Michigan Technological University Digital Commons @ Michigan Tech Dissertations, Master's Theses and Master's Dissertations, Master's Theses and Master's Reports - Open Reports 2013 Oligodeoxynucleotide synthesis using protecting groups and a linker cleavable under non-nucleophilic conditions Xi Lin Michigan Technological University Follow this and additional works at: https://digitalcommons.mtu.edu/etds Part of the Biochemistry Commons, Molecular Biology Commons, and the Organic Chemistry Commons Copyright 2013 Xi Lin Recommended Citation Lin, Xi, "Oligodeoxynucleotide synthesis using protecting groups and a linker cleavable under non- nucleophilic conditions", Dissertation, Michigan Technological University, 2013. https://doi.org/10.37099/mtu.dc.etds/676 Follow this and additional works at: https://digitalcommons.mtu.edu/etds Part of the Biochemistry Commons, Molecular Biology Commons, and the Organic Chemistry Commons OLIGODEOXYNUCLEOTIDE SYNTHESIS USING PROTECTING GROUPS AND A LINKER CLEAVABLE UNDER NON- NUCLEOPHILIC CONDITIONS By Xi Lin A DISSERTATION Submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY In Chemistry MICHIGAN TECHNOLOGICAL UNIVERSITY 2013 ©2013 Xi Lin This dissertation has been approved in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY in Chemistry Department of Chemistry Dissertation Advisor: Dr. Shiyue Fang Committee Member: Dr. Dallas K. Bates Committee Member: Dr. Haiying Liu Committee Member: Dr. Yun Hang Hu Department Chair: Dr. Cary F. Chabalowski Table of Contents List of Figures………………………………………………………………………....... vi List of Tables……………………………………………………………………………. x List of Schemes…………………………………………………………………………. xi Preface…………………………………………………………………………………. xii Acknowledgements…………………………………………………………………… xiii Abstract………………………………………………………………………………… xv Chapter 1 Introduction………………………………………………………………… 1 1.1 General Introduction………………………………………………............................. 1 1.2 Our Technology…………………………………………………………………….... 2 References……………………………………………………………………………… 4 Chapter 2 History and Background…………………………………………………… 6 2.1 The Overview of ODNs……………………………………………………………… 6 2.2 ODN Synthesis…………………………………………………………………..…. 10 2.3 The Modifications of ODN………………………………………………………… 23 2.4 Perspectives…………………………………………………………………………. 28 References………………………………………………………………………………. 29 Chapter 3 Feasibility Studies…………………………………………………………. 36 Abstract………………………………………………………………………………… 36 3.1 Introduction………………………………………………………………………… 36 3.2 Results and Discussion…………………………………………………………....... 38 3.3 Conclusion………………………………………………………………………….. 40 References………………………………………………………………………………. 41 iii Chapter 4 Synthesis of dT-Dmoc Linker and Evaluation of Its Application in ODN Synthesis……………………………………………………………………………… 42 Abstract…………………………………………………………………………………. 42 4.1 Introduction…………………………………………………………………………. 42 4.2 Results and Discussion…………………………………………………………… 47 4.3 Conclusion……………………………………………………………….................. 56 4.4 Experimental Section……………………………………………………………… 56 References………………………………………………………………………………. 66 Chapter 5 Synthesis of Dmoc-protected Phosphoramidite Monomers and Their Applications in ODN Synthesis………………………..……………………………… 69 Abstract………………………………………………………………............................. 69 5.1 Introduction…………………………………………………………………………. 69 5.2 Results and Discussion…………………………………………………………… 74 5.3 Conclusion………………………………………………………………………… 89 5.4 Experimental Section……………………………………………………………… 89 References…………………………………………………………………………… 106 Chapter 6 Improved Synthesis of Dmoc-dG-amidite……………………………… 110 Abstract……………………………………………………………………………… 110 6.1 Introduction……………………………………………………………………… 110 6.2 Results and Discussion…………………………………………………………… 111 6.3 Conclusion………………………………………………………………………… 114 6.4 Experimental Section……………………………………………………………… 114 References…………………………………………………………………………… 120 Chapter 7 Future Research………………………………………………………… 121 iv 7.1 Dim Protecting Group for Phosphoramidites…………………………………… 122 7.2 Deoc Protecting Groups for Nucleoside Bases…………………………………… 123 7.3 Conclusion………………………………………………………………………… 125 Appendix A. Supporting Information for Chapter 4……………………………… 126 Appendix B. Supporting Information for Chapter 5……………………………… 142 Appendix C. Supporting Information for Chapter 6……………………………… 167 v List of Figures Figure 1.1. CPG with dT-Dmoc linker and Dmoc-protected phosphoramidites……… 2 Figure 2.1. Complementary base pairing………………………………………………... 6 Figure 2.2. Synthesis of first dinucleotide…………………………………………… 10 Figure 2.3. Phosphodiester approach…………………………………………………... 11 Figure 2.4. Coupling of dC to polymer solid support………………………………… 12 Figure 2.5. Phosphotriester Approach………………………………………………… 13 Figure 2.6. Phosphite-triester approach……………………………………………… 14 Figure 2.7. ODN synthesis cycle using phosphoramidite chemistry………………… 15 Figure 2.8. Detritylation………………………………………………………………... 16 Figure 2.9. Coupling…………………………………………………………………… 17 Figure 2.10. Capping failure sequence………………………………………………… 17 Figure 2.11. Oxidation of P(III) to P(V)……………………………………………… 19 Figure 2.12. Cleavage and deprotection of ODN……………………………………… 21 Figure 2.13. Michael addition to nucleoside base……………………………………… 22 Figure 2.14. Synthesis of phosphothioates……………………………………………... 24 Figure 2.15. Modifications on sugar moiety…………………………………………… 24 Figure 2.16. Synthesis of PNA…………………………………………………………. 25 Figure 2.17. Biotinylation on 3ʹ and 5ʹ terminus……………………………………… 26 Figure 2.18. ODNs with linkers……………………………………………………… 27 Figure 2.19. Modifications on nucleoside bases……………………………………… 28 Figure 3.1. Incubation of authentic ODN with NaIO4………………………………… 39 Figure 4.1. Solid supports for OGN synthesis………………………………………… 43 vi Figure 4.2. Linkers for electrophilic ODN synthesis…………………………………... 45 Figure 4.3. dT-Dmoc Linker…………………………………………………………… 46 Figure 4.4. RP HPLC profiles of ODN 4.13…………………………………………… 49 Figure 4.5. Mechanism of the removal of β-cyanoethyl groups by DBU……………… 50 Figure 4.6. RP HPLC profile of all dTs ODN 4.14……………………………………. 53 Figure 4.7. RP HPLC profile of ODN 4.13..................................................................... 55 Figure 5.1. The trityl family…………………………………………………………… 70 Figure 5.2. Standard protecting groups for nucleoside bases………………………… 71 Figure 5.3. Sekine’s activated phosphite method…………………………………… 72 Figure 5.4. Allylic protecting groups for ODN synthesis……………………………… 73 Figure 5.5. Dmoc-protected nucleoside phosphoramidite monomers………………… 74 Figure 5.6. HPLC profile of ODN 5.16……………………………………………… 77 Figure 5.7. RP HPLC profile of ODN 5.19 from the unsuccessful deprotection……… 80 Figure 5.8. RP HPLC profiles of ODN 5.19 from the successful deprotection………... 84 Figure 5.9. HPLC profiles of ODN 5.20-5.22………………………………………… 86 Figure 6.1. Dmoc-dG-amidite 1.4…………………………………………………… 110 Figure 7.1. Dim-protected phosphoramidites………………………………………… 122 Figure 7.2. Deoc-protected phosphoramidite monomers…………………………… 124 Figure A.1. 1H NMR of compound 4.6……………………………………………… 127 Figure A.2. 13C NMR of compound 4.6………………………………………………. 128 Figure A.3. 1H NMR of compound 4.7……………………………………………… 129 Figure A.4. 13C NMR of compound 4.7……………………………………………… 130 Figure A.5. 1H NMR of compound 4.8……………………………………………… 131 Figure A.6. 13C NMR of compound 4.8……………………………………………… 132 vii Figure A.7. 1H NMR of compound 4.9……………………………………………… 133 Figure A.8. 13C NMR of compound 4.9……………………………………………… 134 Figure A.9. 1H NMR of compound 4.10……………………………………………… 135 Figure A.10. 13C NMR of compound 4.10…………………………………………… 136 Figure A.11. 1H NMR of compound 4.11…………………………………………… 137 Figure A.12. 13C NMR of compound 4.11…………………………………………… 138 Figure A.13. 1H NMR of compound 4.12…………………………………………… 139 Figure A.14. 13C NMR of compound 4.12…………………………………………… 140 Figure A.15. MALDI-TOF spectrum of compound 4.13…………………………….. 141 Figure B.1. 1H NMR of compound 1.2…………………………………………… 143 Figure B.2. 13C NMR of compound 1.2…………………………………………… 144 Figure B.3. 31P NMR of compound 1.2…………………………………………… 145 Figure B.4. 1H NMR of compound 1.3…………………………………………… 146 Figure B.5. 13C NMR of compound 1.3…………………………………………… 147 Figure B.6. 31P NMR of compound 1.3…………………………………………… 148 Figure B.7. 1H NMR of compound 1.4…………………………………………… 149 Figure B.8. 13C NMR of compound 1.4…………………………………………… 150 Figure B.9. 31P NMR of compound 1.4…………………………………………… 151 Figure B.10. 1H NMR of compound 5.9…………………………………………… 152 Figure B.11. 13C NMR of compound 5.9…………………………………………… 153 Figure B.12. 1H NMR of compound 5.10…………………………………………… 154 Figure B.13. 13C NMR of compound 5.10…………………………………………… 155 Figure B.14. 1H NMR of compound 5.12…………………………………………… 156 Figure B.15. 13C NMR of compound 5.12…………………………………………… 157 Figure B.16. 1H NMR of compound 5.14…………………………………………… 158 Figure B.17. 13C NMR of compound 5.14…………………………………………… 159 Figure B.18. 1H NMR of compound 5.18…………………………………………… 160 viii Figure B.19. 13C NMR of compound 5.18…………………………………………… 161 Figure B.20. MALDI-TOF spectrum of compound 5.16……………………………... 162 Figure B.21. MALDI-TOF spectrum of compound 5.19……………………………... 163 Figure B.22. MALDI-TOF spectrum of compound 5.20……………………………... 164 Figure B.23. MALDI-TOF spectrum of compound 5.21…………………………… 165 Figure B.24. MALDI-TOF spectrum of compound 5.22…………………………… 166 Figure C.1. 1H NMR of compound 6.1……………………………………………….. 168 Figure C.2. 13C NMR of compound 6.1…………………………………………… 169 Figure C.3. 1HNMR of compound 6.2…………………………………………… 170 Figure C.4. 13C NMR of compound 6.2…………………………………………… 171 ix List of Tables Table 2.1. Step durations in a typical cycle using