 2-OH of GlcA and IdoA residues can be sulfated. The GlcN monosaccharide can be either N-acetylated (GlcNAc) or N-sulfated (GlcNS), while O-sulfation could be at 6-OH and 3- OH (Figure 1.1).1 Figure 1.1. Structure of the disaccharide repeating unit of heparin and HS. Heparin has been used as an anticoagulant drug for over 80 years. Doyon firstly recorded heparin as an anticoagulant in 1912, and the discovery of heparin was also reported by the physiologist William Howell when he investigated the purification of tissue thromboplastin. In 1918-1920, Howell improved the isolation method and named the polysaccharide anticoagulant “heparin”. It was not until the 1930s that preparations of heparin were ready for clinical trials. One of the most famous landmarks in heparin analysis was the identification of L- 1 iduronic acid in an acid hydrolysate of heparin by Cifonelli and Dorfman in 1962, as early on, it was mistakenly considered that D-glucuronic acid was the only uronic acid in the heparin structure. The configuration of glucosaminidic and uronidic linkages was established by Roden in 1989.2-5 The sulfation positions, especially 3-O sulfation, were discussed in his report as well. The description of the minimal binding requirement for antithrombin is another important landmark for heparin structure research. Lindahl’s group was able to identify a unique pentasaccharide sequence from an isolated octasaccharide, which was thought to be the smallest fragment required for antithrombin binding.6 There are three forms of heparin approved by US Food and Drug Administration (FDA) as anti-coagulant drugs: unfractionated heparin (UFH, average molecular weight (MW) 14,000 Da), low-molecular-weight heparin (LMWH, MW 3500–6500 Da) (12-20 saccharide units) and the synthetic pentasaccharide, fondaparinux (MW 1508.3 Da).1 As a well-known anticoagulant drug, UFH is a mixture of polysaccharides in different lengths with various sulfation patterns.7, 8 As it is sourced from animals (primarily from porcine intestinal mucosa with some from bovine), with the heterogeneity of compounds from nature, the reliability and safety of heparin preparations have received increasing attention. This is especially the case after the incident caused by the worldwide distribution of oversulfated chondroitin sulfate contaminated heparin in 2007-2008, with adverse reactions identified in over 100 patients in 13 states alone in less than three months.9, 10 As a result, less heterogeneous or even homogenous heparin derivatives are highly desired. Bemiparin and M-Enoxaparin are currently marketed in Europe and the U.S. as LMWH anticoagulant drugs.11-13 Ultralow molecular weight (ULMW) heparin pentasaccharide fondaparinux as a chemically pure anticoagulant is the most expensive drug among heparins as results of its challenging chemical synthesis (~50 steps) with an overall low yield (~0.1%).14, 15 2 Besides the anticoagulant activities, heparin can mediate many other protein functions. It is involved in tumor growth and metastasis in part as a result of its inhibition of heparanase, a β-endoglycosidase capable of cleaving HS side chains of heparan sulfate proteoglycans (HSPGs) on the cell surface and in the extracellular matrix (ECM). Furthermore, it can interact with angiogenesis mediators such as fibroblast growth factors (FGFs) and vascular endothelial growth factor (VEGF) in the ECM to promote cell proliferation.16-19 Synthesis of homogeneous heparin polysaccharides is a formidable challenge due to their length and structural complexity. Heparin’s polypharmacy and anticoagulant properties suggest that they need to be tailored for clinical use in applications other than anticoagulation. Synthetic or semi-synthetic heparin mimetics could be designed to address these challenges associated with heparin. In this review, we focus on compounds that can mimic the structural characters and activities of heparin in three common functions, i.e., anti-coagulant, growth factor binding, and heparanase inhibition. Due to space limitation, more native-like heparin oligo and longer saccharide sequences prepared through innovative chemical20-25 as well as chemoenzymatic syntheses,26-33 are not included. Interested readers are referred to several excellent reviews covering those compounds.32, 34-36 1.2. Heparin Mimetics as Anticoagulants According to the World Health Organization (WHO) 2017 fact sheets, cardiovascular diseases (CVDs) are the number one cause of death (31%) globally, accounting for 17.9 million deaths in 2016. Thrombosis and inflammation are strongly related to different types of CVDs, including heart attack, stroke, arterial thrombosis, and venous thromboemlism.37-39 UFH, as a commonly used anticoagulant with low costs, is rapid-onset, and its effects can be reversed with 3 the administration of protamine.40 However, heparin-induced thrombocytopenia (HIT) is a significant and life-threatening side effect caused by the interaction of UFH with platelet factor 4 (PF4), a positively charged chemokine.41-44 Besides the side effects, short half-life (< 1 h) and low dose-response relationships encourage scientists to discover improved anticoagulants.45 LMWH and ULMH are currently approved anticoagulant drugs. LMWH was introduced in the 1940s with its subcutaneous administration route, improved bioavailability, and longer half-life.1, 46 With those advantages, LMWH has been the most prescribed heparin in the US.47 However, LMWH preparation still depends on the source of UFH, and incomplete neutralization with protamine occurs with risks of bleeding.31 On the other hand, fondaparinux, the chemically synthesized pentasaccharide, reduces the risk of contamination and limits the interactions with other plasma proteins. Nevertheless, the laborious chemical synthesis and high cost impede it being widely prescribed. Thus, heparin mimetics are developed in recent years to overcome these limitations of heparin and heparin derivatives. “Non-glycosamino” glycan analogs of heparins were reported in 1992 to reduce the synthetic steps of antithrombin III binding pentasaccharide.48 Glucose was used to substitute the glucosamine unit (Compounds 1-6, Figure 1.2), which simplified building block preparations, and avoided the need for selective N-sulfation. Furthermore, only acyl and benzyl esters were applied as temporary protecting groups for building blocks used in synthesis, which reduced the number of deprotection steps needed. Overall, the synthetic route was shortened to approximate 25 steps in comparison to ~50 steps for fondaparinux preparation. Idraparinux 4 was found as a potent pentasaccharide derivative, which showed inhibition of thrombin generation via both the extrinsic and intrinsic pathways. 1611 anti-Xa units/mg and a long in vivo half-life (9.2 hours in rats and 61.9 hours in baboons) were observed after intravenous (IV) and subcutaneous (SC) 4 administration with a linear pharmacokinetic profile. Idraparinux had been tested to prevent stroke in atrial fibrillation and venous thromboembolism (VTE). However, its antithrombotic activity cannot be reversed due to a lack of neutralizing agents. Idrabiotaparinux 7 (Figure 1.3) is a biotinylated Idraparinux derivative to bestow the reversibility with avidin, which has no reported toxicity and a very short pharmacokinetic half-life.49-52 The pharmacodynamic results from the Phase I study confirmed that by forming an avidin-idrabiotaparinux complex, avidin effectively reversed the anti-FXa activity of idrabiotaparinux.53, 54 Phase III clinical trial study of idrabiotaparinux for arterial fibrillation was halted because it did not show better results than standard long-term warfarin treatment for VTE.55 Figure 1.2. “Non-glycosamino” glycan analogs. 5 Figure 1.3. Idrabiotaparinux. To obtain more potent antithrombic drugs, Petitou et al. synthesized a series of “non- glycosamino” glycan heparin mimetics to reach full anticoagulant activity, including thrombin inhibitory properties.56 As discussed above, UFH binds with many plasma proteins causing significant side effects, particularly PF4. In this study, various lengths of oligosaccharides were designed to discriminate between thrombin and PF4. 6-mer to 14-mer 8-11 (Figure 1.4) did not display high antithrombin activities. The size-dependent increases in activities were observed for 16-mer (12) to 20-mer (14), with the 20-mer (14) being half as potent as UFH. To mimic the full anticoagulant activity of heparin, the structure of an oligosaccharide or even longer saccharide sequence would include an antithrombin-binding domain (A-domain) coupled to a thrombin- binding domain (T-domain). Another important issue is how to link these two domains. Repeating α and β-linked 3-O-methyl-2,6-di-O-sulfo-D-glucose units were attached to both the reducing and non-reducing ends as a thrombin-binding domain. Compounds 15-17 with T- domain at the non-reducing end were inhibitors of both factor Xa and thrombin. Thrombin inhibition was also size-dependent, and compound 17 was about twice more potent than heparin according to their IC50 values. With the A- domain at the non-reducing end and T-domain at the reducing end, 18-mer (18) showed 30 to 100 times weaker activities for thrombin inhibition than 6 the corresponding 17/19-mer saccharides 16 and 17 bearing the T-domain at the non-reducing end instead. All compounds 8-18 showed effective in vivo anti-FXa activities, and dose- dependent thrombin inhibition activities were observed for compounds 12-17. Even though compounds 15-17 inhibited both factor Xa and thrombin, the cross-reactivity with PF4 was hardly overcome, with the compounds failing to abolish the interactions with PF4. 7 Figure 1.4. A series of “non-glycosamino” glycan heparin mimetics. 8 To reduce PF4 binding, the charge of a molecule is an essential parameter for consideration to avoid the nonspecific binding. Compound 19 was designed with a pentasaccharide as A-binding domain and a hexasaccharide at the non-reducing end as the T domain, which was connected by a neutral methylated hexasaccharide. Both domains contain oligosaccharides that are shorter than 8-mer, the minimum unit required for PF4 binding.57 Compound 19 showed no significant interactions with PF4 even at high concentrations (100 ug/ml) and exhibited higher antithrombotic activity compared to heparin.56 SA123781A, compound 20, is another “non-glycosaminoglycan” synthetic heparin mimetics based on the strategy of charge reduction for polyanionic compounds, which is a structural variant of compound 19.58 SR123781A exhibited high affinity for human antithrombin and potent factor Xa and thrombin inhibition in multiple animal models for arterial-venous thrombosis. However, its development was halted during phase IIb clinical study due to the success of the LMWH.59 To display full anticoagulant activities, both antithrombin and thrombin binding domains are required to be present on the same polysaccharide chain. Glycoconjugate was designed to link both domains to facilitate the anticoagulant activity of the compound. Glycoconjugate 24 (Figure 1.5) comprises the antithrombin binding pentasaccharide, a linear spacer, and a persulfated maltotrioside thrombin binding region.60 Polysulfated maltotrisaccharide 1-amino-hexaethylene glycol derivative (21) was prepared for thrombin binding, and a thioacetyl-substituted spacer was linked to a pentasaccharide as the antithrombin binding domain 22. Sulfosuccinimidyl (4-iodoacetylamino) benzoate (sulfo-SIAB) (23) was applied to connect 21 with 22 to form glycoconjugate 24, which has the similar length as a heparin 18-mer oligosaccharide. In order to study the binding requirement for thrombin, cellobiose heptasulfate and multipennate hexadecasulfate were attached to synthesize 9 antithrombin binding pentasaccharide (25, 26). Symmetric pentasaccharide glycoconjugates were also prepared with various sulfation patterns. All the compounds showed good to potent in vitro anti-FXa and antithrombin inhibitory activity, and higher antithrombin activities were observed with the glycoconjugate 26 bearing higher charges. However, the binding with PF4 was not completely avoided. 10 Figure 1.5. Maltotrioside 21, antithrombin binding domain 22, sulfo-SIAB 23, and glycoconjugates 24, 25 and 26. 11 Glycopolymers with well-defined pendants have been developed in recent years. Free radical polymerization and copolymerization of glucosyloxyethyl methacrylate (GEMA) were reported for the preparation of highly water-soluble polymers.61, 62 Akashi and colleagues utilized the poly(GEMA) 27 with pendant glucoside residue to synthesize poly(GEMA) sulfate 28 (Figure 1.6). The degree of sulfation in 28 varied from 1.91 to 3.75 per sugar according to the amount of DMF/sulfur trioxide complex added and the reaction time.63 The anticoagulant activity of poly(GEMA) sulfate polymer 28 was tested, which showed anticoagulant activity with prolonged coagulation time. However, the anticoagulant effect was about 180 times weaker than that of heparin.64 The Hsieh-Wilson group utilized ring-opening metathesis polymerization (ROMP) to generate heparin glycopolymers 29 and 30 carrying disaccharide units (Figure 1.7).65 Anti-FXa and anti-FIIa activities of these glycopolymers were measured via chromogenic substrate assays.66 Impressively, glycopolymer 29-45 (45-mer) showed high potency against FIIa and FXa, with activities 100 fold higher than those of UFH or LMWH. The lack of 3-O-sulfation on 30-155 (155-mer) led to significantly attenuated FXa and FIIa inhibition reaffirming the importance of 3-O-sulfation. However, the anticoagulant activity of 29-45 was neutralized by the addition of PF4.65 Interestingly, the anti-FIIa activity of glycopolymer 29-30 (polymer with an average length of 30 monomers) was only partially neutralized by PF4, suggesting that the PF4 reactivity can be reduced by modulating the length of the glycopolymer. Figure 1.6. Structures of poly(GEMA) 27 and poly(GEMA) sulfate 28. 12 Figure 1.7. Heparin-based glycopolymers 29 and 30 prepared through ROMP. 1.3 Heparin Mimetics as Growth Factor Binders FGFs and vascular endothelial growth factor (VEGF) initiate cell signaling pathways, and regulate cell proliferation and angiogenesis by forming ternary complexes with heparin/HS and cell surface receptors.67 These growth factors were also found sequestered by HS in the ECM. Therefore, reducing heparin and HS binding with these growth factors by heparin mimetics has been investigated in recent years as a novel approach to block cell proliferation. Parish and coworkers reported structurally well-defined cyclitol-based heparin mimetics with a wide variety of linker chain lengths and extent of sulfation.68 The sulfated tetrameric cyclitols mimicked heparin disaccharide units linked through a 2 to 8 carbon atom spacer (Figure 1.8). Growth factors exhibited distinct binding patterns with various spacer lengths. Cyclitols with a short spacer of 2 carbon atoms (31) effectively bound with FGF-1 but poorly inhibited FGF-2 and VEGF. Compound 32 with a longer linker possessed highest inhibitory activity for the latter two growth factors. This study indicated subtle changes in the spacer could affect the binding ability toward proteins and supported the idea that different domains of heparin account for the binding of various proteins. 13 Figure 1.8. Cyclitol-based heparin mimetics 31 and 32. A 6-azido-6-deoxy-α-D-mannopyranoside was employed as a heparin mimetic core structure template to investigate the binding affinity and selectivity against growth factors FGF-1, FGF-2, and VEGF. Preliminary binding studies showed 2,3-disulfated mannoside has a similar binding ability with FGF-1 and VEGF as a trisulfated monosaccharide.69 Therefore, [3+2] copper-catalyzed alkyne azide cycloaddition and Swern oxidation-Wittig reactions were applied to further decorate the 2,3-disulfate template by selected hydrophobic or polar groups. An 18- membered library, including the lead compound 33, was successfully prepared (Figure 1.9).70 All the mimetics had good binding affinity towards FGF-1 and VEGF and selectivity over FGF-2. The attachment of phenyl groups improved the binding to FGF-1 and VEGF, and an extra anionic group such as carboxylate could enhance binding. Compound 33 with a trifluoromethyl substituted aromatic linker exhibited the best binding affinity with FGF-1 and VEGF (KD = 84 and 49 uM, respectively) and good selectivity over FGF-2 (29- and 51-fold, respectively). Ugi four-component condensation reaction was used for a monosaccharide library preparation to introduce additional hydrophobic groups (Figure 1.10).71 The library was used to examine the effect of the hydrophobic groups on binding with growth factors (FGF-1, FGF-2, and VEGF). Surface plasmon resonance (SPR) binding assay showed the compounds with either an aromatic group or an extra negative charge is preferred over an aliphatic group for increased affinity with 14 growth factors, especially FGF-2 and VEGF. Figure 1.9. Compound 33 synthesized via the click reaction. Figure 1.10. Representative heparin mimetic compounds synthesized via the Ugi reaction for growth factor binding. Nontoxic polyanionic compounds were developed to inhibit FGF-2 induced biological activities.72 RG-13577 is a synthetic poly-4-hydroxyphenyl acetic acid aromatic compound with an average of 5,000 molecular weight. Effect of RG-13577 on FGF-2 binding was tested in competition with the binding of heparin to FGF-2, and 50% competition was observed at 3 ug/ml. Heparin-induced FGF receptor dimerization and formation of a ternary FGF-2-FGFR-heparin complex are essential steps in FGF-2 signaling.73 Compound RG-13577 alone did not promote the binding between FGF-2 and soluble or cell-surface FGFR1 and abrogated heparin-mediated dimerization of FGF-2 and FGFR1 at 10 ug/ml. In the presence of 5–10 ug/ml compound RG- 13577, vascular endothelial cell proliferation was dramatically inhibited. Sulfated peptide combinatorial libraries are another novel polyanionic structures that mimic heparin, and its biologic activity toward FGFs.74 Peptides were chosen as templates for heparin mimetics due to the ease in accessing a range of peptide structures via automated solid- 15 phase peptide synthesis. The structures can be readily modified by split–pool synthesis to create combinatorial peptide libraries. O-Sulfation of heparin was mimicked by sulfated serine, threonine, and hydroxyproline residues, and N-sulfated amino acid resembled N-sulfation of heparin. The sulfated peptide library (240,000 members) was screened with fluorescence-labeled FGF-1 to pinpoint heparin mimetic candidates. Eight peptides were identified and further investigated by SPR. However, only 2 out of 8 peptides showed low micromolar IC50 value of FGF-1 binding in competition assays against heparin immobilized on the sensor chip. To improve binding with growth factors, multivalent constructs of heparin oligosaccharide such as dendrimers have been investigated. Hexasaccharide 36 could bind strongly with FGF-2, as demonstrated by microarray analysis (Figure 1.11).25 Disaccharide 37 and monosaccharide 38 also exhibited binding with FGF-2. The Seeberger group synthesized glycodendrimers incorporating synthetic heparin analogs (36-39) to improve the interactions with FGFs. FGF-2 was incubated with heparin glycodendrimers for competitive microarray assay against immobilized heparin. Dendrimers 41 and 42 (IC50 = 43 and 165 uM respectively) showed weaker inhibitory activities than 40 (IC50 = 1.4 uM), but remarkably better than the monovalent counterpart 37 and 38, indicating the increased avidity associated with the multivalent dendrimers. The binding results were confirmed by SPR experiments. In order to validate the performance of heparin dendrimers in the heparin-mediated biological process, dendrimer 40 was tested for the ability to mediate FGF-FGFR complex formation, which successfully activated FGF-2-mediated signaling in L6 myoblasts.75 16 Figure 1.11. Structures of heparin oligosaccharides and heparin glycodendrimers. 17 Besides dendrimers, glycopolymers have been used to enhance FGF binding. The Godula group reported synthetic neoproteoglycans (neoPGs) utilizing the poly(acrylamide) scaffold and disaccharides prepared by HS depolymerization.76 A library of tetramethylrhodamine (TAMRA)-labeled 12 disaccharides and 5 monosaccharide glycopolymers were successfully generated. The trends of structure dependence of FGF-2 binding obtained from microarray screening were consistent with those previously reported for HS.24 2-O-sulfations on disaccharide motifs were required for FGF-2 binding, and neoPGs 46 was the best FGF-2 binder within mimetics 45-50 (Figure 1.12). In order to introduce neoPGs to the mouse embryonic stem cells (mESCs), phospholipid tail and Alex Fluor 488 (AF488) tag were installed for membrane insertion and imaging. With neoPGs inserted on cell membrane, increased FGF-2 binding was observed with mESCs remodeled with 49, while NeoPGs 48 and 50 failed to bind FGF-2 on the cell surface. To confirm FGF-2 binding, a growth factor stimulation assay was performed. The cells remodeled with 47 successfully formed neural rosettes in neural monolayer differentiation experiments.77 Furthermore, several kinases isolated from these mESCs showed significantly increased degree of phosphorylation by Western blot suggesting FGF-2 indeed facilitated signaling in cells. 18 Figure 1.12. A library of neoPGs 45 - 50. While current glycopolymers with well-defined saccharide moieties and glycomimetic clusters of heparin provided multivalency in receptor binding, these compounds do not mimic the natural linear connection in heparin. A new generation of linear mimetics was designed using modified GlcN monosaccharide (compounds 51 – 57) (Figure 1.13).78, 79 A serine was added to GlcN to mimic the uronic acid residue. The oligomers without sulfate groups and those with 6-O and 3-O sulfate groups on the GlcN moiety were synthesized through amide bond coupling. The interactions of these compounds with FGF-2 were performed by SPR, and results showed higher binding affinity with increased chain length and 3-O-sulfation. Compounds without sulfate groups and short 6-O-sulfated oligomer exhibited little interactions with FGF-2, and stronger binding was observed with 3-O-sulfation and longer chain length. Overall, the binding affinities were still modest with the most potent compound 57 having a KD value of 448 uM. 19 Figure 1.13. Linear heparin mimetics with glyco-amino acid oligomers 51-57. 1.4. Heparin Mimetics as Heparanase Inhibitors With their presence on cell surface and in the ECM, heparin and HS can function as a reservoir of growth factors, chemokines, receptors and lipoproteins.16, 80 Moreover, by binding with ECM components such as laminin, fibronectin, and collagens I and IV, heparan sulfate linked to proteins, also known as heparan sulfate proteoglycans, contribute to the structural integrity of the ECM and basement membrane, and are involved in cell survival, proliferation, and migration.81 Heparanase is the sole endoglycosidase degrading HS side chains of heparan sulfate proteoglycans. Due to its impact on cancer, inflammation, and other disease processes, heparanase has been a potential target for anti-cancer therapeutics. Heparin shares the same polysaccharide backbone structure as HS, which makes it the potential heparanase inhibitor candidate. While heparin exhibited potent anti-heparanase activity, its anticoagulant property restrains its clinical use as heparanase inhibitors. Heparin mimetics without any anticoagulant activities are considered as potential therapeutic drugs for cancers. PI-88 (58), a mixture of highly sulfated phosphosulfomannan, was isolated from the 20 yeast Pichia holstii NRRL Y-2448 (Figure 1.14). PI-88 contains heterogeneous oligosaccharides ranging from di- to hexa-saccharides, with major components being tetra- (~30%) and penta- saccharides (~60%).82 PI-88 showed 2 ug/ml heparanase inhibitory IC50 value, which is comparable to that of heparin (1 ug/ml). Besides its inhibition of heparanase, PI-88 inhibited angiogenesis by abolishing the interactions between growth factors and their receptors.83-85 In vivo study of PI-88 showed that it was effective in inhibiting tumor growth, metastasis, and angiogenesis.83 PI-88 also minimized the malignant cell load in rodent models of human myeloid leukemia.86 In addition to heparanase, Hsulf-1 and Hsulf-2, two human extracellular endoglucosamine 6-sulfatases, which are upregulated in several types of cancers, are inhibited by PI-88 in a concentration-dependent manner.87 However, a phase III study for hepatocellular carcinoma has been terminated in 2015 due to a lack of significant improvements in disease-free survival compared to other treatment methods suggesting further development of PI-88 like mimetics is needed. Figure 1.14. PI-88, a heparin mimetic with heparanase inhibitory activities. PG 500 is a library of synthetic, fully sulfated, and anomerically pure oligosaccharides with the reducing ends modified by various lipophilic aglycones.88 PG500 compound series were reported as dual inhibitors of heparanase and angiogenesis via inhibition of the enzyme and growth factors. They exhibited low nanomolar binding with FGF-1, FGF-2, and VEGH through 21 the BIAcore binding assay. Both tube formation and rat aortic in vivo assays indicated the antiangiogenic activity of PG500 compounds. B16 solid tumor and metastatic model studies also showed the inhibition of tumor development by PG500. Based on the biological results and accessibility of the compounds, a member of the PG500 series, PG545 (59) (Figure 1.15), had been selected as the lead clinical candidate for further research.88 The Hammond group reported the inhibition of tumor growth and metastasis in the orthotopic 4T1 breast cancer model by PG545.89 Recently, PG545 was reported to attenuate the growth of patient-derived lung cancer xenografts from fifteen patients.90 Phase I clinical trial (NCT02042781) demonstrated the safety and tolerability of PG545 in patients with advanced solid tumors.91 Figure 1.15. Structure of PG545. Oligomannurarate sulfate (JG3) (60) are derived from sodium alginate, ranging from tetra- to deca-saccharides (Figure 1.16).92 JG3 inhibited heparanase activity by binding to the KKDC and QPLK domains of the heparanase molecule. In vitro studies showed the inhibition of heparanase derived from NIH 3T3 and MDA-MB-435 human breast cancer cells by JG3. In an ex vivo aorta sprout outgrowth assay, 100 ug/ml JG3 inhibited 77% microvessel growth, and the antiangiogenic property of JG3 was confirmed in an in vivo chorioallantoic membrane model and the direct binding with FGF-2. JG3 modulated tumor angiogenesis and metastasis.92 Moreover, it inhibited nuclear factor kappa B (NF-KB) transcription factor activation, which is aberrantly 22 regulated in cancer development and involved in chemotherapy resistance. The inhibition mechanism is attributed to antagonizing the doxorubicin triggered Ataxia-telangiectasia-mutated kinase (ATM) and the successive MEK/ERK/p90Rsk/IKK signaling pathway. Thus, JG3 could be a potential chemotherapy sensitizer and NF-KB inhibitor.93 Figure 1.16. Structure of JG3. Suramin (61), a polysulfated naphthylurea, has been tested to treat acquired immunodeficiency syndrome (Figure 1.17). Suramin was investigated as a heparanase inhibitor due to its potential inhibitive activity of glycosaminoglycan catabolism. Melanoma-derived heparanase was completely inhibited by suramin at ~100 uM (IC50 = 46 uM), which was more potent than similar-sized oversulfated heparin tetrasaccharide. B16 melanoma cell invasion was effectively inhibited by suramin as well.94 Suramin inhibited the human ovarian and cervical cancer cell growth with modest potencies (IC50 values around 350 ug/ml) and significantly downregulated heparanase expression.95 However, suramin had been associated with multitoxicity at the therapeutic concentration in patients,96-98 which led to the disapproval of its use by the FDA. Several suramin analogs have been reported to address the toxicity issue.99, 100 23 Figure 1.17. Structure of suramin. Glycopolymers that could mimic the multivalent property of HS for heparanase inhibition have been reported by the Nguyen group.101 [GlcNS(6S)α(1,4)GlcA] disaccharide unit was applied on diantennary monomer 62 (Figure 1.18) with a carboxylate group on the scaffold. The monomer 63 served as a control to shine light on the effect of the carboxylate group on heparanase inhibitory activity. ROMP was applied to form glycopolymers from monomers 62 and 63. Degrees of polymerization (DP = 5-12) were controlled by fine-tuning the amount of Grubbs third-generation catalyst (9-20 mol%). The heparanase inhibitory activities were measured by TR-FRET assay against fluorescently tagged HS.102 Monomer 62 exhibited ~4.3- fold higher inhibition than monomer 63, suggesting the additional carboxylate moiety can enhance inhibition. While the polymer backbone did not show any heparanase inhibitory activity, the polymer of monomer 62 (n = 12) had stronger heparanase inhibition (IC50 = 0.10 ± 0.036 nM) than heparin (IC50 = 0.54 ± 0.028 nM). To investigate the effect of local saccharide density, an additional N- and O- sulfated GlcN unit was placed generating the diantennary monomer 64. ROMP of diantennary monomer 64 afforded its corresponding glycopolymer (DP =8). The diantennary polymer showed similar activity against heparanase as the polymer from monoantennary monomer (DP = 9). The lack of further enhancement with the diantennary polymer could be a result of the steric crash and electronic repulsion of the additional GlcNS moiety based on docking study results.103 The glycopolymer with 12 repeating monomer 62 has the most potent heparanase inhibitory activity. Next, the binding of this glycopolymer was 24 investigated with a variety of other potential targets for heparin interactions through biolayer interferometry (BLI). The glycopolymers showed very low binding affinity to growth factors (FGF-1, FGF-2, and VEGF) and PF4, suggesting the high selectivity of the glycopolymer as heparanase inhibitors. The glycopolymer was effective in reducing cancer metastasis when evaluated in a mouse 4T1 breast cancer model, demonstrating its high translational potential. Figure 1.18. Diantennary and monoantennary monomers 62, 63 and 64. In order to investigate the relationship between the sulfation pattern on the pedant disaccharides and their biological functions, a systematic study on sulfation patterns of glycopolymers was reported.104 Structurally, the GlcN unit in the disaccharide module may be the key for heparanase recognition. Disaccharide motifs bearing different sulfation patterns on GlcN were designed (65-70) (Figure 1.19). The correlation between sulfation patterns of the GlcN and heparanase inhibition had been investigated. 6-O-sulfation was found crucial for heparanase inhibition, as removal of 6-O sulfate (65 vs 66) drastically reduced the inhibition. The addition of 3-O-sulfate was detrimental to heparanase inhibition. Replacement of N-sulfate with acetamide or ammonium (compounds 68 and 69) did not significantly affect the inhibitory activities. 25 Figure 1.19. Disaccharide motifs 65-70 with various sulfation patterns used for glycopolymer construction. Recently, Turnbull and coworkers employed multivalent single-entity heparin sulfate dendrimers to enhance their heparanase inhibitory activities (Figure 1.20).105 Since N-sulfated heparin oligosaccharide dendrimer did not exhibit high potency in heparanase inhibition (IC50 ~ 1 uM), glucose and maltose with higher sulfation levels were chosen as dendritic pendants with various dendritic core length. Glucose tetramers moderately inhibited heparanase with IC50 values over 1 uM. In comparison, maltose tetramers, especially “short-armed” ones such as 74 and 75, showed similar potencies (IC50 = 11 and 23 nM respectively) as PG 545 (IC50 = 8 nM) as the positive control. The glycodendrimer 75 significantly reduced tumor growth in a xenograft mouse model with human myeloma cells (88.5% inhibition at 4 weeks). The dendrimers 74 and 75 also showed potential inhibitory activity against angiogenesis. Importantly, unlike PG 545, the glycodendrimers exhibited little anticoagulant capabilities, reducing the potential concerns of anti-coagulation side effects, a hurdle for clinical applications as heparanase inhibitors. 26 Figure 1.20. Dendrimer glycomimetics 71-76 for heparanase inhibition. 1.5. Conclusions Heparin, a well-known anticoagulant drug, has been widely prescribed. However, in some patients, heparin can induce life-threatening side effects such as HIT. The heparin contamination crisis in 2007 and 2008 has further stimulated the interests in discovery of more structurally defined heparin compounds for biomedical applications. LMWH has improved pharmacokinetic profiles, but its heterogeneity and the reliance on animal source for heparin are still concerns for wide applications. Fondaparinux, a synthetic heparin oligosaccharide of defined structures, demonstrated the feasibility of obtaining pure heparin structures through organic synthesis. However, the long synthesis needed (around 50 steps) to prepare fondaparinux hinders its wide applications. To expedite biomedical applications, heparin mimetics have been investigated as attractive substitutes of native heparin, with some of the innovative design and applications of heparin mimetics summarized in this chapter. Three major types of biological functions have been discussed, which include anti-coagulants, growth factor binding, and heparanase inhibition. 27 For anti-coagulation applications, “non-glycosamino” glycan analogs exemplified by idraparinux 4 have been designed, which contains glucose moiety in lieu of the glucosamine unit. As a result, the synthesis was significantly shortened with idraparinux taking about 25 steps to prepare, half of those required for fondaparinux. At the same time, these synthetic analogs can maintain potent anti-coagulant activities. Furthermore, structural features can be built into these mimetics to recruit further factors involved in anti-coagulation and to modulate neutralization of the compounds if necessary. Growth factors play important roles in regulating cell proliferation and angiogenesis. A variety of sulfated glycans and cyclitols have been investigated for growth factor binding. To expand structural diversity, multicomponent Ugi reaction has been utilized to introduce additional structural features such as hydrophobic groups and anions into the mimetics, which can help enhance growth factor binding. Furthermore, with the ease in automated solid-phase synthesis, sulfated peptide libraries were screened for growth factor binding leading to binders with modest affinity to FGF-1. To enhance the avidity towards growth factors, glycopolymers and glycodendrimers bearing sulfated mono- or oligo-saccharides have been prepared. These mimetics can present sugar moieties in a multivalent manner, which can be used to modulate heparin-protein interactions, including the regulation of embryonic stem cell differentiation. Heparanase plays important roles in cell survival, proliferation and migration. Heparin is a potent heparanase inhibitor, which can potentially inhibit tumor growth and metastasis. Nevertheless, the anticoagulant activities of heparin impede its clinical use as heparanase inhibitors. A variety of sulfated glycans including phophosulfomannan PI-88, hydrophobic aglycon bearing PG500 series, oligomannurarate sulfate JG3, as well as polysulfated naphthylurea suramin have been investigated. However, they have relatively modest efficacy 28 and/or potential toxicities, which need to be improved to enable further clinical evaluations. Glycodendrimers and glycopolymers bearing sulfated mono- and di-saccharides have shown promising heparanase inhibitory activities with potency (IC50 < 1 nM) of some compounds higher than that by native heparin. Furthermore, the structures of these mimetics can be tuned to avoid the interactions with other heparin-binding proteins including growth factors, and blood coagulating factors. When evaluated in mouse tumor models, these novel mimetics could significantly reduce tumor development, thus providing promising leads for further development. Compared to native heparin, heparin mimetics can be synthesized with more homogeneous structures. While the field has witnessed great progress, there are still some limitations with current heparin mimetics. Native heparin contains sulfated and non-sulfated domains connected linearly. Current mimetics have not been able to approach the structural complexity and diversity present in heparin yet. For example, glycopolymers and glycodendrimers are only constructed in a branched fashion. Linearly linking the sulfated and non-sulfated modules may more closely mimic the native heparin structures. With the great advances achieved in synthesis, especially chemoenzymatic synthesis of heparin like oligosaccharides, more sophisticated heparin mimetics will be sure to be prepared in the near future to aid in more thorough understanding of the structure-activity relationship of heparin and the development of advanced agents for biomedical applications. 29 Chapter 2. Chemical Synthesis of Heparin Like Head to Tail Multimers 2.1. Introduction Heparin is a highly sulfated, complex linear polysaccharide that belongs to the family of glycosaminoglycans (GAG). It is composed of disaccharide repeating units consisting of D- glucosamine (GlcN) and α-(1-4)-linked to a uronic acid (90% L-iduronic acid (IdoA) and 10% D- glucuronic acid (GlcA)).106 The most common disaccharide unit includes three sulfate groups, which makes heparin one of the biological macromolecules with the highest density of negative charges. 2-OH of GlcA and IdoA residues may be sulfated. The glucosamine monosaccharide can be either N-acetylated (GlcNAc) or N-sulfated (GlcNS), while O-sulfation normally occurs at 6-OH and/or 3-OH. Due to the heterogeneity and variability of its structure, heparin can interact with a wide variety of proteins.107 Heparin has been the most widely used anticoagulant drug in the world since the 1930s. Heparin exerts its anticoagulant activities by binding with serine protease inhibitor antithrombin III (AT III), resulting in a conformational change and facilitating its interaction with thrombin and activated factor X (FXa). A unique pentasaccharide sequence with 3-O-sulfated GlcN is the minimum requirement for FXa inhibition. On the other hand, heparin fragments with at least 14-18 saccharides long can inhibit the action of thrombin.108-111 The heparin-AT III interaction is the most thoroughly studied among heparin- binding proteins. However, the interpretation of detailed structure-activity relationships has been stymied by the chemical complexity of heparin polysaccharide. Heparin chains are elongated and modified in the Golgi compartment of the mast cell with alternating addition of glucuronate and N-acetyl-glucosamine residues to form a linear polymer, which is attached to a serine residue in a core protein. A series of modifications of 30 heparin backbone begin with N-deacetylation/N-sulfation of the N-acetylglucosamine in the presence of 3’-phosphoadenosine 5’-phosphosulfate (PAPS), followed by C5-epimerization of GlcA to IdoA and stepwise O-sulfations of the component sugars.112 A total of 12 enzymes is involved in heparin biosynthesis. However, the sulfation and epimerization of the backone are often incomplete, accounting for the heterogeneity of heparin. The molecular weights of commercially available heparins could range from 7,000 to 25,000 Da. The availability of large heparin oligosaccharide libraries is an important tool in understanding the interactions of heparin and a plethora of proteins. The libraries of well-defined heparin/heparan sulfate (HS) di- and tetra-saccharides have been built through chemical synthesis by the Hung and Boons groups.24, 113 The structure-activity relationships between heparin and binding proteins, especially fibroblast growth factors (FGFs), have been revealed. However, disaccharide and even tetrasaccharide may not represent full binding epitopes. Extended heparin-based libraries are needed to establish more extensive structure and activity relationship.114 Building libraries with longer heparin oligosaccharides is hampered by tedious and challenging heparin chemical syntheses. Synthesis of the most common trisulfated heparin disaccharide takes about 20 steps from monosaccharide building blocks. Thus, efficient strategies that can quickly elongate the heparin sequence and provide a variety of sulfation patterns are required. To achieve this goal, we developed a novel “head-to-tail” mimetics design based on well-defined disaccharide modules containing various sulfation patterns. Instead of glycosidic linkage, the disaccharide modules were connected from the reducing end to the non- reducing end. The deprotection and sulfation sequences are applied to disaccharide modules instead of longer oligosaccharides, which significantly reduces the complexity of chemical synthesis. Furthermore, the disaccharide modular approach permits the easy access to different 31 sulfation patterns on each disaccharide module of pseudo-oligosaccharides and facile preparations of long pseudo-oligosaccharide sequences. Here, we report the synthesis of a library of 27 heparin pseudo-hexasaccharides through the “head-to-tail” strategy, and binding studies with FGF-2 confirm that the pseudo- hexasaccharides can mimic the natural heparin oligosaccharides. With 23 different members, the FGF family is involved in cell proliferation, migration, differentiation, morphogenesis, and angiogenesis.115, 116 The most extensively investigated members of FGF family are FGF-1 and FGF-2. High-resolution X-ray crystal structure of a dimeric 2:2:2 FGF-FGFR-heparin ternary complex explained the dual role for heparin in augmenting FGF-FGFR binding and promoting FGFR dimerization.73, 117-119 The heparin pseudo-oligosaccharide library will provide insights into structure-function studies. 2.2. Synthetic Challenges in Preparation of Disaccharide Modules In order to build our heparin mimetics library, we first investigated a modular strategy with ketone and hydroxylamine-functionalized heparin tetrasaccharide modules (Figure 2.1). The tetrasaccharide modules could be linked through chemoselective oxime ligation between the hydroxylamine bearing non-reducing end module with the ketone moiety on the elongation sequence (Scheme 2.1).120 The pseudo heparin oligosaccharides generated could be further elongated by repeating TFA deprotection and oxime formation sequence. The pseudo oligosaccharides mimic the natural linear connection of heparin, and the sulfation pattern could be readily modified on individual modules to achieve great diversity in structures. Moreover, the modular strategy significantly reduced the complexity of oligosaccharide synthesis. Partial 32 deprotection and sulfation could be performed on the short modules rather than the long final oligosaccharides, leading to higher overall yields. Furthermore, the chain length of the mimetics would be readily elongated. Figure 2.1. Target tetrasaccharide modules. 33 Scheme 2.1. “Head-to-tail” strategy via chemoselective oxime ligation. To obtain the tetrasaccharide modules, we designed a [2 + 2] synthetic route. 2-Amino- 2-deoxy-D-glucopyranose (GlcN) and L-idose were applied for disaccharide formation. Methallyl group was installed at the 4-O position of the GlcN 1, which could be oxidized to ketone for oxime synthesis. 6-O-Levulinoyl (6-O-Lev) of idose 2 would be selectively removed, and the remaining primary alcohol would be oxidized to carboxylate after completion of the chain assembly. However, the preactivation-based glycosylation121-123 failed to generate the desired disaccharide product in high yields. We observed decomposed donor 1, and most receptor 2 was recovered (Scheme 2.2). Then, we investigated glucosamine donors with different protective groups at 4-OH. The methallyl group was oxidized to a vicinal diol by treatment with osmium tetroxide and 4-methylmorpholine N-oxide, which was subsequently protected by Ac (5) or tert-butyldimethylsilyl (TBS) group (6). The diol was also converted to a ketone (4) under sodium periodate oxidation condition. Unfortunately, all the glycosylation reactions with 4-6 yielded less than 10% of the desired disaccharides with recovered acceptor 2 34 and decomposed donors (Scheme 2.3). Since various donors were tested, we assumed the failure was attributed to the electron-withdrawing property of the Lev group, which reduced the nucleophilicity power of acceptor 2. Scheme 2.2. Failed glycosylation with idose acceptor 2. Scheme 2.3. Glycosylation with various donors. For the purpose of increasing the nucleophilicity of idose acceptor, we chose to replace the 6-O-Lev with an electron-donating group, such as p-methoxylbenzyl (PMB).124 The donor 5 was activated by p-TolSCl/AgOTf at -78 oC and followed by the addition of 2,4,6-tri-tert- butylpyrimidine (TTBP) and acceptor 7. The consumption of acceptor 7 was indicated by TLC after the reaction was warmed up to room temperature. 1,6-anhydro disaccharide 8 was formed as the sole product. We proposed that the PMB group increased the nucleophilicity of 6-O on the idose. The glycosylation was carefully monitored with a steadily raised temperature. The 35 disaccharide 8 started to form at -40 °C, and most of the desired product 9 was converted to 8 when the temperature reached 0 °C. Finally, the desired disaccharide 9 was successfully produced in 68% yield when the reaction temperature was kept at -78 °C with recovered acceptor 7 (~20%) (Scheme 2.4). Scheme 2.4. Preparation of disaccharide 9. With the desired tetrasaccharide in hand, deprotection and sulfation were carried out. However, we found the hydroxylamine group might not survive under Pd/C catalyzed hydrogenation conditions, typically utilized to remove benzyl protective groups.125 Disaccharide 10 was used as a model to test the stability of the hydroxylamine group. The O-N bond was found to be cleaved before all the O- and N-benzyl (Bn) were removed (Scheme 2.5). Next, disaccharide 10 was treated with H2 in DMSO/TEA/IPA mixture, which was reported as the reduction condition for N-aryl hydroxylamines synthesis from nitroaromatics.126 We observed significant O-N bond cleavage byproduct 11 along with incomplete Bn removal. 36 Scheme 2.5. Hydroxylamine bond cleavage under hydrogenation conditions. With the aim of solving the hydroxylamine stability issue and reducing the complexity of synthesis, we started to prepare disaccharide modules instead of tetrasaccharides and attempted to install hydroxylamine after hydrogenolysis. After we had disaccharide 12 in hand, Ac and benzoyl (Bz) groups were removed by sodium metal in MeOH. The diol at the non- reducing end was protected by benzylidene acetal initially and followed by O-sulfation and hydrogenolysis to afford compound 13 (Scheme 2.6). Scheme 2.6. Disaccharide module deprotection sequence. Next, the hydroxylamine was installed by the amidation reaction. The free amine at the reducing end of disaccharide 13 coupled with p-nitrophenol activated ester 14 to generate disaccharide 15 successfully (Scheme 2.7). Unfortunately, TFA protecting group could not be removed from 15 in high yields under either saponification or NaBH4 conditions. A major byproduct was isolated with the amide bond on the linker cleaved. We assumed the electronics of hydroxylamine stabilized the TFA protective group, rendering it challenging to selectively remove the TFA. 37 Scheme 2.7. Hydroxylamine group installation. Since TFA selective deprotection was not feasible, 9-fluorenylmethoxycarbonyl (Fmoc) was explored as an alternative to TFA protective group (Scheme 2.8). The free amine bearing disaccharide 13 was treated with Fmoc protected hydroxylamine NHS ester 16, which led to the desired Fmoc protected disaccharide 17. Scheme 2.8. Synthesis of Fmoc protected hydroxylamine disaccharide 17. The Fmoc protecting group was removed by piperidine, and the tetrasaccharide 18 was successfully prepared by oxime formation via coupling between hydroxylamine and ketone from disaccharide modules under HOAc conditions. Nevertheless, the ketoxime group was not stable under [4 + 2] hexasaccharide formation. Besides the desired hexasaccharide (20) (< 20% yield), a mixture of tetrasaccharides (21, 22) was generated (Scheme 2.9). G-15 gel column failed to separate the hexa- from tetra-saccharides due to the similar molecular weight of the compounds. 38 To overcome this issue, we attempted to reduce the ketoxime of pseudo tetrasaccharides. We investigated various ketoxime reduction conditions. Unfortunately, neither NaBH4 nor NaCNBH3 could afford the desired reduced product as the major component in the reaction mixture. The reductive reactions were sluggish under low concentrations of HOAc (< 20%), but higher concentrations resulted in ketoxime bond cleavage. Furthermore, acetone adduct to hydroxylamine was a persistent byproduct of oxime formation reactions presumably due to trace contamination of acetone in the reaction mixture. Scheme 2.9. Hydrolysis of ketoxime under pseudo-hexasaccharide formation condition. Since the ketoxime formation has become a difficult hurdle for pseudo hexasaccharide synthesis, we next tried reductive amination reaction between ketone 13 and amine 23 to connect disaccharide modules.127 Unfortunately, the desired tetrasaccharide was not produced as the major product. Under both NaBH4 and NaCNBH3 conditions, ketone was mostly reduced to an 39 alcohol. NaBH(OAc)3 was reported for ketone reductive amination reaction in DCE or THF.128 Nevertheless, the disaccharides (13, 23) could dissolve in neither of the solvents with, no desired products generated. The solubility issue was resolved by using DMF. Unfortunately, no reactions occurred and the disaccharides were recovered (Scheme 2.10). Scheme 2.10. Failure of reductive amination reaction to link the amine bearing disaccharide module and a ketone bearing disaccharide. Since the ketoxime formation has become a difficult hurdle for pseudo hexasaccharide synthesis, we switched to reductive amination reactions with an aldehyde at the reducing end of disaccharide modules. However, similar to the results for ketone reactions, the aldehyde was partially reduced under acidic conditions. Oxime formation and reductive amination failed to give us our target pseudo hexasaccharides. These difficulties prompted us to shift our focus to amide bond formation. The 40 carboxylate group was introduced at the non-reducing end of disaccharide 26 for HATU mediated coupling with amine 25. In addition to the desired pseudo tetrasaccharide 27 (<10%), the six-membered lactone 28 was also formed as the major byproduct during the HATU coupling reaction (Scheme 2.11). Scheme 2.11. Lactone formation from carboxylic acid 26 during HTAU mediated amide formation reaction. 2.3. Monosaccharide Building Block Preparation To overcome the lactone formation, we envisioned that if we can extend the carboxylic acid linker by one more carbon, the seven-membered ring would be more difficult to form than the six-membered lactone, thus favoring the desired amide product formation. To fulfill this idea, we started with GlcN donor modification. 4-Methoxybenzylidene acetal was utilized to protect 41 the 4-O and 6-O positions of compound 29, and the 3-O was protected by the Bn group. NaCNBH3-mediated reductive 4-methoxybenzylidene 4-O-ring opening afforded compound 30. Allyl group was installed at 4-O position of 30 followed by a hydroboration-oxidation reaction to obtain the primary alcohol linker. The alcohol was then oxidized to a carboxylate group, which was subsequently protected as a benzyl ester. The 6-O-PMB protecting group was replaced with Fmoc as a potential O-sulfation site to form the GlcN donor 31 (Scheme 2.12). Scheme 2.12. Glucosamine donor synthesis. 6-OH on idopyranosyluronate 32 was oxidized by a catalytic amount of 2,2,6,6- tetramethyl-1-piperidinyloxy (TEMPO) with bis(acetoxy)iodobenzene (BAIB), followed by methyl ester formation to afford idopyranosyluronate 33. Glycosylation of 33 with N-Cbz and N- Bn protected ethanolamine linker provided α product in 87% yield. The stereochemistry was confirmed by one-bond coupling constants between the anomeric carbon and hydrogen atoms (1JCH = 172 Hz). 2-O-Bz conferred better neighboring group assistance than 2-O-Fmoc, which only afforded α/β stereoselectivity in 3:1 ratio. 2-O-Bz was removed by NaOMe in DCM and 42 MeOH cosolvent system followed by 2-O-Fmoc protection and 4-O-PMB deprotection to provide idopyranosyluronate acceptor 34 (Scheme 2.13). Scheme 2.13. Idopyranosyluronate acceptor 34 preparation. 2.4. Synthesis of Disaccharide modules with Various Sulfation Patterns The presence of non-participating azide group at C2 of glucosamine donor 31 assisted the formation of the desired 1,2-cis disaccharide 35 (1JCH = 173 Hz) via promotion by p- TolSCl/AgOTf (Scheme 2.14). Disaccharide 36 was originally obtained by a two-step procedure of azide reduction with Zn/HOAc followed by Fmoc deprotection. Instead, treatment of 36 with 1,3-propanedithiol in Et3N furnished 36 in one step with an 81% yield (Scheme 2.14).129 43 Scheme 2.14. Disaccharide synthesis and modification. Next, we explored whether different sulfation pattern disaccharides could be generated from 36 under various sulfation conditions. N-sulfation of 37 was achieved by treatment with SO3·Et3N in a H2O and THF cosolvent system. The amine of 36 was converted to acetamide followed by O-sulfation via SO3·Et3N at 55 °C in DMF to obtain O-sulfated disaccharide 38. SO3·pyridine complex in pyridine solution was implemented for fully sulfated compound 39 preparation. However, incomplete sulfation byproduct was observed even with extended reaction time and an increased amount of sulfation reagents. Two-step sulfation was performed next with O-sulfation followed by N-sulfation to furnish 39 in 92% yield over two steps. The Bn and Cbz protecting groups were removed by hydrogenation with Pd/C to afford compounds 40-42. The elongation sequence disaccharides 43-45 were prepared by N-Fmoc protection of amines. FmocCl gave the Fmoc protected carboxylate byproduct, and Fmoc-OSu was used to afford selectively N-Fmoc protected 43-45 (Scheme 2.15).130 44 Scheme 2.15. Preparation of disaccharides bearing various sulfation patterns. Next, the reducing end sequence disaccharides were designed with a masked amine as part of the aglycon linker. Treatment with acetic anhydride in MeOH enabled acetylation of amines to furnish desired disaccharides 46-48 (Scheme 2.16). 45 Scheme 2.16. Reducing end sequence disaccharides 46-48. 2.5. Synthesis of a Library of 27 Pseudo-hexasaccharide Heparin Mimetics In order to build a pseudo tetrasaccharide library, the carboxylate groups of disaccharides 43-45 were blocked by methyl ester followed by N-Fmoc deprotection via DBU. Nine pseudo- tetrasaccharides with different combinations of sulfation patterns on disaccharide modules were prepared by HATU or HBTU mediated coupling reactions. We observed seven-membered lactone byproduct (10-20%) produced with fully sulfated disaccharide 45. In this case, HBTU was used for amidation reactions (Scheme 2.17). 46 Scheme 2.17. Pseudo tetrasaccharide formation. As each disaccharide module possesses three different sulfation patterns, 27 various pseudo hexasaccharide mimetics could be generated. Developing such a library is regarded as a potential tool to understand the heparin structure and its biological functions. The pseudo tetrasaccharides (49a-i) were treated with DBU to deprotect the N-Fmoc group followed by HATU or HBTU coupling reaction to furnish the desired pseudo hexasaccharides. Saponification of methyl esters afforded the fully deprotected 27 pseudo hexasaccharides (50, 50a-z) (Scheme 2.18). 47 Scheme 2.18. A library of 27 pseudo-hexasaccharides. 2.6. Conclusions In conclusion, a library of 27 pseudo-hexasaccharide heparin mimetics has been rapidly created through disaccharide modules. Oxime formation failed due to the instability of the O-N bond under both hydrogenation and acidic conditions. Reductive amination could not afford the desired pseudo-tetrasaccharide. Finally, the disaccharide modules were linked through amide 48 bonds. With our “head-to-tail” synthetic strategies, the pseudo heparin oligosaccharides could be easily extended to longer sequences, and each disaccharide unit could have distinct sulfation pattern. Next, the bioactivity of pseudo-hexasaccharides will be studied. 2.7. Experimental Section 2.7.1. General synthetic procedures. All reactions were performed under a nitrogen atmosphere with anhydrous solvents. Solvents were dried using a solvent purification system. Glycosylation reactions were performed with 4Å molecular sieves that were flamed dried under high vacuum. Chemicals used were reagent grade unless noted. Reactions were visualized by UV light (254 nm) and by staining with either Ce(NH4)2(NO3)6 (0.5g) and (NH4)Mo7O24·4H2O (24.0 g) in 6% H2SO4 (500 mL), 5% H2SO4 in EtOH. Flash chromatography was performed on silica gel 601 (230-400 Mesh). NMR spectra were referenced using residual CHCl3, CO3OD, and D2O. Peak and coupling constants assignments are based on 1H-NMR, 1H-1H gCOSY, 1H and 1H-1H TOCSY, 1H-1H NOESY, 1H-13C gHSQC, 1H-13C gHMBC. 2.7.2. General procedure for pre-activation Based Single-step Glycosylation. A solution of the donor (1.0 equiv) and freshly activated molecular sieve MS 4 Å in DCM was stirred for 20 minutes under room temperature and then cooled to -78 °C. A solution of AgOTf (3.0 equiv) in anhydrous Et2O/DCM (10/1, v/v) was added to the reaction solution without touching the wall of the flask. After 5 minutes, orange-colored p-TolSCl (1.0 equiv) was added to the solution through a microsyringe. p-TolSCl should be added directly to the reaction 49 solution to prevent it from freezing on the flask wall and stir bar. The characteristic orange color of p-TolSCl should dissipate within a few seconds indicating consumption of p-TolSCl promoter. TLC analysis could confirm the complete activation of the donor in 5 minutes. A solution of acceptor (1.0 equiv) with TTBP (1.0 equiv) in DCM was added to reaction solution (0.3 M). The reaction mixture can be warmed up to 0 °C under stirring in 2 hours, depending on the reactivity of both donor and acceptor. The reaction mixture was quenched by Et3N (NaHCO3 saturated solution for Fmoc protected compounds) and filtered over Celite with DCM. DCM solution was washed by NaHCO3 and NaCl saturated solutions. The organic layer was collected and dried over Na2SO4, concentrated and purified by silica gel flash chromatography. 2.7.3. General procedure for deprotection of PMB. The PMB-protected compound (1.0 equiv) was dissolved in DCM/H2O (0.1 M, 10:1, v/v). The mixture was cooled to 0 °C, followed by the addition of DDQ (1.5 equiv). The mixture was stirred at room temperature for 1 hour. The residue was diluted with DCM and washed with saturated solutions of NaHCO3 and NaCl, dried (Na2SO4), and filtered. The filtrate was concentrated in vacuo, and the residue was purified by silica gel flash chromatography. 2.7.4. General procedure for protection of 6-OH with Lev. The compound containing 6-OH (1.0 equiv) was dissolved in DCM (0.3 M), followed by the addition of EDC-HCl (3.0 equiv), levulinic acid (1.4 equiv) and DMAP (1.0 equiv). The mixture was stirred under room temperature for 1 hour. The residue was diluted with DCM and washed with 10% HCl, saturated solutions of NaHCO3 and NaCl. The organic solution was dried 50 over Na2SO4 and concentrated in vacuo, then purified by silica gel flash chromatography. 2.7.5. General procedure for O-Acetylation. The compound containing -OH (1.0 equiv) was dissolved in pyridine (1.2 M), followed by the addition of acetic anhydride (7.0 equiv for each hydroxyl group) at 0 °C. The mixture was stirred at room temperature overnight and then washed with 3 x 1.0 M HCl and saturated solutions of NaHCO3 and NaCl. The organic solution was dried over Na2SO4, filtered and concentrated in vacuo, then purified by silica gel flash chromatography to afford the desired compounds. 2.7.6. General procedure for N-Acetylation. The compound containing -NH2 (1.0 equiv) was dissolved in MeOH (0.04 M), followed by the addition of acetic anhydride (3.0 equiv) and Et3N (3.0 equiv). The mixture was stirred at room temperature for 1 h and then was passed through LH-20 gel column with MeOH to afford the desired compounds. 2.7.7. General procedure for protection of OH with Fmoc. To a solution of starting material in DCM (0.3 M), FmocCl (3.0 equiv) and pyridine (4.0 equiv) were added. The reaction mixture was stirred at room temperature for 1 h and diluted with DCM. The organic mixture was washed with 3 x 1 M HCl, NaHCO3 saturated solution, and brine. The organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated by rotary evaporation and the desired product was purified by silica gel column 51 chromatography. 2.7.8. General procedure for protection of NH2 with Fmoc. To a solution of starting material in H2O/DMF (1/1, v/v, 0.3 M), Fmoc-OSu (3.0 equiv) and DIPEA (1.5 equiv) were added. The reaction mixture was stirred at room temperature for 2 h. The organic mixture was washed with 3 x 1 M HCl, NaHCO3 saturated solution and brine. The organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated by rotary evaporation and the desired product was purified by silica gel column chromatography. For disaccharide Fmoc protection, after the reaction completed as indicated by TLC (EtOAc/MeOH/H2O 3/1/1), the reaction mixture was passed through an LH-20 gel column with MeOH. A column of Dowex 50WX4 Na+ resin was used for ion exchange for giving products as sodium salts. 2.7.9. General procedure for preparation of methyl ester. K2CO3 (10.0 equiv) and CH3I (6.0 equiv) was added to a solution of starting material (1.0 equiv) in DMF (0.13M). The reaction mixture was stirred at room temperature for 4 h. After completion, the mixture was passed through an LH-20 gel column to afford pure compounds. 2.7.10. General procedure for NH-Fmoc Deprotection. A mixture of starting material (1.0 equiv) and DBU (5.0 equiv) in DMF (0.15 M) was stirred for 1 h at room temperature. After TLC (EtOAc/MeOH/H2O, 3/1/1, v/v/v) indicating that 52 the reaction was complete, the reaction mixture was passed through an LH-20 gel column to give pure compounds. 2.7.11. General procedure for preparation of pseudo-tetrasaccharides/hexasaccharides. A solution of carboxyethyl disaccharides (1.0 equiv), HATU or HBTU (1.0 equiv) and DIPEA (2.0 equiv) in DMF was added to starting material (1.0 equiv) in DMF (0.013 M). After stirring 1 h at room temperature, TLC (EtOAc/MeOH/H2O, 2.5/1/1, v/v/v) indicated completion of the reaction. Then the reaction mixture was passed through an LH-20 gel column, and the fractions containing product were collected and concentrated under reduced pressure. A column of Dowex 50WX4 Na+ resin was used for ion exchange to give products as sodium salts. 2.7.12. General procedure for hydrogenolysis. A solution of the compound in mixed tBuOH and water [1/1 (v/v), 3 mL] in the presence of 10% Pd(OH)2/C (80 mg) at room temperature was exposed to H2 gas. After overnight, the suspension was filtered through Celite, and the filtrate was concentrated in vacuo. The residue was passed through an LH-20 gel column using MeOH as eluent. The product fractions were concentrated under reduced pressure to give the target molecule. 2.7.13. General procedure for saponification of methyl esters. The starting material (1.0 equiv) was added to a LiOH (10.0 equiv per CO2Me) solution (0.006 M) at 0 °C. The reaction mixture was stirred at 0 °C for 8 h. After TLC (EtOAc/MeOH/H2O, 1.5/1/1, v/v/v) indicated the reaction completion, Amberlite H+ resin was 53 added until pH ~ 7. The reaction mixture was passed through G-15, and Dowex 50WX4 Na+ gel columns to give pure products as the sodium salts. Product Preparation and Characterization Data p-Tolyl 2-azido-3-O-benzyl-2-deoxy-6-O-para-methoxylbenzyl-1-thio-β-D-glucopyranoside (30) The starting material 29 (3.8 g, 12.2 mmol) was dissolved in CH3CN (0.4 M, 32.0 ml), and anisaldehyde dimethyl acetal (4.1 ml, 24.4 mmol) and CSA (1.1 g, 4.9 mmol) were added. After stirring at room temperature overnight, the reaction mixture was neutralized with Et3N and concentrated under reduced pressure. The residue was diluted with DCM and washed with saturated NaHCO3 solution and brine. The organic solution was dried over Na2SO4 and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using a gradient of DCM and methanol (from 20/1 to 12/1, v/v). A solution of the above compound 30 (4.2 g, 9.9 mmol), NaH (0.6 g, 14.8 mmol) and BnBr (2.3 ml, 19.7 mmol) in DMF (15 ml, 0.7 M) was kept stirring at room temperature for 2 h. After the starting material was completely consumed, the reaction mixture was neutralized with NH4Cl saturated solution and sequentially washed with NaHCO3 saturated solution (3 x 70 ml) and brine (75 ml). The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo, 54 and the residue was purified by flash column chromatography to afford the desired product. The above compound (4.9 g, 9.5 mmol) was dissolved in DMF (30 ml, 0.3 M), followed by the addition of NaCNBH3 (6.0 g, 95 mmol) and TFA (7.3 ml, 95 mmol) at 0 °C. The reaction mixture was warmed to room temperature and stirred overnight, and then diluted with EtOAc. The organic mixture was washed with water (3 x 70 ml) to get rid of DMF, NaHCO3 saturated solution (3 x 70 ml) and brine (50 ml). The organic layer was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using a gradient of hexanes and EtOAc (from 3/1 to 2/1, v/v) to give the desired product (30) (4.2 g, 66% for 3 steps). 1H NMR (500 MHz, CDCl3) δ 7.51 – 7.47 (m, 2H; ArCH2-), 7.43 – 7.31 (m, 5H; Ar-H), 7.29 – 7.25 (m, 2H; ArCH2-), 7.11 (d, J = 8.0 Hz, 2H; ArCH-2-), 6.95 – 6.88 (m, 2H; ArCH2-), 4.92 – 4.83 (m, 2H; BnCH2), 4.57 – 4.48 (m, 2H; PMBCH2), 4.39 (d, J = 9.0 Hz, 1H; 1-H), 3.84 (s, 3H; OCH3), 3.78 (dd, J = 10.3, 4.9 Hz, 1H; 6-H), 3.73 (dd, J = 10.3, 4.9 Hz, 1H; 6’-H), 3.65 – 3.59 (m, 1H; 4-H), 3.47 – 3.41 (m, 1H; 5-H), 3.38 (t, J = 9.0 Hz, 1H; 3-H), 3.30 (t, J = 9.0 Hz, 1H; 2-H), 2.85 (d, J = 2.5 Hz, 1H; OH), 2.35 (s, 3H; STolCH3). 13C NMR (125 MHz, CDCl3) δ 159.37, 138.75, 137.88, 134.20, 129.81, 129.74, 129.44, 128.63, 128.27, 128.12, 127.07, 113.88, 86.14, 84.57, 77.86, 75.50, 73.44, 72.24, 70.11, 64.30, 55.32, 21.22. HRMS: m/z calc. for C28H31N3O5S [M+NH4 +]+: 539.2323; found: 539.2336. 55 p-Tolyl 2-azido-3-O-benzyl-4-O-benzyl propionate-2-deoxy-6-O- fluorenylmethyloxycarbonyl-1-thio-β-D-glucopyranoside (31) The starting material 30 (11 g, 21 mmol) was dissolved in DMF (30 ml, 0.7 M), then NaH (1.6 g, 63 mmol) and allyl bromide (3.8 ml, 42 mmol) were added at 0 °C. The reaction mixture was warmed up to room temperature and stirred for 1 h until TLC (Hexanes/EtOAc 3:1, v/v) indicated completion of the reaction. After neutralization with saturated NH4Cl solution, the reaction mixture was diluted with DCM and washed with saturated NaHCO3 solution (3 x 200 ml) and brine (200 ml), dried (Na2SO4). The organic layer was filtered, and the filtrate was concentrated in vacuo, then purified by silica gel column chromatography using a gradient of hexanes and EtOAc (from 15/1 to 12/1, v/v). The above compound (11 g, 20 mmol) was dissolved in THF (87 ml, 0.2 M) and cooled to 0 °C. A solution of 9-BBN in THF (0.5 M, 77 ml, 38 mmol) was added and the reaction mixture was warmed to room temperature and stirred overnight. After hydroboration was complete, ethanol (50 ml) was added slowly to the reaction mixture at 0 °C, followed by the addition of NaOH (1.0 M aq, 98 ml) and H2O2 (30 wt% aq, 98 ml). The reaction mixture was warmed to 60 °C for 3 h, then quenched with saturated NH4Cl solution and extracted into EtOAc (3 x 100 ml). The combined organic extracts were washed with NaHCO3 saturated solution (3 x 100 ml) and brine (100 ml), dried over Na2SO4 and filtered. The filtrate was concentrated by rotary evaporation, and the residue was purified by silica gel column chromatography from pure hexanes to hexanes/EtOAc (10/1, v/v). The above compound (6.7 g, 11.6 mmol), BAIB (8.2 g, 25.5 mmol) and TEMPO (0.5 g, 3.5 mmol) were dissolved in DCM/H2O (4/1, v/v, 1.2 M). After stirring at room temperature for 2 h, the reaction mixture was diluted in DCM and washed with water (3 x 75 ml) and Na2S2O3 (40 ml) solution. The organic solvent was evaporated under reduced pressure. The residue (6.8 g, 11.5 mmol) was dissolved in 56 DMF (23 ml, 0.5 M), followed by the addition of K2CO3 (9.5 g, 69 mmol) and BnBr (8.2 ml, 69 mmol). The reaction mixture was stirred at room temperature for 4 h and diluted with EtOAc and washed with 1M HCl (3 x 75 ml), NaHCO3 saturated solution (3 x 75 ml) and brine (75 ml). The organic solution was dried over Na2SO4, and the filtrate was concentrated by rotary evaporation. The residue was purified by silica gel column chromatography from pure hexanes to hexanes/EtOAc (15/1 to 12/1, v/v). The above compound (5.4 g, 8.0 mmol) was treated according to the general procedures of PMB deprotection and Fmoc protection to give compound 31 (4.3 g, 26% for six steps). 1H NMR (500 MHz, CDCl3) δ 7.82 – 7.76 (m, 2H; ArCH2-), 7.65 (ddd, J = 7.6, 4.5, 1.0 Hz, 2H; ArCH2-), 7.52 – 7.22 (m, 15H; ArCH-), 7.12 – 7.06 (m, 2H; ArCH2-), 5.14 (s, 2H; BnCH2-), 4.85 – 4.76 (m, 2H; BnCH2-), 4.54 – 4.41 (m, 3H; 6-H, FmocCH2-), 4.36 – 4.26 (m, 3H; 1-H, 6’- H, FmocCH-), 4.14 – 4.04 (m, 1H; -OCH2-), 3.88 – 3.81 (m, 1H; -OCH2-), 3.47 – 3.39 (m, 2H; 4-H, 5-H), 3.34 – 3.21 (m, 2H; 2-H, 3-H), 2.64 – 2.51 (m, 2H; CO2BnCH2-), 2.30 (s, 3H; STolCH3). 13C NMR (125 MHz, CDCl3) δ 171.02, 154.96, 143.40, 143.33, 141.29, 138.88, 137.44, 135.72, 134.31, 129.79, 128.57, 128.54, 128.37, 128.31, 128.11, 127.91, 127.23, 127.21, 125.22, 120.07, 85.85, 84.74, 77.62, 76.93, 75.79, 70.04, 68.22, 66.50, 66.27, 64.70, 46.77, 35.36, 21.19. HRMS: m/z calc. for C45H43N3O8S [M+NH4 +]+: 803.3109; found: 803.3099. p-Tolyl idopyranosiduronate (33) methyl-2-O-benzoyl-3-O-benzyl-4-O-para-methoxylbenzyl-1-thio-α-L- A mixture of starting material 32 (9.6 g, 16.0 mmol), BAIB (11.3 g, 35.2 mmol) and 57 TEMPO (0.74 g, 4.8 mmol) was dissolved in DCM/H2O (4/1, 0.64 M). After stirring at room temperature for 4 h, the reaction mixture was diluted in DCM and washed with water (3 x 100 ml) and Na2S2O3 (50 ml) solution. The organic solvent was evaporated under reduced pressure. The residue (9.8 g, 15.9 mmol) was dissolved in DMF (40 ml, 0.4 M), followed by the addition of K2CO3 (13.2 g, 95.4 mmol) and CH3I (6.0 ml, 95.4 mmol). The reaction mixture was stirred at room temperature for 4 h and diluted with EtOAc and washed with 1M HCl (3 x 100 ml), NaHCO3 saturated solution (3 x 100 ml) and brine (100 ml). The organic solution was dried over Na2SO4, and the filtrate was concentrated by rotary evaporation. The residue was purified by silica gel column chromatography from pure hexanes to hexanes/EtOAc (9/1 to 8/1) to yield pure compound 33 (7.2 g, 72% yield for two steps). 1H NMR (500 MHz, CDCl3) δ 7.99 (dd, J = 8.2, 1.5 Hz, 2H; ArCH2-), 7.55 – 7.19 (m, 9H; ArCH-), 7.11 (d, J = 7.9 Hz, 2H; ArCH2-), 7.07 – 7.00 (m, 2H; ArCH2-), 6.83 – 6.66 (m, 2H; ArCH2-), 5.77 – 5.65 (m, 1H; 1-H), 5.45 – 5.43 (m, 1H; 2- H), 5.34 – 5.25 (m, 1H; 5-H), 4.90 (d, J = 11.9 Hz, 1H; BnCH2-), 4.66 (d, J = 11.9 Hz, 1H; BnCH2-), 4.48 – 4.30 (m, 2H; PMBCH2-), 3.99 – 3.92 (m, 3H; 3-H, 4-H, 5-H), 3.84 – 3.76 (m, 6H), 2.32 (s, 3H; SPhCH3). N-(Benzyl)-benzyloxycarbonyl-2-aminoethyl methyl-2-O-fluorenylmethyloxycarbonyl-3-O- benzyl-4-O-para-methoxylbenzyl-1-thio-α-L-idopyranosiduronate (34) 58 Compound 33 (2.4 g, 3.7 mmol) was treated according to the general procedures of pre-activation based single-step glycosylation with the acceptor (1.5 equiv) to give the desired compound. A mixture of the above compound (2.66 g, 3.37 mmol) and NaOMe (25% in MeOH) (0.31 ml, 1.7 mmol) in DCM/MeOH (1/2, v/v, 0.2M) was stirred at room temperature for 1 h. Then the reaction mixture was neutralized with Amberlite-H+ resin and filtered. The crude was evaporated under reduced pressure and subjected to 2-OH Fmoc protection and 4-OH PMB deprotection according to the general procedures to give compound 34 (1.9 g, 72% yield for 3 steps). 1H NMR (500 MHz, CDCl3) δ 7.77 (dt, J = 7.6, 1.0 Hz, 2H; ArCH2-), 7.57 (ddd, J = 7.7, 4.2, 1.0 Hz, 2H; ArCH2-), 7.41 (tt, J = 7.6, 1.4 Hz, 2H; ArCH2-), 7.38 – 7.19 (m, 13H; ArCH-), 7.16 – 6.99 (m, 2H; ArCH-2-), 5.22 – 5.11 (m, 2H; 2-H, CbzCH2-), 5.03 – 4.95 (m, 1H; 1-H), 4.83 – 4.76 (m, 2H; 5-H, CbzCH2-), 4.75 – 4.70 (m, 1H; OBnCH2-), 4.66 –4.59 (m, 1H; OBnCH2-), 4.57 – 4.37 (m, 4H; FmocCH-, NBnCH2-), 4.24 (t, J = 7.4 Hz, 1H; FmocCH-), 4.07 (br, 1H; 3-H), 3.99 – 3.87 (m, 1H; -OCH2-), 3.86 – 3.81 (m, 3H), 3.74 – 3.51 (m, 2H; - CH2NBnCbz, -OCH2-), 3.46 – 3.35 (m, 2H; 4-H, -CH2NBnCbz), 2.78 (s, 1H). 13C NMR (125 MHz, CDCl3) δ 169.68, 156.34, 153.75, 143.04, 141.33, 137.82, 137.23, 136.62, 129.06, 128.55, 128.53, 128.25, 128.08, 127.98, 127.87, 127.76, 127.32, 127.23, 125.33, 125.17, 125.13, 120.16, 98.49, 74.39, 72.32, 70.77, 70.60, 68.02, 67.74, 67.45, 52.46, 51.61, 46.69, 45.67. HRMS: m/z calc. for C46H45NO11 [M+NH4 +]+: 805.3331; found: 805.3311. 59 N-(Benzyl)-benzyloxycarbonyl-2-aminoethyl 3-O-benzyl-4-O-benzylpropionate-2-azido-2- deoxy-6-O-fluorenylmethyloxycarbonyl-1-thio-α-D-glucopyranoside-(1→4)-methyl-3-O- benzyl-2-O-fluorenylmethyloxycarbonyl-1-thio-α-L-idopyranosiduronate (35). Compound 31 (1.8 g, 2.3 mmol) and 34 (1.8 g, 2.3 mmol) were treated according to the general procedures of pre-activation based single-step glycosylation to give compound 35 (2.05 g, 62%, α/β, 3/1). 1H NMR (500 MHz, CDCl3) δ 7.81 – 7.66 (m, 3H; Ar-H), 7.66 – 7.50 (m, 4H; Ar-H), 7.46 – 7.18 (m, 25H; Ar-H), 7.18 – 7.01 (m, 4H; Ar-H), 5.24 – 5.04 (m, 5H, 1-H, 5-H, CbzCH2-), 4.93 – 4.86 (m, 1H, 1’-H), 4.86 – 4.69 (m, 5H), 4.63 – 4.44 (m, 4H; 2-H), 4.43 – 4.29 (m, 4H), 4.27 – 4.01 (m, 5H), 4.01 – 3.81 (m, 4H; anomeric linker-OCH2-), 3.78 – 3.71 (m, 3H; - CO2CH3), 3.68 – 3.59 (m, 1H), 3.57 – 3.35 (m, 4H; 2’-H, 3’-H, 4-H, 5’-H), 3.34 – 3.21 (m, 1H), 2.60 – 2.49 (m, 2H; BnCO2CH2-), 13C NMR (125 MHz, CDCl3) δ 171.00, 169.39, 156.18, 154.99, 154.58, 144.24, 143.35, 143.26, 141.27, 140.22, 137.75, 137.67, 137.34, 135.46, 131.39, 130.39, 130.20, 130.08, 128.77, 128.58, 128.47, 128.45, 128.35, 128.25, 128.00, 127.97, 127.91, 127.84, 127.80, 127.40, 127.29, 127.23, 127.19, 127.15, 125.36, 125.26, 125.15, 120.60, 120.09, 120.04, 98.50, 79.62, 76.78, 75.09, 73.89, 73.24, 70.42, 70.00, 69.61, 68.07, 67.70, 67.32, 66.44, 65.81, 63.30, 52.36, 51.59, 46.72, 46.56, 35.34. N-(Benzyl)-benzyloxycarbonyl-2-aminoethyl 2-amino-3-O-benzyl-4-O-benzylpropionate-2- deoxy-1-thio-α-D-glucopyranoside-(1→4)-methyl-3-O-benzyl-1-thio-α-L- 60 idopyranosiduronate (36). At 50 °C under N2 atmosphere, 1.3-propanedithiol (2.8 ml, 27.0 mmol) and Et3N (3.7 ml, 27.0 mmol) were added to a solution of starting material 35 (0.7 g, 0.48 mmol) in pyridine/H2O (4/1, 0.04 M) and the reaction mixture was stirred for 2 h. The reaction mixture was concentrated by rotary evaporation and purified by silica gel column chromatography using a gradient of DCM and MeOH (from 50/1 to 40/1 to 20/1, v/v) to give the desired product 36 (0.38 g, 81%). 1H NMR (500 MHz, CDCl3) δ 7.45 – 6.95 (m, 19H; Ar-H), 7.12 – 7.04 (m, 2H; ArCH2-), 5.21 – 5.06 (m, 4H; 1-H, 1’-H), 5.00 – 4.90 (m, 2H), 4.88 – 4.80 (m, 2H; BnCH-), 4.70 (d, J = 11.9 Hz, 1H; BnCH-), 4.62 – 4.43 (m, 4H), 4.15 (s, 1H), 4.05 – 3.98 (m, 1H), 3.96-3.84 (m, 3H), 3.78 – 3.64 (m, 6H; -CO2CH3, -NCH2-), 3.64 – 3.51 (m, 1H), 3.46 – 3.32 (m, 4H), 2.83 (dd, J = 10.1, 3.7 Hz, 1H), 2.63 – 2.52 (m, 2H; BnCO2CH2-). 13C NMR (125 MHz, CDCl3) δ 171.56, 138.09, 135.60, 128.60, 128.53, 128.49, 128.46, 128.42, 128.36, 128.32, 127.94, 127.82, 127.74, 127.66, 127.18, 101.88, 96.50, 82.19, 78.58, 76.77, 75.57, 72.23, 72.03, 7.87, 67.80, 67.36, 67.22, 66.54, 66.43, 61.22, 55.04, 52.37, 51.61, 46.68, 45.70, 35.41. HRMS: m/z calc. for C54H62N2O15 [M+H+]+: 978.4150; found: 978.4205. N-(Benzyl)-benzyloxycarbonyl-2-aminoethyl 3-O-benzyl-4-O-benzylpropionate-2-deoxy-2- sulfoamino-1-thio-α-D-glucopyranoside-(1→4)-methyl-3-O-benzyl-1-thio-α-L- idopyranosiduronate (37). 61 The starting material 36 (90 mg, 0.09 mmol) was dissolved in H2O/THF (1/1, v/v, 0.05 M) mixture, followed by addition of NaHCO3 (115 mg, 1.37 mmol) and sulfur trioxide triethylamine complex (142 mg, 0.9 mmol). The reaction mixture was stirred at room temperature for 2 h and diluted with DCM. The organic mixture was washed with water and brine, and then filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography using a mixture of DCM and methanol to give desired product 37 (20/1, v/v). 1H NMR (500 MHz, CD3OD) δ 7.45 (d, J = 7.4 Hz, 2H; ArCH2-), 7.40 – 7.10 (m, 16H; Ar-H), 7.01-6.91 (m, 2H; ArCH2-), 5.41 (d, J = 3.4 Hz, 1H; 1’-H), 5.16 – 5.06 (m, 3H; CO2BnCH2-, CbzCH-), 4.99 (d, J = 10.8 Hz, 1H; CbzCH-), 4.88 (s, 2H; 1-H, 5-H), 4.81 (d, J = 9.5 Hz, 1H; OBnCH-), 4.75 – 4.59 (m, 3H; OBnCH2-), 4.58 – 4.33 (m, 2H; NBnCH2-), 4.18 (s, 2H; 2-H, 4-H), 4.05 – 3.97 (m, 1H; 4’-linker-OCH-), 3.95 – 3.80 (m, 3H; 4’- linker-OCH-, 6’-H), 3.78 – 3.51 (m, 5H; 5’-H, 3-H, 4’-H, anomeric linker-OCH2-), 3.49 – 3.25 (m, 5H; NCH2-, 2’-H, 3’-H), 2.54 – 2.48 (m, 2H; BnCO2CH2-). 13C NMR (125 MHz, CD3OD) δ 171.81, 170.46, 156.65, 139.00, 138.10, 137.68, 128.24, 128.13, 128.10, 128.06, 128.01, 127.86, 127.84, 127.79, 127.77, 127.56, 127.52, 127.47, 127.38, 127.34, 127.26, 127.02, 126.90, 126.87, 101.38, 96.25, 79.92, 77.36, 74.71, 73.59, 72.15, 71.56, 67.64, 67.42, 66.47, 65.88, 60.36, 58.11, 51.55, 51.28, 46.67, 45.78, 35.15. HRMS: m/z calc. for C54H61N2O18S- [M]-: 1057.3646; found: 1057.3646. N-(Benzyl)-benzyloxycarbonyl-2-aminoethyl 2-acetamido-3-O-benzyl-4-O- 62 benzylpropionate-2-deoxy-6-O-sulfonato-1-thio-α-D-glucopyranoside-(1→4)-methyl-3-O- benzyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate (38). To a solution of starting material 36 (225 mg, 0.22 mmol) in DMF (0.08 M) was added sulfur trioxide triethylamine complex (800mg, 4.4 mmol) and the resulting reaction mixture was stirred at 55 °C for 2 h. The reaction mixture was purified by a LH-20 gel column with CH3OH. Fractions containing the product were collected under reduced pressure and the residue was passed through a column of Dowex 50WX4 Na+ resin using H2O as the eluent to give sodium form product 38 (223 mg, 89%). 1H NMR (500 MHz, CD3OD) δ 7.49 – 7.60 (m, 1H; Ar-H), 7.41 – 7.14 (m, 16H; Ar-H), 7.12-6.96 (m, 2H; ArCH2-), 5.22 – 5.09 (m, 4H; CO2BnCH2-, 1-H, 1’-H), 4.83 – 4.73 (m, 3H; 5-H, CbzCH2-), 4.73 – 4.57 (m, 4H; OBnCH2-, OBnCH2-), 4.56 – 4.41 (m, 2H; NBnCH2-), 4.29 – 4.13 (m, 3H; 2-H, 6’-H), 4.12 – 4.03 (m, 3H; 4-H, 7-H), 4.00 – 3.85 (m, 2H; NCH2-), 3.80-3.74 (m, 3H, CO2CH3), 3.70 – 3.61 (m, 3H; 5’-H, 3-H, 4’-H), 3.45 – 3.36 (m, 2H; 2’-H, 3’-H), 2.64 – 2.52 (m, 2H; BnCO2CH2-). HRMS: m/z calc. for C56H62N2O22S2 2- [M]2-: 589.1623; found: 589.1611. N-(Benzyl)-benzyloxycarbonyl-2-aminoethyl 3-O-benzyl-4-O-benzylpropionate- 2-deoxy-2- sulfoamino-6-O-sulfonato-α-D-glucopyranoside-(1→4)-methyl-3-O-benzyl-2-O-sulfonato-1- thio-α-L-idopyranosiduronate (39). To a solution of starting material 36 (0.3 g, 0.3 mmol) in DMF (2.0 ml, 0.15 M) was 63 added sulfur trioxide triethylamine complex (1.12 g, 6.0 mmol) and the resulting reaction mixture was stirred at 55 °C for 2 h. The reaction mixture was purified by a LH-20 gel column with CH3OH. Fractions containing product were collected under reduced pressure and the crude was dissolved in pyridine/Et3N (10/1, v/v, 0.15M). Sulfur trioxide pyridine complex (0.45 g, 3 mmol) was added to the reaction mixture at 55 °C and stirred for 2 h. After reaction completion as indicated by TLC (EtOAc/MeOH/H2O 6/1/1), the reaction mixture was passed through LH-20 gel column with CH3OH. A column of Dowex 50WX4 Na+ resin was used for ion exchange to give sodium form product 39 (0.34 g, 92% for two steps). 1H NMR (500 MHz, CD3OD) δ 7.42 – 7.48 (d, J = 7.5 Hz, 2H; ArCH2-), 7.41 – 7.27 (m, 8H; Ar-H), 7.25 – 7.07 (m, 8H; Ar-H), 6.98 – 6.85 (m, 2H; ArCH2-), 5.40 (d, J = 3.4 Hz, 1H; 1’-H), 5.21 – 5.16 (m, 1H), 5.13 – 5.07 (m, 4H; 1-H), 5.05 – 5.01 (m, 1H), 4.81 – 4.71 (m, 3H; 5-H), 4.66 (d, J = 11.4, 1H), 4.50 – 4.37 (m, 1H; 6’-H), 4.34 – 4.19 (m, 4H; 2-H, 6’-H), 4.07-4.01 (m, 1H), 3.95–3.85 (m, 2H), 3.82 – 3.71 (m, 4H; 4’-linker-OCH2-), 3.70 – 3.56 (m, 2H), 3.46 – 3.38 (m, 3H; 2’-H), 2.51 (t, J = 6.4 Hz, 2H). 13C NMR (125 MHz, CD3OD) δ 172.29, 170.58, 156.68, 138.91, 137.97, 137.64, 137.43, 136.15, 128.34, 128.32, 128.24, 128.18, 128.11, 128.07, 128.00, 127.97, 127.89, 127.87, 127.61, 127.54, 127.46, 127.43, 127.32, 126.97, 99.33, 98.72, 79.70, 77.42, 75.01, 73.47, 73.06, 72.41, 70.87, 70.03, 67.90, 67.25, 66.75, 66.37, 66.13, 65.90, 58.15, 58.07, 52.18, 51.45, 51.38, 46.69, 45.72, 35.26. HRMS: m/z calc. for C54H59N2O24S3 3- [M+H+]2-: 608.1355; found: 608.1370. Aminoethyl 4-O-carboxyethyl-2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside-(1→4)- 64 methyl-1-thio-α-L-idopyranosiduronate (40). Compound 37 (170 mg, 0.16 mmol) was treated according to the general procedures of hydrogenolysis to give compound 40 (80.5 mg, 87%). 1H NMR (500 MHz, CD3OD) δ 5.36 (d, J = 3.7 Hz, 1H; 1’-H), 4.95 (d, J = 3.0 Hz, 1H; 1-H), 4.80 (d, J = 3.2 Hz, 1H; 5-H), 4.18 (t, J = 4.3 Hz, 1H; 3-H), 4.06 – 3.97 (m, 2H; 2-H, 4-H), 3.92 – 3.84 (m, 2H; 6’-CH2-), 3.75 (s, 3H; - CO2CH3), 3.73 – 3.68 (m, 2H; 5’-H, 4’-linker-OCH2-), 3.67 – 3.58 (m, 3H; 4’-linker-OCH2-, anomeric linker-OCH2-), 3.57 – 3.53 (m, 1H; 4’-H), 3.44 – 3.38 (m, 1H, 3’-H), 3.27 – 3.22 (m, 1H; 2’-H), 3.07-2.99 (m, 2H; NH2CH2-), 2.39 (t, J = 5.9 Hz, 2H; CO2HCH2-). 13C NMR (125 MHz, CD3OD) δ 170.39, 101.45, 96.63, 78.53, 74.10, 71.71, 69.08, 68.65, 68.01, 67.12, 64.71, 60.57, 58.06, 51.62, 46.32, 42.68, 39.26, 36.85. HRMS: m/z calc. for C18H31N2O16S- [M]-: 563.1400; found: 563.1401. Aminoethyl 2-acetamido-4-O-carboxyethyl-2-deoxy-6-O-sulfonato-α-D-glucopyranoside- (1→4)-methyl-2-O-sulfonato-α-L-idopyranosiduronate (41). Compound 38 (290 mg, 0.25 mmol) was treated according to the general procedures of hydrogenolysis to give compound 41 (138 mg, 82%). 1H NMR (500 MHz, D2O) δ 7.72 (d, J = 9.6 Hz, 1H; NHAc), 5.02 (s, 1H; 1’-H), 4.95 (d, J = 3.4 Hz, 1H, 1-H), 4.83 (d, J = 1.9 Hz, 1H; 5- H), 4.20 (d, J = 3.0 Hz, 1H; 3-H), 4.15 – 4.06 (m, 3H; 2-H, 6’-CH2-), 3.99 – 3.94 (m, 1H; 4-H), 3.92 – 3.82 (m, 3H; 5’-H, 4’-linker-OCH2-), 3.80 – 3.74 (m, 1H; anomeric linker-OCH2-), 3.66 65 (s, 3H; -CO2CH3), 3.64 – 3.59 (m, 1H; anomeric linker-OCH2-), 3.50 – 3.41 (m, 2H; 3’-H, 4’-H), 3.25 – 3.16 (m, 1H; 2’-H), 3.25 – 3.00 (m, 2H; NH2CH2-), 2.47 (t, J = 5.9 Hz, 2H; CO2HCH2-), 1.88 (s, 3H; -NHCOCH3). 13C NMR (125 MHz, D2O) δ 171.11, 98.96, 94.39, 77.66, 72.93, 71.10, 70.95, 69.64, 68.33, 66.58, 66.42, 64.67, 62.93, 52.96, 52.85, 38.83, 22.03. HRMS: m/z calc. for C20H32N2O20S2 2- [M+H+]-: 685.1074; found: 685.1086. Aminoethyl 2-acetamido-4-O-carboxyethyl-2-deoxy-6-O-sulfonato-1-thio-α-D- glucopyranoside-(1→4)-methyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate (42). Compound 39 (350 mg, 0.29 mmol) was treated according to the general procedures of Hydrogenolysis to give compound 42 (180 mg, 87%). 1H NMR (500 MHz, D2O) δ 5.19 (d, J = 3.4 Hz, 1H, 1’-H), 5.02 (d, J = 2.0 Hz, 1H, 1-H), 4.77 (d, J = 2.1 Hz, 1H; 5-H), 4.26 (t, J = 4.0 Hz, 1H; 3-H), 4.19 – 4.05 (m, 3H; 2-H, 6’-CH2-), 4.04 – 4.00 (m, 1H, 4-H), 3.90 – 3.83 (m, 2H; 5’-H, 4’-linker-OCH2-), 3.81 – 3.75 (m, 1H; 4’-linker-OCH2-), 3.69 (s, 3H; -CO2CH3), 3.65 – 3.56 (m, 2H; anomeric linker-OCH2-), 3.48 (t, J = 9.8 Hz, 1H, 4’-H), 3.25 (t, J = 9.8 Hz, 1H, 3’-H), 3.16 – 3.05 (m, 3H; 2’-H, NH2CH2-), 2.43 – 2.29 (m, 2H; CO2HCH2-). 13C NMR (125 MHz, D2O) δ 170.99, 99.30, 98.96, 77.76, 76.90, 74.53, 70.46, 69.25, 69.07, 67.26, 67.11, 66.16, 64.60, 57.61, 53.01, 38.89. HRMS: m/z calc. for C18H29N2O22S3 3- [M+H+]2-: 361.0232; found: 361.0238. 66 N-Fluorenylmethyloxycarbonyl-2-aminoethyl 4-O-carboxyethyl-2-deoxy-2-sulfoamino-1- thio-α-D-glucopyranoside-(1→4)-methyl-1-thio-α-L-idopyranosiduronate (43). Compound 40 (80 mg, 0.14 mmol) was treated according to the general procedures of protection of NH2 with Fmoc to give compound 43 (79 mg, 71%). 1H NMR (500 MHz, CD3OD) δ 7.79 (d, J = 7.5 Hz, 2H, Ar-H), 7.64 (d, J = 7.5 Hz, 2H, Ar-H), 7.39 (dt, J = 7.5 Hz, 1.2 Hz, 2H, Ar-H), 7.31 (td, J = 7.5, 1.2 Hz, 2H, Ar-H), 5.35 (d, J = 3.7 Hz, 1H; 1’-H), 4.91 – 4.89 (m, 1H; 1- H), 4.79 (d, J = 2.9 Hz, 1H; 5-H), 4.37 – 4.28 (m, 2H; FmocCH2-), 4.22 – 4.13 (m, 2H; FmocCH-, 3-H), 4.04 – 3.95 (m, 2H; 2-H, 6’-CH2-), 3.90 – 3.82 (m, 1H; 6’-CH2-), 3.78 – 3.70 (m, 3H; 5’-H, 4’-linker-OCH2-), 3.68 (s, 3H; -CO2CH3), 3.61 – 3.47 (m, 3H; 4’-H, anomeric linker-OCH2-), 3.43 – 3.35 (m, 1H; 3’-H), 3.35 – 3.32 (m, 1H; -CH2NHFmoc), 3.28 – 3.21 (m, 2H; 2’-H, -CH2NHFmoc), 2.40 (t, J = 5.9 Hz, 2H; CO2HCH2-). 13C NMR (125 MHz, CD3OD) δ 178.87, 170.39, 157.50, 143.88, 141.15, 127.36, 126.77, 124.76, 119.48, 101.33, 96.54, 78.64, 74.02, 71.71, 71.48, 69.30, 69.10, 67.67, 67.59, 67.00, 66.37, 60.46, 57.79, 51.53, 51.51, 40.21, 37.97. HRMS: m/z calc. for C33H41N2O18S- [M]-: 785.2081; found: 785.2103. N-Fluornylmethyloxycarbonyl-2-aminoethyl 2-acetamido-4-O-carboxyethyl-2-deoxy-6-O- 67 sulfonato-1-thio-α-D-glucopyranoside-(1→4)-methyl-2-O-sulfonato-1-thio-α-L- idopyranosiduronate (44). Compound 41 (167 mg, 0.24 mmol) was treated according to the general procedures of protection of NH2 with Fmoc to give compound 44 (190 mg, 86%). 1H NMR (500 MHz, CD3OD) δ 7.80 (d, J = 7.5 Hz, 2H; Ar-CH), 7.67 (d, J = 7.5 Hz, 2H; Ar-CH), 7.50 (d, J = 9.2 Hz, 1H; NHAc), 7.40 (dt, J = 7.5, 1.7 Hz, 2H; Ar-CH), 7.33 (dt, J = 7.5, 1.7 Hz, 2H; Ar-CH), 5.16 (s, 1H; 1’-H), 5.03 (d, J = 3.4 Hz, 1H; 1-H), 4.86 (d, J = 2.1 Hz, 1H; 5-H), 4.34 – 4.28 (m, 4H; 3-H, 2-H, FmocCH2-), 4.25 – 4.17 (m, 3H; 6’-CH2-, FmocCH-), 4.07 – 3.91 (m, 5H; 4-H, 5’-H, 4’-linker- OCH2-, anomeric linker-OCH2-), 3.84-3.75 (m, 2H; anomeric linker-OCH2-, -CH2NHFmoc), 3.73 (s, 3H; CO2CH3), 3.69-3.65 (m, 1H; 3’-H), 3.64-3.55 (m, 2H; 4’-H, -CH2NHFmoc), 3.28- 3.24 (m, 1H, 2’-H), 2.44 (dd, J = 6.8, 5.1 Hz, 2H; CO2HCH2-), 2.07 (s, 3H, NHCOCH3). 13C NMR (125 MHz, CD3OD) δ 178.30, 172.95, 170.30, 157.48, 143.88, 141.14, 127.37, 126.80, 124.86, 119.46, 99.57, 95.93, 78.75, 73.28, 72.93, 71.48, 69.91, 69.14, 67.55, 66.48, 66.36, 66.03, 64.34, 52.95, 51.75, 40.18, 37.44, 21.75. HRMS: m/z calc. for C35H42N2O22S2 2- [M]2-: 453.0841; found: 453.0840. N-Fluorenylmethyloxycarbonyl-2-aminoethyl 2-acetamido-4-O-carboxyethyl-2-deoxy-6-O- sulfonato-1-thio-α-D-glucopyranoside-(1→4)-methyl-2-O-sulfonato-1-thio-α-L- idopyranosiduronate (45). 68 Compound 42 (220 mg, 0.31 mmol) was treated according to the general procedures of protection of NH2 with Fmoc to give compound 45 (235 mg, 82%). 1H NMR (500 MHz, CD3OD) δ 7.80 (d, J = 7.8 Hz, 2H; ArCH-), 7.67 (d, J = 7.5 Hz, 2H; ArCH-), 7.40 (dt, J = 7.8, 2.6 Hz, 2H; ArCH-), 7.33 (td, J = 7.5 Hz, 2.6 Hz, 2H; ArCH-), 5.26 (d, J = 3.4 Hz, 1H, 1’-H), 5.21 (s, 1H, 1- H), 4.84 (d, J = 2.2 Hz, 1H; 5-H), 4.43 – 4.39 (m, 1H; 3-H), 4.34 – 4.28 (m, 3H; 2-H, FmocCH2-), 4.25 – 4.17 (m, 3H; 6’-CH2-, FmocCH-), 4.10 – 4.04 (m, 1H, 4-H), 4.01 – 3.94 (m, 2H, 5’-H, 4’- linker-OCH2-), 3.82 – 3.76 (m, 1H, 4’-linker-OCH2-), 3.74 (s, 3H, -CO2CH3), 3.68 – 3.61 (m, 2H; anomeric linker-OCH2-), 3.60 –3.53 (m, 1H; 4’-H), 3.37-3.34 (m, 2H; -CH2NHFmoc), 3.30-3.25 (m, 1H; 2’-H), 2.61 – 2.50 (m, 2H; CO2HCH2-). 13C NMR (125 MHz, CD3OD) δ 170.51, 143.88, 141.13, 127.39, 126.80, 124.88, 124.85, 119.48, 99.31, 98.68, 78.87, 75.80, 73.53, 71.03, 69.75, 67.70, 66.60, 66.54, 66.14, 65.95, 57.62, 51.88, 40.12, 37.32. HRMS: m/z calc. for C33H39N2O24S3 3- [M]3-: 314.3690; found: 314.3696. Acetmidoethyl 4-O-carboxyethyl-2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside-(1→4)- methyl-1-thio-α-L-idopyranosiduronate (46). Compound 40 (40 mg, 0.07 mmol) was treated according to the general procedures of hydrogenolysis to give compound 46 (40 mg, 93%). 1H NMR (500 MHz, CD3OD) δ 5.36 (d, J = 3.6 Hz, 1H, 1’-H), 4.89 (s, 1H, 1-H), 4.80 (d, J = 3.0 Hz, 1H; 5-H), 4.16 (t, J = 4.3 Hz, 1H; 3-H), 4.08 – 3.97 (m, 2H; 2-H, 4-H), 3.93 – 3.85 (m, 1H; 6’-CH2-), 3.80 – 3.69 (m, 6H; -CO2CH3, 5’-H, 69 6’-CH2-, 4’-linker-OCH2-), 3.62 – 3.50 (m, 3H; 4’-linker-OCH2-, 4’-H, anomeric linker-OCH2-), 3.45 – 3.34 (m, 3H; anomeric linker-OCH2-, 3’-H, -CH2NHAc), 3.30 – 3.22 (m, 2H; 2’-H, - CH2NHAc), 2.49 – 2.42 (m, 2H; CO2HCH2-), 1.94 (s, 3H; -NHCOCH3). 13C NMR (125 MHz, CD3OD) δ 177.09, 171.96, 170.45, 101.41, 96.58, 78.48, 74.12, 71.67, 71.62, 69.30, 68.55, 67.89, 67.27, 67.22, 60.53, 57.92, 51.55, 38.94, 36.65, 21.13. HRMS: m/z calc. for C20H33N2O17S- [M]-: 605.1505; found: 605.1513. Acetmidoethyl 2-acetamido-4-O-carboxyethyl-2-deoxy-6-O-sulfonato-1-thio-α-D- glucopyranoside-(1→4)-methyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate (47). Compound 41 (19 mg, 0.03 mmol) was treated according to the general procedures of Hydrogenolysis to give compound 47 (15 mg, 75%). 1H NMR (500 MHz, D2O) δ 4.98 (s, 1H, 1’- H), 4.96 (d, J = 3.5 Hz, 1H, 1-H), 4.81 (d, J = 2.2 Hz, 1H; 5-H), 4.18 (d, J = 3.0 Hz, 1H, 3-H), 4.14 – 4.10 (m, 3H; 2-H, 6’-CH2-), 3.93 (m, 1H; 4-H), 3.91 – 3.82 (m, 2H; 4’-linker-OCH2-), 3.79 – 3.72 (m, 1H; anomeric linker-OCH2-), 3.71 – 3.63 (m, 4H; -CO2CH3, 5’-H), 3.58 – 3.46 (m, 3H; 3’-H, 4’-H, anomeric linker-OCH2-), 3.26 – 3.19 (m, 3H; 2’-H, -CH2NHAc), 2.37 – 2.31 (m, 2H; CO2HCH2-), 1.89 (s, 3H; NHAc on linker), 1.82 (s, 3H; NHAc). 13C NMR (125 MHz, D2O) δ 174.64, 174.16, 171.21, 99.07, 94.35, 77.88, 73.08, 71.02, 69.63, 69.41, 67.33, 66.56, 66.32, 63.00, 53.07, 52.71, 48.75, 39.00, 37.35, 22.07, 21.68. HRMS: m/z calc. for C22H34N2O21S2 2- [M]2-: 363.0553; found: 363.0555. 70 Acetmidoethyl 2-acetamido-4-O-carboxyethyl-2-deoxy-6-O-sulfonato-1-thio-α-D- glucopyranoside-(1→4)-methyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate (48). Compound 42 (20 mg, 0.03 mmol) was treated according to the general procedures of N-acetylation to give compound 48 (13 mg, 62%). 1H NMR (500 MHz, CD3OD) δ 5.32 (d, J = 3.5 Hz, 1H, 1’-H), 5.15 (d, J = 2.7 Hz, 1H, 1-H), 4.80 (d, J = 2.9 Hz, 1H; 5-H), 4.36 – 4.32 (m, 1H; 3-H), 4.30 – 4.26 (m, 1H; 2-H), 4.21 (d, J = 2.8 Hz, 2H; 6’-CH2-), 4.14 – 3.92 (m, 3H; 4-H, 4’-linker-OCH2-), 3.81 (s, 4H; CO2CH3, anomeric linker-OCH2-), 3.72 – 3.67 (m, 1H; 5’-H), 3.64 – 3.59 (m, 1H; 4’-H), 3.58 – 3.56 – 3.49 (m, 1H; anomeric linker-OCH2-), 3.47 – 3.38 (m, 1H; 3’-H), 3.37-3.30 (m, 2H; -CH2NHAc), 3.28-3.23 (m, 1H, 2’-H) 2.64 – 2.47 (m, 2H, CO2HCH2-), 1.95 (s, 3H; -NHCOCH3). 13C NMR (125 MHz, CD3OD) δ 175.49, 171.98, 170.26, 99.76, 98.97, 78.53, 76.58, 75.02, 71.53, 69.71, 67.96, 67.85, 67.54, 67.41, 66.03, 58.04, 51.76, 38.81, 35.41, 21.13. HRMS: m/z calc. for C20H31N2O23S3 3- [M+Na+]2-: 393.0194; found: 393.0200. N-Fluorenylmethyloxycarbonyl-2-aminoethyl O-(4-O-methylpropionate-2-deoxy-2- 71 sulfoamino-6-O-sulfoamino-1-thio-α-D-glucopyranoside)-(1→4)-O-(methyl-2-O-sulfonato-1- thio-α-L-idopyranosiduronate)-(1→4)-O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-6-O- sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-methyl-2-O-sulfonato-1-thio-α-L- idopyranosiduronate (49a). Starting material (9 mg, 0.012 mmol) was treated according to the general procedures of pseudo-tetrasaccharide preparation with HBTU to give compound 49a (15 mg, 76%). 1H NMR (500 MHz, D2O) δ 7.79 (d, J = 7.5 Hz, 2H), 7.56 (t, J = 7.5 Hz, 2H), 7.40 – 7.33 (m, 2H), 7.32 – 7.23 (m, 2H), 5.17 – 5.08 (m, 2H; A-1, C-1), 4.94 (s, 1H; B-1), 4.89 (s, 1H; D-1), 4.54 (s, 1H), 4.42 – 4.34 (m, 1H), 4.31 – 4.24 (m, 2H), 4.22 – 4.00 (m, 7H), 3.95 – 3.79 (m, 5H), 3.75 (s, 1H), 3.67 – 3.61 (m, 3H), 3.55 (s, 3H), 3.52 – 3.38 (m, 6H), 3.28 – 3.22 (m, 2H), 3.19 (s, 3H), 3.12 – 3.03 (m, 3H), 2.55 – 2.47 (m, 2H), 2.39 – 2.29 (m, 2H). 13C NMR (125 MHz, D2O) δ 174.62, 173.99, 171.17, 148.60, 143.64, 140.89, 128.16, 128.02, 127.44, 125.17, 124.96, 120.70, 120.10, 99.61, 99.20, 77.44, 77.36, 77.18, 73.69, 70.62, 70.27, 69.13, 69.05, 68.27, 67.48, 67.31, 67.03, 66.40, 66.26, 66.05, 65.86, 57.91, 52.90, 52.67, 52.18, 46.91, 40.09, 38.89, 36.46, 34.74. N-Fluorenylmethyloxycarbonyl-2-aminoethyl O-(2-deoxy-4-O-methylpropionate- 2- sulfoamino-6-O-sulfoamino-1-thio-α-D-glucopyranoside)-(1→4)-O-(methyl-2-O-sulfonato-1- thio-α-L-idopyranosiduronate)-(1→4)-O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-1-thio-α- D-glucopyranoside)-(1→4)-O-methyl-1-thio-α-L-idopyranosiduronate (49b). 72 Starting material (13.5 mg, 0.018 mmol) was treated according to the general procedures of pseudo-tetrasaccharide preparation with HBTU to give compound 49b (17 mg, 65%). 1H NMR (500 MHz, D2O) δ 7.77 (d, J = 7.5 Hz, 2H), 7.55 (t, J = 6.9 Hz, 2H), 7.36 (t, J = 7.5 Hz, 2H), 7.29 – 7.22 (m, 2H), 5.18 (d, J = 3.5 Hz, 1H; A-1), 5.13 (d, J = 3.5 Hz, 1H; C-1), 4.95 (s, 1H; B-1), 4.74 – 4.71 (m, 1H; D-1), 4.68 – 4.67 (m, 1H), 4.58 (d, J = 2.6 Hz, 1H), 4.42 – 4.35 (m, 1H), 4.31 – 4.23 (m, 2H), 4.16 – 4.00 (m, 5H), 3.95 (t, J = 2.6 Hz, 2H), 3.92 – 3.78 (m, 5H), 3.76 – 3.65 (m, 3H), 3.64 (s, 3H), 3.61 – 3.51 (m, 9H), 3.51 – 3.45 (m, 3H), 3.31 – 3.22 (m, 3H), 3.21 – 3.15 (m, 3H), 3.13 – 3.00 (m, 5H), 2.53 – 2.45 (m, 2H), 2.37 – 2.26 (m, 2H). 13C NMR (125 MHz, CD3OD) δ 173.05, 170.54, 170.44, 157.55, 143.90, 141.15, 127.38, 126.79, 124.84, 124.81, 119.50, 101.24, 99.58, 98.79, 96.69, 78.29, 78.16, 76.10, 74.19, 71.60, 71.30, 69.65, 69.00, 67.88, 67.66, 67.44, 67.08, 66.86, 66.42, 65.95, 60.37, 58.12, 51.84, 51.70, 50.85, 40.21, 38.75, 36.47, 34.77. HRMS: m/z calc. for C37H60N4O37S4 4- [M+H+]3-: 427.0638; found: 427.0630. N-Fluorenylmethyloxycarbonyl-2-aminoethyl O-(4-O-methylpropionate-2-deoxy- 2-sulfoamino-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(methyl-2-O-sulfonato-1- thio-α-L-idopyranosiduronate)-(1→4)-O-(4-O-carboxyethyl-2-deoxy-2-acetamido-6-O- sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-methyl-2-O-sulfonato-1-thio-α-L- idopyranosiduronate (49c). 73 Starting material (18 mg, 0.024 mmol) was treated according to the general procedures of pseudo-tetrasaccharide preparation with HBTU to give compound 49c (25 mg, 63%). 1H NMR (500 MHz, CD3OD) δ 7.80 (d, J = 7.5 Hz, 2H), 7.67 (d, J = 7.5 Hz, 2H), 7.44 – 7.37 (m, 2H), 7.35 – 7.29 (m, 2H), 5.28 (d, J = 3.6 Hz, 1H; A-1), 5.18 (d, J = 4.4 Hz, 2H; C-1, B-1), 5.00 (d, J = 3.4 Hz, 1H; D-1), 4.86 (d, J = 1.9 Hz, 1H), 4.84 (d, J = 2.4 Hz, 1H), 4.40 (t, J = 3.7 Hz, 1H), 4.35 – 4.27 (m, 5H), 4.25 – 4.15 (m, 5H), 4.13 – 4.05 (m, 2H), 4.03 – 3.94 (m, 5H), 3.84 – 3.76 (m, 5H), 3.74 – 3.71 (m, 2H), 3.69 (s, 3H), 3.66 – 3.54 (m, 5H), 3.48 – 3.34 (m, 5H), 3.26 (dd, J = 10.6, 3.4 Hz, 1H), 2.66 – 2.56 (m, 2H), 2.48 (t, J = 5.9 Hz, 2H), 2.08 (s, 3H). 13C NMR (125 MHz, CD3OD) δ 173.02, 170.48, 157.50, 143.89, 141.14, 127.39, 126.81, 124.89, 119.48, 99.55, 98.86, 96.42, 78.29, 76.15, 73.75, 73.44, 73.08, 71.53, 69.95, 69.64, 68.35, 67.56, 67.42, 67.37, 66.49, 66.21, 65.93, 64.31, 58.15, 53.35, 51.85, 50.84, 40.15, 38.62, 36.53, 34.76, 21.79. HRMS: m/z calc. for C39H61N4O41S5 5- [M+H+]4-: 350.5379; found: 350.5379. N-Fluorenylmethyloxycarbonyl-2-aminoethyl-O-(2-deoxy-4-O-methylpropionate- -2- sulfoamino-1-thio-α-D-glucopyranoside)-(1→4)-O-(methyl-1-thio-α-L- idopyranosiduronate)-(1→4)-O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-6-O-sulfonato-1- thio-α-D-glucopyranoside)-(1→4)-O-methyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate (49d). 74 Starting material (21 mg, 0.036 mmol) was treated according to the general procedures of pseudo-tetrasaccharide preparation with HATU to give compound 49d (32 mg, 59%). 1H NMR (500 MHz, CD3OD) δ 7.80 (d, J = 7.5 Hz, 2H), 7.67 (d, J = 7.4 Hz, 2H), 7.43 – 7.38 (m, 2H), 7.33 (td, J = 7.5, 1.2 Hz, 2H), 5.41 (d, J = 3.6 Hz, 1H; A-1), 5.28 – 5.19 (m, 2H; C-1, B-1), 4.97 (d, J = 2.8 Hz, 1H; D-1), 4.84 (t, J = 2.9 Hz, 2H), 4.43 (d, J = 3.2 Hz, 1H), 4.35 – 4.28 (m, 3H), 4.23 – 4.15 (m, 4H), 4.10 – 4.03 (m, 2H), 4.02 – 3.96 (m, 3H), 3.93 – 3.86 (m, 2H), 3.81 – 3.77 (m, 3H), 3.76 – 3.73 (m, 6H), 3.73 – 3.70 (m, 2H), 3.68 (s, 3H), 3.66 – 3.55 (m, 6H), 3.50 – 3.45 (m, 1H), 3.43 – 3.34 (m, 5H), 3.30 – 3.21 (m, 3H), 2.65 – 2.50 (m, 4H), 2.46 – 2.36 (m, 2H). 13C NMR (125 MHz, CD3OD) δ 173.21, 172.85, 170.56, 157.50, 143.90, 141.13, 127.38, 126.82, 124.91, 124.87, 119.47, 101.44, 99.36, 99.17, 96.17, 78.16, 77.94, 76.57, 73.61, 73.43, 71.74, 71.48, 71.35, 69.59, 68.99, 67.99, 67.92, 67.57, 67.33, 66.75, 66.50, 66.36, 66.10, 65.65, 60.51, 58.27, 58.03, 51.83, 51.52, 50.72, 40.17, 38. 86, 36.63, 34.74. HRMS: m/z calc. for C37H60N4O37S4 4- [M]4-: 320.0460; found: 320.0446. N-Fluorenylmethyloxycarbonyl-2-aminoethyl 4-O-methylpropionate-2-deoxy-2- sulfoamino-1-thio-α-D-glucopyranoside)-(1→4)-(methyl-1-thio-α-L-idopyranosiduronate- (1→4)-4-O-carboxyethyl-2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)-(1→4)-methyl- 1-thio-α-L-idopyranosiduronate (49e). 75 Starting material (12 mg, 0.020 mmol) was treated according to the general procedures of pseudo-tetrasaccharide preparation with HATU to give compound 49e (20 mg, 70%). 1H NMR (500 MHz, CD3OD) δ 7.81 (d, J = 7.5 Hz, 2H), 7.66 (d, J = 7.5 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H), 5.35 (d, J = 3.5 Hz, 1H; A-1), 5.32 (d, J = 3.5 Hz, 1H; C-1), 4.97 (d, J = 2.6 Hz, 1H; B-1), 4.82 – 4.77 (m, 2H; D-1), 4.37 – 4.27 (m, 2H), 4.22 – 4.16 (m, 3H), 4.10 – 3.95 (m, 5H), 3.92 – 3.85 (m, 3H), 3.76 – 3.72 (m, 5H), 3.73 – 3.68 (m, 8H), 3.67 (s, 3H), 3.63 – 3.59 (m, 4H), 3.58 – 3.53 (m, 4H), 3.43 – 3.48 (m, 3H), 3.32 – 3.29 (m, 6H), 2.62 – 2.47 (m, 5H), 2.41 – 2.32 (m, 2H). 13C NMR (125 MHz, CD3OD) δ 173.03, 172.75, 170.43, 143.93, 141.17, 127.38, 126.80, 124.84, 119.50, 101.54, 101.37, 96.93, 96.39, 78.14, 78.08, 74.57, 74.04, 72.13, 71.63, 71.44, 69.30, 69.01, 67.92, 67.88, 67.46, 67.27, 67.21, 66.40, 60.62, 60.46, 58.45, 58.30, 54.42, 53.98, 51.50, 51.41, 50.66, 46.48, 42.39, 40.27, 38.88, 37.97, 36.57, 34.78. HRMS: m/z calc. for C37H62N4O31S2 2- [M]2-: 561.1425; found: 561.1426. N-Fluorenylmethyloxycarbonyl-2-aminoethyl O-(4-O-methylpropionate-2-deoxy-2- sulfoamino-1-thio-α-D-glucopyranoside)-(1→4)-O-(methyl-1-thio-α-L- idopyranosiduronate)-(1→4)-O-(2-acetamido-4-O-carboxyethyl-2-deoxy-6-O-sulfonato-1- thio-α-D-glucopyranoside)-(1→4)-O-methyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate (49f). 76 Starting material (8 mg, 0.013 mmol) was treated according to the general procedures of pseudo-tetrasaccharide preparation with HATU to give compound 49f (20 mg, 61%). 1H NMR (500 MHz, CD3OD) δ 7.81 (d, J = 7.5 Hz, 2H), 7.67 (d, J = 7.6 Hz, 2H), 7.40 (tt, J = 7.6, 1.6 Hz, 2H), 7.33 (tt, J = 7.5, 1.4 Hz, 2H), 5.37 (d, J = 3.6 Hz, 1H; A-1), 5.18 (s, 1H; C-1), 4.96 (d, J = 3.4 Hz, 1H; B-1), 4.92 (d, J = 2.9 Hz, 1H; D-1), 4.85 (d, J = 2.0 Hz, 1H), 4.83 (d, J = 3.0 Hz, 1H), 4.35 – 4.27 (m, 4H), 4.24 – 4.15 (m, 4H), 4.11 – 4.04 (m, 2H), 4.03 – 3.94 (m, 4H), 3.82 – 3.85 (m, 2H), 3.81 – 3.74 (m, 5H), 3.73 – 3.66 (m, 8H), 3.64 – 3.52 (m, 5H), 3.43 – 3.37 (m, 2H), 3.29 – 3.19 (m, 2H), 2.64 – 2.52 (m, 3H), 2.49 – 2.39 (m, 3H), 2.09 (s, 3H). 13C NMR (125 MHz, CD3OD) δ 173.03, 172.97, 172.79, 170.47, 170.40, 143.90, 141.14, 127.37, 126.81, 124.90, 124.87, 119.47, 101.36, 99.53, 97.08, 96.29, 77.28, 78.12, 74.22, 73.87, 73.06, 71.87, 71.48, 71.42, 69.92, 69.17, 68.31, 67.87, 67.50, 67.29, 67.16, 66.48, 66.10, 65.84, 64.69, 60.55, 58.16, 53.60, 51.76, 51.51, 50.69, 40.18, 38.75, 36.62, 34.74, 21.75. HRMS: m/z calc. for C39H63N4O35S3 3- [M+H+]2-: 622.1262; found: 622.1260. N-Fluorenylmethyloxycarbonyl-2-aminoethyl O-(2-acetamido-2-deoxy-4-O- methylpropionate-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(methyl-2-O- sulfonato-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(4-O-carboxyethyl-2-deoxy-2- sulfoamino-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-methyl-2-O-sulfonato-1- thio-α-L-idopyranosiduronate (49g). 77 Starting material (22 mg, 0.03 mmol) was treated according to the general procedures of pseudo-tetrasaccharide preparation with HATU to give compound 49g (26 mg, 62%). 1H NMR (500 MHz, CD3OD) δ 8.00 (d, J = 7.6 Hz, 2H), 7.78 (t, J = 7.0 Hz, 2H), 7.58 (td, J = 7.6, 3.6 Hz, 2H), 7.52 – 7.46 (m, 2H), 5.37 (d, J = 3.6 Hz, 1H; A-1), 5.19 (s, 1H; C-1), 5.15 – 5.07 (m, 2H; B-1, D-1), 4.98 (s, 1H), 4.76 (d, J = 2.2 Hz, 1H), 4.64 – 4.59 (m, 1H), 4.52 – 4.47 (m, 1H), 4.45 – 4.20 (m, 9H), 4.18 – 3.94 (m, 8H), 3.89 – 4.81 (m, 5H), 3.76 (s, 4H), 3.72 – 3.55 (m, 7H), 3.51 – 3.41 (m, 4H), 3.33 – 3.22 (m, 3H), 2.77 – 2.66 (m, 2H), 2.56 (t, J = 5.9 Hz, 2H), 2.10 (s, 3H). 13C NMR (125 MHz, CD3OD) δ 173.39, 173.31, 172.68, 169.77, 169.41, 142.34, 139.55, 126.65, 126.07, 123.81, 123.58, 118.73, 98.01, 97.65, 97.22, 93.48, 76.05, 75.93, 75.20, 73.28, 71.48, 70.13, 69.61, 69.24, 68.15, 67.75, 66.88, 66.52, 65.98, 65.82, 65.42, 64.86, 64.74, 64.66, 61.61, 56.36, 51.32, 51.62, 51.49, 50.81, 47.38, 45.57, 38.71, 37.54, 35.12, 33.52, 20.69. HRMS: m/z calc. for C39H61N4O41S5 5- [M]5-: 280.2288; found: 280.2278. N-Fluorenylmethyloxycarbonyl-2-aminoethyl O-(2-acetamido-2-deoxy-4-O- methylpropionate-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(methyl-2-O- sulfonato-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(4-O-carboxyethyl-2-deoxy-2- sulfoamino-1-thio-α-D-glucopyranoside)-(1→4)-O-methyl-1-thio-α-L-idopyranosiduronate (49h). 78 Starting material (8.5 mg, 0.01 mmol) was treated according to the general procedures of pseudo-tetrasaccharide preparation with HATU to give compound 49h (11.7 mg, 80%). 1H NMR (500 MHz, CD3OD) δ 7.81 (d, J = 7.5 Hz, 2H), 7.66 (d, J = 7.5 Hz, 2H), 7.44 – 7.38 (m, 2H), 7.33 (td, J = 7.5, 1.2 Hz, 2H), 5.35 (d, J = 3.7 Hz, 1H; A-1), 5.16 (s, 1H; C-1), 4.99 (d, J = 3.5 Hz, 1H; B-1), 4.92 (d, J = 2.7 Hz, 1H; D-1), 4.87 (d, J = 2.0 Hz, 1H), 4.80 (d, J = 2.9 Hz, 1H), 4.36 – 4.24 (m, 4H), 4.22 – 4.13 (m, 4H), 4.12 – 3.93 (m, 7H), 3.93 – 3.85 (m, 2H), 3.85 – 3.74 (m, 6H), 3.65 – 3.52 (m, 6H), 3.48 – 3.38 (m, 4H), 3.27 – 3.19 (m, 1H), 2.63 – 2.56 (m, 2H), 2.55 – 2.38 (m, 3H), 2.08 (s, 3H). 13C NMR (125 MHz, CD3OD) δ 173.03, 172.96, 172.89, 170.43, 143.92, 141.18, 127.38, 126.79, 124.84, 119.50, 101.37, 99.69, 96.98, 96.69, 78.32, 78.27, 74.30, 74.12, 73.31, 71.59, 71.56, 71.50, 69.98, 69.13, 67.87, 67.78, 67.66, 67.60, 67.27, 66.39, 66.34, 65.93, 64.82, 60.48, 58.19, 53.59, 51.78, 51.60, 40.24, 38.79, 36.43, 34.86, 21.74. HRMS: m/z calc. for C39H63N4O35S3 3- [M+H+]2-: 622.1262; found: 622.1260. N-Fluorenylmethyloxycarbonyl-2-aminoethyl O-(2-acetamido-2-deoxy-4-O- methylpropionate-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(methyl-2-O- sulfonato-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-acetamido-4-O-carboxyethyl-2- deoxy-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-methyl-2-O-sulfonato-1-thio-α- L-idopyranosiduronate (49i). 79 Starting material (8.0 mg, 0.01 mmol) was treated according to the general procedures of pseudo-tetrasaccharide preparation with HATU to give compound 49i (12.7 mg, 80%). 1H NMR (500 MHz, CD3OD) δ 7.81 (d, J = 7.5 Hz, 2H), 7.67 (d, J = 7.5 Hz, 2H), 7.40 (tt, J = 7.5, 1.5 Hz, 2H), 7.33 (tt, J = 7.5, 1.5 Hz, 2H), 5.16 (d, J = 4.8 Hz, 2H; A-1, C-1), 4.99 (d, J = 3.5 Hz, 1H; B-1), 4.97 (d, J = 3.4 Hz, 1H; D-1), 4.86 (dd, J = 5.6, 1.9 Hz, 2H), 4.34 – 4.25 (m, 6H), 4.22 – 4.18 (m, 5H), 4.11 – 4.05 (m, 3H), 4.02 – 3.93 (m, 7H), 3.82 (s, 3H), 3.74 – 3.66 (m, 7H), 3.66 – 3.54 (m, 6H), 3.43 – 3.37 (m, 3H), 3.33 – 3.35 (m, 2H), 3.30 (d, J = 2.7 Hz, 1H), 2.62 – 2.56 (m, 2H), 2.49 – 2.43 (m, 2H), 2.07 (d, J = 1.7 Hz, 6H). 13C NMR (125 MHz, CD3OD) δ 172.96, 172.92, 170.49, 170.33, 157.48, 143.90, 141.14, 127.38, 126.81, 124.89, 119.48, 99.61, 99.56, 97.23, 96.43, 78.34, 78.26, 74.36, 73.47, 73.09, 71.52, 71.42, 69.99, 69.91, 68.22, 67.76, 67.55, 67.25, 66.49, 66.27, 66.22, 65.91, 64.79, 64.40, 53.60, 53.40, 51.83, 51.75, 50.82, 40.17, 38.67, 36.46, 34.85, 21.74. HRMS: m/z calc. for C41H64N4O39S4 4- [M+H+]3-: 455.0708; found: 455.0688. Acetamidoethyl O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-deoxy-2-sulfoamino-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O- carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfoamino- 6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α- L-idopyranosiduronate) (50). 80 The starting material (5.8 mg, 0.004 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50 (3.8 mg, 51% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.22 (d, J = 3.6 Hz, 1H; A-1), 5.16 (t, J = 4.2 Hz, 2H; C-1, F-1), 5.00 (d, J = 3.6 Hz, 2H; B-1, D-1), 4.95 (d, J = 2.8 Hz, 1H; E-1), 4.45 (dd, J = 11.5, 2.3 Hz, 2H), 4.41 (d, J = 2.8 Hz, 1H), 4.17 (dd, J = 11.0, 2.1 Hz, 1H), 4.13-4.08 (m, 5H), 4.07 – 3.99 (m, 4H), 3.94 – 3.62 (m, 14H), 3.57 – 3.44 (m, 6H), 3.32-3.16 (m, 8H), 3.13-3.05 (m, 3H), 2.53 – 2.34 (m, 6H), 1.82 (s, 3H). 13C NMR (125 MHz, D2O) δ 173.83, 99.07, 98.72, 96.85, 77.81, 77.78, 77.59, 76.06, 75.94, 74.68, 74.55, 70.48, 70.43, 68.84, 68.82, 68.77, 68.62, 68.40, 68.29, 68.08, 67.61, 67.52, 67.30, 66.92, 66.81, 66.20, 66.12, 57.78, 57.77, 57.71, 39.04, 38.91, 36.22, 36.20, 35.30, 21.69. HRMS: m/z calc. for C53H79N6O65S9 9- [M+4Na+]5-: 443.8033; found: 443.8041. Acetamidoethyl O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-deoxy-2-sulfoamino-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O- carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfoamino- 1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate) (50a). The starting material (6.5 mg, 0.004 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and 81 saponification of methyl esters to give compound 50a (3.7 mg, 48% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.19 (d, J = 3.6 Hz, 1H; A-1), 5.15 (dd, J = 6.2, 3.6 Hz, 2H; C-1, F-1), 5.01 – 4.96 (m, 2H; B-1, D-1), 4.73 – 4.70 (m, 1H; E-1), 4.44 –4.33 (m, 3H), 4.18 – 4.13 (m, 1H), 4.12 – 4.05 (m, 5H), 4.04-3.99 (m, 2H), 3.98-3.93 (m, 1H), 3.92 – 3.71 (m, 12H), 3.70 – 3.44 (m, 14H), 3.32 – 3.15 (m, 9H), 3.12 – 3.00 (m, 4H), 2.50 – 2.35 (m, 6H), 1.82 (s, 3H). 13C NMR (125 MHz, D2O) δ 173.84, 100.61, 98.90, 98.70, 97.47, 97.09, 95.60, 78.04, 77.82, 77.64, 75.98, 75.95, 75.38, 74.69, 74.43, 70.68, 70.54, 70.47, 68.79, 68.46, 68.27, 68.22, 68.18, 67.78, 67.66, 67.52, 66.77, 66.71, 66.16, 59.75, 57.77, 57.75, 57.72, 39.05, 38.97, 38.91, 36.19, 21.70. HRMS: m/z calc. for C53H81N6O59S7 7- [M+2Na+]5-: 403.0278; found: 403.0291. Acetamidoethyl O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-deoxy-2-sulfoamino-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O- carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-acetamido-2-deoxy-6- O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L- idopyranosiduronate) (50b). The starting material (5.8 mg, 0.004 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50b (1.4 mg, 19% for 3 steps). 1H NMR (500 82 MHz, D2O) δ 5.17 (dd, J = 8.9, 3.5 Hz, 2H; A-1, C-1), 4.98 (s, 2H; F-1, B-1), 4.94 (d, J = 3.7 Hz, 1H; D-1), 4.91 (s, 1H; E-1), 4.43 – 4.34 (m, 4H), 4.19 – 3.99 (m, 13H), 3.94 – 3.72 (m, 14H), 3.71 – 3.43 (m, 13H), 3.35 – 3.16 (m, 10H), 3.13 – 3.03 (m, 3H), 2.47 – 2.35 (m, 7H), 1.88 (s, 3H), 1.82 (s, 3H). 13C NMR (125 MHz, D2O) δ 77.87, 75.88, 73.73, 70.49, 70.81, 68.86, 68.76, 68.61, 66.85, 66.91, 66.28, 66.16, 57.76, 39.01, 38.90, 36.18, 27.45, 22.07, 21.70. HRMS: m/z calc. for C55H82N6O63S8 8- [M+3Na+]5-: 431.8177; found: 431.8189. Acetamidoethyl O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-1- thio-α-L-idopyranosiduronate)-(1→4)-O-(2-acetamido-2-deoxy-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate) (50c). The starting material (4.5 mg, 0.003 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50c (1.4 mg, 51% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.22 (d, J = 3.5 Hz, 1H; A-1), 5.18 (d, J = 3.6 Hz, 1H; C-1), 5.14 (d, J = 3.9 Hz, 1H; E-1), 4.97 (d, J = 2.4 Hz, 1H; B-1), 4.94 (d, J = 2.9 Hz, 1H; D-1), 4.73 (d, J = 2.7 Hz, 1H; F-1), 4.41 – 4.32 (m, 3H), 4.19 – 3.99 (m, 9H), 3.97 – 3.92 (m, 1H), 3.92 – 3.73 (m, 12H), 3.71 – 3.42 83 (m, 15H), 3.37 – 3.16 (m, 10H), 3.12 – 3.01 (m, 4H), 2.52 – 2.30 (m, 7H), 1.82 (s, 3H). 13C NMR (125 MHz, D2O) δ 177.77, 173.87, 122.73, 119.89, 109.99, 100.57, 98.83, 97.48, 97.10, 96.74, 95.65, 78.05, 77.75, 77.69, 76.04, 75.93, 74.32, 70.73, 70.58, 70.49, 68.75, 68.70, 68.41, 68.34, 66.87, 66.75, 66.47, 66.20, 57.78, 57.73, 39.05, 38.98, 38.85, 36.35, 21.69. HRMS: m/z calc. for C53H81N6O59S7 7- [M+2Na+]5-: 403.0278; found: 403.0296. Acetamidoethyl O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-1- thio-α-L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfonato-1-thio-α-D-glucopyranoside)- (1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate) (50d). The starting material (4.5 mg, 0.003 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50d (3.0 mg, 56% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.18 (d, J = 3.7 Hz, 1H; A-1), 5.14 (dd, J = 3.9, 1.4 Hz, 2H; C-1, E-1), 4.97 (d, J = 2.5 Hz, 1H; B-1), 4.74 – 4.71 (m, 2H; D-1, F-1), 4.39 – 4.32 (m, 3H), 4.18 – 4.14 (m, 1H), 4.12 – 3.99 (m, 4H), 3.97 – 3.92 (m, 2H), 3.91 – 3.72 (m, 11H), 3.70 – 3.45 (m, 18H), 3.31 – 3.16 (m, 10H), 3.10 – 2.98 (m, 4H), 2.48 – 2.33 (m, 7H), 1.82 (s, 3H). 13C NMR (125 MHz, D2O) δ 173.93, 173.85, 100.61, 98.85, 97.11, 95.65, 95.57, 78.12, 78.05, 77.97, 75.94, 74.45, 74.30, 84 70.71, 70.55, 70.50, 68.78, 68.39, 68.27, 68.12, 67.83, 67.58, 67.38, 66.75, 66.69, 66.49, 66.19, 59.76, 57.78, 57.74, 38.99, 39.06, 36.26, 36.15, 21.70. HRMS: m/z calc. for C53H83N6O53S5 5- [M+Na+]4-: 458.5627; found: 458.5641. Acetamidoethyl O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-1- thio-α-L-idopyranosiduronate)-(1→4)-O-(2-acetamido-2-deoxy-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate) (50e). The starting material (4.5 mg, 0.003 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50e (2.9 mg, 50% for 3 steps). 1H NMR (500 MHz, D2O) δ 7.96 (d, J = 9.8 Hz, 1H; NHAc), 5.18 (d, J = 3.7 Hz, 1H; A-1), 5.14 (d, J = 3.7 Hz, 1H; C-1), 4.98 – 4.90 (m, 3H; D-1, B-1, E-1), 4.75 – 4.71 (m, 1H; F-1), 4.39 (d, J = 2.3 Hz, 1H), 4.35 (dd, J = 6.9, 2.4 Hz, 2H), 4.17 – 4.00 (m, 8H), 3.97 – 3.74 (m, 13H), 3.70 – 3.44 (m, 13H), 3.35 – 3.17 (m, 9H), 3.10 – 3.01 (m, 2H), 2.46 – 2.34 (m, 6H), 1.88 (s, 3H), 1.83 (s, 3H). 13C NMR (125 MHz, D2O) δ 190.14, 109.99, 109.71, 99.00, 98.65, 98.64, 96.97, 95.61, 93.51, 78.11, 77.77, 75.88, 75.41, 74.39, 73.67, 70.85, 70.73, 70.81, 70.53, 68.83, 68.71, 68.59, 68.26, 68.10, 67.42, 66.85, 66.44, 66.18, 63.81, 57.70, 53.04, 39.00, 38.87, 36.28, 36.13, 22.07, 21.70. HRMS: 85 m/z calc. for C55H84N6O57S6 6- [M+Na+]5-: 391.0422; found: 391.0433. Acetamidoethyl O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-acetamido-2-deoxy-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O- carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfonato-6- O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L- idopyranosiduronate) (50f). The starting material (5.2 mg, 0.003 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50f (3.7 mg, 55% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.22 (d, J = 3.5 Hz, 1H; A-1), 5.16 (d, J = 3.5 Hz, 1H; C-1), 5.00 – 4.97 (m, 1H; E- 1), 4.94 (m, 3H; B-1, D-1, F-1), 4.46 – 4.38 (m, 3H), 4.20 – 3.98 (m, 11H), 3.97 – 3.71 (m, 12H), 3.66 (m, 3H), 3.60 – 3.44 (m, 6H), 3.38 – 3.15 (m, 9H), 3.09 (m, 2H), 2.54 – 2.45 (m, 2H), 2.44 – 2.35 (m, 4H), 1.88 (s, 3H), 1.82 (s, 3H). 13C NMR (125 MHz, D2O) δ 99.08, 98.74, 98.58, 97.49, 96.82, 93.69, 90.46, 77.83, 77.61, 76.03, 75.95, 73.19, 70.78, 70.51, 70.45, 68.80, 68.76, 68.67, 68.63, 68.41, 68.14, 67.70, 66.80, 66.91, 66.71, 66.20, 66.12, 57.79, 57.73, 53.01, 39.04, 38.93, 38.84, 36.24, 36.15, 22.11, 21.69. HRMS: m/z calc. for C55H82N6O63S8 8- [M+Na+]7-: 301.8728; found: 301.8744. 86 Acetamidoethyl O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-acetamido-2-deoxy-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O- carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfonato-1- thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate) (50g). The starting material (4.8 mg, 0.003 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50g (2.5 mg, 44% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.17 (d, J = 3.7 Hz, 1H; A-1), 5.14 (d, J = 3.6 Hz, 1H; C-1), 4.99 – 4.97 (m, 1H; E- 1), 4.93 (d, J = 3.1 Hz, 2H; B-1, D-1), 4.73 – 4.69 (m, 1H; F-1), 4.39 (dd, J = 13.7, 2.3 Hz, 2H), 4.33 (d, J = 2.4 Hz, 1H), 4.18 – 3.99 (m, 8H), 3.95 (t, J = 3.9 Hz, 2H), 3.92 – 3.72 (m, 11H), 3.70 – 3.42 (m, 13H), 3.36 – 3.17 (m, 9H), 3.13-3.01 (m, 3H), 2.51 – 2.32 (m, 6H), 1.88 (s, 3H), 1.81 (s, 3H). 13C NMR (125 MHz, D2O) δ 174.21, 173.90, 109.99, 100.60, 97.21, 95.57, 93.54, 78.03, 77.85, 77.70, 75.91, 75.02, 74.45, 73.45, 70.69, 70.49, 68.75, 68.32, 68.17, 67.60, 66.69, 66.15, 63.59, 59.74, 57.74, 39.05, 38.93, 36.13, 22.09, 21.70. HRMS: m/z calc. for C55H84N6O57S6 6- [M+Na+]5-: 391.0422; found: 391.0435. 87 Acetamidoethyl O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-acetamido-2-deoxy-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O- carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-acetamido-2-deoxy-6- O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L- idopyranosiduronate) (50h). The starting material (5.2 mg, 0.003 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50h (2.8 mg, 42% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.16 (d, J = 3.7 Hz, 1H; A-1), 4.98 (s, 1H; C-1), 4.93 (m, 4H; B-1, D-1, E-1, F-1), 4.46 – 4.38 (m, 3H), 4.22 – 3.99 (m, 12H), 3.94 – 3.71 (m, 14H), 3.69 – 3.62 (m, 3H), 3.60 – 3.41 (m, 7H), 3.35 – 3.19 (m, 9H), 3.17 (s, 1H), 3.08 (dd, J = 10.6, 3.5 Hz, 1H), 2.52 – 2.28 (m, 7H), 1.88 (s, 6H), 1.82 (s, 3H). 13C NMR (125 MHz, D2O) δ 174.62, 174.15, 173.87, 98.73, 98.67, 98.58, 97.46, 93.65, 93.54, 77.82, 77.62, 76.01, 74.77, 73.65, 73.26, 70.83, 70.78, 70.45, 68.88, 68.79, 68.62, 68.58, 68.48, 68.15, 67.72, 66.87, 66.79, 66.70, 66.22, 66.10, 63.79, 57.77, 53.01, 39.00, 38.93, 38.86, 36.20, 36.15, 35.39, 22.09, 21.69. HRMS: m/z calc. for C57H85N6O61S7 7- [M+2Na+]5-: 419.8320; found: 419.8341. 88 Acetamidoethyl O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)- (1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfoamino- 6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α- L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfoamino-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate) (50i). The starting material (4.6 mg, 0.003 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50i (1.2 mg, 20% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.24 (s, 1H; A-1), 5.15 (d, J = 12.9 Hz, 2H; C-1, E-1), 4.99 (s, 1H; B-1), 4.93 (s, 2H; D-1, F-1), 4.38 – 4.31 (m, 3H), 4.14 – 3.99 (m, 8H), 3.96 – 3.71 (m, 13H), 3.69 – 3.42 (m, 14H), 3.36 – 3.15 (m, 9H), 3.12 – 3.01 (m, 4H), 2.44 – 2.32 (m, 6H), 1.82 (d, J = 1.8 Hz, 3H). HRMS: m/z calc. for C53H81N6O59S7 7- [M+2Na+]5-: 403.0278; found: 403.0296. Acetamidoethyl O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)- 89 (1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfoamino- 6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α- L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)- (1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate) (50j). The starting material (4.2 mg, 0.003 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50j (2.4 mg, 48% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.17 (d, J = 2.9 Hz, 1H; A-1), 5.14 (t, J = 2.5 Hz, 2H; C-1, E-1), 4.98 (s, 1H; B-1), 4.72 (dd, J = 5.4, 2.5 Hz, 2H; D-1, F-1), 4.46 – 4.35 (m, 3H), 4.12 – 4.05 (m, 3H), 4.03 – 3.83 (m, 9H), 3.83 – 3.69 (m, 5H), 3.68 – 3.44 (m, 15H), 3.36 – 3.13 (m, 9H), 3.11 – 2.99 (m, 3H), 2.46 – 2.34 (m, 6H), 1.81 (d, J = 1.9 Hz, 3H). 13C NMR (125 MHz, D2O) δ 173.88, 170.07, 100.62, 100.60, 98.86, 97.38, 95.63, 77.98, 77.81, 77.66, 76.06, 75.08, 74.38, 74.22, 70.75, 70.66, 70.57, 70.51, 68.79, 68.19, 68.15, 68.09, 67.97, 67.91, 67.64, 67.41, 67.28, 66.82, 66.73, 66.54, 66.11, 59.73, 57.81, 57.70, 39.04, 38.98, 38.88, 36.33, 36.18, 35.58, 21.69. HRMS: m/z calc. for C53H83N6O53S5 5- [M+Na+]4-: 458.5627; found: 458.5641. Acetamidoethyl O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)- (1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfoamino- 6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α- 90 L-idopyranosiduronate)-(1→4)-O-(2-acetamido-2-deoxy-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate) (50k). The starting material (6.6 mg, 0.004 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50k (5.0 mg, 59% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.15 (m, 2H; A-1, C-1), 4.99 (s, 1H; E-1), 4.94 – 4.90 (m, 2H; D-1, B-1), 4.72 (s, 1H; F-1), 4.45 – 4.36 (m, 3H), 4.13 – 3.99 (m, 8H), 3.95 (t, J = 3.7 Hz, 1H), 3.91 – 3.70 (m, 12H), 3.70 – 3.60 (m, 5H), 3.60 – 3.43 (m, 8H), 3.36 – 3.17 (m, 9H), 3.12 – 2.96 (m, 3H), 2.44 – 2.34 (m, 6H), 1.88 (s, 3H), 1.82 (s, 3H). 13C NMR (125 MHz, D2O) δ 174.62, 173.88, 100.60, 98.77, 98.68, 95.61, 93.54, 77.84, 77.68, 76.04, 74.81, 74.70, 74.23, 73.63, 70.82, 70.76, 70.56, 70.49, 68.88, 68.80, 68.58, 68.40, 68.20, 68.00, 67.72, 67.32, 66.88, 66.81, 66.53, 66.25, 66.14, 63.77, 59.70, 57.82, 57.69, 53.00, 39.00, 38.88, 36.33, 36.17, 22.07, 21.69. HRMS: m/z calc. for C55H84N6O57S6 6- [M+Na+]5-: 391.0422; found: 391.0430. Acetamidoethyl O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)- (1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfoamino- 1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate)- 91 (1→4)-O-(2-deoxy-2-sulfoamino-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O- carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate) (50l). The starting material (2.5 mg, 0.002 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50l (1.4 mg, 42% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.22 (d, J = 3.5 Hz, 1H; A-1), 5.19 – 5.06 (m, 2H; C-1, E-1), 4.94 (s, 1H; B-1), 4.77 – 4.71 (m, 2H; D-1, F-1), 4.38 – 4.30 (m, 3H), 4.13 – 4.07 (m, 2H), 4.05 – 3.99 (m, 2H), 3.95 (t, J = 3.9 Hz, 2H), 3.93 – 3.83 (m, 6H), 3.82 – 3.71 (m, 4H), 3.70 – 3.41 (m, 16H), 3.34 – 3.15 (m, 9H), 3.11 – 3.01 (m, 4H), 2.44 – 2.31 (m, 6H), 1.82 (s, 3H). HRMS: m/z calc. for C53H83N6O53S5 5- [M+Na+]4-: 458.5627; found: 458.5623. Acetamidoethyl O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)- (1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfoamino- 1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-1- thio-α-L-idopyranosiduronate) (50m). The starting material (3.0 mg, 0.002 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharides preparation with HATU and 92 saponification of methyl esters to give compound 50m (0.8 mg, 22% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.15 (d, J = 3.6 Hz, 3H; A-1, C-1, E-1), 4.73 (d, J = 4.1 Hz, 3H; B-1, D-1, F-1), 4.38 – 4.28 (m, 3H), 3.93 – 3.99 (m, 3H), 3.91-3.82 (m, 6H), 3.80 – 3.73 (m, 4H), 3.70 – 3.44 (m, 20H), 3.33 – 3.17 (m, 9H), 3.11 – 2.99 (m, 4H), 2.46 – 2.33 (m, 6H), 1.82 (s, 2H). HRMS: m/z calc. for C53H85N6O47S3 3- [M]3-: 551.1208; found: 551.1201. Acetamidoethyl O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)- (1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfoamino- 1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-acetamido-2-deoxy-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O- carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate) (50n). The starting material (3.8 mg, 0.003 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HATU and saponification of methyl esters to give compound 50n (2.6 mg, 52% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.19 – 5.05 (m, 2H; A-1, C-1), 4.95 – 4.91 (m, 2H; E-1, B-1), 4.73 (m, 2H; D-1, F- 1), 4.43 – 4.35 (m, 3H), 4.12 – 4.01 (m, 4H), 3.95 (d, J = 4.0 Hz, 2H), 3.92 – 3.72 (m, 12H), 3.70 – 3.42 (m, 18H), 3.32 – 3.19 (m, 9H), 3.08-2.98 (m, 3H), 2.47 – 2.33 (m, 7H), 1.88 (s, 3H), 1.81 (s, 3H). 13C NMR (125 MHz, D2O) δ 174.62, 173.88, 100.61, 98.65, 95.67, 95.59, 93.61, 77.90, 93 77.86, 77.75, 74.30, 74.27, 73.59, 70.85, 70.75, 70.71, 70.55, 68.87, 68.56, 68.18, 68.08, 67.37, 67.28, 66.88, 66.52, 63.77, 59.72, 57.77, 57.70, 53.04, 39.00, 38.89, 36.29, 36.23, 22.07, 21.70. HRMS: m/z calc. for C55H86N6O51S4 4- [M]4-: 443.5806; found: 443.5810. Acetamidoethyl O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)- (1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-acetamido-2-deoxy-1- thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate)-(1→4)- O-(2-acetamido-2-deoxy-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O- carboxyl-2-O-sulfonato 1-thio-α-L-idopyranosiduronate) (50o). The starting material (3.9 mg, 0.003 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HATU and saponification of methyl esters to give compound 50o (1.7 mg, 34% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.21 (d, J = 3.7 Hz, 1H; A-1), 5.14 (d, J = 3.7 Hz, 1H; C-1), 4.97 – 4.91 (m, 3H; B- 1, D-1, E-1), 4.73 (s, 1H; F-1), 4.48 – 4.36 (m, 3H), 4.14 – 3.99 (m, 8H), 3.98 – 3.71 (m, 13H), 3.70 – 3.44 (m, 13H), 3.36 – 3.15 (m, 9H), 3.14 – 3.00 (m, 3H), 2.47 – 2.33 (m, 6H), 1.88 (s, 3H), 1.82 (s, 3H). HRMS: m/z calc. for C55H84N6O57S6 6- [M+2Na+]4-: 494.5500; found: 494.5502. 94 Acetamidoethyl O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)- (1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-acetamido-2-deoxy-6- O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L- idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)-(1→4)- O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate) (50p). The starting material (7.1 mg, 0.005 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HATU and saponification of methyl esters to give compound 50p (4.4 mg, 51% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.19 – 5.04 (m, 2H; A-1, C-1), 4.96 – 4.92 (m, 2H; B-1, E-1), 4.74 – 4.71 (m, 2H; D-1, F-1), 4.45 (d, J = 2.1 Hz, 1H), 4.40 (dd, J = 4.4, 2.5 Hz, 2H), 4.12 – 4.00 (m, 5H), 3.95 (d, J = 4.3 Hz, 2H), 3.91 – 3.73 (m, 12H), 3.71 – 3.44 (m, 19H), 3.38 – 3.17 (m, 10H), 3.07 – 3.01 (m, 3H), 2.47 – 2.36 (m, 6H), 1.89 (s, 3H), 1.81 (s, 3H). 13C NMR (125 MHz, D2O) δ 174.65, 174.47, 174.15, 173.94, 100.65, 98.65, 95.66, 93.79, 77.94, 77.78, 77.71, 74.37, 74.22, 73.35, 70.92, 70.82, 70.73, 70.67, 70.59, 68.92, 68.54, 68.17, 68.13, 67.98, 67.40, 67.27, 66.79, 66.75, 66.59, 66.19, 63.57, 59.73, 59.70, 57.72, 53.05, 39.04, 38.93, 36.28, 36.21, 35.40, 22.09, 21.69. HRMS: m/z calc. for C55H86N6O51S4 4- [M]4-: 443.5806; found: 443.5805. 95 Acetamidoethyl O-(4-O-carboxyethyl-2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)- (1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-acetamido-2-deoxy-6- O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L- idopyranosiduronate)-(1→4)-O-(2-acetamido-2-deoxy-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate) (50q). The starting material (6.9 mg, 0.005 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HATU and saponification of methyl esters to give compound 50q (4.0 mg, 45% for 3 steps). 1H NMR (500 MHz, D2O) δ 7.99 – 7.88 (m, 3H, NHAc), 5.14 (d, J = 3.6 Hz, 1H; A-1), 4.96 – 4.91 (m, 4H; B-1, C-1, D-1, E-1), 4.39 (d, J = 2.4 Hz, 2H; F-1), 4.32 (s, 1H), 4.15 – 4.02 (m, 9H), 3.96 – 3.73 (m, 16H), 3.69 – 3.42 (m, 16H), 3.38 – 3.19 (m, 11H), 3.05 (dd, J = 10.4, 3.5 Hz, 2H), 2.46 – 2.31 (m, 6H), 1.89 (s, 6H), 1.83 (s, 3H). 13C NMR (125 MHz, D2O) δ 174.63, 98.68, 98.54, 95.59, 93.55, 93.50, 78.13, 77.86, 77.75, 74.38, 73.74, 73.33, 70.85, 70.72, 70.57, 69.28, 68.85, 68.63, 68.18, 67.52, 66.86, 66.65, 66.46, 66.27, 63.89, 63.47, 59.81, 59.70, 57.66, 53.05, 39.03, 38.89, 36.28, 36.21, 22.12, 22.09, 21.72. HRMS: m/z calc. for C57H87N6O55S5 5- [M+Li+]4-: 475.5745; found: 475.5755. 96 Acetamidoethyl O-(2-acetamido-4-O-carboxyethyl-2-deoxy-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-deoxy-2-sulfoamino-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O- carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfoamino- 6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α- L-idopyranosiduronate) (50r). The starting material (5.5 mg, 0.003 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50r (2.4 mg, 34% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.22 (d, J = 3.3 Hz, 1H; A-1), 5.16 (d, J = 3.6 Hz, 1H; C-1), 5.00 (s, 2H; E-1), 4.96 – 4.89 (m, 3H; B-1, D-1, F-1), 4.48 – 4.39 (m, 3H), 4.19 – 3.98 (m, 10H), 3.93 – 3.45 (m, 20H), 3.34 – 3.04 (m, 11H), 2.52 – 2.33 (m, 6H), 1.89 (s, 3H), 1.82 (s, 3H). 13C NMR (125 MHz, D2O) δ 173.79, 171.71, 109.99, 99.08, 98.56, 97.59, 93.80, 91.93, 91.61, 77.81, 77.76, 77.61, 76.07, 75.96, 74.67, 74.22, 73.18, 72.85, 70.86, 70.79, 70.49, 68.91, 68.78, 68.37, 67.62, 66.93, 66.13, 57.72, 53.00, 45.75, 38.87, 57.72, 39.05, 38.87, 36.23, 22.10, 21.70. HRMS: m/z calc. for C55H82N6O63S8 8- [M+3Na+]5-: 431.8177; found: 431.8196. 97 Acetamidoethyl O-(2-acetamido-4-O-carboxyethyl-2-deoxy-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-deoxy-2-sulfoamino-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O- carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfoamino- 1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate) (50s). The starting material (8.2 mg, 0.005 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50s (5.0 mg, 51% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.19 (d, J = 3.6 Hz, 1H; A-1), 5.14 (d, J = 3.7 Hz, 1H; C-1), 4.98 (d, J = 2.1 Hz, 1H; E-1), 4.96 – 4.91 (m, 2H; B-1, D-1), 4.73 – 4.70 (m, 1H; F-1), 4.43 (d, J = 2.0 Hz, 2H), 4.37 (d, J = 2.4 Hz, 1H), 4.19 – 3.99 (m, 9H), 3.96 (t, J = 3.9 Hz, 2H), 3.92 – 3.44 (m, 29H), 3.35 – 3.16 (m, 10H), 3.12 – 3.02 (m, 3H), 2.50 – 2.33 (m, 6H), 1.89 (s, 3H), 1.81 (s, 3H). 13C NMR (125 MHz, D2O) δ 174.66, 174.16, 173.87, 100.63, 98.91, 98.56, 97.22, 95.62, 93.76, 78.01, 77.77, 77.61, 76.01, 75.29, 74.41, 73.18, 70.83, 70.79, 70.67, 70.56, 68.90, 68.81, 68.41, 68.24, 68.17, 68.09, 67.72, 67.48, 66.81, 66.73, 66.16, 66.13, 63.42, 59.74, 57.77, 57.72, 52.99, 39.05, 38.97, 38.87, 36.25, 36.18, 35.51, 22.09, 21.70. HRMS: m/z calc. for C55H84N6O57S6 6- [M+2Na+]4-: 494.5500; found: 494.5519. 98 Acetamidoethyl O-(2-acetamido-4-O-carboxyethyl-2-deoxy-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-deoxy-2-sulfoamino-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O- carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-acetamido-2-deoxy-6- O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L- idopyranosiduronate) (50t). The starting material (7.1 mg, 0.004 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50t (4.5 mg, 50% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.17 (d, J = 3.5 Hz, 1H; A-1), 4.99 (s, 1H; C-1), 4.97 – 4.90 (m, 4H; B-1, D-1, E-1, F-1), 4.47 – 4.40 (m, 3H), 4.16 (dd, J = 11.1, 2.7 Hz, 1H), 4.13 – 3.99 (m, 10H), 3.95 – 3.42 (m, 24H), 3.35 – 3.18 (m, 9H), 3.09 (dd, J = 10.5, 3.5 Hz, 2H), 2.53 – 2.32 (m, 6H), 1.88 (s, 6H), 1.82 (s, 3H). 13C NMR (125 MHz, D2O) δ 199.58, 177.66, 174.62, 174.16, 173.84, 98.77, 98.68, 98.56, 97.48, 93.80, 93.57, 77.84, 77.75, 77.61, 76.04, 74.83, 73.63, 73.17, 70.85, 70.78, 70.46, 68.90, 68.84, 68.58, 68.44, 68.36, 67.74, 66.89, 66.83, 66.75, 66.26, 66.13, 63.79, 63.43, 57.78, 53.00, 39.00, 38.93, 36.24, 36.18, 35.43, 22.09, 21.70. HRMS: m/z calc. for C57H85N6O61S7 7- [M+2Na+]5-: 419.8320; found: 419.8333. 99 Acetamidoethyl O-(2-acetamido-4-O-carboxyethyl-2-deoxy-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-thio- α-L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfoamino-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate) (50u). The starting material (6.8 mg, 0.005 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50u (4.0 mg, 45% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.21 (d, J = 3.5 Hz, 1H; A-1), 5.14 (d, J = 3.7 Hz, 1H; C-1), 4.96 – 4.91 (m, 3H; E- 1, B-1, D-1), 4.75 – 4.71 (m, 1H; F-1), 4.39 (dd, J = 13.1, 2.4 Hz, 3H), 4.20 – 3.99 (m, 9H), 3.96 (t, J = 3.8 Hz, 1H), 3.93 – 3.73 (m, 13H), 3.70 – 3.43 (m, 15H), 3.39 – 3.16 (m, 11H), 3.06 (m, 3H), 2.49 – 2.33 (m, 6H), 1.89 (s, 3H), 1.82 (s, 3H). 13C NMR (125 MHz, D2O) δ 174.64, 173.89, 99.03, 98.60, 96.80, 95.65, 93.68, 78.02, 77.74, 77.66, 75.95, 74.31, 73.36, 70.84, 70.57, 68.86, 68.72, 68.57, 68.41, 68.19, 66.89, 66.74, 66.48, 66.15, 63.58, 59.74, 57.78, 57.74, 52.97, 39.05, 36.35, 35.78, 22.09, 21.69. HRMS: m/z calc. for C55H84N6O57S66- [M+Na+]5-: 391.0422; found: 391.0437. 100 Acetamidoethyl O-(2-acetamido-4-O-carboxyethyl-2-deoxy-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-thio- α-L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)- (1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate) (50v). The starting material (7.3 mg, 0.005 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50v (4.1 mg, 46% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.14 (s, 2H; A-1, C-1), 4.93 (s, 2H; B-1, E-1), 4.75 – 4.70 (m, 2H; D-1, F-1), 4.43 – 4.30 (m, 4H), 4.20 – 4.02 (m, 5H), 3.95 (q, J = 4.1 Hz, 2H), 3.91 – 3.71 (m, 12H), 3.69 – 3.42 (m, 18H), 3.33 – 3.13 (m, 10H), 3.04 (dd, J = 10.5, 3.7 Hz, 3H), 2.48 – 2.30 (m, 6H), 1.88 (s, 3H), 1.81 (s, 3H). 13C NMR (125 MHz, D2O) δ 174.63, 173.93, 100.60, 98.60, 95.63, 95.58, 93.67, 78.00, 77.94, 77.65, 74.42, 74.33, 73.36, 70.79, 70.69, 70.50, 68.86, 68.54, 68.23, 68.10, 67.52, 66.69, 66.51, 66.14, 63.57, 59.74, 57.77, 57.74, 52.96, 39.05, 38.95, 36.25, 36.17, 22.08, 21.70. HRMS: m/z calc. for C55H86N6O51S4 4- [M]4-: 443.5806; found: 443.5793. 101 Acetamidoethyl O-(2-acetamido-4-O-carboxyethyl-2-deoxy-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-deoxy-2-sulfoamino-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-thio- α-L-idopyranosiduronate)-(1→4)-O-(2-acetamido-2-deoxy-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate) (50w). The starting material (7.0 mg, 0.005 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50w (4.5 mg, 50% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.14 (d, J = 3.7 Hz, 1H; A-1), 4.95 – 4.90 (m, 4H; B-1, C-1, D-1, E-1), 4.75 – 4.71 (m, 1H; F-1), 4.42 – 4.35 (m, 3H), 4.18 – 4.01 (m, 8H), 3.96 (t, J = 3.7 Hz, 1H), 3.92 – 3.73 (m, 13H), 3.70 – 3.44 (m, 14H), 3.37 – 3.16 (m, 10H), 3.04 (dd, J = 10.4, 3.6 Hz, 1H), 2.51 – 2.30 (m, 6H), 1.88 (s, 6H), 1.82 (s, 3H). 13C NMR (125 MHz, D2O) δ 174.63, 173.87, 100.56, 98.65, 98.60, 95.66, 93.68, 93.56, 78.00, 77.75, 77.65, 74.32, 73.61, 73.35, 70.84, 70.80, 70.72, 70.58, 68.85, 68.51, 68.16, 66.86, 66.74, 66.49, 66.14, 63.78, 63.57, 59.73, 57.74, 53.04, 52.97, 39.00, 36.29, 36.16, 35.67, 22.07, 21.70. HRMS: m/z calc. for C57H87N6O55S5 5- [M]5-: 379.0565; found: 379.0584. 102 Acetamidoethyl O-(2-acetamido-4-O-carboxyethyl-2-deoxy-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-acetamido-2-deoxy-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O- carboxyl-2-O-sulfonato-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfoamino-6- O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L- idopyranosiduronate) (50x). The starting material (5.3 mg, 0.003 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50x (1.0 mg, 15% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.24 (d, J = 3.6 Hz, 1H; A-1), 4.92 (d, J = 3.4 Hz, 4H; C-1, B-1, D-1, E-1), 4.40 – 4.32 (m, 3H), 4.16 – 3.97 (m, 10H), 3.95 – 3.73 (m, 12H), 3.70 – 3.43 (m, 11H), 3.39 – 3.13 (m, 11H), 2.42 – 2.31 (m, 6H), 1.85 (s, 7H). HRMS: m/z calc. for C57H85N6O61S7 7- [M+3Na+]4-: 530.5373; found: 530.5389. 103 Acetamidoethyl O-(2-acetamido-4-O-carboxyethyl-2-deoxy-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)- (1→4)-O-(2-acetamido-2-deoxy-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O- carboxyl-2-O-sulfonato-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-deoxy-2-sulfoamino-1- thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-1-thio-α-L-idopyranosiduronate) (50y). The starting material (6.7 mg, 0.004 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50y (4.4 mg, 55% for 3 steps). 1H NMR (500 MHz, D2O) δ 5.13 (d, J = 3.8 Hz, 1H; A-1), 4.92 (s, 3H; B-1, C-1, E-1), 4.73 – 4.69 (m, 1H; D-1), 4.46 – 4.32 (m, 3H), 4.17 – 3.99 (m, 7H), 3.94 (t, J = 3.9 Hz, 1H), 3.91 – 3.70 (m, 11H), 3.69 – 3.43 (m, 11H), 3.34 – 3.14 (m, 8H), 3.03 (dd, J = 10.5, 3.6 Hz, 1H), 2.50 – 2.31 (m, 6H), 1.87 (s, 4H), 1.80 (s, 3H). 13C NMR (125 MHz, D2O) δ 174.64, 173.91, 100.61, 98.63, 98.55, 95.61, 93.74, 93.65, 77.96, 77.78, 77.59, 74.38, 73.37, 73.20, 70.81, 70.66, 70.54, 68.89, 68.33, 68.20, 67.45, 66.73, 66.11, 63.53, 63.42, 59.71, 57.71, 52.98, 39.03, 38.92, 36.19, 35.39, 22.07, 21.68. HRMS: m/z calc. for C57H87N6O55S5 5- [M]5-: 379.0565; found: 379.0574. Acetamidoethyl O-(2-acetamido-4-O-carboxyethyl-2-deoxy-6-O-sulfonato-1-thio-α-D- glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L-idopyranosiduronate)- 104 (1→4)-O-(2-acetamido-2-deoxy-6-O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O- carboxyl-2-O-sulfonato-thio-α-L-idopyranosiduronate)-(1→4)-O-(2-acetamido-2-deoxy-6- O-sulfonato-1-thio-α-D-glucopyranoside)-(1→4)-O-(6-O-carboxyl-2-O-sulfonato-1-thio-α-L- idopyranosiduronate) (50z). The starting material (8.0 mg, 0.005 mmol) was treated according to the general procedures of NH-Fmoc deprotection, pseudo-hexasaccharide preparation with HBTU and saponification of methyl esters to give compound 50z (4.5 mg, 44% for 3 steps). 1H NMR (500 MHz, D2O) δ 4.98 – 4.90 (m, 6H; A-1, B-1, C-1, D-1, E-1, F-1), 4.42 – 4.35 (m, 3H), 4.17 – 4.01 (m, 12H), 3.93 – 3.74 (m, 15H), 3.70 – 3.45 (m, 11H), 3.36 – 3.21 (m, 10H), 2.44 – 2.31 (m, 6H), 1.89 (s, 8H), 1.83 (s, 3H). 13C NMR (125 MHz, D2O) δ 174.67, 170.33, 109.99, 98.68, 98.57, 93.63, 77.86, 73.74, 73.35, 70.86, 69.21, 68.87, 68.67, 66.87, 66.65, 66.28, 63.90, 63.55, 53.05, 52.91, 39.03, 38.89, 36.22, 22.12, 22.09, 21.72. HRMS: m/z calc. for C59H88N6O59S6 6- [M]6-: 336.0405; found: 336.0412. 105 APPENDIX 106 Product Characterization Spectra Figure 2.2. 1H-NMR of 30 (500 MHz CDCl3) Figure 2.3. 13C-NMR of 30 (125 MHz CDCl3) 107 Figure 2.4. 1H-1H gCOSY of 30 (500 MHz CDCl3) Figure 2.5. 1H-NMR of 31 (500 MHz CDCl3) 108 Figure 2.6. 13C-NMR of 31 (125 MHz CDCl3) Figure 2.7. 1H-1H gCOSY of 31 (500 MHz CDCl3) 109 Figure 2.8. 1H-NMR of 33 (500 MHz CDCl3) Figure 2.9. 13C-NMR of 33 (125 MHz CDCl3) 110 Figure 2.10. 1H-1H gCOSY of 33 (500 MHz CDCl3) Figure 2.11. 1H-13C gHSQCAD of 33 (500 MHz CDCl3) 111 Figure 2.12. 1H-NMR of 34 (500 MHz CDCl3) Figure 2.13. 13C-NMR of 34 (125 MHz CDCl3) 112 Figure 2.14. 1H-1H gCOSY of 34 (500 MHz CDCl3) Figure 2.15. 1H-13C gHSQCAD of 34 (500 MHz CDCl3) 113 Figure 2.16. 1H-NMR of 35 (500 MHz CDCl3) Figure 2.17. 13C-NMR of 35 (125 MHz CDCl3) 114 Figure 2.18. 1H-1H gCOSY of 35 (500 MHz CDCl3) Figure 2.19. 1H-13C gHSQCAD of 35 (500 MHz CDCl3) 115 Figure 2.20. 1H-13C gHMBCAD of 35 (500 MHz CDCl3) Figure 2.21. 1H-NMR of 36 (500 MHz CDCl3) 116 Figure 2.22. 13C-NMR of 36 (125 MHz CDCl3) Figure 2.23. 1H-NMR of 37 (500 MHz CD3OD) 117 Figure 2.24. 13C-NMR of 37 (125 MHz CD3OD) Figure 2.25. 1H-1H gCOSY of 37 (500 MHz CD3OD) 118 Figure 2.26. 1H-NMR of 38 (500 MHz CD3OD) Figure 2.27. 1H-NMR of 39 (500 MHz CD3OD) 119 Figure 2.28. 13C-NMR of 39 (125 MHz CD3OD) Figure 2.29. 1H-1H gCOSY of 39 (500 MHz CD3OD) 120 Figure 2.30. 1H-NMR of 40 (500 MHz CD3OD) Figure 2.31. 13C-NMR of 40 (125 MHz CD3OD) 121 Figure 2.32. 1H-NMR of 41(500 MHz D2O) Figure 2.33. 13C-NMR of 41 (125 MHz D2O) 122 Figure 2.34. 1H-NMR of 42 (500 MHz D2O) Figure 2.35. 13C-NMR of 42 (125 MHz D2O) 123 Figure 2.36. 1H-13C gHSQCAD of 42 (500 MHz D2O) Figure 2.37. 1H-NMR of 43 (500 MHz CD3OD) 124 Figure 2.38. 13C-NMR of 43 (125 MHz CD3OD) Figure 2.39. 1H-NMR of 44 (500 MHz CD3OD) 125 Figure 2.40. 13C-NMR of 44 (125 MHz CD3OD) Figure 2.41. 1H-NMR of 45 (500 MHz CD3OD) 126 Figure 2.42. 13C-NMR of 45 (125 MHz CD3OD) Figure 2.43. 1H-NMR of 46 (500 MHz CD3OD) 127 Figure 2.44. 13C-NMR of 46 (125 MHz CD3OD) Figure 2.45. 1H-13C gHSQCAD of 46 (500 MHz CD3OD) 128 Figure 2.46. 1H-NMR of 47 (500 MHz D2O) Figure 2.47. 13C-NMR of 47 (125 MHz D2O) 129 Figure 2.48. 1H-NMR of 48 (500 MHz CD3OD) Figure 2.49. 13C-NMR of 48 (125 MHz CD3OD) 130 Figure 2.50. 1H-1H gCOSY of 48 (500 MHz CD3OD) Figure 2.51. 1H-13C gHSQCAD of 48 (500 MHz CD3OD) 131 Figure 2.52. 1H-NMR of 49a (500 MHz D2O) Figure 2.53. 13C-NMR of 49a (125 MHz D2O) 132 Figure 2.54. 1H-NMR of 49b (500 MHz D2O) Figure 2.55. 13C-NMR of 49b (125 MHz D2O) 133 Figure 2.56. 1H-1H gCOSY of 49b (500 MHz D2O) Figure 2.57. 1H-NMR of 49c (500 MHz CD3OD) 134 Figure 2.58. 13C-NMR of 49c (125 MHz CD3OD) Figure 2.59. 1H-NMR of 49d (500 MHz CD3OD) 135 Figure 2.60. 13C-NMR of 49d (125 MHz CD3OD) Figure 2.61. 1H-NMR of 49e (500 MHz CD3OD) 136 Figure 2.62. 13C-NMR of 49e (125 MHz CD3OD) Figure 2.63. 1H-NMR of 49f (500 MHz CD3OD) 137 Figure 2.64. 13C-NMR of 49f (125 MHz CD3OD) Figure 2.65. 1H-1H gCOSY of 49f (500 MHz CD3OD) 138 Figure 2.66. 1H-13C gHSQCAD of 49f (500 MHz CD3OD) Figure 2.67. 1H-13C gHMBCAD of 49f (500 MHz CD3OD) 139 Figure 2.68. 1H-NMR of 49g (500 MHz CD3OD) Figure 2.69. 13C-NMR of 49g (125 MHz CD3OD) 140 Figure 2.70. 1H-NMR of 49h (500 MHz CD3OD) Figure 2.71. 13C-NMR of 49h (125 MHz CD3OD) 141 Figure 2.72. 1H-1H gCOSY of 49h (500 MHz CD3OD) Figure 2.73. 1H-13C gHSQCAD of 49h (500 MHz CD3OD) 142 Figure 2.74. 1H-13C gHMBCAD of 49h (500 MHz CD3OD) Figure 2.75. 1H-NMR of 49i (500 MHz CD3OD) 143 Figure 2.76. 13C-NMR of 49i (125 MHz CD3OD) Figure 2.77. 1H-1H gCOSY of 49i (500 MHz CD3OD) 144 Figure 2.78. 1H-13C gHSQCAD of 49i (500 MHz CD3OD) Figure 2.79. 1H-13C gHSQCAD of 49i (500 MHz CD3OD) 145 Figure 2.80. 1H-13C gHMBCAD of 49i (500 MHz CD3OD) Figure 2.81. 1H-NMR of 50 (500 MHz D2O) 146 Figure 2.82. 13C-NMR of 50 (125 MHz D2O) Figure 2.83. 1H-1H gCOSY of 50 (500 MHz D2O) 147 Figure 2.84. 1H-NMR of 50a (500 MHz D2O) Figure 2.85. 13C-NMR of 50a (125 MHz D2O) 148 Figure 2.86. 1H-13C gHSQCAD of 50a (500 MHz D2O) Figure 2.87. 1H-NMR of 50b (500 MHz D2O) 149 Figure 2.88. 13C-NMR of 50b (125 MHz D2O) Figure 2.89. 1H-1H gCOSY of 50b (500 MHz D2O) 150 Figure 2.90. 1H-NMR of 50c (500 MHz D2O) Figure 2.91. 13C-NMR of 50c (125 MHz D2O) 151 Figure 2.92. 1H-13C gHSQCAD of 50c (500 MHz D2O) Figure 2.93. 1H-NMR of 50d (500 MHz D2O) 152 Figure 2.94. 13C-NMR of 50d (125 MHz D2O) Figure 2.95. 1H-13C gHSQCAD of 50d (500 MHz D2O) 153 Figure 2.96. 1H-NMR of 50e (500 MHz D2O) Figure 2.97. 13C-NMR of 50e (125 MHz D2O) 154 Figure 2.98. 1H-NMR of 50f (500 MHz D2O) Figure 2.99. 13C-NMR of 50f (125 MHz D2O) 155 Figure 2.100. 1H-13C gHSQCAD of 50f (500 MHz D2O) Figure 2.101. 1H-NMR of 50g (500 MHz D2O) 156 Figure 2.102. 13C-NMR of 50g (125 MHz D2O) Figure 2.103. 1H-13C gHSQCAD of 50g (500 MHz D2O) 157 Figure 2.104. 1H-NMR of 50h (500 MHz D2O) Figure 2.105. 13C-NMR of 50h (125 MHz D2O) 158 Figure 2.106. 1H-13C gHSQCAD of 50h (500 MHz D2O) Figure 2.107. 1H-NMR of 50i (500 MHz D2O) 159 Figure 2.108. 1H-NMR of 50j (500 MHz D2O) Figure 2.109. 13C-NMR of 50j (125 MHz D2O) 160 Figure 2.110. 1H-13C gHSQCAD of 50j (500 MHz D2O) Figure 2.111. 1H-NMR of 50k (500 MHz D2O) 161 Figure 2.112. 13C-NMR of 50k (125 MHz D2O) Figure 2.113. 1H-13C gHSQCAD of 50k (500 MHz D2O) 162 Figure 2.114. 1H-NMR of 50l (500 MHz D2O) Figure 2.115. 1H-1H gCOSY of 50l (500 MHz D2O) 163 Figure 2.116. 1H-NMR of 50m (500 MHz D2O) Figure 2.117. 1H-NMR of 50n (500 MHz D2O) 164 Figure 2.118. 13C-NMR of 50n (125 MHz D2O) Figure 2.119. 1H-13C gHSQCAD of 50n (500 MHz D2O) 165 Figure 2.120. 1H-NMR of 50o (500 MHz D2O) Figure 2.121. 1H-13C gHSQCAD of 50o (500 MHz D2O) 166 Figure 2.122. 1H-NMR of 50p (500 MHz D2O) Figure 2.123. 13C-NMR of 50p (125 MHz D2O) 167 Figure 2.124. 1H-13C gHSQCAD of 50p (500 MHz D2O) Figure 2.125. 1H-NMR of 50q (500 MHz D2O) 168 Figure 2.126. 13C-NMR of 50q (125 MHz D2O) Figure 2.127. 1H-NMR of 50r (500 MHz D2O) 169 Figure 2.128. 13C-NMR of 50r (125 MHz D2O) Figure 2.129. 1H-13C gHSQCAD of 50r (500 MHz D2O) 170 Figure 2.130. 1H-NMR of 50s (500 MHz D2O) Figure 2.131. 13C-NMR of 50s (125 MHz D2O) 171 Figure 2.132. 1H-13C gHSQCAD of 50s (500 MHz D2O) Figure 2.133. 1H-NMR of 50t (500 MHz D2O) 172 Figure 2.134. 13C-NMR of 50t (125 MHz D2O) Figure 2.135. 1H-13C gHSQCAD of 50t (500 MHz D2O) 173 Figure 2.136. 1H-NMR of 50u (500 MHz D2O) Figure 2.137. 13C-NMR of 50u (125 MHz D2O) 174 Figure 2.138. 1H-13C gHSQCAD of 50u (500 MHz D2O) Figure 2.139. 1H-NMR of 50v (500 MHz D2O) 175 Figure 2.140. 13C-NMR of 50v (125 MHz D2O) Figure 2.141. 1H-13C gHSQCAD of 50v (500 MHz D2O) 176 Figure 2.142. 1H-NMR of 50w (500 MHz D2O) Figure 2.143. 13C-NMR of 50w (125 MHz D2O) 177 Figure 2.144. 1H-13C gHSQCAD of 50w (500 MHz D2O) Figure 2.145. 1H-NMR of 50x (500 MHz D2O) 178 Figure 2.146. 1H-NMR of 50y (500 MHz D2O) Figure 2.147. 13C-NMR of 50y (125 MHz D2O) 179 Figure 2.148. 1H-13C gHSQCAD of 50y (500 MHz D2O) Figure 2.149. 1H-NMR of 50z (500 MHz D2O) 180 Figure 2.150. 13C-NMR of 50z (125 MHz D2O) 181 Chapter 3. Biological Studies of Pseudo-hexasaccharide Heparin Mimetics 3.1. Bio-Layer Interferometry (BLI) Competition Assay on FGF-2-heparin interaction With the successful preparation of the library of heparin mimetics, we explored whether the library of pseudo-hexasaccharides could mimic the natural heparin oligosaccharides. We examined the binding with FGF-2 that is essential for normal and cancer biology.131 FGF-2 is one of the most extensively studied heparin-binding proteins, which can mediate cell growth, differentiation, survival, and patterning.116 FGF-2 has low and high molecular weight isoforms,132 and low molecular weight FGF-2 binds with both FGFR and HSPGs on cell surface. The formation of the FGF-2-FGFR-HSPGs ternary structure activates the downstream signaling pathways.133 FGF-2-FGFR1 complexes are also found in the nuclear matrix, promoting cell proliferation.134 Dysregulation of FGF-2 signaling is associated with aggressive cancer phenotypes, enhanced chemotherapy resistance, and many acquired forms of cancers.135-137 The binding studies were performed by competition bio-layer interferometry (BLI). Before testing the pseudo-hexasaccharides, heparin-FGF-2 interaction was studied. Biotinylated heparin (27.5 nM) was immobilized on streptavidin (SA) biosensors, and various concentrations of FGF-2 were prepared as analytes. The titration curves fitted well to a 1:1 binding mode, and strong binding was observed as expected with KD values of 2.7 nM (Figure 3.1). 182 Figure 3.1. The sensorgram of heparin-FGF-2 interaction. Next, heparin, compounds 50k, and fondaparinux were selected as inhibitors for competition BLI. Different concentrations of each inhibitor were mixed with FGF-2 (75 nM), and biotinylated heparin was used as the ligand (Figure 3.2). Heparin showed potent inhibitory activity against heparin-FGF-2 interaction, and ~80% inhibition was achieved for 6.5 nM heparin. However, both compound 50k and fondaparinux only exhibited inhibitory activity at high concentrations (> 4 uM). Figure 3.2. The sensorgrams of competition BLI. A: heparin as the inhibitor. Concentrations from top to bottom: 0, 3.2, 6.5, 13.1, 26.1, 51.7, 77.8 nM. B: compound 50k. Concentrations from top to bottom: 0, 12.5, 25, 50, 100, 500, 1000 uM. C: fondaparinux. Concentrations from top to bottom: 0, 1.0, 10.0, 25, 50, 1000, 2000 uM. 183 3.2. Binding Studies of Pseudo-hexasaccharides and FGF-2 Using Surface Plasmon Resonance (SPR) Since the pseudo-hexasaccharides did not exhibit potent inhibitory activity against heparin-FGF-2 binding through competition experiments, the direct binding studies were performed by surface plasmon resonance (SPR). FGF-2 was immobilized on CDH sensor chip surface through EDC/NHS coupling, and fully sulfated (50), O-sulfated (50z) and N-sulfated (50m) pseudo-hexasaccharides were selected as analytes. 50 and 50z showed strong and moderate binding to FGF-2 with KD values of 19.6 and 940 nM respectively, and the only weak signal was observed for 50m (Figure 3.3). Delightfully, the SPR results correlated well with that of heparin hexasaccharides25 (36, 51, 52). Fully sulfated hexasaccharide 36 showed comparable FGF-2 binding ability as that of heparin through microarray assay, and no significant binding signal was observed for N-sulfated hexasaccharide 52. The pseudo-hexasaccharides also exhibited much stronger FGF-2 binding ability in comparison to that of heparin di- and tetra- saccharides (54, 53) (Figure 3.4).20, 113 50 is about 20 times more potent than the strongest tetrasaccharide binder 53 based on KD values acquired from SPR assay. Figure 3.3. The SPR sensorgram of FGF-2 and compound 50 interaction at different concentrations 184 Figure 3.4. Heparin hexa-, tetra- and disaccharides. In order to gain more insights into the structure-activity relationship between heparin and FGF-2, the competition assay was performed by the Linhardt group (Figure 3.5). Biotinylated heparin was immobilized on the biosensor, and 27 pseudo-hexasaccharides (10 uM) was mixed with FGF-2 (50 nM) individually. As expected, the fully sulfated pseudo- hexasaccharide (50) showed the most potent inhibitory activity. Compounds with more sulfate groups showed relatively higher potency. Among 27 pseudo-hexasaccharides, the best inhibitors (50, 50a, 50b, 50i, 50r), which showed about 50-65% inhibition of binding between FGF-2 and heparin contain successive fully sulfated disaccharide modules. For IC50 measurements, biotinylated heparin was immobilized on the SA sensor chip as described above, and FGF-2 (50 nM) was pre-mixed with different concentrations of pseudo-hexasaccharides. Fully sulfated pseudo-hexasaccharide 50 displayed the highest IC50 value of 1.7 uM, and 50a, 50b, and 50r 185 also exhibited potent competitive effect (IC50 = 10.8, 4.0, and 3.5 uM, respectively) (Figure 3.6). According to competition SPR results, successive fully sulfated disaccharide modules are required for potent competitive activity. Figure 3.5. Measurement of inhibition of 27 pseudo-hexasaccharides on FGF-2- heparin interaction using competition SPR 186 Figure 3.6. Competition SPR for IC50 measurements for pseudo-hexasaccharides 50 (A), 50a (B), 50b (C), and 50r (D) 3.3. Heparanase Inhibitory Activity Tests Through the Colorimetric Assay Next, we investigated the anti-tumor activity of pseudo-hexasaccharides by inhibiting heparanase enzyme that is strongly related to tumor formation, including tumor initiation, growth and metastasis.138 Proheparanase was synthesized on the endoplasmic reticulum (ER) and transported to the Golgi apparatus and secreted into the extracellular space. Proheparanase may interact with receptors on the cell surface and upregulate the expression of VEGF, resulting in cell proliferation. On the other hand, proheparanase could also be activated by internalization through binding with cell surface syndecans, and active heparanase may be translocated into the nucleus and secreted into the ECM.139, 140 Heparanase is the sole endoglycosidase that degrades HS side chains of HSPGs. The cleavage of HS releases the sequestered growth factors and degrades the structural integrity of the basement membrane and ECM. Thus, heparanase is strongly related to tumor growth and metastasis. Since heparin and HS have the same backbone 187 arrangement of glucosamine and uronic acid disaccharide repeating units, heparin was considered as a potential potent heparanase inhibitor.141-143 Heparanase activity was measured by a colorimetric assay. Fondaparinux 55 was used as the substrate, which would be cleaved by heparanase, and the appearance of disaccharide product 56 was measured by the tetrazolium salt WST-1 58 (Scheme 3.1).144 Heparin exhibited potent heparanase activity, and 100% inhibition was achieved with ~20 nM (Figure 3.7). However, our pseudo-hexasaccharides did not exhibit significant heparanase inhibitory activity (Figure 3.8). Compound 50a and 50f were added up to 1 and 10 uM, respectively, and the absorbance did not decrease dramatically. Scheme. 3.1. Heparanase colorimetric assay. 188 Figure 3.7. Inhibition of heparanase by heparin Figure 3.8. Inhibition of heparanase by A: compound 50a (10, 50, 100, 1000, and 1000 nM). B: compound 50f (0.1, and 10 uM). 3.4. Experimental Section 3.4.1. Bio-Layer Interferometry (BLI). Samples or buffers were dispensed into 96-well microtiter plates (Millipore, Billerica, 189 0204060801000.00.51.01.5heparin as inhibitor nMheparin as inhibitor nM584nm Abs MA) at a volume of 200 uL per well. The operating temperature was maintained at 25 ºC. Streptavidin-coated biosensor tips (FortéBio, Inc., Molecular Devices) were pre-wetted with PBS buffer for 10 min, and biotinylated heparin (27.5 nM) was loaded for 5 min. Various concentrations of analytes were then immobilized on the biosensor tips while agitating for 10 min at 1000 rpm, and biosensors was dipped into PBS buffer for 10 min for dissociation. NaCl solution (2.0 M) was employed for regeneration to achieve baseline status. All measurements were performed using 0.05% surfactant P-20 PBS buffer. 3.4.2. Surface Plasmon Resonance (SPR). FGF-2 was immobilized on sensor chip by primary amine coupling reaction. The surface of a CDH sensor chip (FortéBio, Inc., Molecular Devices, USA) was activated using freshly mixed N-hydroxysuccimide (NHS; 0.05 M) and 1-(3-(dimethylamino)propyl)-ethylcarbodiimide (EDC; 0.2 M) (1/1,v/v) in water, followed by the injection of FGF-2 (50 ug/ml) in PBS buffer. The remaining active esters were quenched by ethanolamine (1.0 M). All measurements were performed using 0.01% Tween 20 PBS buffer. Aqueous NaCl (2.0 M) was employed for regeneration to achieve baseline status. 3.4.3. Heparanase Colorimetric Assays. Fondaparinux colorimetric heparanase assays were carried out in 96 well microplates (NEST Scientific 96 Well Cell Culture Plate, 701002) pre-treated with a solution of 4% BSA in phosphate-buffered saline containing 0.05% Tween 20 (PBST) for 2 h at 37 °C. 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