University of Southern California Dissertations and Theses

Synthetic studies of chemical probes for i) DNA, ii) RNA polymerases and iii) tropomyosin receptor kinase

(USC Thesis Other)

Synthetic studies of chemical probes for i) DNA, ii) RNA polymerases and iii) tropomyosin receptor kinase

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content Synthetic studies of chemical probes for i) DNA, ii) RNA polymerases and iii) tropomyosin receptor kinase by Carolina D. Amador _____________________________________________________________ A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY (CHEMISTRY) May 2019 ii DEDICATION To my godmother and grandmothers. For being the greatest examples of women leaders, for fighting for their rights, and for empowering me to be the independent woman I am today. iii ACKNOWLEDGMENTS On Spring 2014, I moved to the US to start my graduate studies at USC. At the time, I knew little about the US and even less about Los Angeles in particular, but I did know that I wanted to receive training at a prestigious university and study early stage drug development. I have always been fascinated with the power of medicinal and bioorganic chemistry, and how we are able to create molecules that can positively impact peoples’ lives. My PhD program has been an adventurous ride and I am thrilled that I had the chance to be part of such a dynamic team of scientists and innovators. I couldn’t have done it without the unconditional support of my mentors, friends, and family. Firstly, I would like to express my deepest gratitude to my adviser, Prof. Charles E. Mckenna, for his continuous support and guidance over the past five years. Thank you for your patience, immense knowledge, and for showing me how to be a better chemist. I would also like to thank Dr. Boris Kashemirov of all his guidance, patience, and support. Dr. Kashemirov spends endless hours providing research guidance to students and helps us solve day-to-day chemistry challenges, so I thank him for all the brainstorming and problem-solving sessions we had together. It has been a pleasure to work with such inspiring and hard-working collaborators; therefore I would like to thank Prof. Dong Wang, Prof. Myron Goodman, Dr. David Jung, Dr. Judith Kempfle and Dr. Samuel Wilson for being such great co-investigators and pleasant to work with. My gratitude also goes to the faculty and staff of the Chemistry Department: Prof. Stephen Bradforth, Prof. G. K. Surya Prakash, Prof. Hanna Reisler, Michele Dea and Magnolia Benitez for all their support. A special thank you to my dissertation committee, Prof. Smaranda Marinescu and Prof. Vsevolod Katritch for their guidance and feedback on my thesis. iv A warm shout-out to the Mckenna Lab, my friends and partners in crime during this journey. Inah Kang, you have given me the utmost support and friendship. Dr. Corinne Minard, you have been my best friend and the best lab partner I could have ever asked for. Dr. Candy Hwang, thank you for empowering me and for all the advice on how to best develop my skills. Thank you to all the other lab members for the good times, especially Dr. Eric Richard, Marlon Duro, and Hammond Sun. I would like to thank my fellow graduate students, Paymaneh, Kyle, Arunika, and Gözde for their friendship, lunch breaks, and happy hours! To the amazing BCLA team: Stefanie, Katrina, Evgeny, Yari, Adhi, Valentina, Arthela, Radhika, and many others—we have successfully turned long hours of volunteering into a thriving organization, strong friendships, and fun meetings. Finally, I would like to give a special thank you to my family. Their unconditional support and love have made me who I am. To Hugo, thank you for sharing your life with me, for reminding me what I am made of when I cease to remember, and for your constant encouragement on all my crazy ideas. You have been my greatest support and you inspire me to be a better person. To my sister, for always being there and taking my side every time I needed it. To my nephews for keeping things interesting and reminding me how great it is to be a kid. Thanks Mom and Dad for never doubting my abilities and for encouraging me to pursue my dreams, even when that meant to leave my country. With a combination of my dad’s entrepreneurial and hard-working essence, and my mom’s kindness and resilient spirit, I have come this far, and this is just the beginning. My accomplishments are yours too! v TABLE OF CONTENTS DEDICATION ................................................................................................................... ii ACKNOWLEDGMENTS ................................................................................................. iii LIST OF TABLES .............................................................................................................. x LIST OF FIGURES ............................................................................................................ xi LIST OF SCHEMES ......................................................................................................... xv ABSTRACT ..................................................................................................................... xvi Chapter 1. DNA and RNA polymerases: functionality and structure, fidelity mechanisms and cancer therapeutics ................................................................................... 1 1.1 Functionality and Structure of Polymerases ........................................................ 1 1.1.1 DNA Polymerase b ............................................................................................. 1 1.1.2 RNA Polymerase II ............................................................................................ 3 1.2 Fidelity Mechanisms ........................................................................................... 6 1.2.1 DNA Polymerase b Fidelity ............................................................................... 6 1.2.2 RNA Polymerase II Transcription Fidelity ........................................................ 7 1.3 Using Probes to Study Polymerase Kinetics ............................................................. 8 1.4 Using Polymerases and cancer therapeutics .............................................................. 9 1.4.1 DNA Polymerase b and Cancer Therapeutics .................................................... 9 1.4.2 RNA Polymerase II and Cancer Therapeutics .................................................. 10 1.5 Chapter References .................................................................................................. 11 Chapter 2. Synthesis of b,g-CHF dCTP probes and small molecule inhibitors for DNA polymerase b ............................................................................................................ 15 2.1 Part I: Using deoxyribonucleotide triphosphate (dNTP) as probes ......................... 15 2.1.1 Introduction .......................................................................................................... 15 2.1.2 Results and Discussion ......................................................................................... 16 2.1.2.1 Synthesis of β,g-CHF dCTP .......................................................................... 16 2.1.2.2 LFER Analysis of β,g-CHF dCTP ................................................................. 19 2.1.2.3 NMR Spike Experiments of (S)- and (R)-β,γ-CHF dCTP ............................. 21 2.1.3 Conclusion ............................................................................................................ 26 2.1.4 Experimental Procedure ....................................................................................... 27 2.1.4.1 Materials and Methods .................................................................................. 27 2.1.4.2 Synthesis of (fluoro(hydroxy((2-nitrobenzyl)oxy)phosphoryl)methyl) phosphonic acid 1 ...................................................................................................... 28 vi 2.1.4.3 Synthesis of [(S)-fluoro[hydroxy({[(1R)-1-phenylpropyl]amino})phosphor- ryl]methyl][(2-nitrophenyl)methoxy]phosphinic acid and [(R)-fluoro[hydroxy ({[(1R)-1-phenylpropyl]amino})phosphoryl]methyl][(2-nitrophenyl)methoxy]- phosphinic acid - 2 ..................................................................................................... 29 2.1.4.4 Synthesis of [(S)-fluoro({hydroxy[(2-nitrophenyl)methoxy]phosphoryl}) methyl]phosphonic acid and [(R)-fluoro({hydroxy[(2-nitrophenyl)methoxy]- phosphoryl}) methyl]phosphonic acid – 3 ................................................................ 30 2.1.4.5 Synthesis of [(R)-{[({[(2R,5R)-5-(4-amino-2-oxo-1,2-dihydropyrimidin-1- yl)-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)oxy][fluoro({hydroxy[(2- nitrophenyl)methoxy]phosphoryl})methyl]phosphinic acid and [(S)-{[({[(2R,5R)-5- (4-amino-2-oxo-1,2-dihydropyrimidin-1-yl)-3-hydroxyoxolan-2- yl]methoxy}(hydro-xy)phosphoryl)oxy][fluoro({hydroxy[(2- nitrophenyl)methoxy]phosphoryl})methyl] phosphinic acid – 4 ............................. 32 2.1.4.6 Synthesis of [(R)-{[({[(2R,5R)-5-(4-amino-2-oxo-1,2-dihydropyrimidin-1- yl)-3-hydroxyoxolan-2- yl]methoxy}(hydroxy)phosphoryl)oxy](hydroxy)phosphoryl} (fluo- ro)methyl]phosphonic acid and [(S)-{[({[(2R,5R)-5-(4-amino-2-oxo-1,2-dihydro- pyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)oxy]- (hydroxy)-phosphoryl}(fluoro)methyl]phosphonic acid – (R)-CHF dCTP and (S)- CHF dCTP ............................................................................................................... 33 2.2 - Part II: Pamoic acid derivatives as small molecule inhibitors .............................. 35 2.2.1 Introduction .......................................................................................................... 35 2.2.2 Results and Discussion ......................................................................................... 36 2.2.2.1 Choosing the Leading Compound ................................................................. 36 2.2.2.2 Synthesis of PA Derivatives .......................................................................... 38 2.2.2.3 Docking Experiments with PA Derivatives .................................................. 42 2.2.2.4 DNA Pol β dRP-Lyase Activity of Compound 8 .......................................... 45 2.2.3 Conclusion and future directions .......................................................................... 46 2.2.4 Experimental procedure ........................................................................................ 47 2.2.4.1 Materials and Methods .................................................................................. 47 2.2.4.2 Synthesis of diethyl [(naphthalen-2-yl)methyl]phosphonate 1 ..................... 48 2.2.4.3 Synthesis of diethyl (3-hydroxynaphthalen-2-yl)phosphonate 2 .................. 49 2.2.4.5 Synthesis of (4-{[3-(diethoxyphosphoryl)-2-hydroxynaphthalen-1- yl]methyl}-3-hydroxynaphthalen-2-yl)(ethoxy)phosphinic acid 4 ........................... 50 2.2.4.6 Synthesis of diethyl {3-hydroxy-4-[(2-hydroxynaphthalen-1- yl)methyl]naphthalen-2-yl}phosphonate 7 ................................................................ 50 2.2.4.7 Synthesis of ammonium ethyl {3-hydroxy-4-[(2-hydroxynaphthalen-1- yl)methyl]naphthalen-2-yl}phosphonate 8 ................................................................ 52 vii 2.2.4.8 Tentative Syntheses of 4-[(3-carboxy-2-hydroxynaphthalen-1- yl)(phenyl)methyl]-3-hydroxynaphthalene-2-carboxylic acid PA-Ph ...................... 53 2.3 Chapter References .................................................................................................. 54 Chapter 3. Synthesis of b,g-CXY UTP probes to study the mechanism and fidelity of RNA polymerase II ............................................................................................................ 57 3.1 Introduction ............................................................................................................. 57 3.2 Results and Discussion ............................................................................................ 59 3.2.1 Synthesis of β,γ-CXY NTP .............................................................................. 59 3.2.2 Finding the Optimal Coupling Step Conditions ............................................... 60 3.2.3 Nucleotide Incorporation: In vitro Transcription Assays ................................. 64 3.3 Conclusion and Future Directions ........................................................................... 66 3.4 Experimental Procedure .......................................................................................... 67 3.4.1 Materials and Methods ..................................................................................... 67 3.4.2 Synthesis of β,γ-CXY U/ATP derivatives: General Method 1 ......................... 69 3.4.3 Synthesis of ({[({[(2S,5S)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxyoxolan-2- yl]methoxy}(hydroxy)phosphoryl)oxy](hydroxy)phosphoryl}dichloromethyl)phosp honic acid - β,γ-CCl2 ATP ........................................................................................ 69 3.4.4 Synthesis of [dichloro({[({[(2S,5S)-5-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin- 1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)oxy](hydroxy) phosphoryl})methyl]phosphonic acid - β,γ-CCl2 UTP ............................................ 70 3.4.5 Synthesis of ({[({[(2S,5S)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxyoxolan-2- yl]methoxy}(hydroxy)phosphoryl)oxy](hydroxy)phosphoryl}(chloro)methyl)phosph onic acid - β,γ-CHCl ATP ........................................................................................ 71 3.4.6 Synthesis of [chloro({[({[(2S,5S)-5-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1- yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)oxy](hydroxy) phosphoryl})methyl]phosphonic acid - β,γ-CHCl UTP ........................................... 72 3.4.7 Synthesis of [chloro({hydroxy[(2-nitrophenyl)methoxy]phosphoryl})methyl] phosphonic acid 1 ..................................................................................................... 73 3.4.8 Synthesis of [(S)-chloro[hydroxy({[(1R)-1-phenylpropyl]amino}) phosphoryl] methyl][(2-nitrophenyl)methoxy]phosphinic acid and [(R)-chloro[hydroxy ({[(1R)- 1-phenylpropyl]amino})phosphoryl]methyl][(2-nitrophenyl)methoxy]phosphinic acid - 2 ...................................................................................................................... 74 3.4.9 Synthesis of [(S)-chloro({hydroxy[(2-nitrophenyl)methoxy]phosphoryl}) methyl]phosphonic acid and [(R)-chloro({hydroxy[(2-nitrophenyl)methoxy]- phosphoryl}) methyl]phosphonic acid - 3 ................................................................. 76 3.4.10 Synthesis of ({[(S)-chloro({hydroxy[(2-nitrophenyl)methoxy]phosphoryl}) methyl](hydroxy)phosphoryl}oxy)({[(2S,5S)-5-(2,4-dioxo-1,2,3,4-tetrahydropyri- midin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy})phosphinic acid and ({[(R)- viii chloro({hydroxy[(2-nitrophenyl)methoxy]phosphoryl})methyl](hydroxy)phos- phoryl}oxy)({[(2S,5S)-5-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-3,4-dihydro- xyoxolan-2-yl]methoxy})phosphinic acid – 4 ........................................................... 77 3.4.11 Synthesis of [(S)-chloro({[({[(2S,5S)-5-(2,4-dioxo-1,2,3,4-tetrahydro pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)oxy] (hydroxy)phosphoryl})methyl]phosphonic acid and [(R)-chloro({[({[(2S,5S)-5-(2,4- dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy} (hydroxy)phosphoryl)oxy] (hydroxy)phosphoryl})methyl]phosphonic acid - (R)- and (S)-β,γ-CHCl UTP ............................................................................................. 78 3.5 Chapter References .................................................................................................. 79 Chapter 4. Solid-phase synthesis of BP conjugate 1Aa to target Tropomyosin receptor kinases (TrkB and TrkC) ................................................................................................... 81 4.1 Introduction ............................................................................................................. 81 4.1.1 The Human Ear and Hearing Loss ................................................................... 81 4.1.2 Neurotrophic Properties .................................................................................... 82 4.2 Results and Discussion ............................................................................................ 84 4.2.1 Synthesis of 1Aa ............................................................................................... 84 4.2.2 Tentative Synthesis of RIS-1Aa ...................................................................... 87 4.2.3 Spiral Ganglion Neurite Outgrowth in Vitro .................................................... 94 4.2.4 Regeneration of Cochlear Ribbon Synapses in Vitro ....................................... 96 4.3 Conclusion and Future Directions ........................................................................... 98 4.4 Experimental Procedure .......................................................................................... 99 4.4.1 Materials and Methods ..................................................................................... 99 4.4.2 Synthesis of 1-[(4-carboxy-2-nitrophenyl)methyl]triaza-1,2-dien-2-ium 2 ... 100 4.4.3 Synthesis of 4-(aminomethyl)-3-nitrobenzoic acid 3 ..................................... 101 4.4.4 Synthesis of 4-({[(4-methylphenyl)diphenylmethyl]amino}methyl)-3- nitrobenzoic acid 4 .................................................................................................. 101 4.4.5 Synthesis of 3-amino-4-({[(4-methylphenyl)diphenylmethyl]amino} methyl)benzoic acid 5 .............................................................................................. 102 4.4.6 Synthesis of Intermediate 6: Coupling Compound 5 to the Rink resin .......... 103 4.4.7 Synthesis of intermediate 7: Incorporation of Fmoc-Lys(Boc)-OH ............... 104 4.4.8 Synthesis of intermediate 8: Incorporation of Fmoc-Ile-OH ......................... 104 4.4.9 Synthesis of 2-fluoro-5-nitrobenzoyl chloride 9 ............................................ 105 4.4.10 Synthesis of intermediate 10: Incorporation of 2-fluoro-5-nitrobenzoyl chloride .................................................................................................................... 106 4.4.11 Synthesis of 1Aa-NO2 .................................................................................. 107 ix 4.4.12 Synthesis of 1Aa (on SP) ................................................................................. 108 4.4.13 Synthesis of (12S,15S)-20-amino-12-(4-aminobutyl)-15-(butan-2-yl)- 11,14,17-trioxo-2,10,13,16-tetraazatricyclo[16.4.0.04,9]docosa-1(18),4(9),5,7,19, 21-hexaene-7-carboxamide 1Aa .............................................................................. 108 4.4.14 Synthesis of 1Aa-COOH (no Boc) .............................................................. 109 4.4.15 Synthesis of 1Aa-COOH ............................................................................. 110 4.5 Chapter References ................................................................................................ 110 Appendix A. Chapter 2 Supporting Data ........................................................................ 113 Appendix B. Chapter 3 Supporting Data ......................................................................... 141 Appendix C. Chapter 4 Supporting Data ......................................................................... 168 x LIST OF TABLES Table 2.1 - Summary of relative 31 P and 19 F NMR (D2O, pH 10.0) chemical shifts of Pγ, according to the absolute configuration of β,γ-CHF dCTP……………………………..24 Table 2.2- Energy scores in kcal/mol obtained after docking with AutoDock Vina ........ 44 Table 3.1- Study of the optimal conditions for the coupling step ..................................... 62 Table 4.1- Trials of different combinations of reaction conditions in solid phase for coupling of RIS-linker with intermediate 1Aa-COOH to produce RIS-1Aa-(no PEG) . 89 Table 4.2- Trials of different combinations of reaction conditions in solution phase for coupling of RIS-linker with the intermediate 1Aa-COOH to produce RIS-1Aa ............. 90 Table 4.3- Trials of different combinations of reaction conditions for coupling of the model compound 11 with RIS-linker to produce compound 14 .................................................. 93 xi LIST OF FIGURES Figure 1.1- X-ray crystallography structure of a DNA polymerase enzyme in an open conformation state. .............................................................................................................. 2 Figure 1.2- The transcription machinery during initiation and elongation. ........................ 4 Figure 2.1- Structure of β,g-CXY dNTP. ......................................................................... 16 Figure 2.2- Preparative RP-C18 HPLC separation of compounds (R)- and (S)-2. .......... 18 Figure 2.3- Brønsted correlations of β,γ-CXY dCTP analogues. .................................... 20 Figure 2.4- 31 P NMR (203 MHz, D2O, pH 10.0) of β,γ-CHF dCTP mixture – only Pg is shown. ................................................................................................................................ 21 Figure 2.5- 31 P NMR (203 MHz, D2O, pH 10.0) of β,γ-CHF dCTP mixture spiked with isolated (S)-β,γ-CHF dCTP analogue (ratio 1:2) – only Pg is shown. ............................. 22 Figure 2.6- 19 F NMR (470 MHz, D2O, pH 10.0) of β,γ-CHF dCTP mixture spiked with isolated (R)-β,γ-CHF dCTP analogue (ratio 1:3) - only Pg is shown. ............................. 23 Figure 2.7- 19 F NMR (470 MHz) spectra simulated on MestReNova (Mnova 11.0.3- 18688). ............................................................................................................................... 25 Figure 2.8- DNA polymerase β. ....................................................................................... 36 Figure 2.9- A. Structure of PA and possible modifications. B. Generations of compounds as potential inhibitors. ....................................................................................................... 37 Figure 2.10- Structure of compound 9. ............................................................................. 42 Figure 2.11- A. Interaction of compound 9 with the lyase pocket. B. Interaction of –COOH of compound 9 with threonine 67. ..................................................................................... 43 Figure 2.12- DNA pol β dRP-lyase activity assay for compound 8. ................................ 46 Figure 3.1- Nucleotide recognition network in the RNA pol II catalytic site. ................. 57 Figure 3.2- Structure of β,γ-CXY NTP. ........................................................................... 58 Figure 3.3- Coupling reaction and HPLC chromatogram of the UTP analogue. ............. 61 Figure 3.4- Correlation of pH, bases tested and area % of the HPLC peak product. ....... 62 Figure 3.5- Correlation of pH, reaction time and area % of the HPLC peak product. ..... 63 Figure 3.6- Incorporation rate of β,γ -CXY UTPs by RNA pol II, with different β,γ- bridging atoms. .................................................................................................................. 65 Figure 3.7- Incorporation rate of β,γ-CHCl UTPs by RNA pol II. ................................. 66 Figure 4.1- The auditory system. ...................................................................................... 82 Figure 4.2- Structure of desired compounds RIS-1Aa and RIS-1Aa-(no PEG).. .......... 87 Figure 4.3- Spiral ganglion neurite outgrowth in vitro data. ............................................ 95 Figure 4.4- Cochlear synapse regeneration in vitro data. ................................................. 97 Figure A1- 1 H NMR (500 MHz, D2O, pH 10.3) of compound 1. .................................. 113 ......................................................................................................................................... 113 Figure A2- 19 F NMR (376 MHz, D2O, pH 10.0) of compound 1. ................................. 113 Figure A3- 31 P NMR (162 MHz, D2O, pH 10.0) of compound 1. ................................. 114 Figure A4- MS (ESI) [M-H] – of compound 1. ............................................................... 114 Figure A5- 1 H NMR (500 MHz, CD3OD) of (R)-isomer of compound 2. ..................... 115 Figure A6- 31 P NMR (202 MHz, CD3OD) of (R)-isomer of compound 2. .................... 115 Figure A7- 19 F NMR (470 MHz, CD3OD) of (R)-isomer of compound 2. .................... 116 Figure A8- MS (ESI) [M-H] – of (R)-isomer of compound 2. ........................................ 117 Figure A9- 1 H NMR (400 MHz, CD3OD) of (S)-isomer of compound 2. ..................... 118 xii Figure A10- 19 F NMR (376 MHz, CD3OD) of (S)-isomer of compound 2. ................... 118 Figure A11- 31 P NMR (162 MHz, CD3OD) of (S)-isomer of compound 2. ................... 119 Figure A12- MS (ESI) [M-H] – of (S)-isomer of compound 2. ....................................... 120 Figure A13- 1 H NMR (400 MHz, CD3OD) of (R)-isomer of compound 3. ................... 121 Figure A14- 19 F NMR (376 MHz, CD3OD) of (R)-isomer of compound 3. .................. 121 Figure A15- 31 P NMR (162 MHz, D2O, pH 10.0) of (R)-isomer of compound 3. ......... 122 Figure A16- MS (ESI) [M-H] – of (R)-isomer of compound 3. ...................................... 123 Figure A17- 1 H NMR (500 MHz, D2O, pH 10.0) of (S)-isomer of compound 3. .......... 124 Figure A18- 19 F NMR (470 MHz, D2O, pH 10.0) of (S)-isomer of compound 3. ......... 124 Figure A19- 31 P NMR (202 MHz, D2O, pH 10.0) of (S)-isomer of compound 3. ......... 125 Figure A20- MS (ESI) [M-H] – of (S)-isomer of compound 3. ....................................... 126 Figure A21- 1 H NMR (400 MHz, D2O, pH 10.0) of (R)-β,γ-CHF dCTP. ................... 127 Figure A22- 19 F NMR (564 MHz, D2O, pH 10.0) of (R)-β,γ-CHF dCTP. ................... 127 Figure A23- 31 P NMR (243 MHz, D2O, pH 10.0) of (R)-β,γ-CHF dCTP. ................... 128 Figure A25- Preparative SAX HPLC (second pass) of (R)-β,γ-CHF dCTP. ................ 129 Figure A26- 1 H NMR (400 MHz, D2O, pH 10.0) of (S)-β,γ-CHF dCTP. .................... 130 Figure A27- 19 F NMR (564 MHz, D2O, pH 10.0) of (S)-β,γ-CHF dCTP. ................... 130 Figure A28- 31 P NMR (243 MHz, D2O, pH 10.0) of (S)-β,γ-CHF dCTP. ................... 131 Figure A29- MS (ESI) [M-H] – of (S)-β,γ-CHF dCTP. ................................................. 132 Figure A30- Preparative SAX HPLC (second pass) of (S)-β,γ-CHF dCTP. ................ 132 Figure A31- 1 H NMR (500 MHz, CDCl3) of compound 1. ........................................... 133 Figure A32- 31 P NMR (202 MHz, CDCl3) of compound 1. ........................................... 133 Figure A33- 31 P NMR (202 MHz, CDCl3) of compounds 2 and 3. ............................... 134 Figure A34- 31 P NMR (202 MHz, CDCl3) of compounds 4. ......................................... 134 Figure A35- 31 P NMR (202 MHz, CDCl3) of compound 7. ........................................... 135 Figure A36- MS (ESI) [M-H] – of compound 7. ............................................................. 136 Figure A37- 1 H NMR (500 MHz, CD3OD) of compound 8. ......................................... 137 Figure A38- 31 P NMR (202 MHz, CD3OD) of compound 8. ......................................... 137 Figure A39- MS (ESI) [M-H] – of compound 8. ............................................................. 138 Figure A40- Docking experiment and analysis of Pamoic acid (PA). ........................... 138 Figure A41- Docking experiment and analysis of a tyrosine derivative of PA. ............. 139 Figure A42- Docking experiment and analysis of a phenyl derivative of PA. ............... 139 Figure A43- Docking experiment and analysis of a phenyl and tyrosine derivatives of PA. ......................................................................................................................................... 140 Figure A44- Docking experiment and analysis of a phenyl and n-butyl amide tyrosine derivatives of PA. ............................................................................................................ 140 Figure B1- 1 H NMR (400 MHz, D2O, pH 8.0) of β,γ-CCl2 ATP. ................................ 141 Figure B2- 31 P NMR (162 MHz, D2O, pH 8.0) of β,γ-CCl2 ATP. ................................ 141 Figure B3- MS (ESI) [M-H] - of β,γ-CCl2 ATP. ............................................................ 142 Figure B4- 1 H NMR (500 MHz, D2O, pH 7.5) of β,γ-CCl2 UTP. ................................. 143 Figure B5- 31 P NMR (202 MHz, D2O, pH 7.5) of β,γ-CCl2 UTP. ................................ 143 Figure B6- MS (ESI) [M-H] - of β,γ-CCl2 UTP. ............................................................ 144 Figure B7- 1 H NMR (400 MHz, D2O, pH 8.0) of β,γ-CHCl ATP. ............................... 145 Figure B8- 1 H NMR (243 MHz, D2O, pH 10.0) of β,γ-CHCl ATP. ............................. 145 Figure B9- 1 H NMR (600 MHz, D2O, pH 7.5) of β,γ-CHCl UTP. ............................... 146 xiii Figure B10- 31 P NMR (243 MHz, D2O, pH 7.5) of β,γ-CHCl UTP. ............................ 146 Figure B11- MS (ESI) [M-H] - of β,γ-CHCl UTP. ........................................................ 147 Figure B12- Purity analysis of β,γ-CHCl UTP .............................................................. 148 Figure B13- 31 P NMR of compound 1 (162 MHz, D2O, pH 10.0). ................................ 148 Figure B14- Purification of individual diastereomers (R)- and (S)-2 ............................ 149 Figure B15- 1 H NMR (400 MHz, CD3OD) of compound 2 - HPLC fast. ..................... 149 Figure B16- 31 P NMR (162 MHz, CD3OD) of compound 2 - HPLC fast. ..................... 150 Figure B17- MS (ESI) [M-H]- of compound 2 - HPLC fast. ......................................... 151 Figure B18- 1 H NMR (400 MHz, CD3OD) of compound 2 - HPLC slow. .................... 152 Figure B19- 1 H 2D NMR (400 MHz, CD3OD) of the aromatic area of compound 2 - HPLC slow. ................................................................................................................................. 153 Figure B20- 31 P NMR (162 MHz, CD3OD) of compound 2 - HPLC slow. ................... 154 Figure B21- MS (ESI) [M-H] - of compound 2 - HPLC slow. ........................................ 155 Figure B22- 1 H NMR (400 MHz, D2O, pH 1.0) of compound 3 - HPLC fast. .............. 156 Figure B23- 31 P NMR (243 MHz, D2O, pH 9.1) of compound 3 - HPLC fast. ............. 156 Figure B24- 1 H NMR (400 MHz, D2O, pH 1.0) of compound 3 - HPLC slow. ............ 157 Figure B25- 31 P NMR (162 MHz, D2O, pH 1.0) of compound 3 - HPLC slow. ............ 157 Figure B26- MS (ESI) [M-H] - of compound 3 - HPLC slow. ........................................ 158 Figure B27- 31 P NMR (162 MHz, D2O, pH 9.7) of compound 4 - HPLC fast. ............. 159 Figure B28- MS (ESI) [M-H] - of compound 4 - HPLC fast. ......................................... 160 Figure B29- 31 P NMR (162 MHz, D2O, pH 9.2) of compound 4 - HPLC slow. ............ 161 Figure B30- MS (ESI) [M-H] - of compound 4 - HPLC slow. ........................................ 162 Figure B31- 1 H NMR (400 MHz, D2O, pH 6.5) of β,γ-CHCl UTP – HPLC fast. ........ 163 Figure B32- 31 P NMR (162 MHz, D2O, pH 6.5) of β,γ-CHCl UTP – HPLC fast. ....... 163 Figure B33- MS (ESI) [M-H] - of β,γ-CHCl UTP – HPLC fast. ................................... 164 Figure B34- Purity check of β,γ-CHCl UTP – HPLC fast. ........................................... 164 Figure B35- 1 H NMR (400 MHz, D2O, pH 6.5) of β,γ-CHCl UTP – HPLC slow. ...... 165 Figure B36- 31 P NMR (162 MHz, D2O, pH 6.5) of β,γ-CHCl UTP – HPLC slow. ..... 165 Figure B37- MS (ESI) [M-H] - of β,γ-CHCl UTP – HPLC slow. .................................. 166 Figure B38- Purity check of β,γ-CHCl UTP – HPLC slow. ......................................... 167 Figure C1- 1 H NMR (400 MHz, CD3OD) of azide 2. .................................................... 168 Figure C2- 1 H NMR (500 MHz, CD3OD) studies of the different chemical shift between the desired product 2 (P) and the starting material (SM) 4‐(bromomethyl)‐3‐nitrobenzoic acid. ................................................................................................................................. 168 Figure C3- 13 C NMR (101 MHz, CD3OD). .................................................................... 169 Figure C4- MS ESI [M-H] - for azide 2. ......................................................................... 170 Figure C5- 1 H NMR (600 MHz, D2O pH 10.7) of 3. ..................................................... 171 Figure C6- MS ESI [M-H] - for compound 3. ................................................................. 171 Figure C7- ISCO chromatography of compound 4. ....................................................... 172 Figure C8- 1 H NMR (500 MHz, d-acetone) of 4. .......................................................... 172 Figure C9- MS ESI [M-H] - for compound 4. ................................................................. 173 Figure C10- 1 H NMR (600 MHz, CD3OD) of 5. ........................................................... 174 Figure C11- MS ESI [M-H]- for compound 5. ............................................................... 174 Figure C12- 1 H NMR (400 MHz, CDCl3) of benzoyl chloride 9.. ................................. 175 xiv Figure C13- 1 H NMR (400 MHz, CDCl3) studies of the different chemical shift between the desired product benzoyl chloride 9 (P) and the starting material 2-fluoro-5-nitrobenzoic acid (SM). ........................................................................................................................ 175 Figure C14- LCMS of 1Aa-NO2. .................................................................................. 176 Figure C15- Semi-preparative RP-C18 HPLC of 1Aa (tr = 25.9 min). ......................... 177 Figure C16- LCMS of 1Aa. ........................................................................................... 178 Figure C17- 1 H NMR (400 MHz, D2O) of 1Aa. ............................................................ 179 Figure C18- MS ESI [M-H] - for 1Aa-COOH (no Boc). ............................................... 180 Figure C19- MS ESI [M-H] - for 1Aa-COOH. .............................................................. 181 Figure C20- LCMS of compound 14 (entry 5 from Table 4.3). ..................................... 182 Figure C21- LCMS of compound 14 (entry 6 from Table 4.3). ..................................... 183 Figure C22- MS ESI [M-H] - of compound 14. .............................................................. 184 xv LIST OF SCHEMES Scheme 1.1- Overview of the single nucleotide base excision repair (BER) mechanism. . 3 Scheme 1.2- The involvement of trigger loop (TL) in the transcription catalysis .............. 5 Scheme 2.1- Synthesis of b,g-CHF dCTP diastereomers. ............................................... 18 Scheme 2.2- Synthesis of di-phosphonate compound 5 and 6. ......................................... 39 Scheme 2.3- Naphthalene reactivity showing different behaviors for isomer 2 and 3. .... 39 Scheme 2.4- Synthesis of novel mono-ethly phosphonate 8. ........................................... 40 Scheme 2.5- Methods used for the synthesis of PA-Ph. .................................................. 41 Scheme 3.1- Synthesis of b,g-CYCl U/ATP. ................................................................... 59 Scheme 3.2- Synthesis of individual diastereomers (R)- and (S)-b,g-CHCl UTP. .......... 60 Scheme 4.1- Synthesis of desired product 1Aa. ............................................................... 86 Scheme 4.2- Synthesis of intermediate 1Aa-COOH. ....................................................... 88 Scheme 4.3- Structure of 1Aa-NHS, hypothesized intra-cyclization within 1Aa-COOH... ………………………………………………………………………...………………….90 Scheme 4.4- Use of model compound 11 to test reaction conditions for the coupling step. ……………………………………………………………………………………………92 xvi ABSTRACT DNA polymerase b (DNA pol b) is responsible for DNA repair mechanisms that are involved with gap-filling DNA synthesis in the single nucleotide base excision repair (BER) pathways. BER is very important in maintaining healthy cells because it removes the damaged base, avoiding further mutations. On another hand, RNA polymerases are involved in the transcription process, where a single-stranded mRNA is created from a dsDNA chain, in combination with a diverse number of general transcription factors. This process is crucial for cell growth and differentiation and therefore studying the mechanism of action of these enzymes is of extreme importance. The functionality and structure of both DNA pol b and RNA polymerase II (RNA pol II), as well as their fidelity mechanism and the use of these enzymes as potential cancer therapeutic targets, is explored in detail in the introductory chapter, Chapter 1. In a complementary manner, Chapter 2 is focused on the synthesis of b,g-CHF dCTP probes for DNA pol b. A series of β,g-CXY dNTP compounds have been extensively synthesized by the McKenna lab over the years in order to provide an accessible tool kit of dNTPs to study how DNA pol β cleaves the bisphosphonate moiety of the nucleotide depending on the different CXY derivatives they possess. The diastereomers of β,g-CHF dCTP were synthesized, analyzed and both kinetically and structurally studied. When observing similar electronegative effects of CXY , it can be seen that the dihalo derivatives display a lower k pol when compared with the monohalo and non-halo line (Figure 2.3). In addition, Chapter 2 further explores the use of small molecule inhibitors to target the lyase domain of DNA pol b. Previous studies have shown that pamoic acid has lyase xvii inhibitory properties, therefore we aimed to synthesize modified pamoic acid derivatives, which possess a phosphorus moiety, and test their inhibitory effect. Compound 8 from Generation 2 (Scheme 2.4) was successfully synthesized and characterized and shown to inhibit the lyase domain of DNA pol β at a concentration of 500 μM (Figure 2.12). Moreover, the synthesis of b,g-CXY UTP probes to study the mechanism and fidelity of RNA pol II is analyzed in Chapter 3. This unique enzyme possesses a well-organized network, using a crucially conserved motif called the trigger loop. β,γ-CCl 2 UTP, β,γ- CCl 2 ATP, β,γ-CHCl UTP and β,γ-CHCl ATP were synthesized and the individual diastereomers β,γ-CHCl-1 and β,γ-CHCl-2 UTP were successfully isolated (Scheme 3.2). According to in vitro transcription assays, β,γ-CCl 2 UTP showed a lower incorporation rate when compared with the fluorine derivative, but higher than the methylene version (Figure 3.6). The optimal coupling step conditions were also examined. Lastly, Chapter 4 is focused on the solid-phase synthesis of the bisphosphonate conjugate 1Aa to target Tropomyosin receptor kinase B and C (TrkB and TrkC) in the inner ear. Recently, our group has published preliminary data regarding the use of a bisphosphonate-linked TrkB agonist as a delivery method to reach the cochlea. 1Aa was synthesized over 12 steps, 7 of them being in solid-phase support with an overall yield of 16% (Scheme 4.1). 1Aa promotes spiral ganglion neurite outgrowth in vitro and also bolsters the regeneration of cochlear ribbon synapses in vitro (Figures 4.4 and 4.5). Further studies involve the synthesis of the bisphosphonate counterpart, RIS-1Aa, and its effect on the spiral ganglion neurite outgrowth and the regeneration of cochlear ribbon synapses. 1 Chapter 1. DNA and RNA polymerases: functionality and structure, fidelity mechanisms and cancer therapeutics 1.1 Functionality and Structure of Polymerases 1.1.1 DNA Polymerase b Humans carry at least 15 different of DNA polymerases, known to be essential for the proper function of cells. DNA polymerases are involved in several biochemical pathways from synthesizing DNA to protecting the cell and repairing damaged DNA. Structurally, these enzymes display several similarities to one’s right hand in terms of structure, where the highly conserved ‘palm’ domain possesses critical amino acids that are involved in metal coordination, the ‘fingers’ subdomain interacts with the incoming dNTP whereas the ‘thumb’ subdomain is responsible for correctly positioning the dsDNA in the enzyme’s active site and it is crucial during the translocation of the polymerase onto the next nucleotide incorporation base(Figure 1.1). 1–4 DNA polymerase b (DNA pol b) in particular is responsible for DNA repair mechanisms that are involved with gap-filling DNA synthesis in the single nucleotide base excision repair (BER) pathways. BER is very important in maintaining healthy cells because it removes the damaged base, avoiding further mutations. 1,2 This process starts with a DNA glycosylase, specific for the damaged base, creating an AP site (apurinic or apyrimidinic site) followed by the action of an AP endonuclease that cleaves the sugar- phosphate backbone, exposing the 3’-OH on the adjacent nucleotide (Scheme 1.1). The lyase domain of the DNA pol b will then remove the 5’-deoxyribose phosphate (5’-dRP) 2 moiety leaving the chain with a one nucleotide gap, which is subsequently filled by the action of the nucleotide ‘gap-filling’ activity of DNA pol b and lastly the final repair of the nicked DNA is accomplished by DNA ligase (Scheme 1.1). Figure 1.1- X-ray crystallography structure of a DNA polymerase enzyme in an open conformation state. 1 DNA pol b is a 39 kDa enzyme with 335 amino acid residues and is divided into two different domains that confer distinct functions to the enzyme: a 31 kDa C-terminal polymerase domain responsible for the synthesis of DNA and for the nucleotidyl transferase activity, and a 8 kDa N-terminal lyase domain involved in the binding of the ssDNA, 5’-dRP recognition and removal during BER. 3,4 DNA pol b lacks proofreading activity, also known as 3’-5’ exonuclease activity, and therefore this enzyme is not able to remove misincorporate nucleotides on its own. 3 Scheme 1.1- Overview of the single nucleotide base excision repair (BER) mechanism. 27 1.1.2 RNA Polymerase II RNA polymerases are involved in the transcription process, where a single-stranded mRNA is created from a dsDNA chain, in combination with a diverse number of general transcription factors. This process is crucial for cell growth and differentiation and therefore studying the mechanism of action of these enzymes is of extreme importance. The transcription machinery involves a multicomplex process and utilizes different RNA polymerases in different organisms. In eukaryotes, the most complex one is the transcription system provided by RNA polymerase II (RNA pol II). 5 4 The RNA pol II transcription cycle encompasses three main stages. Gene-specific regulatory factors, alongside with the core promoter, are recruited to take part in this process. The enzyme and the factors bound together to the core promoter (preinitiation complex, PIC) subsequently go through a tremendous conformational change where the promoter’s template chain is placed inside the active site of the polymerase (Open Complex) and the first RNA bond is synthesized, marking the start of the initiation stage. After the insertion of 20-30 bases, it is known that the elongation process starts by the removal of the core promoter and factors from the active machinery and the recruitment of new factors (Figure 1.2) 5,6 . Once the machinery reaches the terminator portion of the gene, the transcription process is completed, and the mRNA chain is released. Structurally, RNA pol II is a 550 kDa protein containing 12 subunits, in eukaryotes. This unique enzyme possesses a well-organized network around the enzyme, including two essential magnesium ions for catalysis and a very crucial conserved motif called the trigger loop (TL). 7–10 Figure 1.2- The transcription machinery during initiation and elongation. 5 5 The TL, and its conformational changes, are involved in substrate selection and catalysis of the transcription process as well as during the elongation step. It is known that the TL can adopt different conformations, and its correct structural reorganization is dependent on the incorporation of the matched substrate. 7–10 Once the correct nucleotide is incorporated, the TL gets altered from an open inactive conformation to a close active conformation, where the substrate is stabilized and the incorporation of the adjacent nucleotide gets expedited. The TL catalysis acts in a stepwise manner, and if the non- complementary NTP (ncNTP) is added, it is believed that the TL will not close and will expel the ncNTP from the active pocket (Scheme 2.2). 11 It was also investigated that two amino acids, R1239 and H1242, are responsible for stabilizing the reaction transition state, being considered the main catalytic sources of the TL. 11,12 Scheme 1.2- The involvement of trigger loop (TL) in the transcription catalysis. Legend: complementary nucleotide (cNTP), non-complementary nucleotide (ncNTP). 11 Selectivity of binding kinetic discrimination in open active center TL folding and catalysis Substrate expelled from the active center 6 1.2 Fidelity Mechanisms 1.2.1 DNA Polymerase b Fidelity Fidelity of a polymerase is represented by the ability of the enzyme to select the correct and complementary nucleotide to be incorporated during DNA synthesis, over several of other highly similar dNTPs. Naturally, during the process of DNA synthesis several types of errors can be produced and the enzymes have mechanisms in order to correct those faulty steps. When a non-complementary nucleotide is incorporated, compromising the Watson-Crick base pairing, the enzyme tends to incorporate it in a very slow fashion, allowing time for the wrong nucleotide to leave and re-start the base filling process. In other cases, after a mispaired nucleotide is incorporated, the enzyme utilizes its intrinsic proofreading activity to correct the mistake, or it dissociates to allow an extrinsic enzyme (e.g. AP endonuclease) to come in and repair that error. 3,13 DNA pol b in particular has an error rate, for the single-nucleotide gap filling, of 1 error per 3,000 nucleotides incorporated 3,13 – a moderate fidelity value for the DNA polymerases that lack proofreading activity. Fidelity values are known to widely vary between different polymerases, however catalytic efficiency does not. Studies 14 have shown that the fidelity of DNA polymerases is associated with the efficiency for correct – and not incorrect – dNTP insertion. In other words, low-fidelity DNA polymerases insert the correct dNTP slowly, exhibiting low efficiency. However, not every low-efficiency DNA pol possesses low-fidelity. 14 7 1.2.2 RNA Polymerase II Transcription Fidelity RNA polymerase II incorporates NTPs with high fidelity and efficiency 5,12 . This is extremely important due to the natural function of this enzyme: synthesizing an RNA template from DNA. Any transcription error can have severe consequences such as phenotypic changes in the cell, leading to possible tumor creation. 15–17 Transcription mutagenesis can be created by either the RNA pol II while selecting and incorporating NTPs or induced by damaged DNA already incorporated in the DNA strand. During transcription, RNA pol II moves along the DNA strand and selects the correct and complementary NTP in order to synthesize the RNA template. When a mistake is found in the DNA strand, pol II is able to recognize it and either bypasses it or stalls at that particular lesion. If the enzyme bypasses the DNA error, transcriptional mutageneses are created. 5,18 However, if the enzyme stalls at the lesion, a specialized DNA repair mechanism is invoked – transcription-coupled repair (TCR) 19,20 – and the error is cleared. When considering the synthesis of the RNA transcript, RNA pol II utilizes at least three checkpoints in order to maintain the chain error-free. The checkpoints comprise: 1) insertion step, by selecting the correct NTP: 2) the extension step, incorporation of the complementary NTP to obey the Watson-Crick base pairing rules; and 3) the proofreading process, removal of any misincorporated NTP by 3’ to 5’ exonuclease activity. RNA pol II has evolved to display high fidelity and an efficiency mechanism and therefore its error rate is very low, less than 0.001%. 18 Apart from having the capability of synthesizing RNA it also plays an extremely important role in reading the DNA template, by recognizing DNA errors. 8 1.3 Using Probes to Study Polymerase Kinetics Probes have been widely used to study in detail the molecular mechanism of polymerases. Dissecting all the interactions within the polymerase as well as the enzyme and DNA/RNA templates, nucleotides and non-covalent interactions have brought us a better understanding of the overall molecular mechanism of polymerases and how DNA modifications and lesions can affect the fidelity of the enzyme. 7,21–25 Stereoisomeric chemical probes to target the transition state (TS) of DNA pol b and its interaction with the dNTP substrate has been a method introduced by Professor C. E. McKenna in collaboration with Professor M. F. Goodman. 23,24,26 Analyzing the TS of the enzyme provides great insights into chemical vs conformational steps, as well as indications how base pairing and mispairing affects the relative energy involved in the TS. Pre-steady state kinetic experiments using chemical probes show the correlation between the formation and consumption of enzyme-substrate intermediates, right before their steady-state concentrations are achieved. For polymerases in specific, in vitro experiments can be performed where the kinetics of a single nucleotide insertion (correct vs incorrect base) are analyzed. Pre-steady state kinetics has the advantage of having the DNA/RNA substrate in excess in regards to the concentration of the polymerase. When focusing on single-turnover kinetic experiments, the polymerase itself is in excess when compared with the concentration of the substrate, converting all the available DNA/RNA substrate into substrate-polymerase complex (active site), right before the start of the reaction, and therefore the enzyme does not recycle. Alongside with X-ray techniques and computational analysis, a vast number of structures corresponding to the intermediates formed during the polymerase reaction 9 pathways have been generated. As a consequence of having a better understanding of the exact interaction and intermediates, selective inhibitors and other therapeutic approaches have also been investigated. 1,27–31 1.4 Using Polymerases and Cancer Therapeutics 1.4.1 DNA Polymerase b and Cancer Therapeutics DNA polymerase b is the primary enzyme involved in the BER mechanism, that protects cells against single-nucleotide misincorporation. It is known that DNA pol b is overexpressed in cancer cells – high levels of the enzyme have been found in several carcinomas such as gastric, prostate, ovarian and uterine cancers 34–36 – due to the constant need mutated cells have to invoke DNA repair pathways. 2,32,33 In order to better understand the effect of fidelity in cells where DNA pol b is overexpressed, several research studies have been conducted. For example, Chan et al 33 found that the Human (Ha) cell line from B-cell lymphoma expressed DNA pol b with higher rates (~6-fold) when compared with normal human cells. In vitro experiments showed a ~9-fold increase in mutation frequency in the Ha cell line than in the wild type cells. In addition, to confirm that the increase in mutation rate is directly associated with the overexpression of DNA pol b in the Ha cells, the experiment was supplemented with an extra addition of DNA pol b (excess), and the mutation frequency was again increased by a ~6-fold, in relation to the cell line that was not supplemented. 33 A majority of tumors have been found to express enhanced levels of DNA pol b, therefore DNA pol b is an important therapeutic target for cancer. 1,29,37,38 10 DNA pol b inhibitors have been found that encompass a broad variety of chemical functional groups: fatty acids, triterpenoids, flavonoid derivatives, pamoic acid and many others. The main challenge for these inhibitors is lack of selectivity and/or potency. 39–42 As an example, 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) has shown a moderate potency against binding to the 8-KDa domain of the polymerase with IC 50 value of 28 µM, however it also targets other polymerases and glycosilases. 27,43 Toxicity is also a major concern when dealing with inhibitors for pol b. Several studies have been developed in order to find non-toxic and selective inhibitors of DNA pol b that can also increase the sensitivity of the cell to chemotherapeutic agents. 27,41,44,48 1.4.2 RNA Polymerase II and Cancer Therapeutics An accurate transcription process is at the core of a well-functioning cell, and it is necessary for basic processes such as cell growth, survival and differentiation. In cancer cells, certain factors involved in the transcription machinery are overexpressed due to the constant need these cells have to proliferate, therefore they require an active and high- paced transcription process. 45,46 Several drugs used to inhibit carcinogenic cell proliferation have been shown to interfere with the transcription complex. For instance, cisplatin has been shown to induce DNA damage in cancer cells, and also to interfere with the transcription process. 28,47 During transcription inhibition, oncogenetic cells are more susceptible to undergo apoptosis when compared with normal healthy cells. One study showed that ARC nucleotide, a RNA pol II inhibitor that blocks the elongation transcription process, induced highly potent apoptosis in human tumors but does not affect healthy normal cells, in vitro. 47,48 Other 11 studies showed that cancer cells are more dependent on certain specific factors than healthy normal cells. For example, depletion of heat shock factor 1 (HSF1), a transcription factor involved in cell survival, potently affects cancer cell functionality but only minimally impacts healthy cells. 47,49 These findings support the idea that inhibiting the transcription machinery, specifically RNA pol II, the main enzyme involved in this process, is a potential selective therapeutic approach for cancer research. To date there are only a few drugs that directly target RNA pol II, and not the other factors involved in transcription. a-Amanitin, one of those drugs, is a natural product isolated from Amanita mushrooms and binds to the polymerase, inhibiting the translocation process, but it is not used in cancer therapy due to its high liver toxicity. 47,50 Several other drugs target the associated transcriptional factors which indirectly affects the functionality of the RNA pol II. 47,51,52 1.5 Chapter References (1) Berdis, A. J. Inhibiting DNA Polymerases as a Therapeutic Intervention against Cancer. Front. Mol. Biosci. 2017, 4, 78, 1-12. (2) Lange, S. S.; Takata, K.; Wood, R. D. DNA Polymerases and Cancer. Nat. Rev. Cancer 2011, 11 (2), 96–110. (3) Beard, W. A.; Wilson, S. H. Structure and Mechanism of DNA Polymerase β. Chem. Rev. 2006, 106 (2), 361–382. (4) Idriss, H. T.; Al-Assar, O.; Wilson, S. H. DNA Polymerase β. Int. J. Biochem. Cell Biol. 2002, 34 (4), 321–324. (5) Hahn, S. Structure and Mechanism of the RNA Polymerase II Transcription Machinery. Nat. Struct. Mol. Biol. 2004, 11 (5), 394–403. (6) Wang, D.; Bushnell, D. A.; Huang, X.; Westover, K. D.; Levitt, M.; Kornberg, R. D. Structural Basis of Transcription: Backtracked RNA Polymerase II at 3.4 Angstrom Resolution. Science 2009, 324 (5931), 1203–1206. (7) Hwang, C. S.; Xu, L.; Wang, W.; Ulrich, S.; Zhang, L.; Chong, J.; Shin, J. H.; Huang, X.; Kool, E. T.; McKenna, C. E.; et al. Functional Interplay between NTP Leaving Group and Base Pair Recognition during RNA Polymerase II Nucleotide Incorporation Revealed by Methylene Substitution. Nucleic Acids Res. 2016, 44 (8), 3820–3828. 12 (8) Kaplan, C. D.; Larsson, K.-M.; Kornberg, R. D. The RNA Polymerase II Trigger Loop Functions in Substrate Selection and Is Directly Targeted by α-Amanitin. Mol. Cell 2008, 30 (5), 547–556. (9) Toulokhonov, I.; Zhang, J.; Palangat, M.; Landick, R. A Central Role of the RNA Polymerase Trigger Loop in Active-Site Rearrangement during Transcriptional Pausing. Mol. Cell 2007, 27 (3), 406–419. (10) Zhang, J.; Palangat, M.; Landick, R. Role of the RNA Polymerase Trigger Loop in Catalysis and Pausing. Nat. Struct. Mol. Biol. 2010, 17 (1), 99–104. (11) Yuzenkova, Y.; Bochkareva, A.; Tadigotla, V. R.; Roghanian, M.; Zorov, S.; Severinov, K.; Zenkin, N. Stepwise Mechanism for Transcription Fidelity. BMC Biol. 2010, 8 (1), 54. (12) Kaplan, C. D. The Architecture of RNA Polymerase Fidelity. BMC Biol. 2010, 8 (1), 85. (13) Beard, W. A.; Prasad, R.; Wilson, S. H. Activities and Mechanism of DNA Polymerase β. In Methods in Enzymology; Academic Press, 2006; Vol. 408, pp 91– 107. (14) Beard, W. A.; Shock, D. D.; Vande Berg, B. J.; Wilson, S. H. Efficiency of Correct Nucleotide Insertion Governs DNA Polymerase Fidelity. J. Biol. Chem. 2002, 277 (49), 47393–47398. (15) Gordon, A. J. E.; Halliday, J. A.; Blankschien, M. D.; Burns, P. A.; Yatagai, F.; Herman, C. Transcriptional Infidelity Promotes Heritable Phenotypic Change in a Bistable Gene Network. PLoS Biol. 2009, 7 (2), e44. (16) Gordon, A. J. E.; Satory, D.; Halliday, J. A.; Herman, C. Lost in Transcription: Transient Errors in Information Transfer. Curr. Opin. Microbiol. 2015, 24, 80–87. (17) Brégeon, D.; Doetsch, P. W. Transcriptional Mutagenesis: Causes and Involvement in Tumor Development. Nat. Rev. Cancer 2011, 11 (3), 218–227. (18) Xu, L.; Wang, W.; Chong, J.; Shin, J. H.; Xu, J.; Wang, D. RNA Polymerase II Transcriptional Fidelity Control and Its Functional Interplay with DNA Modifications. Crit. Rev. Biochem. Mol. Biol. 2015, 50 (6), 503–519. (19) Hanawalt, P. C.; Spivak, G. Transcription-Coupled DNA Repair: Two Decades of Progress and Surprises. Nat. Rev. Mol. Cell Biol. 2008, 9 (12), 958–970. (20) Lagerwerf, S.; Vrouwe, M. G.; Overmeer, R. M.; Fousteri, M. I.; Mullenders, L. H. F. DNA Damage Response and Transcription. DNA Repair 2011, 10 (7), 743–750. (21) Joyce, C. M. Techniques Used to Study the DNA Polymerase Reaction Pathway. Biochim. Biophys. Acta BBA - Proteins Proteomics 2010, 1804 (5), 1032–1040. (22) Hwang, C. S. Fluorinates Probes of Enzyme Mechanisms, Doctoral dissertation, University of Southern California: Los Angeles, US, 2016. (23) Oertell, K.; Chamberlain, B. T.; Wu, Y.; Ferri, E.; Kashemirov, B. A.; Beard, W. A.; Wilson, S. H.; McKenna, C. E.; Goodman, M. F. Transition State in DNA Polymerase β Catalysis: Rate-Limiting Chemistry Altered by Base-Pair Configuration. Biochemistry 2014, 53 (11), 1842–1848. (24) Oertell, K.; Kashemirov, B. A.; Negahbani, A.; Minard, C.; Haratipour, P.; Alnajjar, K. S.; Sweasy, J. B.; Batra, V. K.; Beard, W. A.; Wilson, S. H.; et al. Probing DNA Base-Dependent Leaving Group Kinetic Effects on the DNA Polymerase Transition State. Biochemistry 2018, 57 (26), 3925–3933. 13 (25) McKenna, C. E.; Kashemirov, B. A.; Upton, T. G.; Batra, V. K.; Goodman, M. F.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H. (R)-β,γ-Fluoromethylene-DGTP-DNA Ternary Complex with DNA Polymerase β. J. Am. Chem. Soc. 2007, 129 (50), 15412–15413. (26) Wu, Y.; Zakharova, V. M.; Kashemirov, B. A.; Goodman, M. F.; Batra, V. K.; Wilson, S. H.; McKenna, C. E. β,γ-CHF- and β,γ-CHCl-DGTP Diastereomers: Synthesis, Discrete 31P NMR Signatures, and Absolute Configurations of New Stereochemical Probes for DNA Polymerases. J. Am. Chem. Soc. 2012, 134 (21), 8734–8737. (27) Barakat, K. H.; Gajewski, M. M.; Tuszynski, J. A. DNA Polymerase Beta (Pol β) Inhibitors: A Comprehensive Overview. Drug Discov. Today 2012, 17 (15–16), 913– 920. (28) Todd, R. C.; Lippard, S. J. Inhibition of Transcription by Platinum Antitumor Compounds. Metallomics 2009, 1 (4), 280-291. (29) Martin, S. A.; McCabe, N.; Mullarkey, M.; Cummins, R.; Burgess, D. J.; Nakabeppu, Y.; Oka, S.; Kay, E.; Lord, C. J.; Ashworth, A. DNA Polymerases as Potential Therapeutic Targets for Cancers Deficient in the DNA Mismatch Repair Proteins MSH2 or MLH1. Cancer Cell 2010, 17 (3), 235–248. (30) Hecht, S. Inhibitors of the Lyase Activity of DNA Polymerase β. Pharm. Biol. 2003, 41, 68–77. (31) Gao, Z.; Maloney, D. J.; Dedkova, L. M.; Hecht, S. M. Inhibitors of DNA Polymerase Beta: Activity and Mechanism. Bioorg. Med. Chem. 2008, 16 (8), 4331–4340. (32) Pillaire, M.-J.; Betous, R.; Conti, C.; Czaplicki, J.; Pasero, P.; Bensimon, A.; Cazaux, C.; Hoffmann, J.-S. Upregulation of Error-Prone DNA Polymerases Beta and Kappa Slows Down Fork Progression Without Activating the Replication Checkpoint. Cell Cycle 2007, 6 (4), 471–477. (33) Chan, K.; Houlbrook, S.; Zhang, Q.-M.; Harrison, M.; Hickson, I. D.; Dianov, G. L. Overexpression of DNA Polymerase Results in an Increased Rate of Frameshift Mutations during Base Excision Repair. Mutagenesis 2007, 22 (3), 183–188. (34) Albertella, M. R.; Lau, A.; O’Connor, M. J. The Overexpression of Specialized DNA Polymerases in Cancer. DNA Repair 2005, 4 (5), 583–593. (35) Tan, X.-H.; Zhao, M.; Pan, K.-F.; Dong, Y.; Dong, B.; Feng, G.-J.; Jia, G.; Lu, Y.-Y. Frequent Mutation Related with Overexpression of DNA Polymerase Beta in Primary Tumors and Precancerous Lesions of Human Stomach. Cancer Lett. 2005, 220 (1), 101–114. (36) Yoshizawa, K.; Jelezcova, E.; Brown, A. R.; Foley, J. F.; Nyska, A.; Hofseth, L. J.; Maronpot, R. M.; Wilson, S. H.; Sepulveda, A. R. Gastrointestinal Hyperplasia with Altered Expression of DNA Polymerase B. PLoS ONE 2009, 4 (8), 1-11. (37) Yang, J.; Parsons, J.; Nicolay, N. H.; Caporali, S.; Harrington, C. F.; Singh, R.; Finch, D.; D’Atri, S.; Farmer, P. B.; Johnston, P. G.; et al. Cells Deficient in the Base Excision Repair Protein, DNA Polymerase Beta, Are Hypersensitive to Oxaliplatin Chemotherapy. Oncogene 2010, 29 (3), 463–468. (38) Shanbhag, V.; Sachdev, S.; Flores, J.; Modak, M.; Singh, K. Family A and B DNA Polymerases in Cancer: Opportunities for Therapeutic Interventions. Biology 2018, 7 (1), 5, 1-16. 14 (39) Mizushina, Y.; Tanaka, N.; Yagi, H.; Kurosawa, T.; Onoue, M.; Seto, H.; Horie, T.; Aoyagi, N.; Yamaoka, M.; Matsukage, A.; et al. Fatty Acids Selectively Inhibit Eukaryotic DNA Polymerase Activities in Vitro. Biochim. Biophys. Acta BBA - Gene Struct. Expr. 1996, 1308 (3), 256–262. (40) Tanaka, N.; Kitamura, A.; Mizushina, Y.; Sugawara, F.; Sakaguchi, K. Fomitellic Acids, Triterpenoid Inhibitors of Eukaryotic DNA Polymerases from a Basidiomycete, Fomitella Fraxinea. J. Nat. Prod. 1998, 61 (2), 193–197. (41) Maloney, D. J.; Deng, J.-Z.; Starck, S. R.; Gao, Z.; Hecht, S. M. (+)-Myristinin A, a Naturally Occurring DNA Polymerase Beta Inhibitor and Potent DNA-Damaging Agent. J. Am. Chem. Soc. 2005, 127 (12), 4140–4141. (42) Hu, H.-Y.; Horton, J. K.; Gryk, M. R.; Prasad, R.; Naron, J. M.; Sun, D.-A.; Hecht, S. M.; Wilson, S. H.; Mullen, G. P. Identification of Small Molecule Synthetic Inhibitors of DNA Polymerase β by NMR Chemical Shift Mapping. J. Biol. Chem. 2004, 279 (38), 39736–39744. (43) Mizushina, Y.; Xu, X.; Asano, N.; Kasai, N.; Kato, A.; Takemura, M.; Asahara, H.; Linn, S.; Sugawara, F.; Yoshida, H.; et al. The Inhibitory Action of Pyrrolidine Alkaloid, 1,4-Dideoxy-1,4-Imino-D-Ribitol, on Eukaryotic DNA Polymerases. Biochem. Biophys. Res. Commun. 2003, 304 (1), 78–85. (44) Kumamoto-Yonezawa, Y.; Sasaki, R.; Suzuki, Y.; Matsui, Y.; Hada, T.; Uryu, K.; Sugimura, K.; Yoshida, H.; Mizushina, Y. Enhancement of Human Cancer Cell Radiosensitivity by Conjugated Eicosapentaenoic Acid - a Mammalian DNA Polymerase Inhibitor. Int. J. Oncol. 2010, 36 (3), 577–584. (45) Cabarcas, S.; Schramm, L. RNA Polymerase III Transcription in Cancer: The BRF2 Connection. Mol. Cancer 2011, 10 (1), 47, 1-15. (46) Drygin, D.; Rice, W. G.; Grummt, I. The RNA Polymerase I Transcription Machinery: An Emerging Target for the Treatment of Cancer. Annu. Rev. Pharmacol. Toxicol. 2010, 50 (1), 131–156. (47) Villicaña, C.; Cruz, G.; Zurita, M. The Basal Transcription Machinery as a Target for Cancer Therapy. Cancer Cell Int. 2014, 14 (1), 18. (48) Radhakrishnan, S. K.; Gartel, A. L. A Novel Transcriptional Inhibitor Induces Apoptosis in Tumor Cells and Exhibits Antiangiogenic Activity. Cancer Res. 2006, 66 (6), 3264–3270. (49) Dai, C.; Whitesell, L.; Rogers, A. B.; Lindquist, S. Heat Shock Factor 1 Is a Powerful Multifaceted Modifier of Carcinogenesis. Cell 2007, 130 (6), 1005–1018. (50) Zheleva, A.; Tolekova, A.; Zhelev, M.; Uzunova, V.; Platikanova, M.; Gadzheva, V. Free Radical Reactions Might Contribute to Severe Alpha Amanitin Hepatotoxicity- -a Hypothesis. Med. Hypotheses 2007, 69 (2), 361–367. (51) Manzo, S. G.; Zhou, Z.-L.; Wang, Y.-Q.; Marinello, J.; He, J.-X.; Li, Y.-C.; Ding, J.; Capranico, G.; Miao, Z.-H. Natural Product Triptolide Mediates Cancer Cell Death by Triggering CDK7-Dependent Degradation of RNA Polymerase II. Cancer Res. 2012, 72 (20), 5363–5373. (52) Titov, D. V.; Gilman, B.; He, Q.-L.; Bhat, S.; Low, W.-K.; Dang, Y.; Smeaton, M.; Demain, A. L.; Miller, P. S.; Kugel, J. F.; et al. XPB, a Subunit of TFIIH, Is a Target of the Natural Product Triptolide. Nat. Chem. Biol. 2011, 7 (3), 182–188. 15 Chapter 2. Synthesis of b,g-CHF dCTP probes and small molecule inhibitors for DNA polymerase b 2.1 Part I: Using Deoxyribonucleotide Triphosphate (dNTP) as Probes 2.1.1 Introduction In order to understand how DNA pol β behaves mechanistically in the transitional state (TS), deoxyribonucleotide incorporation and fidelity rates of pol β have been correlated with structure and electronegativity of the nucleotide’s leaving group. 1–3 The transition state is the state of a reaction that possesses the highest energy and the least stable complex. Probing the TS of DNA pol β gives insight about the structure and characteristics of the rate-determining step (RDS) of DNA pol β. Studies have shown two possibilities for the RDS of the TS. One possibility is that the TS might be the chemical step in which the RDS corresponds to the formation and/or cleavage of the O – P nucleotide bond. The second possibility is based on the conformational change of the protein as being the rate- determining step. It is now proven that DNA pol β is one of the polymerases with a chemical step as the RDS. 1–3 This brings a particular interest to this enzyme because DNA pol β plays a unique role during base excision repair (BER). Linear Free Energy Relationship (LFER) analysis is the model used to verify that in fact PaO-Pb bond cleavage (leaving-group effect) in DNA pol β catalysis corresponds to the RDS. The LFER graph show a Brønsted dependence of the log of the catalytic rate constant (k pol ) versus the pK a4 of the leaving group, the pK a4 can be varied by replacing the natural pyrophosphate leaving group. In order to affirm that the chemical step is the RDS, rate of breaking of the PaO – Pb bond in the dNTP by a different CXY-bisphosphonates, must be the same or slower 16 when compared with its formation. 3 The Brønsted plot is expected to display a linear correlation, with a negative slope whose magnitude reflects how sensitive the TS is to charge stabilization from the X and Y substituents in the bisphosphonate moiety. 1–3 A series of β,g-CXY dNTP compounds (Figure 2.1) was synthesized by the McKenna lab over the years in order to provide an accessible tool kit of dNTPs to study how DNA pol β cleaves the bisphosphonate moiety of the nucleotide depending on the CXY structure. The diastereomers of several compounds having X¹Y were also synthesized, characterized and studied kinetically in the DNA pol β ternary complex 1,4–8 For the purpose of this chapter, the goal of ther project was to study the rate of incorporation of the β,g-CHF dCTP derivative by DNA pol β and compare it with the behavior of other β,g-CXY nucleotide derivatives as well. Figure 2.1- Structure of β,g-CXY dNTP. 2.1.2 Results and Discussion 2.1.2.1 Synthesis of β,g-CHF dCTP In this chapter, I will focus on my synthesis of the β,g-CHF dCTP diastereomers, performed in collaboration with Dr. Corinne Minard. The standard literature method was used to synthesize the mono-fluoro bisphosphonate precursor. 6,9 Individual diastereomers O P HO P O OH OH O P HO O O OH O Base X Y 17 (R)- and (S)-β,g-CHF dGTP have been succefully synthesized in the past 8 using (R)-methyl mandelate as a chiral auxiliary, however this method was not compatible with the cytosine nucleotide derivatives due to the fact that the cytosine base is not stable under the final hydrogenolysis conditions described in the literature. A new and alternative synthetic approach was therefore created in order to overcome this challenge. The use of (R)-1- phenylpropan-1-amine as the chiral auxiliary and the insertion of a photo-cleaved moiety avoids the use of the hydrogenolysis step (Scheme 2.1). The mono-fluoro bisphosphonate precursor was reacted with 2-nitrobenzylbromide in DMF for 24 hours at 125 ºC yielding intermediate 1 with 69% yield after HPLC purification. Subsequently, compound 1 was reacted with the chiral (R)-1-phenylpropan-1-amine, in the presence of 2,2’- dithiodipyridine (PyS) 2 and triphenyl phosphine (PPh 3 ) in DMF. The diastereomers were separated and purified by reverse phase HPLC using a C18 column. The retention time of the HPLC-fast isomer 2 was 25.4 min versus the HPLC-slow isomer 2 eluted from the column at 27.7 min (Figure 2.2). Following the separation of the diastereomers, each individual diastereomer was dissolved in HCl 1M and stirred for 3 hours. The amine moiety was cleaved off and the bisphosphonate acids (R)- and (S)-3 obtained. For the formation of the triphosphonate counterpart, each bisphosphonic acid was reacted with tributylamine, in a mixture of ethanol and water, followed by the addition of 2’-deoxycytidine 5’- phosphoromorpholidate 10,11 in dry DMSO and reacted for 7 days. The final product was purified by SAX and both isomers (R)- and (S)-4 were obtained. The final synthetic step involved the removal of the nitro-benzoyl group by light irradiation at 365 nm for 2 days. After HPLC purification, the triethylammonium salt of diastereomers (R)- and (S)-β,g- CHF dCTP were obtained. 18 Scheme 2.1- Synthesis of b,g-CHF dCTP diastereomers. Conditions: a. DIEA, 2- NO 2 BnBr, DMF, 125 ºC, 24 h; b. (R)-1-phenylpropan-1-amine, (PyS) 2 , PPh 3 , DMF, RT, 3 h; c. HCl [1M], RT, 3 h; d. Bu 3 N, EtOH/H 2 O then dCMP-morpholidate, DMSO, RT, 7 d; e. hv 365 nm, H 2 O, 2 d. Figure 2.2- Preparative RP-C18 HPLC separation of compounds (R)- and (S)-2. Conditions: preparative RP-C18 column (8.0 mL/min, 280 nm) in isocratic mode with 0.1 HO P O HO P F O O OH NO 2 HO P O HO P F OH O OH N H P (S) O HO P F O O OH NO 2 (R) or (R) HO P (S) O HO P F O O OH NO 2 or (R) O P (S) HO P O OH O F O P HO O O OH O N N O NH 2 NO 2 O P (S) HO P O OH OH F O P HO O O OH O N N O NH 2 or (R) or (R) 1 2 (R) or (S) a b c d e 69% 56% isomer ratio 1:1 qt. 9% (HPLC fast) 35% (HPLC slow) (R)- and (S)-CHF dCTP 22% (HPLC fast) 25% (HPLC slow) 3 (R) or (S) 4 (R) or (S) 19 M triethylammonium bicarbonate 27% acetonitrile pH 8.5 buffer (t r fast isomer = 25.4 min, t r slow isomer = 27.7 min). 2.1.2.2 LFER Analysis of β,g-CHF dCTP A total of 55 compounds were synthesized for this full project encompassing the 4 different DNA bases (adenine, guanine, cytosine and thymine) and the work was divided between several students. 1,4,7,8 As mentioned above, I have been involved in the synthesis and characterization of (R)- and (S)-β,g-CHF dCTP. The pK a4 of the bisphosphonate moiety of the nucleotide’s leaving group ranges between 7.8 [CF 2 ] and 10.5 [CH 2 ], with the value for β,g-CHF dCTP derivative being near 9, which confers a broad range of data points around the pK a4 value (8.9) of the natural leaving group pyrophosphoric acid. 4 When the electronegativity of the CXY increases, the pK a4 value decreases making the BP a better leaving group because the negative charge is better stabilized, therefore increasing the incorporation rate of the NTPs, if the RDS is ‘chemical’ as defined above. When analyzing the Brønsted correlation for the dCTP analogues (Figure 2.3), we observe three lines rather than the usual two 4 on the graph of the base pairing (C opp. G), we see that the dihalogens CF 2 and CCl 2 fit in one line, and the CBr 2 and CClF fit on a distinct line (Figure 2.3A). It is also seen that the parent dCTP (O) has moved and it now sits between CClF and CBr 2 in the dihalo line - when compared with previous studies of dATP and dTTP, (O) is placed on the upper part of the monohalo line. 1 These findings show the first case of a compound that does not possess two halogen atoms, but it still fits in the same Brønsted line as the dihalo compounds. The slope of all the three lines on C opp. G 20 are very similar which leads to the conclusion that the mechanism of each grouping is analogous. When analyzing the mispair graph (C opp. A) (Figure 2.3B), it is evident that CHF and (O) are situated together between the dihalo and the monohalo lines. The slopes of these three lines differ, leading to the conclusion that a different enzyme mechanism might be responsible for the observed grouping of data. Evaluating the performance of the individual diastereomers for the C•G base pairing, (R)-CHF isomer shows the fastest incorporation rate when compared to the CHF mixture and the (S)-CHF. The CHF mixture point plot close to the (R)-isomer, and the (S)-isomer shows the slowest incorporation rate between these three analogues. On the other hand, when assessing the mispairing C•A, it is seen that even though the (R)-CHF has the fastest rate, the CHF mixture and the (S)-CHF are located much closer to each other. Figure 2.3- Brønsted correlations of β,γ-CXY dCTP analogues. A. Base pairing: the correct opposite G, B. Mispairing: opposite A. 4 m1= -0.91 m2= -0.85 m3= -0.76 1 2 3 m4= -1.4 m5= -2.3 m6= -0.98 4 5 6 21 2.1.2.3 NMR Spike Experiments of (S)- and (R)-β,γ-CHF dCTP In order to analyze the correlation between the diastereomers’ NMR chemical shifts and the exact (S)- and (R)-isomer absolute configuration determined by X-ray crystallography, 31 P and 19 F NMR spike experiments were conducted (by Dr. Corinne Minard). Starting with a 1:1 ratio of the mixture of isomers (Figure 2.4) we see that the ∆d constant for Pg is 4.0 Hz. Increasing the ratio of (S)-isomer in the previous sample (Figure 2.5), we see that the peaks in blue go up and show the same ratio proportion (1:2), and ∆d is kept constant. Figure 2.4- 31 P NMR (203 MHz, D 2 O, pH 10.0) of β,γ-CHF dCTP mixture – only Pg is shown. 22 In the same line of thought, the 19F NMR spike experiment was analyzed and the peaks corresponding to the (S)-isomer (–216.70 ppm, blue dots) and the (R)-isomer (- 216.74 ppm, pink dots) were identified (Figure 2.6). Figure 2.5- 31 P NMR (203 MHz, D 2 O, pH 10.0) of β,γ-CHF dCTP mixture spiked with isolated (S)-β,γ-CHF dCTP analogue (ratio 1:2) – only Pg is shown. A theoretical 19 F NMR spectrum was also created using the MestreNova software and the splitting pattern was recreated considering a 1:3 as well as a 1:1 ratio (Figure 2.7). We were able to get a similar splitting pattern using the following parameters, for Pg: (S)-CHF dCTP: ddd, J FP = 65.0 Hz, J FP = 56.0 Hz Hz, J FH = 46.0 Hz (R)-CHF dCTP: ddd, J FP = 66.0 Hz, J FP = 55.0 Hz Hz, J FH = 46.0 Hz. 23 In terms of chemical shifts of the 31 P NMR for the (R)-isomer, it can be seen that its chemical shift is more downfield than the (S)-isomer. The opposite effect is observed on the 19 F NMR spectrum. This conclusion is summarized on Table 2.1. Figure 2.6- 19 F NMR (470 MHz, D 2 O, pH 10.0) of β,γ-CHF dCTP mixture spiked with isolated (R)-β,γ-CHF dCTP analogue (ratio 1:3) - only Pg is shown. 24 Table 2.1 - Summary of relative 31 P and 19 F NMR (D 2 O, pH 10.0) chemical shifts of P γ , according to the absolute configuration of β,γ-CHF dCTP. Legend: D, downfield; U, upfield. P γ of β,γ-CHF dCTP 31 P NMR 19 F NMR (R)-isomer D U (S)-isomer U D The full length 31 P and 19 F NMR spectra can be found in Appendix A (Figure A22, A23, A27 and A28). Analyzing the splitting patterns, Pa slips into a doublet with a J Pa-b of 29 Hz. Pb is shown as a doublet of doublet of doublets (ddd) because it not only couples with the other phosphorous atoms (J Pb-Pg = 14 Hz and J Pa-Pb = 29 Hz) but also couples with the fluorine atom (J P-F =65 Hz). Considering the 19 F NMR, the splitting pattern is a doublet of doublet of doublets (ddd) because the fluorine atom couples with both phosphorous and the adjacent hydrogen, therefore the spectra displays 8 peaks per diastereomer. 25 Figure 2.7- 19 F NMR (470 MHz) spectra simulated on MestReNova (Mnova 11.0.3- 18688). Parameters used: (S)-CHF dCTP: ddd, J FP = 65.0 Hz, J FP = 56.0 Hz Hz, J FH = 46.0 Hz; (R)-CHF dCTP: ddd, J FP = 66.0 Hz, J FP = 55.0 Hz Hz, J FH = 46.0 Hz. 26 2.1.3 Conclusion The synthesis and characterization of β,g-CHF dCTP was accomplished. The synthetic route encompasses 5 chemical steps with a total yield of 1% and 3% for HPLC fast and slow isomers, respectively. Individual (S)- and (R)-diastereomers were purified, identified and kinetically studied. In addition, NMR spike experiments as well as theoretical splitting patterns were analyzed in order to identify the differences in chemical shifts of the two isomers in both 31 P and 19 F NMRs. Kinetic studies were performed by our collaborators (Prof. M. F. Goodman Lab) and the β,g-CXY dNTP incorporation rate by DNA polymerase β analyzed. The overall LFER analysis suggested that there are important factors to consider regarding the rate- determining step on the transition state. Firstly, an increase of the leaving group pKa4 is strongly associated with a decrease in the incorporation rate by the enzyme (kpol). When observing similar electronegative effects of CXY, it can be seen that the dihalo derivatives display a lower kpol when compared with the monohalo and non-halo lines. Evaluating the performance of the individual diastereomers (R)- and (S)-β,g-CHF dCTP for the C•G base pairing, (R)-CHF isomer shows the fastest incorporation rate when compared to the CHF mixture and the (S)-CHF. When evaluating all the previous results published in the McKenna lab, it is notable that TS energies vary according to the base pair being studied. 1,4,7,8 27 2.1.4 Experimental Procedure The experimental procedure here stated as well as all the characterization data (Appendix A, Part I) was conducted in collaboration with Dr. Corinne Minard. 2.1.4.1 Materials and Methods Cytidine-5'-monophosphoric acid was purchased from Chem-Impex International. Phosphonic esters and bisphosphonic acids were prepared according the literature. 2,6 Purifications of tetraisopropyl (difluoromethylene)bis(phosphonate), and tetraisopropyl (fluoromethylene)bis(phosphonate) were performed using an ISCO CombiFlashRf+ Lumen instrument equipped with an ELSD detector. All other reagents were purchased from Sigma-Aldrich, Fluka and Alfa Aesar (reagent grade) and used as obtained. Synthesis of individual diastereomers of β,γ-CHF dCTP was performed as described herein. 1 H, 31 P, and 19 F NMR spectra were obtained on Varian 400-MR, or NMR-500, or NMR-600 spectrometers. 31 P NMR spectra were proton-decoupled unless stated otherwise. Multiplicities are quoted as singlet (s), doublet (d), triplet (t), unresolved multiplet (m), doublet of doublets (dd), doublet of doublet of doublets (ddd), doublet of triplets (dt) or broad signal (br). All chemical shifts (δ) are reported in parts per million (ppm) relative to residual CDH2OH in CD3OD (δ 3.34, 1 H NMR), CHCl3 in CDCl3 (δ 7.26, 1 H NMR), HDO in D2O (δ 4.80, 1 H NMR), external 85% H3PO4 (δ 0.00, 31 P NMR) or external C6F6 (δ - 164.9, 19 F NMR). NMR samples using D2O were adjusted to pH 10 using sodium carbonate unless otherwise mentioned. The pH meter was calibrated at three different pH (4, 7, and 10). 1-D NMR spectra processing was performed with MestReNova 9.0.0 and 11.0.2. Preparative HPLC was performed using a Varian ProStar or Shimadzu Prominence 28 equipped with a Shimadzu SPD-20A UV detector (0.5 mm path length) with detection at 280 nm for ortho-nitrobenzyl derivative compounds and 260 nm for dNTP analogues and others. Strong Anion Exchange (SAX) HPLC was performed using, a Macherey Nagel 21.4 mm ´ 250 mm SP15/25 Nucleogel column. RP-C18 HPLC was performed using a Phenomenex Luna 5 μm C18(2) 100A 21.2 mm ´ 250 mm column. Mass spectrometry were performed on a Finnigan LCQ Deca XP Max mass spectrometer equipped with an ESI source in the negative ion mode. Compound calculated MS values were obtained using iMass 1.3. Compound IUPAC names were assigned using MarvinSketch 6.1.5. The molar yields of the final products were determined by UV absorbance using the extinction coefficient of dCTP at pH 7.0 (phosphate buffer) at 260 nm (ε cytidine = 9300 M -1 cm -1 ). The slow and fast terms are related to the elution order of the chiral synthons by RP-C18 HPLC. 2.1.4.2 Synthesis of (Fluoro(hydroxy((2-nitrobenzyl)oxy)phosphoryl)methyl) phosphonic acid 1 To a pre-warmed solution of [fluoro(phosphono)methyl]phosphonic acid (1.22 g, 6.3 mmol) in dry DMF (315 mL) at 125 ºC, DIEA (1.2 mL, 6.9 mmol) was added dropwise and the mixture stirred for 15 min. A solution of 2-nitrobenzylbromide (1.49 g, 6.9 mmol) in DMF (35 mL) was then slowly added (over 15 min) through the condenser and the reaction mixture was kept at 125 ºC for 24 h. Progress of the reaction was monitored by HO P O HO P F O O OH NO 2 HO P O HO P F OH O OH 1 DIEA, 2-NO 2 BnBr DMF, 125 º C, 24 hrs 69% 29 31 P NMR and MS. After completion, the resulting mixture was diluted with ethyl acetate at room temperature and evaporated to dryness. The residue was purified by preparative RP-C18, isocratic mode, using 0.1 M triethylammonium carbonate 15% acetonitrile pH 7.5 buffer (8.0 mL/min, 280 nm). The desired fraction was evaporated to dryness and the desired product was obtained as triethylammonium salt (69%). 1 H NMR (500 MHz, D2O, pH 10.3) δ 8.18 (dd, J = 8.2, 1.3 Hz, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.87 – 7.78 (m, 1H), 7.56 (t, J = 7.8 Hz, 1H), 5.45 (d, J = 7.8 Hz, 2H), 4.95 – 4.85 (m, 1H). 19 F NMR (376 MHz, D2O, pH 10.0) δ -217.91 (ddd, J = 63.9, 56.4, 48.9 Hz). 31 P NMR (162 MHz, D2O, pH 10.0) δ 14.19 (dd, J = 63.2, 11.3 Hz, 1P), 7.30 (dd, J = 55.1, 11.3 Hz, 1P). MS (ESI) m/z: Calcd for [M-H] - = C8H9FNO8P2 – 328.0; found: 328.1. 2.1.4.3 Synthesis of [(S)-Fluoro[hydroxy({[(1R)-1-phenylpropyl]amino})phosphor- ryl]methyl][(2-nitrophenyl)methoxy]phosphinic Acid and [(R)-Fluoro[hydroxy ({[(1R)-1-phenylpropyl]amino})phosphoryl]methyl][(2-nitrophenyl)methoxy]- phosphinic Acid - 2 To a solution of bisphosphonate 1 (500 mg, 1.6 mmol) in dry DMF (50 mL), under nitrogen atmosphere, (R)-1-phenylpropan-1-amine (863 μL, 6.0 mmol), 2, 2’- dithiodipyridine (441 mg, 2.0 mmol) and triphenylphosphine (525 mg, 2.0 mmol) were added, following this specific order, and reacted for 3 h. After completion, the resulting HO P O HO P Cl O O OH NO 2 N H P (S) O HO P Cl O O OH NO 2 (R) or (R) 1 chiral amine, (PyS) 2 PPh 3 , DMF RT, 12 hrs 10 % (HPLC fast) 8 % (HPLC slow) 2 (R) or (S) 30 mixture was diluted with water and solvent was removed under vacuum. The residue was purified on preparative RP-C18 (8.0 mL/min, 280 nm) in isocratic mode with 0.1 M triethylammonium bicarbonate 27% acetonitrile pH 8.5 buffer (tr fast isomer = 25.4 min, tr slow isomer = 27.7 min, Figure 2.2). Total [isomer ratio 1:1] yield 56%. (R)-isomer: 1 H NMR (500 MHz, CD3OD) δ 7.84 – 7.75 (m, 2H), 7.43 (ddd, J = 16.0, 10.5, 5.6 Hz, 1H), 7.26 – 7.17 (m, 1H), 7.05 (dd, J = 12.2, 7.4 Hz, 2H), 7.00 – 6.91 (m, 2H), 6.86 (dt, J = 16.4, 5.6 Hz, 1H), 5.13 (dtt, J = 21.8, 15.9, 7.9 Hz, 2H), 4.47 – 4.24 (m, 1H), 3.99 (dq, J = 16.1, 7.2, 5.4 Hz, 1H), 1.61 – 1.38 (m, 2H), 0.59 – 0.47 (m, 3H). 19 F NMR (470 MHz, CD3OD) δ -218.87 (br). 31 P NMR (202 MHz, CD3OD) δ 10.50 (dd, J = 60.6, 10.1 Hz), 9.70 (dd, J = 60.6, 12.1 Hz), MS (ESI) m/z: [M-H] - Calcd for C17H20FN2O7P2 – 445.1; found 445.2. (S)-isomer: 1 H NMR (400 MHz, CD3OD) δ 8.06 (ddd, J = 8.5, 7.7, 1.3 Hz, 1H), 7.71 (td, J = 7.6, 1.3 Hz, 1H), 7.68 – 7.60 (m, 1H), 7.49 (dd, J = 1.5, 0.8 Hz, 1H), 7.39 – 7.33 (m, 2H), 7.24 (dd, J = 8.4, 6.9 Hz, 2H), 7.16 – 7.09 (m, 1H), 5.40 (t, J = 6.6 Hz, 2H), 4.59 (dt, J = 47.0, 11.4 Hz, 1H), 4.27 (q, J = 7.3 Hz, 1H), 1.91 – 1.64 (m, 2H), 0.81 (t, J = 7.4 Hz, 3H). 19 F NMR (376 MHz, CD3OD) δ -218.67 (ddd, J = 60.2, 52.6, 45.1 Hz). 31 P NMR (162 MHz, CD3OD) δ 10.50 (dd, J = 61.5, 11.3 Hz, 1P), 9.75 (dd, J = 58.3, 13.0 Hz, 1P). MS (ESI) m/z: Calcd for [M-H] - = C17H20FN2O7P2 – 445.1; found 445.3. 2.1.4.4 Synthesis of [(S)-Fluoro({hydroxy[(2-nitrophenyl)methoxy]phosphoryl}) methyl]phosphonic Acid and [(R)-Fluoro({hydroxy[(2-nitrophenyl)methoxy]- phosphoryl}) methyl]phosphonic Acid – 3 31 50 mg of each individual diastereomers (R)- and (S)-2 was dissolved in 2 mL of HCl [1M] and stirred for 3 hours at room temperature. The reaction was monitored by MS and after completion, the mixture was concentrated under vacuum. Residual HCl was co- evaporated several times with a mixture of water and methanol. Purification was performed on a pipet column of DOWEX H + using a mixture of methanol/water (1:1) as eluent and allowing the filtration to proceed by gravity only. The bisphosphonic acids (R)- and (S)-3 were obtained in solid form (quantitative yield). (R)-isomer: 1 H NMR (400 MHz, CD3OD) δ 8.15 (dd, J = 8.2, 1.1 Hz, 1H), 7.94 (d, J = 7.8 Hz, 1H), 7.80 – 7.73 (m, 1H), 7.61 – 7.53 (m, 1H), 5.59 (d, J = 7.2 Hz, 2H), 5.15 (dt, J = 45.6, 13.4 Hz, 1H). 19 F NMR (376 MHz, CD3OD) δ -227.65 (ddd, J = 63.9, 54.5, 45.1 Hz). 31 P NMR (162 MHz, D2O, pH 10.0) δ 11.10 (dd, J = 64.8, 14.6 Hz, 1P), 9.05 (dd, J = 63.2, 14.6 Hz, 1P). MS (ESI) m/z: [M-H] – Calcd for C8H9FNO8P2 – 328.0; found 328.1. (S)-isomer: 1 H NMR (500 MHz, D2O, pH 10.0) δ 7.65 – 7.57 (m, 1H), 7.42 – 7.33 (m, 1H), 7.26 (d, J = 7.8 Hz, 1H), 7.08 – 6.97 (m, 1H), 4.94 – 4.82 (m, 2H), 4.39 (dt, J = 45.3, 12.6 Hz, 1H). 19 F NMR (470 MHz, D2O, pH 10.0) δ -221.96 (ddd, J = 61.1, 51.7, 42.3 Hz). 31 P NMR (202 MHz, D2O, pH 10.0) δ 10.17 (dbr, J = 60.6 Hz, 1P), 8.89 (dbr, J = 62.6 Hz, 1P). MS (ESI) m/z: Calcd for [M-H] – = C8H9FNO8P2 – 328.0; found 328.2. N H P (S) O HO P F O O OH NO 2 (R) or (R) HO P (S) O HO P F O O OH NO 2 or (R) HCl [1M] RT, 3 hrs qt. 2 (R) or (S) 3 (R) or (S) 32 2.1.4.5 Synthesis of [(R)-{[({[(2R,5R)-5-(4-Amino-2-oxo-1,2-dihydropyrimidin-1- yl)-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)oxy][fluoro({hydroxy[(2- nitrophenyl)methoxy]phosphoryl})methyl]phosphinic Acid and [(S)-{[({[(2R,5R)-5- (4-Amino-2-oxo-1,2-dihydropyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy}(hydro- xy)phosphoryl)oxy][fluoro({hydroxy[(2-nitrophenyl)methoxy]phosphoryl})methyl] phosphinic Acid – 4 To a solution of individual bisphosphonic acids (R)- and (S)-3 (25.0 mg, 0.07 mmol, 1.0 equiv) in a mixture of 2 mL of EtOH/H2O (1:1) Bu3N was added to adjust the pH to 2.5-3.0 and the mixture stirred for 15 min, forming (R)- and (S)-3 in salt form. Solvents were removed under vacuum and residual solvents were co-evaporated 3x with dry DMF. In parallel, the previously dried 2’-deoxycytidine 5’-phosphoromorpholidate (53.0 mg, 0.14 mmol, 2.0 equiv) was dissolved in dry DMSO 1 mL and added to the dried tri-n-butyl ammonium salt of the bisphosphonic acid. The solution was stirred for 7 days at room temperature and its progress controlled by 31 P NMR. Once the reaction was done, the crude material was purified by preparative SAX HPLC (8.0 mL/min, 280 nm) with a gradient mode of A/ H2O and B/ 0.5 M triethylammonium bicarbonate pH 7.5 buffer: 0-10 min A/ 100%, 10-16 min A/ 45%-B/ 55%, 16-25 min B/ 100%. 15 mg of HPLC-fast and 17 mg of the HPLC-slow isomers was obtained. The products were not characterized, and after HO P (S) O HO P F O O OH NO 2 or (R) O P (S) HO P O OH O F O P HO O O OH O N N O NH 2 NO 2 or (R) dCMP-morpholidate DMSO, RT, 7 da 22% (HPLC fast) 25% (HPLC slow) 3 (R) or (S) 4 (R) or (S) 33 complete solvent evaporation and dryness, (R)- and (S)-4 were immediately used in the next step. 2.1.4.6 Synthesis of [(R)-{[({[(2R,5R)-5-(4-Amino-2-oxo-1,2-dihydropyrimidin-1- yl)-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)oxy](hydroxy)phosphoryl} (fluo-ro)methyl]phosphonic Acid and [(S)-{[({[(2R,5R)-5-(4-Amino-2-oxo-1,2- dihydro-pyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)oxy]- (hydroxy)-phosphoryl}(fluoro)methyl]phosphonic Acid – (R)-CHF dCTP and (S)- CHF dCTP The HPLC fast (15.0 mg, 0.024 mmol) or HPLC slow (17.0 mg, 0.027 mmol) triphosphate analogues were dissolved in 1 mL of water, placed in a quartz cuvette, and irradiated at 365 nm for 2 days. The progress of the reaction was monitored by 31 P NMR. The resulting reddish-brown crude material was purified by preparative RP-C18 Lumina Phenomenex column in isocratic mode (8.0 mL/min, 280 nm), 0.1 N triethylammonium bicarbonate 4% acetonitrile pH 7.3 buffer (tr product 12 minutes). A second purification was carried out using analytical SAX column in gradient mode (1.0 mL/min, 280 nm), with A/ H2O and B/ 0.3 N triethylammonium bicarbonate acetonitrile 4% pH 7.5 buffer. 0 to 20 minutes A/ 100%, 20 to 40 minutes B/ 100% (tr fast 16.27 minutes, tr slow 18.20 minutes). (S)- and (R)-CHF dCTP O P (S) HO P O OH O F O P HO O O OH O N N O NH 2 NO 2 O P (S) HO P O OH OH F O P HO O O OH O N N O NH 2 or (R) or (R) 4a, 4b hv 365 nm water, 2 da 9% (HPLC fast) 35% (HPLC slow) 34 Solvents were removed under vacuum and the concentration of the sample was evaluated by UV. HPLC fast isomer: 1.0 mg (9%) and ‘HPLC slow’ 4.7 mg (35%). HPLC fast Isomer: 1 H NMR (400 MHz, D2O, pH 10.0) δ 7.84 (d, J = 7.6 Hz, 1H), 6.20 (t, J = 6.6 Hz, 1H), 6.01 (d, J = 7.5 Hz, 1H), 3.13 (q, J = 7.2 Hz, 1H), 2.29 (ddd, J = 14.0, 6.3, 3.8 Hz, 1H), 2.18 (dt, J = 13.8, 6.7 Hz, 1H). 19 F NMR (564 MHz, D2O, pH 10.0) δ -216.75 (dd, J = 55.8, 53.8 Hz). 31 P NMR (243 MHz, D2O, pH 10.0) δ 7.02 (dd, J = 55.5, 15.0 Hz), 4.79 (ddd, J = 65.5, 28.6, 14.6 Hz), -10.98 (d, J = 28.6 Hz). MS (ESI) m/z: [M-H] - calcd for C10H16FN3O12P3 - 482.2, found 482.4. HPLC slow isomer: 1 H NMR (400 MHz, D2O, pH 10.0) δ 7.82 (d, J = 7.5 Hz, 1H), 6.19 (t, J = 6.7 Hz, 1H), 6.00 (d, J = 7.5 Hz, 1H), 4.74 (t, J = 12.4 Hz, 0H), 4.48 (dt, J = 6.5, 3.3 Hz, 1H), 2.28 (ddd, J = 14.0, 6.3, 3.8 Hz, 1H), 2.17 (dt, J = 13.7, 6.7 Hz, 1H). 19 F NMR (564 MHz, D2O, pH 10.0) δ -216.93 (dd, J = 56.4, 67.7 Hz). 31 P NMR (243 MHz, D2O, pH 10.0) δ 7.11 (dd, J = 55.3, 14.7 Hz), 4.89 (ddd, J = 65.2, 27.7, 14.3 Hz), -10.89 (d, J = 28.2 Hz). MS (ESI) m/z: [M-H] - calcd for C10H16FN3O12P3 - 482.2, found 482.4. 35 2.2 - Part II: Pamoic Acid Derivatives as Small Molecule Inhibitors 2.2.1 Introduction DNA pole β possesses two different subdomains with two distinct enzymatic activities (Figure 2.8A): a 31-kDa C-terminal polymerase domain responsible for the synthesis of DNA, 12 and a 8-kDa N-terminal lyase domain involved in the binding of single-stranded DNA, 5’- deoxyribose phosphate recognition and removal in the nicked DNA strand (dRP activity). 12-14 Targeting the lyase domain of DNA pol β would confer selectivity since only certain polymerases actually possess the lyase domain, such DNA pol g, i and l. In addition, the lyase domain also encompasses a DNA specific binding site, and if blocked, the DNA substrate will not be able to bind, leading both the lyase and polymerase domains to lose their functionality. With all this considered, our goal is to inhibit the lyase domain of the DNA polymerase β. This particular domain is constituted by four antiparallel alpha helices, with both the DNA binding side and the lyase activity presented between helix 2 and helix 4. Some important residues to consider, for potential interactions with substrates, are the Tyr 39 right in the center of the pocket, and the flexible lysines (Lys 35, 68, 72 and 84) (Figure 2.8B). 15 The Helix-hairpin-Helix (HhH) motif is located on residues 55-61 and is responsible for DNA binding. 15-17 36 Figure 2.8- DNA polymerase β. A. Lyase (orange) and polymerase (blue) domains of DNA pol β. B. Overview of the lyase domain and its important residues (PDB structure 3JPT). 5 2.2.2 Results and Discussion 2.2.2.1 Choosing the Lead Compound Previous studies reported that pamoic acid (PA) is able to inhibit both the lyase and polymerase activities of DNA pol β and it shows a K D for the lyase domain of 9 ± 3 μM. 18 In addition PA is capable of sensitizing mouse fibroblasts (wild-type) to methyl methanesulfonate, a DNA-methylating agent 18 and therefore decreases BER activity. Beginning from these results for PA, our goal was to find a pamoic acid derivative that would more potently inhibit the lyase domain. We speculated that the substitution of one of the carboxylic groups for a phosphonate group would create a stronger bond between the ligand and the lyase based on previous docking studies 19 which have revealed 37 that one of the COO - groups of the PA moiety resembles the phosphoryl on the DNA substrate and establishes hydrogen bonds with the lysines and Tyr39. 19 In addition, in order to increase the possibility of getting a crystal structure of the derivative and the enzyme, we concluded that restricting the conformation of the aromatic groups on the methylene bridge of the PA derivative would be a good approach. With all these aspects in mind we envisioned three generations (Gen 1, 2 and 3) of compounds that would be interesting to synthesize and check their inhibitory potential toward the lyase domain (Figure 2.9B). Figure 2.9- A. Structure of PA and possible modifications. B. Generations of compounds as potential inhibitors. 38 2.2.2.2 Synthesis of PA Derivatives Starting with Generation 1, we focused on the synthesis of the bisphosphonate 4 (Scheme 2.2). Naphtol was reacted with diethyl phosphite in carbon tetrachloride 20 and triethylamine yielding 92% of diethyl phosphate 1, which was then reacted with LDA generated in situ - from diisoproprylamine and n-butyl lithium in THF 20,21 - for a total of 4.5 h forming both phosphonate 2 and its isomer 3 in a 1.7:1.0 ratio. In order to simplify the synthetic method, this mixture was not purified because in the fact that isomer 3 has no reactivity in the following step due to the difference of the intermediate formed during the electrophilic aromatic substitution (Scheme 2.3). In the case of compound 2, the carbocation formed can be stabilized by resonance without interfering with the second aromatic group. On the other hand, the carbocation formed in compound 3 can only be stabilized by resonance if the aromaticity on the second ring is affected, which is a disfavored process (Scheme 2.3). Compound 4 was synthesized by reacting 2 and 3 with an aqueous solution of 37% formaldehyde at 90 ºC. In order to synthesize the mono and di-bisphosphonate salts, compound 4 was reacted with pyridine at 115 ºC. 23 The reaction was monitored by 31 P NMR, and after 65 min, 68% of the mono dealkylated compound 5 was obtained. However, if the reaction continues for 7.5 hours, compound 6 is obtained instead, in 95% yield based on 31 P NMR. Unfortunately, all attempts to purify these compounds through HPLC failed due to the fact that these hydrophobic compounds are strongly retained in the C-18 column. Therefore compounds 5 and 6 were never isolated. 39 Scheme 2.2- Synthesis of di-phosphonate compound 5 and 6. Conditions: a. diethyl phosphite, CCl 4 , NEt 3 , room temperature, 12 h; b. LDA in situ (DIPA, n-BuLi), THF, -78 ºC to room temperature, 4.5 h; c. Formaldehyde 37% sol., H 2 O, 90 ºC, 4.5 h; d. pyridine, 115 ºC, 65 min for 5 or 7.5 h for 6. Scheme 2.3- Naphthalene reactivity, adapted for phosphonate derivatives, showing different behaviors for isomer 2 and 3. Regarding Generation 2 types of compounds, we aimed to synthesize the mono-ethyl phosphate derivative 8. With that in mind, the isomer mixture of 2 and 3 were reacted with OH a 1 92% O P O OEt OEt b 63% OH P O OEt OEt 2 OH OH P O OEt OEt 4 OH P O OEt OEt 3 c 5% P OEt O OEt OH OH P O O OEt 5 P OEt O OEt OH OH P O O OEt 6 P O O OEt d X 65 min 7.5 hrs 2 OH P O OR OR CH 2 O H + OH P O OR OR OH C1 position: The second aromatic ring is not affected. OH CH 2 P O OR OR O X 3 C3 position: For resonance to happen, the aromaticity on second ring needs to be affected. Disfavored. 40 an excess of naphthol in the presence of formaldehyde 37% solution in glacial acetic acid yielding compound 7. 24 The reaction was monitored by 31 P NMR and after 2 h a 66% of product was formed; longer reaction times did not improve the yield. In addition, the presence of the product was detected by mass spectrometry. Without further purification, derivative 7 was reacted with pyridine at 115°C for 4 hours forming the de-methylated phosphate 8. After HPLC purification the ammonium salt of compound 8 was obtained in 20% yield. Scheme 2.4- Synthesis of novel mono-ethyl phosphonate 8. Conditions: e. naphthol (exc), formaldehyde 37% solution, gl. acetic acid, reflux, 2 h; d. (same as Scheme 2.2) pyridine, 115 ºC, 4 h. With reference to compounds of Generation 3, our first synthetic target was PA-Ph compound in order to test how blocking the methylene bridge would affect the inhibitory effect of the PA derivatives. We followed a procedure developed by Brass in 1932 25 for this exact compound. 2-Hydroxynaphthoic acid was reacted with benzaldehyde in acetic acid and 37% HCl solution at 70 °C for 2 hours (Scheme 2.5, method i). According to the published result, PA-Ph was formed with 50% yield, however we obtained 2 mg of impure product. We found ourselves with a non-reproducible method and taking into account that in 1932 NMR analysis was not available, we decided to try a more recent approach. We OH P O OEt OEt 2 OH OH P O OEt OEt 7 OH P O OEt OEt 3 e 66% d 20% OH OH P O O OEt 8 NH 4 41 turned to a method developed by Baghel et al 24 for the synthesis of PA-Ph with 80% yield reported. Therefore, we refluxed naphthoic acid with benzaldehyde 37% solution in the presence of H 2 SO 4 (Scheme 2.5, method ii). However, in our case the desired product was not formed. On another attempt, the reaction condition described above was used under microwave radiation for 30 minutes but again, no reaction occurred (Scheme 2.5, method iii). After these three methods under acidic conditions failed we decided on a different approach. Alizadeh et al 26 reported a simple method where naphthol reacted with several different aromatic aldehydes in the presence of phosphomoybdic acid hydrate (HPA) as a catalyst, yielded similar compounds. In fact, it was stated that the product was obtained with 51% yield when using benzaldehyde. With that in mind, we refluxed naphothoic acid with benzaldehyde in the presence of HPA in dichloromethane for 1 hour. Unfortunately, the desired product was still not formed (Scheme 2.5, method d). Scheme 2.5- Methods used for the synthesis of PA-Ph. Methods: i. benzaldehyde, gl. acetic acid, HCl 37% sol., 70 °C, 5 h 25 ; ii. benzaldehyde 37% sol., gl. acetic acid, H 2 SO 4 (cat.), reflux, 5 h 24 ; iii. benzaldehyde, acetic acid, HCl 37% sol., 70 °C, μW, 30 min; iv. H 3 [P(Mo 3 O 10 ) 4 ]nH 2 O (HPA), DCM, reflux, 1 hr. 26 COOH OH COOH OH OH COOH X Methods i, ii, iii and iv PA-Ph 42 In the HPA method, 26 the authors obtained moderate to excellent yields using naphthol as starting material. It is evident that utilizing a starting material with a carboxylic group contributes to the lack of product formation. Our previous derivatives bearing carboxylic groups have shown poor solubility in common organic solvents (e.g. methanol and chloroform). In addition, when synthesizing PA derivatives, a large amount of PA itself is formed as a side-product, which is extremely difficult to purify from the desired and low- yield product. Therefore, the synthesis of the Generation 3 compound was, unfortunately, not achieved. 2.2.2.3 Docking Experiments with PA Derivatives To understand the challenges that the -COOH groups impose on the synthesis of the desired derivatives, we decided to further study how the –COOH group interacts with the active site of the DNA pol β enzyme. A series of docking experiments were conducted (Appendix B, Figures B39-43). And one of the best hits found was compound 9 (Figure 2.10 and Figure B42). This compound shows interaction with the lyase domain with an energy score of -9.5 kcal/mol compared to that of -8.0 kcal/mol for PA. Analyzing the involvement of compound 9 with the active site of the enzyme, we see good interaction with all four flexible lysines, Tyr 39 and Glu 75 (Figure 2.10A). Figure 2.10- Structure of compound 9. OH OH P O OR O O OH H 2 N OH O 9 43 Figure 2.11- A. Interaction of compound 9 with the lyase pocket. B. Interaction of –COOH of compound 9 with threonine 67. In addition, it can also be seen that the carboxylic group interacts with Thr 67, possibly through a H-bond interaction (Figure 2.10B). This means that it is likely that the carboxylic group is important for the interaction between the ligand and the pocket. To confirm this hypothesis, we decided to run a second round of docking experiment using molecules that do not possess the –COOH (Table 2.1). Both docking analyses were performed using AutoDock Vina, 27 the ligands were prepared using Spartan 08 software, the files prepared with AutoDockTools-1.5.6 28 and the final results analyzed using PyMOL. 29 The PDB structure of pol β used was 3JPT 5 , lysines 35, 68, 72 and 84 were made flexible to facilitate the entrance of the ligand in the pocket and the exhaustiveness parameter used was 256 count, unless otherwise stated. The energy scores obtained from the second docking experiment are shown in Table 1 (entries 1-10). 44 Table 2.2- Energy scores in kcal/mol obtained after docking with AutoDock Vina. *The exhaustiveness value used was 128 rather than 256 count, due to interest of time. The energy scores of compounds 6 and 8, –9.1 and –9.3 kcal/mol, respectively, were encouraging, however when manually analyzing the specific interactions of these molecules with important amino acids in the active pocket, the interactions were not as impressive. Docking experiments with naphthalene derivatives were also made and the results are shown in Table 1 (entries 11-16). Docking techniques are of importance in order OH OH P O O O N N N N H2N O H OH H H H H Structure Entry 1 2 3 4 Vina Score - 8.9 - 8.9* - 9.0* - 9.1* Structure Entry 5 6 7 8 Vina Score - 7.7 - 9.3* - 8.9 - 8.4* Structure Entry 9 10 11 12 Vina Score - 8.9* - 8.8* - 8.1 - 8.7 Structure Entry 13 14 15 16 Vina Score - 6.8 - 7.6 - 7.9 - 8.1 OH OH P O O O H3N O O OH OH P O O O OH OH P O O O COO OH OH P O O O F OH OH P O O O OH OH P O O O N O H OH H H H H NH O O OH OH P O O O O N F OH OH P O O O O N COOH P O O O F O OH P O O O N O H OH H H H H NH O O OH P O O O O H OH H H H H N N N N H2N OH P O O O O H OH H H H H N N N N H 2 N OH P O O O O H OH H H H H N NH O O OH P O O O H 3 N COO OH P O O O COOH 45 to have a better understanding of the enzyme’s active site and to predict structure-activity relationship outcomes. However, a crystal structure of the enzyme was used combined with a dGTP-DNA ternary complexes 5 and therefore the shape of the pocket may be to be different from the one adapted to interact with pamoic acid derivatives. 2.2.2.4 DNA Pol β dRP-Lyase Activity of Compound 8 The biological assays to measure the DNA pol β dRP-lyase activity were performed in the laboratory of Dr. Samuel Wilson at the National Institute of Environmental Health Sciences (NIEHS). The dRP lyase assay was conducted using 50 mM Hepes, pH 7.4, 20 mM KCl, 0.5 mM EDTA, and 2 mM DTT, 10 mM MgCl2, pamoic acid inhibitor at three different concentrations (5, 50 or 500 μM), and 10 nM pol-β, giving a total of 10 μl of reaction mixture. Uracil-DNA glycosylase (UDG) pre-treated DNA (the single-nucleotide gapped DNA including 5’-dRP) was added to the reaction mixture to a final concentration of 100 nM, and incubated at 37°C for 15 min. In order to stabilize the reaction products, 100 mM of NaBH4 was added to the mixture. It was observed that compound 8 only shows inhibition at the concentrations of 500 μM, and no significant effect was observed at 5 nor 50 μM. This result brings us to the conclusion that in fact the carboxylic group in the molecule possesses a critical role in inhibition of the lyase activity. 46 Figure 2.12- DNA pol β dRP-lyase activity assay for compound 8. 2.2.3 Conclusion and Future directions Previous studies have shown that pamoic acid is able to inhibit the lyase domain of pol β, 15,16,18,19 therefore we aimed to synthesize modified pamoic acid derivatives which possess a phosphorous moiety and test their inhibitory effect. Our initial strategy was to synthesize three generations of compounds: Generation 1 included 2 phosphonate moieties; Generation 2 only possessed one phosphonate moiety; and Generation 3 carried one phosphonate moiety, one carboxylic group and a functionalized methylene bridge between the two ring systems (Figure 2.9). Both Gen 1 and Gen 3 strategies turned out to be a unsuccessful because compounds 5 and 6 of Gen 1 could not be isolated and compound PA-Ph (Gen 3) could not be synthesized at all. Compound 8 from Gen 2 was successfully synthesized and characterized and ehas shown to inhibit the lyase domain of DNA pol β at a concentration of 500 μM. MVDPIP36! MVDPIP169! MVDPIP182! MVDPIP183! CAPIP127! DNA!alone! Polβ!alone! PA! Substrate! Product! 1!!!!!!!2!!!!!!!!3!!!!!!!4!!!!!!!!5!!!!!!!!6!!!!!!!!7!!!!!!!!8!!!!!!!9!!!!!!!!10!!!!!!!11!!!!!12!!!!!!13!!!!!!!14!!!!!!!15!!!!!16!!!!!!17!!!!!18!!!!!!!19!!!!!20!!!!!21!!!!!22!!!!!23!!!!! Lanes! 1:!No!enzyme!control! 2:!Polβ!alone!control! 3:!Pamoic!acid!(500!μM)!plus!polβ! ! 4:!MVDPIP36!alone!(500! μM)!! 5P7:!MVDPIP36!(5,!50,!500! μM)!plus!polβ! ! 8:!MVDPIP169!alone!(500! μM)!! 9P11:!MVDPIP169!(5,!50,!500! μM)!plus!polβ! ! 12:!MVDPIP182!alone!(500! μM)!! 13P15:!MVDPIP182!(5,!50,!500! μM)!plus!polβ! ! ! ! ! 16:!MVDPIP183!alone!(500! μM)!! 17P19:!MVDPIP183!(5,!50,!500! μM)!plus!polβ! ! 20:!CAPIP127!alone!(500! μM)!! 21P23:!CAPIP127!(5,!50,!500! μM)!plus!polβ! ! ! MVDPIP36! MVDPIP169! MVDPIP182! MVDPIP183! CAPIP127! DNA!alone! Polβ!alone! PA! Substrate! Product! 1!!!!!!!2!!!!!!!!3!!!!!!!4!!!!!!!!5!!!!!!!!6!!!!!!!!7!!!!!!!!8!!!!!!!9!!!!!!!!10!!!!!!!11!!!!!12!!!!!!13!!!!!!!14!!!!!!!15!!!!!16!!!!!!17!!!!!18!!!!!!!19!!!!!20!!!!!21!!!!!22!!!!!23!!!!! Lanes! 1:!No!enzyme!control! 2:!Polβ!alone!control! 3:!Pamoic!acid!(500!μM)!plus!polβ! ! 4:!MVDPIP36!alone!(500! μM)!! 5P7:!MVDPIP36!(5,!50,!500! μM)!plus!polβ! ! 8:!MVDPIP169!alone!(500! μM)!! 9P11:!MVDPIP169!(5,!50,!500! μM)!plus!polβ! ! 12:!MVDPIP182!alone!(500! μM)!! 13P15:!MVDPIP182!(5,!50,!500! μM)!plus!polβ! ! ! ! ! 16:!MVDPIP183!alone!(500! μM)!! 17P19:!MVDPIP183!(5,!50,!500! μM)!plus!polβ! ! 20:!CAPIP127!alone!(500! μM)!! 21P23:!CAPIP127!(5,!50,!500! μM)!plus!polβ! ! ! DNA + 8 500 μM 5 50 500 μM DNA+Pol-β & 8 + Pol-β DNA+ Pol-b & PA 500 μM DNA control 47 In order to establish a reliable SAR study, we need to synthesize a range of pamoic acid derivatives and compare their DNA pol β dRP-lyase activity. As of now, we understand that both rings in the pamoic acid derivative are needed to possess a carboxylic and/or phosphonate group due to the fact that compound 8, which has one of the ring systems free of –COOH and –P(O)(OR) 2 , was shown not to be a potent inhibitor. In addition, docking studies have predicted a good interaction of the –COOH with threonine 67 of the enzyme. Future work for this project could involve the synthesis of pamoic acid derivatives for which Autodock Vina predicted the best scores, such as entries 4 and 6 (Table 2.1) in order to validate establish a correlation between the docking score and the actual inhibitory effect shown on the pol-β dRP-lyase assay. 2.2.4 Experimental Procedure 2.2.4.1 Materials and Methods The starting materials naphthol and 2-hydroxy-3-naphthoic acid were purchased from Alfa Aesar and Acros Organics, respectively. All the other reagents and solvents were obtained from other commercial sources and use without further purification. THF and liquid amines were distilled before used in the reactions. 1 H and 31 P NMR spectra were obtained on a Varian NMR-500 and NMR-600 NMR spectrometers. Multiplicities are reported as singlet (s), doublet (d), triplet (t), quintet (quint.), unresolved multiplet (m), doublet of doublets (dd), triplet of doublets (td), doublet of triplets (dt), double of quartets (dq) and broad signal (br). All chemical shifts are given on the δ scale in parts per million (ppm), relative to internal CD 3 OD (δ 3.34 1H NMR), CDCl 3 (δ 7.26 1H NMR) or D 2 O (δ 48 4.79 1H NMR). 31 P NMR spectra were proton-decoupled unless otherwise stated. The coupling constants (J values) for 1 H and 31 P spectra are given in Hz. Chromatograph purification were performed in a CombiFlashRf Lumen with pre-packed silica gel columns. Preparative HPLC was performed using a Dynamax Model SD-200 equipped with a Dynamax Rainin absorbance detector Model UV-D II with wavelength detection at 240 or 250 nm. Mass spectrometry (MS) was obtained on a Finnigan LCQ Deca XP Max mass spectrometer equipped with an ESI method in the negative ion mode. The IUPAC names of the compounds presented were assigned using Marvin Sketch 17.28.0. 2.2.4.2 Synthesis of Diethyl [(naphthalen-2-yl)methyl]phosphonate 1 In a round bottom flask, 2.9 g (20 mmol) of naphthol were dissolved in 8.5 mL of tretrachloride carbon and in 2.14 mL (16 mmol) of diethyl phosphite. An ice bath was added and while stirring, 2.90 mL (21 mmol) of triethylamine, previously distilled and dried, was slowly added to the mixture. Once the addition of triethylamine was over, the reaction was left at room temperature for 12 h. Subsequently, 5 mL of chloroform were added to the mixture and an extraction with DI water was made. The organic layer was washed with 1 N HCl, extracted, the organic layer collected and washed with 5 mL of 1 N NaOH (4x). The organic layer was then washed with DI water, dried over K 2 CO 3 and evaporated until dryness. 4.3 g of a brown solid was obtained with 92 % yield. 1 H NMR OH diethyl phosphite CCl 4 , NEt 3 RT, 12 hrs 1 92% O P O OEt OEt 49 (500 MHz, CDCl 3 ) δ 7.85 – 7.79 (m, 3H), 7.70 (s, 1H), 7.52 – 7.42 (m, 2H), 7.38 (dd, J = 8.9, 2.4 Hz, 1H), 4.33 – 4.20 (m, 4H), 1.38 (t, J = 7.1 Hz, 6H). 31 P NMR (202 MHz, CDCl 3 ) δ 6.21 (s, 1P). 2.2.4.3 Synthesis of Diethyl (3-hydroxynaphthalen-2-yl)phosphonate 2 Distilled diisopropylamine (2.4 mL) and distilled and dried THF (7.0 mL) were added into a warm flask flushed with nitrogen gas, the temperature was then decreased to -78 °C. Carefully 10.6 mL of n-BuLi (C= 1.6 M in hexane) was slowly added to the solvent mixture (note: use a glass syringe when handling n-butyl lithium for safety reasons). The mixture was left for 30 min at -78 °C. The remaining of n-BuLi in the syringe was neutralized with isopropanol. Afterwards, a solution of phosphate 1 (2.4 g, 8.56 mmol) in THF (7.0 mL), previously distilled and dried, under nitrogen atmosphere, was added and the mixture left to react for 1 hr at the same temperature. Thereupon, the temperature was slowly increased to room temperature and continued for 3 more hours. The reaction was then poured into a mixture of saturated ammonium chloride (42 mL) and dichloromethane (51 mL), stirring in an ice bath. The organic layer was separated, extracted with DI water (20 mL), dried over Na 2 SO 4 and evaporated until dryness. 2.4 g of an oily mixture was obtained with 63% yield. This final reaction mixture contained isomers 2 and 3 in a ratio of 64% to 36% 1 O P O OEt OEt LDA in situ THF -78 to RT, 4.5 hrs 63% OH P O OEt OEt 2 OH P O OEt OEt 3 50 respectively, measured by 31 P NMR. 31 P NMR (202 MHz, CDCl 3 ) δ 25.22 (s, 1P, compound 2), 21.11 (s, 1P, compound 3). 2.2.4.5 Synthesis of (4-{[3-(Diethoxyphosphoryl)-2-hydroxynaphthalen-1- yl]methyl}-3-hydroxynaphthalen-2-yl)(ethoxy)phosphinic Acid 4 While stirring, a solution of phosphonate 2 (250 mg, 1.20 mmol) dissolved in distilled water (5 mL) was added to a solution of 37% formaldehyde (2.8 mL). The temperature was increased to 90 °C and the reaction progress monitored by 31 P NMR. After 4.5 h the reaction was stopped, and the solvents dried out. Water and sodium bicarbonate were added to the reaction flask and the mixture extracted with chloroform. The organic layer was collected and evaporated until dryness. The crude material was purified by preparative TLC using 5% acetone in chloroform as the elution solvent. The product was 5% pure by 31 P NMR, therefore it was used for the next step without further purification. 31 P NMR (202 MHz, CDCl 3 ) δ 22.17 (s, 1P). 2.2.4.6 Synthesis of Diethyl {3-Hydroxy-4-[(2-hydroxynaphthalen-1- yl)methyl]naphthalen-2-yl}phosphonate 7 OH P O OEt OEt 2 OH OH P O OEt OEt 4 OH P O OEt OEt 3 5% CH 2 O 37% sol. 90 °C, 4.5 hr P OEt O OEt 51 Naphthol (257 mg, 1.8 mmol) was dissolved in glacial acetic acid (0.7 mL). While stirring, a solution of phosphonate 2 (100 mg, 0.36 mmol) dissolved in glacial acetic acid (0.8 mL) was added to the mixture. The temperature was slightly increased to 40 °C to help the dissolution of the reactants. Afterwards, formaldehyde 37% solution (690 μL) was added and the temperature was increased to 90 °C, and the reaction progress monitored by 31 P NMR. After 2 hr the reaction was stopped, and the acetic acid evaporated under vacuum. Water and sodium bicarbonate were added to the reaction flask (pH ~ 7) and the mixture extracted with chloroform. The organic layer was collected and evaporated until dryness. The crude material was purified by ISCO chromatography with dichloromethane and methanol gradient. The product was 66% pure by 31 P NMR and MS showed the presence of the desired product, therefore it was used for the next step without further purification. 31 P NMR (202 MHz, CDCl 3 ) δ 21.81 (s, 1P). MS (ESI) m/z: Calcd for [M-H] - = C 25 H 24 O 5 P – 435.4; found: 435.1. OH P O OEt OEt 2 OH OH P O OEt OEt 7 OH P O OEt OEt 3 66% napththol (exc) CH 2 O 37% sol. gl. acetic acid 90 °C, 2 hr 52 2.2.4.7 Synthesis of Ammonium Ethyl {3-Hydroxy-4-[(2-hydroxynaphthalen-1- yl)methyl]naphthalen-2-yl}phosphonate 8 Anhydrous pyridine (12.5 mL) was added to phosphonate 7 (90 mg, 0.21 mmol). The reaction mixture was stirred, refluxed and controlled every 30 minutes by 31 P NMR. The reaction was stopped after 4 h and the solvent evaporated until dryness. The mixture was studied by LC-MS with 0.1 N ammonium acetate with 85% of methanol, pH = 5.3 and then 0.1 N ammonium acetate with 64% of methanol, pH = 5.3. Thereupon, HPLC separation was preformed using 0.1 N ammonium acetate with 75% of methanol pH = 5.3, with the product being eluted at 15.4 minutes. The solvent was evaporated and 22 mg of the desired product 8 was obtained with 20% yield. 1 H NMR (500 MHz, CD 3 OD) δ 8.27 (d, J = 8.5 Hz, 1H), 8.20 (d, J = 8.6 Hz, 1H), 8.01 (d, J = 15.1 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.59 (dd, J = 8.0, 1.5 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 7.20 – 7.07 (m, 5H), 4.83 (s, 2H), 3.86 (m, 2H), 1.16 (t, J = 7.0 Hz, 3H). 31 P NMR (202 MHz, CD 3 OD) δ 18.72 (s, 1P). MS (ESI) m/z: Calcd for [M-H] - = C 25 H 24 O 5 P – 407.4; found: 407.1. OH OH P O OEt OEt 7 OH OH P O O OEt 8 NH 4 Pyridine 115 °C, 4 hr 20% 53 2.2.4.8 Tentative Syntheses of 4-[(3-Carboxy-2-hydroxynaphthalen-1- yl)(phenyl)methyl]-3-hydroxynaphthalene-2-carboxylic Acid PA-Ph Method i: 2-Hydroxy-1-naphthoic acid (300 mg, 1.59 mmol), dissolved in glacial acetic acid (2.4 mL), and combined with benzaldehyde (100 μL, 0.98 mmol), previously distilled, HCl (300 μL) was added in portions. The mixture was left stirring for 5 h at 70°C. The yellow precipitate was filtered and washed with hot acetic acid for around 10 minutes, 3 times, and then washed with hot water. Afterwards analysis by 1 H NMR showed that no product was formed. Method ii: 2-Hydroxy-1-naphthoic acid (300 mg, 1.59 mmol), dissolved in glacial acetic acid (2.4 mL), was combined with benzaldehyde (0.6 mL, 5.9 mmol), previously distilled, and DI water (0.92 mL). While stirring, a catalytic amount of sulfuric acid was added (around 5 drops) and left refluxing for 5 h. The reaction was controlled by MS but the product was not identified. Method iii: In a pear shape flask, 2-hydroxy-1-naphthoic acid (309 mg, 1.64 mmol), dissolved in glacial acetic acid (2.4 mL), was combined with benzaldehyde (100 μL, 0.98 mmol), previously distilled. Subsequently, HCl (300 μL) was added and the reaction was stirred under μW radiation for 30 minutes at 70 °C. The yellow precipitate was filtered and washed with hot acetic acid for around 10 minutes, 3 times, and then washed with hot COOH OH COOH OH OH COOH X Methods i, ii, iii and iv PA-Ph 54 water. The crude was analyzed by 1 H NMR and MS and only starting material was identified. Method iv: 2-Hydroxy-1-naphthoic acid (376 mg, 2.0 mmol), dissolved in dichloromethane (1 mL), benzaldehyde (102 μL, 1.0 mmol) and phosphomoybdic acid hydrate (H 3 [P(Mo 3 O 10 ) 4 ].nH 2 O, 73 mg, 0.04 mmol) were added. The mixture was left refluxing for 1 hr. The reaction was controlled by MS and there was no evidence of the formation of the desired product. 2.3 Chapter References (1) Oertell, K.; Chamberlain, B. T.; Wu, Y.; Ferri, E.; Kashemirov, B. A.; Beard, W. A.; Wilson, S. H.; McKenna, C. E.; Goodman, M. F. Transition State in DNA Polymerase β Catalysis: Rate-Limiting Chemistry Altered by Base-Pair Configuration. Biochemistry 2014, 53 (11), 1842–1848. (2) Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Batra, V. K.; Martínek, V.; Xiang, Y.; Beard, W. A.; Pedersen, L. C.; Wilson, S. H.; McKenna, C. E.; et al. Modifying the β,γ Leaving-Group Bridging Oxygen Alters Nucleotide Incorporation Efficiency, Fidelity, and the Catalytic Mechanism of DNA Polymerase β. Biochemistry 2007, 46 (2), 461–471. (3) Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Osuna, J.; Oertell, K.; Beard, W. A.; Wilson, S. H.; Florián, J.; Warshel, A.; McKenna, C. E.; et al. DNA Polymerase β Fidelity: Halomethylene-Modified Leaving Groups in Pre-Steady-State Kinetic Analysis Reveal Differences at the Chemical Transition State. Biochemistry 2008, 47 (3), 870–879. (4) Oertell, K.; Kashemirov, B. A.; Negahbani, A.; Minard, C.; Haratipour, P.; Alnajjar, K. S.; Sweasy, J. B.; Batra, V. K.; Beard, W. A.; Wilson, S. H.; et al. Probing DNA Base-Dependent Leaving Group Kinetic Effects on the DNA Polymerase Transition State. Biochemistry 2018, 57 (26), 3925–3933. (5) Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H.; Kashemirov, B. A.; Upton, T. G.; Goodman, M. F.; McKenna, C. E. Halogenated β,γ-Methylene- and Ethylidene-DGTP-DNA Ternary Complexes with DNA Polymerase β: Structural Evidence for Stereospecific Binding of the Fluoromethylene Analogues. J. Am. Chem. Soc. 2010, 132 (22), 7617–7625. (6) McKenna, C. E.; Kashemirov, B. A.; Upton, T. G.; Batra, V. K.; Goodman, M. F.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H. (R)-β,γ-Fluoromethylene-DGTP-DNA Ternary Complex with DNA Polymerase β. J. Am. Chem. Soc. 2007, 129 (50), 15412–15413. 55 (7) Oertell, K.; Wu, Y.; Zakharova, V. M.; Kashemirov, B. A.; Shock, D. D.; Beard, W. A.; Wilson, S. H.; McKenna, C. E.; Goodman, M. F. Effect of β,γ-CHF and β,γ-CHCl DGTP Halogen Atom Stereochemistry on the Transition State of DNA Polymerase β(). Biochemistry 2012, 51 (43), 8491–8501. (8) Wu, Y.; Zakharova, V. M.; Kashemirov, B. A.; Goodman, M. F.; Batra, V. K.; Wilson, S. H.; McKenna, C. E. β,γ-CHF- and β,γ-CHCl-DGTP Diastereomers: Synthesis, Discrete 31P NMR Signatures, and Absolute Configurations of New Stereochemical Probes for DNA Polymerases. J. Am. Chem. Soc. 2012, 134 (21), 8734–8737. (9) McKenna, C. E.; Shen, P.-D. Fluorination of Methanediphosphonate Esters by Perchloryl Fluoride. Synthesis of Fluoromethanediphosphonic Acid and Difluoromethanediphosphonic Acid. J. Org. Chem. 1981, 46 (22), 4573–4576. (10) Blackburn, G. M.; Kent, D. E.; Kolkmann, F. Three New β,γ-Methylene Analogues of Adenosine Triphosphate. J. Chem. Soc. Chem. Commun. 1981, (22), 1188–1190. (11) Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H.; Kashemirov, B. A.; Upton, T. G.; Goodman, M. F.; McKenna, C. E. Halogenated β,γ-Methylene- and Ethylidene-DGTP-DNA Ternary Complexes with DNA Polymerase β: Structural Evidence for Stereospecific Binding of the Fluoromethylene Analogues. J. Am. Chem. Soc. 2010, 132 (22), 7617–7625. (12) Beard, W. A.; Prasad, R.; Wilson, S. H. Activities and Mechanism of DNA Polymerase β. In Methods in Enzymology; Academic Press, 2006; Vol. 408, pp 91– 107. (13) Prasad, R.; Beard, W. A.; Chyan, J. Y.; Maciejewski, M. W.; Mullen, G. P.; Wilson, S. H. Functional Analysis of the Amino-Terminal 8-KDa Domain of DNA Polymerase β as Revealed by Site-Directed Mutagenesis: DNA binding and 5’deoxyribose phosphate lyase activities. J. Biol. Chem. 1998, 273 (18), 11121– 11126. (14) Idriss, H. T.; Al-Assar, O.; Wilson, S. H. DNA Polymerase β. Int. J. Biochem. Cell Biol. 2002, 34 (4), 321–324. (15) Prasad, R.; Batra, V. K.; Yang, X. P.; Krahn, J. M.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H. Structural Insight into the DNA Polymerase Beta Deoxyribose Phosphate Lyase Mechanism. DNA Repair 2005, 4 (12), 1347–1357. (16) Hazan, C.; Boudsocq, F.; Gervais, V.; Saurel, O.; Ciais, M.; Cazaux, C.; Czaplicki, J.; Milon, A. Structural Insights on the Pamoic Acid and the 8 KDa Domain of DNA Polymerase Beta Complex: Towards the Design of Higher-Affinity Inhibitors. BMC Struct. Biol. 2008, 8 (1), 1-12. (17) Maciejewski, M. W.; Liu, D.; Prasad, R.; Wilson, S. H.; Mullen, G. P. Backbone Dynamics and Refined Solution Structure of the N-Terminal Domain of DNA Polymerase β. Correlation with DNA Binding and DRP Lyase Activity. J. Mol. Biol. 2000, 296 (1), 229–253. (18) Hu, H.-Y.; Horton, J. K.; Gryk, M. R.; Prasad, R.; Naron, J. M.; Sun, D.-A.; Hecht, S. M.; Wilson, S. H.; Mullen, G. P. Identification of Small Molecule Synthetic Inhibitors of DNA Polymerase β by NMR Chemical Shift Mapping. J. Biol. Chem. 2004, 279 (38), 39736–39744. 56 (19) Mustafa, D. II. Design and Synthesis of Polymerase β Lyase Domain Inhibitors, Ph.D. Thesis, University of Southern California; 2013. (20) Kenner, G. W.; Williams, N. R. A Method of Reducing Phenols to Aromatic Hydrocarbons. J. Chem. Soc. 1955, 522–525. (21) Hatano, M.; Miyamoto, T.; Ishihara, K. Enantioselective Dialkylzinc Addition to Aldehydes Catalyzed by Chiral Zn(II)-BINOLates Bearing Phosphonates and Phosphoramides in the 3,3′-Positions. Synlett 2006, 1762–1764. (22) Dhawan, B.; Redmore, D. Lithiation-Induced 1,3-Migrations of Phosphorus(IV) Groups from Heteroatom to the Naphthalene Ring. J. Org. Chem. 1991, 56, 833– 835. (23) Vepsäläinen, J.; Nupponen, H.; Pohjala, E. Bisphosphonic Compounds VIII. A Facile and Selective One-Pot Synthesis of P,P-Dialkyl (Dichloromethylene)Bisphosphonate Partial Esters. Tetrahedron Lett. 1996, 37 (20), 3533–3536. (24) Baghel, G. S.; Rao, C. P. Pamoic Acid in Forming Metallo-Organic Framework: Synthesis, Characterization and First Crystal Structure of a Dimeric Ti(IV) Complex. Polyhedron 2009, 28 (16), 3507–3514. (25) Brass, F. Berichte. 1932, 325, 1657–1658. (26) Alizadeh, A.; Khodaei, M. M.; Moradi, K. H. Green and Diasteroselective Oxidative Cyclization of Bisnaphthols to Spirans. J. Iran. Chem. Soc. 2010, 7 (2), 351–358. (27) Trott, O.; Olson, A. J. AutoDock Vina: Improving the Speed and Accuracy of Docking with a New Scoring Function, Efficient Optimization and Multithreading. J. Comput. Chem. 2010, 31 (2), 455–461. (28) Sanner, M. F. Python: A Programming Language for Software Integration and Development. J. Mol. Graphics Mod. 1999, No. 17, 57–61. (29) The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC. 57 Chapter 3. Synthesis of b,g-CXY UTP probes to study the mechanism and fidelity of RNA polymerase II 3.1 Introduction RNA polymerase II (RNA pol II) is a 550 kDa enzyme that contains around 12 subunits in eukaryotes. It is responsible for the transcription of DNA into mRNA and it is known to perform with high transcriptional efficiency and fidelity. 1–3 This unique enzyme possesses a well-organized network around the enzyme, using a crucially conserved motif called the trigger loop (TL). 4–7 When the substrate is incorporated into the enzyme, the TL is modified from an open conformation (inactive state) into a close conformation (active state), which creates the ideal structural orientation so that the incorporated substrate is stabilized and accelerates nucleotide incorporation. The TL, alongside with the bridge helix, together with the nucleoside triphosphate (NTP) substrate and the RNA-DNA hybrid chain form the nucleotide recognition network that confers a high transcriptional fidelity and pairing accuracy to the RNA pol II (Figure 3.1). 3,6–8 Figure 3.1- Nucleotide recognition network in the RNA pol II catalytic site. 4 58 In our previously published results 4 , β,γ-CH 2 ATP and UTP were utilized as probes of RNA pol II and showed a significant difference between the β,γ-methylene substitution in the non-cognate UTP/dT (~3-fold decrease in k pol ) relative to the cognate ATP/dT scaffold (~130-fold decrease in k pol ). The methylene substitution lowers the lower leaving aptitude of the bisphosphonate moiety, which slows down the rate of substrate incorporation. 4 These results also showed that the active site of RNA pol II is sensitive to different electrostatic and steric effects conferred by the CXY substituents on the leaving group moiety. The goal of this research project was to further elucidate the molecular mechanism of RNA pol II during transcription in terms of transcriptional fidelity. In order to exanimate this, we varied the β,γ-CXY substituents of the uridine triphosphate (UTP) and adenosine triphosphate (ATP) nucleotide probes to study how the different CXY electronic and steric effects influence the cleavage rate of the P α -O-P β of the triphosphate backbone by the RNA pol II (Figure 3.2). Figure 3.2- Structure of β,γ-CXY NTP. O P HO P O OH OH O P HO O O OH O Base X Y HO β γ α 59 3.2 Results and Discussion 3.2.1 Synthesis of β,γ-CXY NTP Additional NTP analogues are needed to further understand their effect on the molecular mechanism of RNA pol II. With this in mind, the di-chloro and mono-chloro bisphosphonic acids were synthesized according to the literature procedure 9,10 and coupled with the respective nucleotides (Scheme 3.1). The final products β,γ-CCl 2 UTP (8%), β,γ- CCl 2 ATP (24%), β,γ-CHCl UTP (9%) and β,γ-CHCl ATP (1%) were achieved after a dual pass of HPLC purification. Scheme 3.1- Synthesis of β,γ-CYCl U/ATP. Conditions: a. Bu 3 N, EtOH/H 2 O then NMP-morpholidate, DMSO, RT, 22 h for uracil (U) and 72 h for adenine (A). It is also important to determine the effect of the individual diastereomers and their effect on the enzyme. Therefore, (R)- and (S)-β,γ-CHCl UTP were synthesized and characterized. For the synthesis of the individual diastereomers we used the same method reported in Chapter 2, where (R)-1-phenylpropan-1-amine was used as the chiral auxiliary and a photo-cleavable moiety was attached to the bisphosphonate as described in the synthetic procedure (Scheme 3.2). The synthesis and purification of the individual HO P O HO P Cl OH O OH O P HO P O OH OH Cl O P HO O O OH O a β,γ-CYCl U/ATP Y Y OH N HN O O U or A U Base N N N N NH 2 A Base A U Y= Cl 24 % 8 % Y= H 1 % 9 % 60 diastereomers was successfully achieved with a final step yield of 20% for the HPLC fast isomer, and 16% for the HPLC slow isomer. Scheme 3.2- Synthesis of individual diastereomers (R)- and (S)-β,γ-CHCl UTP. Conditions: b. DIEA, 2-NO 2 BnBr, DMF, 125 ºC, 12 h; c. (R)-1-phenylpropan-1-amine, (PyS) 2 , PPh 3 , DMF, RT, 12 h; d. HCl [1M], RT, 12 h; a. Bu 3 N, EtOH/H 2 O then UMP- morpholidate, DMSO, RT, 2 d; e. hv 365 nm, H 2 O, 2 days. 3.2.2 Finding the Optimal Coupling Step Conditions Optimizing the reaction for coupling conditions can be a very challenging task when it comes to using the individual diastereomers as substrates. Synthesizing, purifying, and obtaining a useful quantity of diastereomers is a time-consuming task. When using the mixture of diastereomers for coupling, the bisphosphonic acid is used with an excess of 4.0 equiv, in relation to the nucleotide. However, when dealing with separated individual diastereomers, we only used 1.1 equiv due to the limited amount of nucleotide isomer available. HO P O HO P Cl O O OH NO 2 HO P O HO P Cl OH O OH 1 21% b N H P (S) O HO P Cl O O OH NO 2 (R) or (R) 2 (R) or (S) 10 % (HPLC fast) 8 % (HPLC slow) c HO P (S) O HO P Cl O O OH NO 2 d qt. 4 (R) or (S) 53 % O P (S) HO P O OH O Cl O P HO O O OH O N HN O O or (R) OH NO 2 a (S)- and (R)-β,γ-CHCl UTP O P (S) HO P O OH OH Cl O P HO O O OH O N HN O O or (R) e 20% (HPLC fast) 16% (HPLC slow) OH 3 (R) or (S) 61 Figure 3.3- Coupling reaction and HPLC chromatogram of the UTP analogue. HPLC conditions: SAX semi prep, flow 4.0 ml/min, buffer A (H 2 O), buffer B (0.5 M TEAB, pH 7.5), method: 0-10 min 100% buffer A; 10-16 min 20% buffer B; 16-25 min 20-45% buffer B; 25-30 min 100% buffer B. With that in mind, we used CHCl-BP to study the optimal coupling conditions, since changes in the pH of the reaction mixture and amount of available BP in solution can be crucial for the coupling reaction to proceed. The parameters analyzed were the pH of the BP solution, the base used to change the pH, equivalents of UMP-morpholidated used and the overall pH of the solution after adding the nucleotide counterpart, over a period of 60 days (Table 3.1). The results were analyzed by measuring the area % of the HPLC peaks (Figure 3.2). The data shows that the higher the pH of the bisphosphonic moiety is, and therefore the higher the overall pH of the final reaction mixture solution, the lower the 62 amount of product formed, in terms of the area % of the HPLC product peak (Figures 3.3 and 3.4). Table 3.1- Study of the optimal conditions for the coupling step. Entry BP pH base used to adjust pH equiv of UMP-morph pH 6.5 Solution pH HPLC area peak % Day 3 Day 20 Day 40 1 4.1 NBu 3 0.6 6.6 40 - 46 2 4.1 NBu 4OH 1.1 6.3 48 - 48 3 2.0 No base 1.1 3.0 73* 73 71 Day 4 Day 11 Day 60 4 3.5 NBu 3 0.6 5.7 68 67 64 5 3.5 NBu 4OH 0.6 5.9 25 39 - Figure 3.4- Correlation of pH, bases tested and area % of the HPLC peak product. 63 Figure 3.5- Correlation of pH, reaction time and area % of the HPLC peak product. pH was adjusted with the base NBu 4 , excepted when marked with ‘*’, representing that pH was not adjusted at all. Overall, when the BP solution is used in its pure acidic form and no base is added, the best coupling results were achieved. It was also observed that there was no substantial difference when the reaction was running for 4 days or 40/60 days. The optimal conditions found in this study was to use the BP in its acidic form and let the reaction run for 2-4 days. In terms of the amount of UMP-morpholidate used, it is not clear where the optimal value lies. Even though, the best result was achieved when using 1.1 equiv of UMP- morpholidate, the other parameter that is mainly responsible for this result is the use of BP in its acidic form. Therefore, to draw any conclusion regarding the optimal equivalents of UMP-morpholidate to use, more studies need to be performed. 64 3.2.3 Nucleotide Incorporation: In Vitro Transcription Assays The in vitro transcription assays were performed as described in literature. 8,11 In summary, an aliquot of 5’- 32 P-labeled RNA was annealed with a 1.5-fold amount of template DNA and 2-fold amount of non-template DNA to form the RNA/DNA scaffold. The elongation buffer used was 20 mM Tris–HCl (pH 7.5), 40 mM KCl, 5 mM MgCl 2 . An aliquot of the annealed scaffold of RNA/DNA was then incubated with a 4-fold amount of pol II at room temperature for 10 min to ensure the formation of a pol II elongation complex. For the in vitro transcription assays, the RNA pol II elongation complex is then mixed with equal volumes of NTP solution at different concentrations. 8,11 During transcription, RNA pol II cleaves the Pa-O-Pb bond of the incoming NTP and catalyzes the formation of the NMP with the 3’-OH end of the RNA chain. A previous published report from our group, in collaboration with Prof. Dang at UCSD, 4 with β,γ-CH 2 UTP showed that the incorporation rate of this nucleotide by the enzyme is slower when compared with the wt UTP incorporation. This result is expected because β,γ-CH 2 BP is a worse leaving group than the natural occurring PP i . As expected, β,γ-CHF and β,γ-CF 2 UTPs 12 , have a higher incorporation rate due to the elevated electronegative effect conferred to the BP moiety due to the presence of the fluorine atom. As for the new results, β,γ-CCl 2 UTP showed a lower incorporation rate when compared with the di-fluorine derivative, but higher than the methylene version. When analyzing the individual diastereomer β,γ-CHCl-2 ((R)-isomer) and β,γ-CCl 2 , it is evident that the incorporation rate is fairly constant and even though another chloride atom raises the electronegativity of the leaving group, that was not reflected in the rate in which the enzyme cleaves the BP moiety. In terms of the individual diastereomers themselves, β,γ- 65 CHCl-1 ((S)-isomer) shows a lower incorporation rate. These findings are consistent with previous research in the McKenna group, while studying the mechanism of DNA polymerase, 13 where the enzyme was proved to be able to select between (R)- and (S)- diastereomers, with a slower incorporation rate for (S)-isomer and with the incorporation rate of the mixture being closer to the (S)-isomer. 13 Figure 3.6- Incorporation rate of β,γ -CXY UTPs by RNA pol II, with different β,γ- bridging atoms. A. Kinetic effect of nucleotide incorporation. B. Gel showing the incorporation of the separated individual diastereomers (R)- and (S)-isomer (CHCl-UTP- 2 and CHCl-UTP-1, respectively). Legend: CH 4 - previously published results; CH 12 - unpublished results from Mckenna, C.E., Wang, D. et al. β,γ-CHCl UTP, the diastereomer mixer, was also analyzed. Intriguing results were obtained, shown in blue in Figure 3.7. The first result corresponds to CHCl-A (Figure 3.7) where an unexpected high incorporation rate is observed. This result was not observed prior 66 from the DNA pol β studies, and the fact that its value is higher than the one observed for the CHF and CF2 derivatives made us very skeptical of this result. In addition, the values of the CHCl-A do not match with the results observed when the artificial mixture of individual diastereomers is created (CHCl-1 + CHCl-2, 1:1) Therefore, we decided to re- test it (CHCl-B, Figure 3.7) and the result achieved was similar. We then decided to re- check the purity and stability of that particular batch, and no impurities nor decomposition were observed on 1 H NMR, 31 P NMR, MS nor LCMS (CHCl-C, Figure 3.7). CHCl-C was again tested, showing the same unexpected result. The individual diastereomer samples were examined, their purity re-checked by LCMS, MS and NMR and nothing unusual was found. To this date, we are not able to explain why CHCl has shown such a high incorporation value and further tests will need to be made in order to clear this result. Figure 3.7- Incorporation rate of β,γ-CHCl UTPs by RNA pol II. 3.3 Conclusion and Future Directions 67 The aim of this project was to study the molecular mechanism of RNA pol II, in particular, how the cleavage rates of the bisphosphonate moiety from the NTPs are affected according to the electronic and steric properties of the β,γ-CXY substituents on the leaving group moiety. β,γ-CCl2 UTP, β,γ-CCl2 ATP, β,γ-CHCl UTP and β,γ-CHCl ATP were synthesized and the individual diastereomers β,γ-CHCl-1 and β,γ-CHCl-2 UTPs were successfully isolated. The optimal coupling step conditions were examined, and the best result was achieved when the BP solution was used in its pure acidic form and the coupling reaction ran for 2-4 days. According to the in vitro transcription assays, β,γ-CCl 2 UTP showed a lower incorporation rate when compared with the fluorine derivative, but higher than the methylene version and when comparing β,γ-CHCl-2 and β,γ-CCl 2 , it is evident that the incorporation rate is fairly constant. In terms of the individual diastereomers themselves, β,γ-CHCl-1 shows a low incorporation rate. β,γ-CHCl UTP, the diastereomer mixture, was also analyzed and inconsistent results were found. In order to clarify this result, further work will be needed. Future work to understand these effects will require crystal structures of the nucleotide-enzyme complexes. 3.4 Experimental Procedure 3.4.1 Materials and Methods Adenine- and uridine-5'-monophosphoric acids were purchased from Chem-Impex International. Phosphonic esters, and bisphosphonic acids were prepared according to the literature. 9,10 All other reagents were purchased from Sigma-Aldrich, Fluka and Alfa Aesar 68 as reagent grade and used as obtained. Synthesis of individual diastereomers of β,γ-CHF UTP was performed as described. 1 H and 31 P, spectra were obtained on Varian 400-MR, or NMR-500, or NMR-600 spectrometers. 31 P NMR spectra were proton-decoupled unless stated otherwise. Multiplicities are quoted as singlet (s), doublet (d), triplet (t), unresolved multiplet (m), doublet of doublets (dd), doublet of doublet of doublets (ddd), doublet of triplets (dt) and broad signal (bs). All chemical shifts (δ) are reported in parts per million (ppm) relative to residual CDH2OH in CD3OD (δ 3.34, 1H NMR), CHCl3 in CDCl3 (δ 7.26, 1H NMR), HDO in D2O (δ 4.80, 1 H NMR), external 85% H3PO4 (δ 0.00, 31 P NMR) or external C6F6 (δ -164.9, 19 F NMR). The pH meter was calibrated at three different pH (4, 7, and 10). 1D NMR spectra processing was performed with MestReNova 9.0.0 and 11.0.2. Preparative HPLC was performed using a Varian ProStar or Shimadzu Prominence equipped with a Shimadzu SPD-20A UV detector (0.5 mm path length) with detection at 280 nm for ortho-nitrobenzyl derivative compounds and 260 nm for NTP analogues. Strong anion exchange (SAX) HPLC was performed using, a Macherey Nagel 21.4 mm ´ 250 mm SP15/25 Nucleogel column. RP-C18 HPLC was performed using a Phenomenex Luna 5 μm C18(2) 100A 21.2 mm ´ 250 mm column. Mass spectrometry was performed on a Finnigan LCQ Deca XP Max mass spectrometer equipped with an ESI source in the negative ion mode. Compounds calculated MS were performed using iMass 1.3. Compound IUPAC names were assigned with the assistance of MarvinSketch 6.1.5. The molar yields of the final products were determined by UV absorbance using the extinction coefficient of UTP at pH 7.0 (phosphate buffer) at 260 nm (ε adenine = 15,400 M -1 cm -1 and ε uridine = 10,000 M -1 cm -1 ). The slow and fast terms are related to elution order of chiral synthons by RP-C18 HPLC. 69 3.4.2 Synthesis of β,γ-CXY U/ATP Derivatives: General Method 1 To a solution of the bisphosphonic acid (4.0 equiv) in a mixture of EtOH/H2O (1:1) was added Bu3N so that pH was adjusted to 2.5-3.0 and the mixture stirred for 15 min. Solvents were removed under vacuum and residual solvents were co-evaporated 3x with dry DMF. In parallel, previously dried 2’-uridine 5’-phosphoromorpholidate or 2’-adenine 5’-phosphoromorpholidate (1.0 equiv) was dissolved in dry DMSO (C = 0.101 M) and added to the dried tri-n-butyl ammonium salt of the bisphosphonic acid. The solution was stirred at room temperature and its progress controlled by 31 P NMR. Once the reaction was done, the crude material was purified by preparative SAX HPLC (8.0 mL/min, 262 nm (U) or 259 nm (A)) with a gradient mode of A (H2O) and B (0.5 M triethylammonium bicarbonate (TEAB), pH 7.4 buffer): 0-20 min A/ 100%, 20-35 min B/ 60-100%, 35-45 min B/ 100%. A second HPLC using a C-18 column was needed (8.0 mL/min, 262 nm (U) or 259 nm (A)) with an isocratic mode using 0.1 M triethylammonium bicarbonate pH 7.4 buffer, 7.5% of acetonitrile. 3.4.3 Synthesis of ({[({[(2S,5S)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxyoxolan-2- yl]methoxy}(hydroxy)phosphoryl)oxy](hydroxy)phosphoryl}dichloromethyl)phosph onic Acid - β,γ-CCl 2 ATP HO P O HO P Cl OH O OH O P HO P O OH OH Cl O P HO O O OH O Cl Cl OH N N N N NH 2 β,γ-CCl 2 ATP AMP-morpholidate DMSO, RT, 72 hrs 24% 70 To a solution of the di-chloro bisphosphonic acid (295 mg, 1.21 mmol, 4.0 equiv) in a mixture of EtOH/H2O (10 mL, 1:1) was added Bu3N (108 μL, 1.5 equiv) so that the pH was adjusted to 2.5-3.0 and the mixture stirred for 15 min. Solvents were removed under vacuum and residual solvents were co-evaporated 3x with dry DMF. In parallel, previously dried 2’-adenine 5’-phosphoromorpholidate (126 mg, 0.303 mmol, 1.0 equiv) was dissolved in dry DMSO (3 mL) and added to the dried tri-n-butyl ammonium salt of the bisphosphonic acid. The solution was stirred at room temperature for 72 h and its progress controlled by 31 P NMR. Once the reaction was done, the crude material was purified by preparative HPLC as indicated in the general Method 1 above. The retention time for SAX HPLC purification was 30.2 min, and for C18 HPLC purification, 9.3 min. The final amount of compound was measured using UV analysis and 41 mg of final product obtained (24% yield). 1 H NMR (400 MHz, D2O, pH 8.0) δ 8.41 (s, 1H), 8.10 (s, 1H), 5.99 (d, J = 5.9 Hz, 1H), 4.52 – 4.48 (m, 1H), 4.28 – 4.17 (m, 2H), 4.14 – 4.06 (m, 2H). 31 P NMR (162 MHz, D2O, pH 8.0) δ 7.91 (d, J = 18.6 Hz), 1.50 (dd, J = 31.3, 17.7 Hz), -10.85 (d, J = 30.9 Hz). MS (ESI) m/z: Calcd for [M-H] - = C11H15Cl2N5O12P3 - 571.93; found: 572.2. 3.4.4 Synthesis of [Dichloro({[({[(2S,5S)-5-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin- 1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)oxy](hydroxy) phosphoryl})methyl]phosphonic acid - β,γ-CCl 2 UTP HO P O HO P Cl OH O OH O P HO P O OH OH Cl O P HO O O OH O Cl Cl OH β,γ-CCl 2 UTP UMP-morpholidate DMSO, RT, 24 hrs N HN O O 8% 71 To a solution of the dichloro bisphosphonic acid (295 mg, 1.21 mmol, 4.0 equiv) in a mixture of EtOH/H 2 O (10 mL, 1:1) was added Bu 3 N (108 μL, 1.5 equiv) so that pH was adjusted to 2.5-3.0 and the mixture stirred for 15 min. Solvents were removed under vacuum and residual solvents were co-evaporated 3x with dry DMF. In parallel, previously dried 2’-uridine 5’-phosphoromorpholidate (119 mg, 0.303 mmol, 1.0 equiv) was dissolved in dry DMSO (3 mL) and added to the dried tri-n-butyl ammonium salt of the bisphosphonic acid. The solution was stirred at room temperature for 24 h and its progress controlled by 31 P NMR. Once the reaction was done, the crude material was purified by preparative HPLC as indicated in the general method 1 above. The retention time for SAX HPLC purification was 29.7 min, and for C18 HPLC purification was 9.2 min. The final amount of compound was measured using UV analysis and 13 mg of final product obtained (8% yield). 1 H NMR (500 MHz, D 2 O, pH 7.5) δ 7.77 (d, J = 7.8 Hz, 1H), 5.90 (d, J = 5.0 Hz, 2H), 5.80 (d, J = 7.9 Hz, 1H), 4.39 – 4.33 (m, 1H), 4.27 – 4.16 (m, 2H), 4.17 – 4.07 (m, 2H). 31 P NMR (202 MHz, D 2 O, pH 7.5) δ 7.91 (d, J = 18.2 Hz), 1.75 (dd, J = 31.4, 17.9 Hz), -10.96 (d, J = 31.5 Hz). MS (ESI) m/z: Calcd for [M-H] - = C 10 H 14 Cl 2 N 2 O 14 P 3 – 548.90; found: 549.1. 3.4.5 Synthesis of ({[({[(2S,5S)-5-(6-Amino-9H-purin-9-yl)-3,4-dihydroxyoxolan-2- yl]methoxy}(hydroxy)phosphoryl)oxy](hydroxy)phosphoryl}(chloro)methyl)phosph onic Acid - β,γ-CHCl ATP 72 To a solution of the monochloro bisphosphonic acid (171 mg, 0.82 mmol, 4.0 equiv) in a mixture of EtOH/H 2 O (6.6 mL, 1:1) was added Bu 3 N (73 μL, 1.5 equiv) so that pH was adjusted to 2.5-3.0 and the mixture stirred for 15 min. Solvents were removed under vacuum and residual solvents were co-evaporated 3x with dry DMF. In parallel, previously dried 2’-adenine 5’-phosphoromorpholidate (110 mg, 0.204 mmol, 1.0 equiv) was dissolved in dry DMSO (2 mL) and added to the dried tri-n-butyl ammonium salt of the bisphosphonic acid. The solution was stirred at room temperature for 72 h and its progress monitored by 31 P NMR. Once the reaction was done, the crude material was purified by preparative HPLC as indicated in the general Method 1 above. The final amount of compound was measured using UV analysis and 7 mg of final product obtained (1% yield). 1 H NMR (400 MHz, D 2 O, pH 8.0) δ 8.41 (s, 1H), 8.12 (s, 1H), 6.02 (d, J = 5.8 Hz, 1H), 4.46 (t, J = 4.3 Hz, 1H), 4.30 – 4.24 (m, 1H), 4.22 – 4.04 (m, 2H), 3.86 (t, J = 15.6 Hz, 1H). 31 P NMR (243 MHz, D 2 O, pH 10.0) δ 8.44 (bs), 7.03 – 6.41 (m), -10.97 (d, J = 25.9 Hz). MS (ESI) m/z: Calcd for [M-H] - = C 11 H 16 ClN 5 O 12 P 3 – 537.97. 3.4.6 Synthesis of [Chloro({[({[(2S,5S)-5-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1- yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)oxy](hydroxy) phosphoryl})methyl]phosphonic Acid - β,γ-CHCl UTP HO P O HO P Cl OH O OH O P HO P O OH OH Cl O P HO O O OH O OH N N N N NH 2 β,γ-CHCl ATP AMP-morpholidate DMSO, RT, 72 hrs 1% 73 To a solution of the monochloro bisphosphonic acid (225 mg, 1.07 mmol, 4.0 equiv) in a mixture of EtOH/H 2 O (8.8 mL, 1:1) was added Bu 3 N (96 μL, 1.5 equiv) so that pH was adjusted to 2.5-3.0 and the mixture stirred for 15 min. Solvents were removed under vacuum and residual solvents were co-evaporated 3x with dry DMF. In parallel, previously dried 2’-uridine 5’-phosphoromorpholidate (105 mg, 0.268 mmol, 1.0 equiv) was dissolved in dry DMSO (2.7 mL) and added to the dried tri-n-butyl ammonium salt of the bisphosphonic acid. The solution was stirred at room temperature for 24 h and its progress monitored by 31 P NMR. Once the reaction was done, the crude material was purified by preparative HPLC as indicated in the general Method 1 above. The final amount of compound was measured using UV analysis and 13 mg of final product obtained (9% yield). 1 H NMR (600 MHz, D 2 O, pH 7.5) δ 7.83 (d, J = 7.9 Hz, 1H), 5.94 – 5.75 (m, 2H), 4.33 – 4.20 (m, 2H), 4.19 – 4.03 (m, 3H), 3.83 (t, J = 16.2 Hz, 1H). 31 P NMR (243 MHz, D 2 O, pH 7.5) δ 9.24 (bs, 1P), 4.12 (bs, 1P), -11.21 (d, J = 26.9 Hz). MS (ESI) m/z: Calcd for [M-H] - = C 10 H 15 ClN 2 O 14 P 3 – 514.94; found: 515.12. 3.4.7 Synthesis of [Chloro({hydroxy[(2-nitrophenyl)methoxy]phosphoryl})methyl] phosphonic Acid 1 HO P O HO P Cl OH O OH O P HO P O OH OH Cl O P HO O O OH O OH β,γ-CHCl UTP UMP-morpholidate DMSO, RT, 24 hrs N HN O O 9% HO P O HO P Cl O O OH NO 2 HO P O HO P Cl OH O OH 1 DIEA, 2-NO 2 BnBr DMF, 125 º C, 12 hrs 21% 74 To a pre-warmed solution of [chloro(phosphono)methyl]phosphonic acid (800 mg, 3.8 mmol) in dry DMF (190 mL) at 125 ºC, was added DIEA (1.3 mL, 7.6 mmol) dropwise and the mixture was stirred for 15 min. A solution of 2-nitrobenzylbromide (903 mg, 4.2 mmol, 1.1 equiv) in DMF (21 mL) was then slowly added (over 15 min) through the condenser and the reaction mixture was kept at 125 ºC for 24 h. Progress of the reaction was monitored by 31 P NMR and MS. After completion, the resulting mixture was diluted with ethyl acetate at room temperature and evaporated to dryness. The residue was purified by preparative RP-C18, isocratic mode, using 0.1 M triethylammonium carbonate 18% acetonitrile pH 7.9 buffer (8.0 mL/min, 280 nm). The desired fraction was evaporated to dryness and the desired product was obtained as triethylammonium salt (21%). 31 P NMR (162 MHz, D2O, pH 10.0) δ 15.89 (s, 1P), 8.47 (s, 1C). MS (ESI) m/z: Calcd for [M-H] - = C8H9ClNO8P2 - 344.56. 3.4.8 Synthesis of [(S)-Chloro[hydroxy({[(1R)-1-phenylpropyl]amino}) phosphoryl] methyl][(2-nitrophenyl)methoxy]phosphinic Acid and [(R)-Chloro[hydroxy ({[(1R)- 1-phenylpropyl]amino})phosphoryl]methyl][(2-nitrophenyl)methoxy]phosphinic Acid - 2 To a solution of bisphosphonate 1 (109 mg, 0.32 mmol) in dry DMF (1.1 mL), under nitrogen atmosphere, was added (R)-1-phenylpropan-1-amine (460 μL, 3.2 mmol, 10.0 HO P O HO P Cl O O OH NO 2 N H P (S) O HO P Cl O O OH NO 2 (R) or (R) 1 chiral amine, SH(Py) 2 PPh 3 , DMF RT, 12 hrs 10 % (HPLC fast) 8 % (HPLC slow) 2 (R) or (S) 75 equiv), 2, 2’-dithiodipyridine (220 mg, 0.95 mmol, 3.0 equiv) and triphenylphosphine (249 mg, 0.95 mmol, 3.0 equiv), following this specific order, and reacted for 12 h. After completion, the resulting mixture was diluted with water and solvent was removed under vacuum. The residue was purified on preparative RP-SAX (8.0 mL/min, 280 nm) in isocratic mode with 0.5 M triethylammonium bicarbonate 10% acetonitrile pH 7.9 buffer. In order to separate the diastereomers, (R)- and (S)-2, HPLC-C18 (3.0 mL/min, 280 nm) was performed using isocratic mode with 0.1 M triethylammonium bicarbonate 32% acetonitrile pH 8.5 (tr fast isomer = 8.5 min, tr slow isomer = 9.4 min). The desired products, HPLC-fast and HPLC-slow isomers, were obtained with 10% and 8% yield, respectively. HPLC-fast: 1 H NMR (400 MHz, CD3OD) δ 8.11 – 8.03 (m, 1H), 7.74 – 7.59 (m, 2H), 7.59 – 7.52 (m, 1H), 7.52 – 7.43 (m, 1H), 7.43 – 7.35 (m, 2H), 7.24 (t, J = 7.7 Hz, 1H), 7.15 – 7.07 (m, 1H), 5.50 – 5.25 (m, 2H), 4.27 (q, J = 7.2 Hz, 1H), 3.50 (t, J = 14.2 Hz, 1H), 1.92 – 1.80 (m, 1H), 1.77 – 1.63 (m, 1H), 0.81 (t, J = 7.4 Hz, 3H). 31 P NMR (162 MHz, CD3OD) δ 11.79 (d, J = 5.0 Hz, 1P), 10.89 (d, J = 5.0 Hz, 1P). MS (ESI) m/z: Calcd for [M-H] - = C17H20ClN2O7P2 - 461.04; found 461.37. HPLC-slow: 1 H NMR (400 MHz, CD3OD) δ 8.10 (dd, J = 15.1, 7.8 Hz, 1H), 7.70 (t, J = 7.5 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.34 (d, J = 7.4 Hz, 2H), 7.22 (t, J = 7.5 Hz, 2H), 7.11 (t, J = 7.3 Hz, 1H), 5.42 (qd, J = 16.1, 7.1 Hz, 2H), 4.31 – 4.17 (m, 1H), 3.64 (t, J = 14.4 Hz, 1H), 3.69 – 3.59 (m, 1H), 2.07 – 1.92 (m, 1H), 1.78 – 1.66 (m, 1H), 0.88 – 0.75 (m, 3H). 31 P NMR (162 MHz, CD3OD) δ 12.28 (d, J = 3.2 Hz, 1P), 11.25 (d, J = 3.5 Hz, 1P). MS (ESI) m/z: Calcd for [M-H] - = C17H20ClN2O7P2 - 461.04; found 461.38. 76 3.4.9 Synthesis of [(S)-Chloro({hydroxy[(2-nitrophenyl)methoxy]phosphoryl}) methyl]phosphonic acid and [(R)-Chloro({hydroxy[(2-nitrophenyl)methoxy]- phosphoryl}) methyl]phosphonic Acid - 3 51 mg of each isomer (R)- and (S)-2 was dissolved in 2.2 mL of aqueous HCl [1M] and stirred for 12 h at room temperature. The reaction was monitored by MS and after completion, the mixture was concentrated under vacuum. Residual HCl was co-evaporated several times with a mixture of water and methanol. Purification was performed on a pipet column of DOWEX H+ using a mixture of methanol/water (1:1) as eluent and allowing the filtration to proceed by gravity only. The bisphosphonic acids (R)- and (S)-3 were obtained in solid form (quantitative yield). HPLC-fast: 1 H NMR (400 MHz, D2O, pH 1.0) δ 8.16 (d, J = 8.2 Hz, 1H), 7.90 (d, J = 7.8 Hz, 1H), 7.78 (t, J = 7.6 Hz, 1H), 7.55 (t, J = 7.8 Hz, 1H), 5.46 (d, J = 7.8 Hz, 2H), 4.14 (t, J = 16 Hz 1H). 31 P NMR (243 MHz, D2O, pH 9.1) δ 15.82 (s, 1P), 8.74 (s, 1P). MS (ESI) m/z: Calcd for [M-H] - = C 8 H 9 ClNO 8 P 2 - 344.56. HPLC-slow: 1 H NMR (400 MHz, D2O, pH 1.0) δ 8.04 (d, J = 9.2 Hz, 1H), 7.78 (d, J = 7.3 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.43 (t, J = 7.7 Hz, 1H), 5.34 (d, J = 8 Hz, 2H), 4.0 (t, J = 16 Hz, 1H). 31 P NMR (162 MHz, D2O, pH 1.0) δ 12.73 (s, 1P), 11.07 (s, 1P). MS (ESI) m/z: Calcd for [M-H] - = C 8 H 9 ClNO 8 P 2 - 344.56; found 344.16. N H P (S) O HO P Cl O O OH NO 2 (R) or (R) HO P (S) O HO P Cl O O OH NO 2 or (R) HCl [1M] RT, 12 hrs qt. 2 (R) or (S) 3 (R) or (S) 77 3.4.10 Synthesis of ({[(S)-Chloro({hydroxy[(2-nitrophenyl)methoxy]phosphoryl}) methyl](hydroxy)phosphoryl}oxy)({[(2S,5S)-5-(2,4-dioxo-1,2,3,4-tetrahydropyri- midin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy})phosphinic Acid and ({[(R)- Chloro({hydroxy[(2-nitrophenyl)methoxy]phosphoryl})methyl](hydroxy)phos- phoryl}oxy)({[(2S,5S)-5-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-3,4-dihydro- xyoxolan-2-yl]methoxy})phosphinic Acid – 4 To a solution of the bisphosphonic acid (R)- and (S)-3 (12 mg (HPLC fast) and 11.4 mg (HPLC slow), 1.0 equiv) in its pure acidic form, previously co-evaporated 3x with dry DMF, was added 2’-uridine 5’-phosphoromorpholidate (1.1 equiv, 160 uµL of UMP- morpholidate stock solution C = 0.24 M in DMSO). The solution was stirred for 2 days at room temperature and its progress controlled by 31 P NMR. Once the reaction was done, the crude material was purified by preparative SAX HPLC (8.0 mL/min, 280 nm) with a gradient mode of A/ H2O and B/ 0.5 M triethylammonium bicarbonate pH 7.5 buffer: 0-10 min A/ 100%, 10-16 min A/ 45%-B/ 55%, 16-25 min B/ 100%. 11.4 mg of HPLC-fast (tr = 36.5 min) and 12 mg of the HPLC-slow (tr = 36.1 min) isomers (R)- and (S)-4 were obtained. HPLC-fast: 31 P NMR (162 MHz, D2O, pH 9.7) δ 11.67 (d, J = 3.2 Hz, 1P), 1.98 (dd, J = 26.7, 3.6 Hz, 1P), -11.30 (d, J = 26.9 Hz, 1P). MS (ESI) m/z: Calcd for [M-H] - = C17H20ClN3O16P3 - 649.97; found 650.12. HPLC-slow: 31 P NMR (162 MHz, D2O, pH 9.2) HO P (S) O HO P Cl O O OH NO 2 or (R) UMP-morpholidate DMSO, RT, 2 da 53 % O P (S) HO P O OH O Cl O P HO O O OH O N HN O O or (R) OH NO 2 3 (R) or (S) 4 (R) or (S) 78 δ 11.57 (bs, 1P), 1.88 - 2.06 (m, 1P), -11.32 (d, J = 26.2 Hz). MS (ESI) m/z: Calcd for [M- H] - = C17H20ClN3O16P3 - 649.97; found 649.89. 3.4.11 Synthesis of [(S)-Chloro({[({[(2S,5S)-5-(2,4-dioxo-1,2,3,4-tetrahydro pyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl)oxy] (hydroxy)phosphoryl})methyl]phosphonic Acid and [(R)-Chloro({[({[(2S,5S)-5- (2,4-dioxo-1,2,3,4-tetrahydropyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy} (hydroxy)phosphoryl)oxy] (hydroxy)phosphoryl})methyl]phosphonic Acid - (R)- and (S)-β,γ-CHCl UTP The HPLC fast (11.4 mg, 0.0175 mmol) and HPLC slow (12.0 mg, 0.018 mmol) (R)- and (S)-4 triphosphate analogues were dissolved in 1 mL of water, placed in a quartz cuvette, and irradiated at 365 nm for 2 days. The progress of the reaction was monitored by 31 P NMR. The resulting reddish-brown crude material was purified by semi-prep RP- HPLC C18-polymeric column in isocratic mode (3.5 mL/min, 254 nm), 0.1 N triethylammonium bicarbonate 2 % acetonitrile pH 7.3 buffer (tr of HPLC-fast = 12.6 minutes and tr of HPLC-slow = 16.7). Solvents were removed under vacuum and the concentration of the sample was evaluated by UV: 1.8 mg of HPLC-fast and 1.5 mg of HPLC-slow isomers were obtained. Final purity was determined by HPLC semi-prep. HPLC-fast: 1 H NMR (400 MHz, D2O, pH 6.5) δ 7.99 (d, J = 8.1 Hz, 1H), 6.01 (d, J = 4.0 (S)- and (R)-CHCl UTP O P (S) HO P O OH OH Cl O P HO O O OH O N HN O O or (R) hv 365 nm water, 2 da 20% (HPLC fast) 16% (HPLC slow) OH O P (S) HO P O OH O Cl O P HO O O OH O N HN O O or (R) OH NO 2 4 (R) or (S) 79 Hz, 1H), 6.00 (d, J = 8.0 Hz, 1H), 4.46 – 4.38 (m, 2H), 4.34 – 4.23 (m, 3H), 4.04 (t, J = 16.3 Hz, 1H). 31 P NMR (162 MHz, D2O) δ 10.66 – 8.90 (m), 4.81 – 2.66 (m), -10.75 (d, J = 26.9 Hz). MS (ESI) m/z: Calcd for [M-H] - = C10H15ClN2O14P3 - 514.94; found 515.12. HPLC-slow: 1 H NMR (600 MHz, D2O, pH 6.5) δ 7.83 (d, J = 8.1 Hz, 1H), 5.87 – 5.83 (m, 2H), 4.31 – 4.22 (m, 2H), 4.18 – 4.08 (m, 3H), 3.85 (t, J = 16.2 Hz, 1H). 31 P NMR (162 MHz, D2O, pH 6.5) δ 10.02 (bs, 1P), 3.89 (m, 1P), -10.69 (d, J = 26.6 Hz). MS (ESI) m/z: Calcd for [M-H] - = C10H15ClN2O14P3 - 514.94; found 515.14. 3.5 Chapter References (1) Xu, L.; Plouffe, S. W.; Chong, J.; Wengel, J.; Wang, D. Inside Cover: A Chemical Perspective on Transcriptional Fidelity: Dominant Contributions of Sugar Integrity Revealed by Unlocked Nucleic Acids (Angew. Chem. Int. Ed. 47/2013). Angew. Chem. Int. Ed. 2013, 52 (47), 12196–12196. (2) Xu, L.; Da, L.; Plouffe, S. W.; Chong, J.; Kool, E.; Wang, D. Molecular Basis of Transcriptional Fidelity and DNA Lesion-Induced Transcriptional Mutagenesis. DNA Repair 2014, 19, 71–83. (3) Xu, L.; Butler, K. V.; Chong, J.; Wengel, J.; Kool, E. T.; Wang, D. Dissecting the Chemical Interactions and Substrate Structural Signatures Governing RNA Polymerase II Trigger Loop Closure by Synthetic Nucleic Acid Analogues. Nucleic Acids Res. 2014, 42 (9), 5863–5870. (4) Hwang, C. S.; Xu, L.; Wang, W.; Ulrich, S.; Zhang, L.; Chong, J.; Shin, J. H.; Huang, X.; Kool, E. T.; McKenna, C. E., Functional Interplay between NTP Leaving Group and Base Pair Recognition during RNA Polymerase II Nucleotide Incorporation Revealed by Methylene Substitution. Nucleic Acids Res. 2016, 44 (8), 3820–3828. (5) Zhang, J.; Palangat, M.; Landick, R. Role of the RNA Polymerase Trigger Loop in Catalysis and Pausing. Nat. Struct. Mol. Biol. 2010, 17 (1), 99–104. (6) Kaplan, C. D.; Larsson, K.-M.; Kornberg, R. D. The RNA Polymerase II Trigger Loop Functions in Substrate Selection and Is Directly Targeted by α-Amanitin. Mol. Cell 2008, 30 (5), 547–556. (7) Toulokhonov, I.; Zhang, J.; Palangat, M.; Landick, R. A Central Role of the RNA Polymerase Trigger Loop in Active-Site Rearrangement during Transcriptional Pausing. Mol. Cell 2007, 27 (3), 406–419. (8) Wang, D.; Bushnell, D. A.; Westover, K. D.; Kaplan, C. D.; Kornberg, R. D. Structural Basis of Transcription: Role of the Trigger Loop in Substrate Specificity and Catalysis. Cell 2006, 127 (5), 941–954. 80 (9) Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Batra, V. K.; Martínek, V.; Xiang, Y.; Beard, W. A.; Pedersen, L. C.; Wilson, S. H.; McKenna, C. E.; et al. Modifying the β,γ Leaving-Group Bridging Oxygen Alters Nucleotide Incorporation Efficiency, Fidelity, and the Catalytic Mechanism of DNA Pol β. Biochem. 2007, 46, 461–471. (10) McKenna, C. E.; Kashemirov, B. A.; Upton, T. G.; Batra, V. K.; Goodman, M. F.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H. (R)-β,γ-Fluoromethylene-DGTP-DNA Ternary Complex with DNA Polymerase β. J. Am. Chem. Soc. 2007, 129 (50), 15412–15413. (11) Wang, D.; Bushnell, D. A.; Huang, X.; Westover, K. D.; Levitt, M.; Kornberg, R. D. Structural Basis of Transcription: Backtracked RNA Polymerase II at 3.4 Angstrom Resolution. Science 2009, 324 (5931), 1203–1206. (12) Hwang, C. S. Fluorinated Probes of Enzyme Mechanisms. Doctoral dissertation, University of Southern California: Los Angeles, US, 2016. (13) Oertell, K.; Kashemirov, B. A.; Negahbani, A.; Minard, C.; Haratipour, P.; Alnajjar, K. S.; Sweasy, J. B.; Batra, V. K.; Beard, W. A.; Wilson, S. H.; et al. Probing DNA Base-Dependent Leaving Group Kinetic Effects on the DNA Polymerase Transition State. Biochemistry 2018, 57 (26), 3925–3933. 81 Chapter 4. Solid-phase synthesis of BP conjugate 1Aa to target Tropomyosin receptor kinases (TrkB and TrkC) 4.1 Introduction 4.1.1 The Human Ear and Hearing Loss The human ear is a complex organ that is divided into 3 main parts: the outer ear, the middle ear and the inner ear (Figure 4.1). Sound waves move from the outside into the ear canal and once they reach the eardrum, it vibrates and passes the vibrations to a tiny chain of ossicle bones in the middle ear: malleus, incus and stapes. 1 The vibrations then reach the cochlea of the inner ear, which is shaped like a snail, and is filled with thousands of hair cells that possess stereocilia (microscopic hair-like projections) sticking up out of the top. Once the hair cells receive the sound vibrations they move up and down and bend, provoking pore-like channels to open up. Certain components are known to enter, creating an electrical signal that will then travel to the brain, through the auditory nerve, to be recognized by the sound we hear. 1 When the sound is too loud, or lasts too long, it can cause damage to the sensory hair cells in the inner ear. Once damaged, hair cells do not grow back and the ability to hear decreases significantly. Other causes that are responsible for damaging the hair cells are exposure to viruses or toxic chemicals such as antibiotics and chemotherapy agents. 2 According to the World Health Organization, 3 1.1 billion young people (from ages between 12-35 years old) are at risk of hearing loss due to their high exposure to noise and loud sounds in recreational settings. 82 Figure 4.1- The auditory system. a) General view. b) Close up of the cochlea. 2 Age-related sensorineural hearing loss (SNHL) affects 65% of adults that are 70 and older, in the US, 4 and it is a progressive and an irreversible condition. Current treatments include the use of hearing aids as sound amplifiers and the use of cochlear implants that electrically stimulate the remaining healthy neurons. 5 The use of these devices significantly improves hearing capability, but does not restore the patient’s hearing aptitude, therefore regenerative therapy could lead to a greater impact in the patient’s life. 4.1.2 Neurotrophic Properties SNHL, either due to aging or noise exposure, is directly associated with the damage and loss of hair cells, cochlear spiral ganglion neurons (SGNs) and ribbon synapses. Research has shown that improving SGN survival, neurite outgrowth and synaptogenesis could lead to great advancements in treating hearing loss. 6-8 Neurotrophins are a large family of proteins that promote the survival of neurons via interaction with tyrosine receptor kinases (Trk). 5 TrkA, TrkB and TrkC are structurally 83 analogous receptors in the Trk family and can be found in neural cells. TrkB is the one with the highest affinity to bind with the brain-derived neurotrophic factor (BDNF), which has a crucial role in the survival and function of neurons, while TrkC binds to neurotrophin- 3, a protein growth factor involved in the survival and differentiation of neurons. 4,9 Trk receptors are responsible for regulating synaptic strength and plasticity and have a significant impact on neuronal development and function in the adult nervous system. 9,10 A large variety of small molecule agonists for Trks have been studied, particularly 7,8-dihydroxyflavone (DHF) 11,12 and the cyclic peptidomimetic 1Aa which were demonstrated to be potent TrkB agonists. 13 The peptidomimetic 1Aa has been confirmed to have trophic selectivity toward TrkC, providing significant trophic survival rates at concentrations of 10 µM and lower. 14 However, efficient delivery methods of drug candidates to the inner ear are lacking. Recently, our group published preliminary data regarding the use of a bisphosphonate-linked TrkB agonist as a delivery method to reach the cochlea. 12 In this study, the bisphosphonate moiety was a risedronate (RIS) linker conjugated with DHF (RIS-DHF), and it was observed that RIS-DHF was able to increase SGN neurite outgrowth between the hair cells and the SGN in vitro. When tested adsorbed on hydroxyapatite, a bone mineral, it was seen that the activity of RIS-DHF was maintained. 12 With these results in mind, our aim with this project was to test the action of 1Aa, which is believed to have greater selectivity towards TrkC 14 , and its bisphosphonate conjugated version, Ris-1Aa, as potential TrkB agonists. 84 4.2 Results and Discussion 4.2.1 Synthesis of 1Aa The synthesis of 1Aa (Scheme 4.1) was accomplished based on a synthetic method developed by Burgess. 13,15 The synthesis of the pre-solid phase compounds, and thus the synthesis of precursor 5 is reported with modifications. Starting with commercially available 4-(bromomethyl)-3-nitrobenzoic acid. By reacting it with sodium azide in DMF, the azide intermediate 2 was formed, which was then combined with triphenyl phosphine in a mixture of THF and water to yield amine 3. In literature reports, 13 when 4- (bromomethyl)-3-nitrobenzoic acid reacts with liquid ammonia in ethanol, yielding amine 3, the hydroxyl counterpart is likely to be formed as a byproduct. This side product is believed to be carried over the course of the synthetic route, since it would be challenging to separate it from the desired amine, and it could potentially be carried out into the solid phase reactions. Therefore, using the method here described, where azide 2 is synthesized as an intermediate, conferred an improved synthetic approach due to the absence of this undesirable side product. Amine 3 was subsequently reacted with 4-methyltrityl chloride to form the protected amine 4, which was then reacted with H2 over platinum (IV) oxide to reduce the nitro group yielding amine 5. 13 Afterwards, compound 5 was coupled to Rink amide resin - previously swollen and the Fmoc group removed - using HBTU and HOBt as coupling reagents and DIEA as base, in DMF. The first amino acid, Fmoc-Lys(Boc)- OH, was coupled to the aromatic amine 6, using PyBrop and 2,6-lutidine in DCM. The Fmoc group was cleaved off using a 20% v/v piperidine in DMF solution obtaining compound 7, which was then coupled with the second amino acid, Fmoc-Ile-OH, through 85 the use of DIC and HOBt as coupling agents and DIEA, in a 4:1 mixture of DCM:DMF. The Fmoc deprotection step was performed, exposing the primary amine 8. In parallel, 2-fluoro-5-nitrobenzoyl chloride 9, was synthesized from 2-fluoro-5- nitrobenzoic acid using thionyl chloride according to the literature procedure. 16 The acyl chloride 9 was then combined with the solid-phase amine 8 in the presence of DIEA, yielding intermediate 10, which was subsequently treated with a suspension of potassium carbonate in DMF to yield the cyclized product 1Aa-NO2. The nitro intermediate was reduced by the use of tin(II) chloride dehydrate, and was then cleaved off the resin using a solution of 90% TFA, 5%TIS and 5% H2O. The desired amine 1Aa was purified by semi- prep RP-C18, gradient mode, using the buffers A, 0.1% formic acid with 5% acetonitrile in water, and B, 0.1% formic acid with 10% acetonitrile in water. 1Aa was obtained with a 16% solid-phase overall yield, affording 7.4 mg of pure product. The desired product was characterized by MS (ESI) and 1 H NMR, and its estimated purity of 90% was assessed by LCMS analysis (Appendix C, Figure C16). 86 Scheme 4.1- Synthesis of desired product 1Aa. Conditions: 13 a. NaN 3 , DMF, 25 °C, 12 h; b. PPh 3 , THF/H 2 O, 25 °C, 12 h; c. MttCl, CHCl 3 /DMF, TEA, 25 °C, 130 min; d. PtO 2 (20 mol%), H 2 , EtOAc, 25 °C, 24 h; e. Rink Amide resin (deprotected), HBTU, HOBt, DIEA, DMF, 2 h; f. i) Fmoc-Lys(Boc)-OH, PyBrop, 2,6-lutidine, DCM, 12 h, ii) 20% OH O O 2 N N 3 OH O O 2 N Br OH O O 2 N H 2 N a b qt. 2 3 50% OH O O 2 N MttHN 4 c 31% OH O H 2 N MttHN 5 d qt. e H 2 N N H O MttHN Resin 6 f (i) (ii) HN N H O MttHN (S) O H 2 N Resin NHBoc 7 HN N H O MttHN O N H O NH 2 NHBoc 8 HN N H O O N H O NH O NH 2 F O 2 N h, i 10 HN N H O O N H O HN O O 2 N H N NHBoc 1Aa-NO 2 HN NH 2 O O N H O HN O H 2 N H N NH 2 1Aa HN N H O O N H O HN O H 2 N H N NHBoc k 1Aa (on SP) g (i) (ii) Resin Resin j Resin Resin l 87 piperidine/DMF; g. i) Fmoc-Ile-OH, DIC, HOBt, DIEA, DCM/DMF (4:1), 4 h, ii) 20% piperidine/DMF; h. benzoyl chloride 9, DIEA, DCM, 50 min; i. 1% TFA, 5% TIS, DCM, 15 min; j. K 2 CO 3 , DMF, 2 d; k. SnCl 2 .2H 2 O, DMF, 22 h; l. 90% TFA, 5% TIS, 5% H 2 O, 2 h. 4.2.2 Tentative Synthesis of RIS-1Aa In order to test the targeted delivery methodology to the cochlea, using a bisphosphonate moiety, our aim was to synthesize RIS-1Aa-(no PEG) and RIS-1Aa (Figure 4.2). We started with the simplest target, RIS-1Aa-(no PEG), by reacting 1Aa(on SP) with succinic anhydride in DMF for 5.5 h, a method adapted from the literature, 17 yielding intermediate 1Aa-COOH (Scheme 4.2), determined by mass spectrometry (Appendix C, Figure C18). Figure 4.2- Structure of desired compounds RIS-1Aa and RIS-1Aa-(no PEG). Legend: RIS-linker (orange), PEG and succinic acid linkers (black) and 1Aa moiety (purple). HN NH 2 O O N H O HN O N H H N NH 2 O O N P HO P H N OH OH OH O OH O OH O O 4 NH (S) (S) HN NH 2 O O N H O HN O N H H N NH 2 O O N P HO P N H OH OH OH O OH O OH RIS-1Aa-(no PEG) (S) (S) RIS-1Aa 88 Scheme 4.2- Synthesis of intermediate 1Aa-COOH. With the intermediate 1Aa-COOH in hand, we focused on the coupling step. There were two possible approaches: either perform it on solid-phase support or cleave it off of the resin and execute the coupling step in solution phase. Since our product was already available in solid support, we chose to start with this solid phase approach. Five different methods were attempted (Table 4.1), but unfortunately, all of them were unsuccessful. As activating agents for the carboxylic group, both 2,3,5,6-tetrafluorophenol (TFP) and N- hydroxysuccinimide (NHS), in the presence of DCC, were used. RIS-linker with pH adjusted to 8.2-8.4 was added to two difference solvent systems: water/DMF and water/THF. By means of analyzing the reaction crude, a portion of the resin beads were cleaved off using 90% TFA, 5% TIS and 5% water, and the results inspected by LCMS, in which only the starting material 1Aa-COOH was found. HN N H O O N H O HN O H 2 N H N NHBoc 1Aa (on SP) Resin HN NH 2 O O N H O HN O H 2 N H N NH 2 90% TFA, 5% TIS, 5% H 2 O 1Aa 89 Table 4.1- Trials of different combinations of reaction conditions in solid phase for coupling of RIS-linker with intermediate 1Aa-COOH to produce RIS-1Aa-(no PEG). Molecular weight: 1Aa-COOH(no Boc) = 623 gmol -1 . Next, the coupling step reaction was investigated in solution phase (Table 4.2). 1Aa- COOH in solid phase was cleaved from the resin by submitting the crude material to a solution of 90% TFA, 5% TIS and 5% water for 2 h. For entries 1-3, the starting material is in TFA salt form, due to the use of TFA in the previous step. The use of TFA is also responsible for removing the Boc group, exposing the primary amine on the molecule. Therefore, a reaction with di-tert-butyl dicarbonate was necessary before proceeding with the synthesis. 1Aa-COOH was reacted with NHS and DCC and the pH of the solution adjusted with addition of triethylamine, but no activation occurred (Table 4.2, entry 1). More drastic conditions were attempted, by using TSTU, and the result is believed to be an intra-cyclization within the molecule (Table 4.2, entries 2-3; Scheme Entry Activation Activ. Solvent Coupling Coupl. Solvents Results & Obs 1 TFP, DCC (2 h) DMF/CHCl 3 (1:1) RIS-linker (pH 8.4, DIEA) Water/DMF (12 h) 𝗫 MS ESI – 622 ESI + 624 2 Added NHS, EDC to previous sample (2 d) 𝗫 MS ESI + 624 3 NHS, DCC (2 h) DMF RIS-linker (pH 8.2, Na 2CO 3) Water/DMF (12 h) 𝗫 MS ESI – 622 4 NHS, DCC (2 h) DMF RIS-linker (pH 8.2, Na 2CO 3) Water/THF (12 h) 5 NEt 3, TSTU (30 min) DMF RIS-linker (pH 9.0, TEA) Water/DMF (3 h) 90 4.3). Since the desired coupled product was not formed, it was decided that 1Aa-COOH needed to be purified in order to remove the excess of TFA present in the sample, which could be involved in the lack of product formation. Scheme 4.3- Structure of 1Aa-NHS, hypothesized intra-cyclization within 1Aa-COOH. Table 4.2- Trials of different combinations of reaction conditions in solution phase for coupling of RIS-linker with the intermediate 1Aa-COOH to produce RIS-1Aa. Molecular weight: 1Aa-COOH = 723 gmol -1 ; 1Aa-NHS (cyclized) = 705 gmol -1 . Entry Activation Activ. Solvent Coupling Coupl. Solvents Results & Obs 1 NHS, DCC (1.5h) then NEt 3 (1.5 h) DMF - - 𝗫 MS ESI – 722 2 Added NEt 3 and TSTU to the sample in entry 1 (12 h) 𝗫 MS ESI – 704 ESI + 728 3 NEt 3, TSTU (30 min) DMF RIS-linker (pH 8.2, Na 2CO 3) Water/DMF (12 h) 𝗫 MS ESI – 704 ESI + 728 4 NHS, DCC (5 h) THF - - 𝗫 MS ESI – 722 5 Added to entry 4: NHS, DCC, DMAP (2 h) THF - - 𝗫 MS ESI – 722 HN NH 2 O O N H O HN O N H N NHBoc 1Aa-NHS O O HN NH 2 O O N H O HN O N H H N NHBoc 1Aa-COOH O HO O 91 1Aa-COOH was purified on HPLC (C18 column, flow 3.5 mL/min, 260 nm) with gradient mode using A/ 0.1 N TEAB pH 7.8-8.3 with 2% acetonitrile and B/ 0.1 N TEAB pH 7.8-8.3 with 60% acetonitrile, method: 0-7 min 100% A; 7-15 min 0-50% of B; 15-25 min 50% B; 25-30 min 50-100% of B and 30-40 min 100% of B (tr = 27.5 min). With purified 1Aa-COOH, the results were similar (Table 4.2, entries 4-5) and no product was observed, only starting material was present. Due to the fact that 1Aa-COOH was available in limited amount, a model compound 11 was used instead so more trials could be performed (Scheme 4.4). Several different reaction conditions were studied (Table 4.3). For entries 1-4, the solvent used for the activation step was 60% water in DMF, using EDC as the coupling agent and NHS or NHS- sulfo as the activating agent. For all entries, the activated intermediate was believed to be formed. When using NHS-sulfo, the presence of compound 13 was confirmed by mass spectrometry. When using NHS, the presence of the neutral compound 12 was identified by the decrease of the MS peak with the value of 192 m/z, which corresponds to compound 11 in MS ESI - . The coupling reaction with the RIS-linker was attempted, shown in entries 1-4, but the desired product was not formed. 92 Scheme 4.4- Use of model compound 11 to test reaction conditions for the coupling step. Legend: NHS and NHS-Sulfo moiety (blue) and RIS-linker (orange). For entries 5-7 (Table 4.3), a few changes were made, such as using DCC as the coupling reagent and decreasing the concentration of the coupling reaction. For entry 5, the concentration of RIS-linker in the total volume of the solution, after the coupling reaction started, was 0.02 M. A small amount of product was detected by MS and further confirmed by LCMS (Appendix C, Figure C20). In order to optimize this result, the concentration of the coupling reaction was diluted. Entry 6 and 7 from Table 4.3 show a concentration of 0.008 M, with either Na2CO3 or TEA as the base used to adjust the pH of the RIS-linker solution. H N O O NH HO N P P HO O HO OH HO O HO H N O OH O H N O O O N O O 11 12 13 H N O O O N O O S O O OH 14 NHS coupl. agent NHS-Sulfo coupl. agent 93 Table 4.3- Trials of different combinations of reaction conditions for coupling of the model compound 11 with RIS-linker to produce compound 14. Molecular weight: 11 = 193 gmol - 1 ; 12 = 290 gmol -1 ; 13 = 370 gmol -1 and 14 = 532 gmol -1 . Activation Activ. Solvent pH Obs. Coupling Coupling solvent Results & Obs. 1 EDC 10 equiv NHS-sulfo 10 equiv H2O (60%), DMF 6 MS ESI – 369 (13) ✔ RIS-linker pH 8.7, TEA 2.5 equiv H2O (60%), DMF 𝗫 MS ESI - : no 14 2 7 MS ESI – 369 (13) ✔ 𝗫 MS ESI - : no 14 3 EDC 10 equiv NHS 10 equiv 6.7 MS ESI – : no 11 ✔ RIS-linker pH 8.7, TEA 2.5 equiv H2O (60%) DMF pH 7.7 (TEA) 𝗫 MS ESI - : no 14 4 EDC 1.1equiv NHS 1.1equiv 6.2 MS ESI – : small amounts of 11 still present RIS-linker pH 8.5, Na2CO3 2.5 equiv H2O (60%), DMF pH 8.1 C= 0.02 M 𝗫 MS ESI - : no 14 5 DCC 0.9 equiv NHS-sulfo 0.9 equiv (3 h) DMF - MS ESI – 369 (13) ✔ RIS-linker pH 8.5, Na2CO3 2 equiv ✔ YES MS ESI – 530 (14) Small amount confirmed by LCMS 6 DCC 1.2 equiv NHS-sulfo 1.2 equiv (20 h) DMF - MS ESI – 369 (13) ✔ RIS-linker pH 8.5, Na2CO3 2 equiv H2O (60%), DMF pH 8.5 C= 0.008 M ✔ YES MS ESI – 530 (14) ~ 50% estimated by LCMS 7 RIS-linker pH 8.5, TEA 2 equiv 8 DCC 1.2 equiv NHS-sulfo (TEA salt) 1.2 equiv DCM - MS ESI – 369 (13) ✔ - - Hydrolysis of 13 observed after 12 h 9 NHS 1.2 equiv DCC 2 equiv (30 min) DCM - MS ESI – 369 (13) ✔ Solvent was evaporated RIS-linker pH 8.0, Na2CO3 2 equiv H2O (60%) DMF C=0.02 M ✔ YES MS ESI – 530 (14) 94 LCMS of entry 6 (Appendix, Figure C21) shows an improvement in the synthesis of compound 14. With these promising results, the conditions of entry 6 in Table 4.3 were used with 1Aa-COOH. However, the results obtained were disappointing: no product was identified, and MS analysis showed mainly starting material and the cyclized byproduct. Literature methods have shown that the undesirable cyclization reaction, between a succinic acid linker and an aromatic amine, has been reported in other groups 18 and in order to decrease the formation of the cyclized side product, DCM was used as the reaction solvent 18,19 . Therefore, we decided to use this approach with the model compound 11 (Table 4.3, entries 8-9). The activation step was controlled by MS over several time periods: 20, 40, 60, 90 min and 12 h (entry 8, Table 4.3) and the best result appeared to be at 40 min, and after 12 h of reaction the complete hydrolysis of the activated ester was achieved. The best result obtained so far was achieved when NHS (1.2 equiv) and DCC (2 equiv) was used to activate the carboxylic group of compound 11, in DCM, for only 30 min. After that, the solvent was evaporated and the RIS-linker (pH 8) was dissolved in water (60%) in DMF and added to the crude with the activated material (Table 4.3, entry 9). After 1 h of coupling reaction, MS was analyzed and a strong peak with the desired m/z value of 530 (MS ESI - ) was observed (Appendix C, Figure C22). However, since this reaction was carried out using a model compound, the purification step was not performed. This method will be applied to the actual desired product. 4.2.3 Spiral Ganglion Neurite Outgrowth in Vitro In order to investigate the effect that 1Aa has on spiral ganglion neurite outgrowth, in vitro outgrowth studies 12 were performed in the laboratory of Dr. David Jung and Dr. 95 Judith Kempfle (ME&I, Harvard Medical School) and the data included here with their permission (unpublished data). Postnatal SGNs were plated and treated with 400 nM of 1Aa, DHF, a combination of both (1Aa+DHF), or DMSO for the control experiment, and incubated over 48 h (Figure 4.3). To determine the average neurite outgrowth, both immunohistochemistry and quantitative analysis of neuron specific class III Tubulin (TuJ) were utilized in this assay. Figure 4.3- Spiral ganglion neurite outgrowth in vitro data. A. Cochlear SGNs treated with 400 nM of 1Aa, DHF, 1Aa+DHF, or DMSO for the control. Represents an immunohistochemical analysis, in which neurons where stained with the neural marker TuJ (red) and the nuclei tagged with DAPI, 4′,6-diamidino-2-phenylindole (blue). The scale bar represents 100 µm. B. Quantitative relative outgrowth ratio with 400 nM of DHF, 1Aa, DHF+1Aa and control (DMSO), measured through the 3D neurite tracer software (sample A B 96 size n= 9). Results are indicated in mean ± SEM. Legend: n.s. represents ‘not significant’, ** stands for p < 0.01). By analyzing the increase in fiber length in the samples, it was found that 1Aa promoted SGN outgrowth, when compared with the control alone. The effect of 1Aa was found to be similar to the action of DHF, showing no statistically significant difference. When 1Aa and DHF were combined, a similar average of neurite outgrowth was also observed. For this experiment, the neurite outgrowth was measured with the aid of 3D neurite tracer software. 4.2.4 Regeneration of Cochlear Ribbon Synapses in Vitro With the goal of examining whether 1Aa promotes regeneration of cochlear ribbon synapses, in vitro experiments 12 utilizing Organ of Corti (OC) explants were performed in the Jung’s lab. OC explants with attached neurons were dissected, plated and incubated with kainic acid (KA, 0.5 mM) for 2 hours. KA is responsible for causing excitotoxic damage to ribbon synapses and neurite retraction. 20 The damaged OC explants were subsequently treated with 400 nM of 1Aa, DHF, 1Aa+DHF, or DMSO for the control over a period of 24 hours. The result obtained were analyzed using immunohistochemistry and confocal microscopy (Figure 4.4). Each experiment was conducted without KA (KA-), to set a baseline control of synapses survival in growth medium, and with KA (KA+) to induce excitotoxic damage. The peptidomimetic 1Aa was able to promote a marked regeneration of the cochlear ribbon synapses, comparing to KA+, and its value was similar with the one observed for 97 DHF, with no statistically significant difference. 1Aa+DHF combined showed a similar effect. Figure 4.4- Cochlear synapse regeneration in vitro data. A. Organ of Corti (OC) explants firstly treated with 0.5 mM KA (KA+) for 2 h, and subsequently incubated with 400 nM of 1Aa, DHF, 1Aa+DHF, or DMSO for the control for 24 h. Both KA+ and KA- (untreated explants) were used as controls. The images represent an immunohistochemical analysis, A B Juxtaposition/IHC 98 in which presynaptic synapses were stained with CtBP2 (red); neural postsynaptic synapse was tagged with PSD95 (green); hair cells were labeled with myosin VIIa, Myo7a (white) and the ‘merge’ represents all 3 stains combined. The scale bar represents 100 µm. B. Quantitative data of synapses regrowth per inner hair cell (IHC) with a sample size of 6 (n = 6) after a 24 h of incubation period. Results are indicated in mean ± SEM. Legend: n.s. represents ‘not significant’, ** stands for p < 0.01, *** stands for p < 0.001) 4.3 Conclusion and Future Directions Previous studies have identified a wide range of small molecules agonists for tropomyosin receptor kinases, TrkB and TrkC, that are able to improve spiral ganglion neuron (SGN) survival, including 7,8-dihydroxyflavone (DHF) 11,12 and the peptidomimetic cyclic compound 1Aa. 13-15 Here we present the synthesis of 1Aa and its use as a neurotrophin receptor agonist. 1Aa was synthesized over 12 steps, 7 of them being in solid- phase support with an overall yield of 16%. We see that 1Aa successfully promotes spiral ganglion neurite outgrowth in vitro and also bolsters the regeneration of cochlear ribbon synapses in vitro. Future studies will include in vivo experiments of both the spiral ganglion neurite outgrowth and the regeneration of cochlear ribbon synapses. Regarding RIS-1Aa-(no PEG), several attempts to perform the coupling step between 1Aa-COOH and the RIS-linker were made, however it has not been successful. The next steps are to transpose the reaction conditions used on Table 4.3, entry 9, to 1Aa- COOH compound. Once RIS-1Aa-(no PEG) is synthesized, the same coupling approach to incorporate the PEG linker will be used in order to get RIS-1Aa. The biological effect 99 of RIS-1Aa, with and without the PEG linker, on the spiral ganglion neurite outgrowth and the regeneration of cochlear ribbon synapses will be tested in both in vitro and in vivo. 4.4 Experimental Procedure 4.4.1 Materials and Methods Rink amide resin (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin) was purchased from Advanced ChemTech. The specification of the resin is: substitution (0.5 mmol/g), mesh size (100-200 mesh) and catalog number (SA5030). All other reagents were purchased from Sigma-Aldrich, Oakwood Chemicals and Alfa Aesar as reagent grades and used as received. Compounds were purified using an ISCO CombiFlashRf+ Lumen instrument equipped with an ELSD detector. Semi-preparative HPLC was performed using a Varian ProStar or Shimadzu Prominence equipped with a Shimadzu SPD-20A UV detector (0.5 mm path length) with detection at 254 and 280 nm. RP-C18 HPLC was performed using a Phenomenex Luna 5 μm C18(2) 100A 250 ´ 10 mm column. LCMS analysis were conducted with an analytical XTerra RP18 3.5 μm 4.6 x 150 mm or a Phenomenex Luna 250 ´ 4.6 mm column. Mass spectrometry was performed on a Finnigan LCQ Deca XP Max mass spectrometer supplied with an ESI source in the negative ion mode. Compounds calculated MS were performed by ChemDraw 16.0 or iMass 1.3. Compound IUPAC names were assigned with the assistance of MarvinSketch 17.28.0. 1 H and 13 C NMR spectra were acquired on Varian 400-MR, VNMRS-500, or VNMRS-600 spectrometers. Multiplicities are listed as singlet (s), doublet (d), triplet (t), unresolved multiplet (m), doublet of doublets (dd) and doublet of doublet of doublets (ddd). All 100 chemical shifts (δ) are reported in parts per million (ppm) relative to residual CDH2OH in CD3OD (δ 3.34, 1 H NMR), CHCl3 in CDCl3 (δ 7.26, 1 H NMR), HDO in D2O (δ 4.79, 1 H NMR) and (CD2H)2CO in (CD3)2CO (δ 2.09, 1 H NMR). The cleavage of the Fmoc group was monitored by UV spectral analysis (lmax = 300 nm, e = 7800 M -1 cm -1 ). 21 4.4.2 Synthesis of 1-[(4-carboxy-2-nitrophenyl)methyl]triaza-1,2-dien-2-ium 2 In a round bottom flask, 4-(bromomethyl)-3-nitrobenzoic acid (502 mg, 1.93 mmol) was dissolved in 9.6 mL of anhydrous DMF. Sodium azide (1 equiv, 125 mg) was slowly added and reacted at 25 °C overnight, under nitrogen atmosphere. The crude reaction mixture was then extracted with ethyl acetate (2 x 20 mL) and aqueous HCl 0.1 M (2 x 20 mL). The organic solvent was dried over MgSO4 and evaporated to dryness. The desired azide 2, an orange viscous oil, was obtained, in a quantitative yield, and used immediately in the next step without further purification. In order to completely understand the chemical shift difference between the bromo derivative starting material and the desired azide product, proton NMR studies were conducted (Appendix C, Figure C2). 1 H NMR (400 MHz, CD3OD) δ 8.55 (s, 1H), 8.25 (d, J = 8.1 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 4.89 (s, 2H). 13 C NMR (101 MHz, CD3OD) δ 165.40 (C1), 147.69 (C4), 135.86, 134.05, 131.83, 130.43, 125.59, 51.36 (C8). MS (ESI) m/z: Calcd for [M-H] - = C8H5N4O4 – 221.0; found: 221.2. OH O O 2 N N 3 OH O O 2 N Br NaN 3 , DMF 25 °C, 12 hrs qt. 2 101 4.4.3 Synthesis of 4-(aminomethyl)-3-nitrobenzoic acid 3 The azide 2 (1.93 mmol) was dissolved in 1.6 mL of water and 5.1 mL of THF and triphenylphosphine (1.4 equiv, 709 mg) was slowly added to the reaction mixture and reacted at 25 °C overnight. The solvent was evaporated to dryness, and the residue re- dissolved in alkaline water (pH adjusted to 11 with NH4 + ), and centrifuged. The solution was evaporated to dryness yielding 192 mg (50% yield) of the desired benzylamine 3 and used in the next step without further purification. 1 H NMR (600 MHz, D2O) δ 8.34 (s, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 3.91 (s, 2H). MS (ESI) m/z: Calcd for [M-H] - = C8H7N2O4 – 195.1; found: 195.1 4.4.4 Synthesis of 4-({[(4-methylphenyl)diphenylmethyl]amino}methyl)-3- nitrobenzoic acid 4 In a round bottom flask, 4-methyltrityl chloride (2.9 g, 10.2 mmol) was dissolved in a mixture of anhydrous chloroform (20 mL) and anhydrous DMF (10 mL) and stirred vigorously. The benzylamine 3 (2.0 g, 10.2 mmol) was added to the reaction mixture at 25 °C and stirred for 30 min. Distilled and anhydrous triethylamine (2 equiv, 2.8 mL) was OH O O 2 N N 3 OH O O 2 N H 2 N PPh 3 , THF/H 2 O 25 °C, 12 hrs 2 3 50% OH O O 2 N H 2 N 3 OH O O 2 N MttHN 4 MttCl, CHCl 3 /DMF TEA, 25 °C 130 min 31% 102 added dropwise and reacted for 100 min. The reaction progress was monitored by TLC. After a total of 130 min the reaction was stopped and evaporated to dryness. ISCO chromatography was performed using: solvent A- hexane, solvent B- ethyl acetate, solvent B4- methanol, flow 40 mL/min, silica column 40 g (Appendix, Figure C7). 1.4 g of the pure desired protected amine 4 was obtained with 31% yield. 1 H NMR (500 MHz, acetone- d6) δ 8.49 (s, 1H), 8.40 – 8.34 (m, 2H), 7.56 (d, J = 9.2 Hz, 4H), 7.43 (d, J = 8.3 Hz, 2H), 7.31 (t, J = 7.2 Hz, 4H), 7.21 (t, J = 7.3 Hz, 2H), 7.13 (d, J = 8.5 Hz, 2H), 3.69 (s, 2H), 2.29 (s, 3H). MS (ESI) m/z: Calcd for [M-H] - = C28H24N2O4 – 451.5; found: 451.2 4.4.5 Synthesis of 3-amino-4-({[(4-methylphenyl)diphenylmethyl]amino} methyl)benzoic acid 5 In a Schlenk flask, the protected amine 4 (700 mg, 1.55 mmol), ethyl acetate (18 mL) and platinum dioxide (70 mg, 20 mol%) were combined and, under nitrogen atmosphere, the oxygen gas in the reaction mixture was removed by using the freeze-pump-thaw method (3x). Once the reaction flask was free of oxygen, the atmosphere in the flask was changed to hydrogen by placing a H2-filled balloon, connected via a needle inserted directly into the solution. The hydrogenation reaction was left stirring for 24 h at 25 °C. The reaction progress was monitored by mass spectrometry. The crude material was then filtered with celite. 655 mg of the desired product 5 was obtained in a quantitative yield, OH O O 2 N MttHN 4 OH O H 2 N MttHN 5 PtO 2 20 mol%, H 2 EtOAc, 25 °C, 24 hrs qt. 103 and used in the next step without further purification. 1 H NMR (600 MHz, CD3OD) δ 7.48 (d, J = 8.2 Hz, 4H), 7.36 – 7.33 (m, 3H), 7.29 – 7.25 (m, 5H), 7.20 – 7.15 (m, 3H), 7.10 (d, J = 8.1 Hz, 2H), 3.35 (s, 2H), 2.29 (s, 3H). MS (ESI) m/z: Calcd for [M-H] - = C28H25N2O2 – 421.5; found: 421.3 4.4.6 Synthesis of Intermediate 6: Coupling Compound 5 to the Rink Amide resin The Rink resin (0.09 mmol, 0.5 mmol/g, 180 mg) was swollen overnight in dry DCM (10 mL/g; 1.8 mL) in a plastic fritted syringe. To remove the Fmoc protective group, the resin was treated with a solution of 20% (v/v) piperidine in anhydrous DMF (1.5 mL) for 1.5 h, and then a fresh portion of the same solution (1.5 mL) was added for another hour. The cleavage of the Fmoc group was monitored by UV spectral analysis (lmax = 300 nm, e = 7800 M -1 cm -1 ). The resin was washed (DMF, MeOH, DMF, MeOH, DCM 2x, MeOH 2x, and dry DCM 3x) and ready for the next step. A solution of starting material 5 (3 equiv); HBTU (3 equiv, 102 mg); HOBt (3 equiv, 36 mg) and DIEA (5 equiv, 79 µL) in anhydrous DMF (1.5 mL) were added to the fritted syringe carrying the rink resin and reacted for 2 h with gentle shaking. The reaction was monitored by the use of the ninhydrin test. The solvents were drained and the resin washed (DMF, MeOH, DMF, MeOH, DCM 2x, MeOH 2x, and dry DCM 3x). OH O H 2 N MttHN 5 HN N H O MttHN (S) O H 2 N Resin NHBoc 6 Rink resin(deprotected) HBTU, HOBt, DIEA DMF, 2 hr 104 4.4.7 Synthesis of intermediate 7: Incorporation of Fmoc-Lys(Boc)-OH The reaction mixture with intermediate 6 was treated with a solution of Fmoc-Lys- (Boc)-OH (4 equiv, 169 mg), PyBrop (6 equiv, 252 mg) and distilled and anhydrous 2,6- lutidine (15 equiv, 157 µL) in anhydrous DCM (1.5 mL) and it was left overnight with gently shaking. To check for completion of reaction, a few beads were removed from the original sample, washed and the FMOC group was removed with a solution of 20% (v/v) piperidine in anhydrous DMF (1.5 mL for 15 min). The ninhydrin test was then performed, and the solution was purple indicating that the free amine was present. Therefore, the solvents of the original crude material were drained, the beads were washed and the Fmoc group cleaved (2 x 1.5 mL, 1 h each turn). The removal of the Fmoc group was monitored by UV spectral analysis (lmax = 300 nm, e = 7800 M -1 cm -1 . The resin was washed (DMF, MeOH, DMF, MeOH, DCM 2x, MeOH 2x, and dry DCM 3x). 4.4.8 Synthesis of intermediate 8: Incorporation of Fmoc-Ile-OH H 2 N N H O MttHN Resin 6 HN N H O MttHN (S) O H 2 N Resin NHBoc 7 i) Fmoc-Lys(Boc)-OH PyBrop, 2,6-lutidine DCM, 12 hr ii) 20% Piperidine/DMF HN N H O MttHN (S) O H 2 N Resin NHBoc 7 HN N H O MttHN O N H O NH 2 NHBoc 8 Resin i) Fmoc-Ile-OH DIC, HOBt, DIEA DCM/DMF (4:1), 4 hr ii) 20% Piperidine/DMF 105 The reaction mixture with intermediate 7 was treated with a solution of Fmoc-Ile-OH (3 equiv, 95 mg), DIC (3 equiv, 42 µL); HOBt (3 equiv, 36 mg) and anhydrous DIEA (5 equiv, 79 µL) in anhydrous DCM/DMF (4:1 – 1.2 mL DCM & 300 µL DMF, Vtotal=1.5 mL) and reacted for 4 h. The reaction was monitored by checking the color of the solution after using the ninhydrin test. The solvents were drained, the beads were washed and the FMOC group cleaved with 20% (v/v) piperidine in anhydrous DMF (2 x 1.5 mL, 10 min first, then 15 min). The removal of the FMOC group was monitored by UV spectral analysis (lmax = 300 nm, e = 7800 M -1 cm -1 ). The resin was washed (DMF, MeOH, DMF, MeOH, DCM 2x, MeOH 2x, and dry DCM 3x). 4.4.9 Synthesis of 2-fluoro-5-nitrobenzoyl chloride 9 2-Fluoro-5-nitrobenzoic acid (500 mg, 2.7 mmol) was dissolved in thionyl chloride (4.3 mL) and refluxed for 135 min. The progress of the reaction was monitored by proton NMR of the reaction mixture, and by combining it with starting material and comparing the differences in chemical shift (Appendix C, Figure C13). The excess of SOCl2 and the HCl formed during the reaction were distilled out of the reaction flask. Benzoyl chloride 9 was then washed with ethyl ether and dried on a desiccator until the weight was stable (approximately 2 h). The desired benzoyl chloride 9 was obtained with a quantitative yield and the off-white solid was used immediately in the next step without further purification. F O 2 N HO O F O 2 N Cl O SOCl 2 , 75 ºC 135 min qt. 9 106 1 H NMR (400 MHz, CDCl3) δ 9.00 (dd, J = 6.2, 2.8 Hz, 1H), 8.54 (ddd, J = 9.1, 3.9, 2.9 Hz, 1H), 7.41 (t, J = 9.2, 1H). 4.4.10 Synthesis of intermediate 10: Incorporation of 2-fluoro-5-nitrobenzoyl chloride The reaction mixture with compound 8 was treated with a solution of 2-fluoro-5- nitrobenzoyl chloride 9 (3 equiv, 55 mg) and anhydrous and distilled DIEA (3 equiv, 47 µL) in anhydrous DCM (1.5 mL) for 50 min. The reaction was monitored by checking the color of the solution after using the ninhydrin test. The solvents were drained, and the beads were washed. The Mtt (methyl trityl) protecting group was removed by treatment with a solution of 1% TFA and 5% TIS in DCM. 2 mL of the solution was used to rinse the beads, 8 times, or until the yellow color became clear. Lastly, the resin was washed (DMF, MeOH, DMF, MeOH, DCM 2x, MeOH 2x, and dry DCM 3x). Cl O F O 2 N HN N H O O N H O NH O NH 2 F O 2 N NHBoc i) ii) 1% TFA, 5% TIS DCM,15 min DIEA, DCM, 50 min 10 9 HN N H O MttHN O N H O NH 2 NHBoc 8 Resin Resin 107 4.4.11 Synthesis of 1Aa-NO 2 Intermediate 10 was treated with a suspension of K2CO3 (10 equiv, 124 mg) in anhydrous DMF (2 mL) at 25 ºC, with gentle shaking, for 2 d. Some beads were removed from the original crude material, washed (H2O 5x before the regular washing sequence), pump dried for 1 h and the solid-phase product was then cleaved from the resin by adding 1 mL of 90% TFA, 5% TIS and 5% H2O for 2 h. The solvent was drained, and the resin rinsed with water (3x) and ethyl ether (3x). The solution was evaporated to dryness. In order to confirm the presence of the desired product 1Aa-NO2, LCMS analysis was run using an analytical RP-C18 (1.0 mL/min, 254 nm, 280 nm), buffer A, 0.1% formic acid in H2O, and buffer B, 0.1% formic acid in acetonitrile, in gradient mode: 0 to 15 min from buffers A to B (Appendix, Figure C14). The MS of the desired compound was identified at 8.13 min. The original reaction mixture was then washed and ready to use in the next step. MS (ESI) m/z: Calcd for [M-H] - = C27H36N7O6 + 554.3, found 554.4. HN N H O O N H O HN O O 2 N H N NHBoc K 2 CO 3 , DMF, 2 da 1Aa-NO 2 HN N H O O N H O NH O NH 2 F O 2 N NHBoc 10 Resin Resin 108 4.4.12 Synthesis of 1Aa (on SP) The solid-phase crude material containing 1Aa-NO2 was treated with a solution of 677 mg of SnCl2•2H2O in 1.5 mL of DMF (2 M in DMF) for 22 h with gentle shaking. The resin was washed (H2O 3x before the regular washing sequence), pump dried for 1 h and ready to be utilized in the next step. 4.4.13 Synthesis of (12S,15S)-20-amino-12-(4-aminobutyl)-15-(butan-2-yl)- 11,14,17-trioxo-2,10,13,16-tetraazatricyclo[16.4.0.04,9]docosa-1(18),4(9),5,7,19, 21-hexaene-7-carboxamide 1Aa A solution of 90% TFA, 5% TIS and 5% H2O was added to the fritted syringe and stirred for 2 h. The solvent was drained, and the resin rinsed with water (3x) and ethyl ether (3x). The solution was evaporated to dryness. The compound 1Aa was purified by semi- prep RP-C18 (3.5 mL/min, 254 nm), buffer A, 0.1% formic acid 5% acetonitrile in H2O, HN N H O O N H O HN O O 2 N H N NHBoc 1Aa-NO 2 Resin HN N H O O N H O HN O H 2 N H N NHBoc SnCl 2 .2H 2 O, DMF 22 hrs 1Aa (on SP) Resin HN N H O O N H O HN O H 2 N H N NHBoc 1Aa (in SP) Resin HN NH 2 O O N H O HN O H 2 N H N NH 2 90% TFA, 5% TIS, 5% H 2 O 1Aa 109 and buffer B, 0.1% formic acid 10% acetonitrile in H2O, in gradient mode: 0 to 20 min (A), 20 to 40 min (B) (Appendix C, Figure C15). The MS of the desired compound was identified at the peak with the retention time of 25.9 min. 7.4 mg (16% overall yield) of the desired product 1Aa was obtained, with an estimated purity of 90%. The purity was confirmed by LCMS analysis (Appendix C, Figure C16) using an analytical RP-C18 column (1.0 mL/min, 254 nm, 280 nm), buffer A, 2% acetonitrile in 0.1% of formic acid in H2O, and buffer B, 10% acetonitrile in 0.1% of formic acid in H2O, gradient mode: 0 to 10 min buffer A and 10 to 25 min buffer B. 1 H NMR (400 MHz, D2O) δ 7.95 (s, 1H), 7.79 – 7.73 (m, 2H), 7.28 (s, 1H), 7.21 (d, J = 8.6 Hz, 1H), 7.10 (d, J = 8.8 Hz, 1H), 4.36 – 4.29 (m, 2H), 4.21 – 4.14 (m, 1H), 4.02 – 3.94 (m, 1H), 3.64 – 3.56 (m, 1H), 3.03 (t, J = 7.6 Hz, 2H), 2.05 – 1.91 (m, 2H), 1.85 – 1.64 (m, 6H), 1.04 (d, J = 6.7 Hz, 3H), 1.03 – 0.93 (m, 3H). MS (ESI) m/z: Calcd for [M-H] + = C27H38N7O4 + 524.6; found: 524.3. 4.4.14 Synthesis of 1Aa-COOH (no Boc) To the 1Aa(on SP), a solution of succinic anhydride (36 equiv, 234 mg) and DMF (1 mL) was added to solid support and the reaction ran for 5.5 h. The resin was then washed (DMF, MeOH, DMF, MeOH, DCM 2x, MeOH 2x, and dry DCM 3x). 1Aa-COOH was HN N H O O N H O HN O H 2 N H N NHBoc HN NH 2 O O N H O HN O N H H N NH 2 O HO O i) Succinic anhydride DMF, 5.5 hrs Resin 1Aa (in SP) 1Aa - COOH (no Boc) ii) 90% TFA, 5% TIS, 5% H 2 O 110 cleaved from the resin by reaction with a solution of 90% TFA, 5% TIS and 5% H2O and stirred for 2 h. The solvent was drained, and the resin rinsed with water (3x) and ethyl ether (3x). The solution was evaporated to dryness. MS (ESI) m/z: Calcd for [M-H] - = C36H48N7O9 - 622.30; found: 622.4. 4.4.15 Synthesis of 1Aa-COOH Compound 1Aa (3 mg, 4.8 µmol) was dissolved in methanol (300 µL) and distilled triethylamine (2 equiv, 9.6 µmol) and Boc2O (1.1 equiv, 5.3 µmol) were added to the reaction flask and stirred for 12 h at room temperature. The reaction was monitored by mass spectrometry and after completion the solvent was evaporated to dryness and the crude material used immediately in the next step. MS (ESI) m/z: Calcd for [M-H] - = C31H40N7O7 - 722.35; found: 722.5. 4.5 Chapter References (1) How Do We Hear? https://www.nidcd.nih.gov/health/how-do-we-hear (accessed Sep 22, 2018) (2) Müller, U.; Barr-Gillespie, P. G. New Treatment Options for Hearing Loss. Nat. Rev. Dru g Discov. 2015, 14, 346-365. (3) Deafness and hearing loss http://www.who.int/news-room/fact-sheets/detail/deafness- and-hearing-loss (accessed Sep 19, 2018). (4) Lin, F. R.; Thorpe, R.; Gordon-Salant, S.; Ferrucci, L. Hearing Loss Prevalence and Risk Factors Among Older Adults in the United States. J. Gerontol. Ser. A 2011, 66A (5), 582–590. HN NH 2 O O N H O HN O N H H N NH 2 O HO O HN NH 2 O O N H O HN O N H H N NHBoc O HO O Boc 2 O, NEt 3 MeOH 12 hrs 1Aa-COOH (no Boc) 1Aa-COOH 111 (5) Nayagam, B. A.; Muniak, M. A.; Ryugo, D. K. The Spiral Ganglion: Connecting the Peripheral and Central Auditory Systems. Spiral Ganglion Neurons 2011, 278 (1), 2– 20. (6) Kujawa, S. G., and Liberman, M. C. Adding insult to injury: cochlear nerve degeneration after ″temporary″ noise-induced hearing loss. J. Neurosci. 2009, 29, 14077−14085. (7) Stankovic, K., Rio, C., Xia, A., Sugawara, M., Adams, J. C., Liberman, M. C., and Corfas, G. Survival of adult spiral ganglion neurons requires erbB receptor signaling in the inner ear. J. Neurosci. 2004, 24, 8651−8661. (8) Ernfors, P., Kucera, J., Lee, K. F., Loring, J., and Jaenisch, R. Studies on the physiological role of brain-derived neurotrophic factor and neurotrophin-3 in knockout mice. Int. J. Dev. Biol. 1995, 39, 799−807. (9) Bailey, J. J.; Schirrmacher, R.; Farrell, K.; Bernard-Gauthier, V. Tropomyosin Receptor Kinase Inhibitors: An Updated Patent Review for 2010-2016 – Part II. Expert Opin. Ther. Pat. 2017, 27 (7), 831–849 (10) Huang, E. J.; Reichardt, L. F. Trk Receptors: Roles in Neuronal Signal Transduction. Annu. Rev. Biochem. 2003, 72 (1), 609–642 (11) Jang, S.-W.; Liu, X.; Yepes, M.; Shepherd, K. R.; Miller, G. W.; Liu, Y.; Wilson, W. D.; Xiao, G.; Blanchi, B.; Sun, Y. E.; et al. A Selective TrkB Agonist with Potent Neurotrophic Activities by 7,8-Dihydroxyflavone. Proc. Natl. Acad. Sci. 2010, 107 (6), 2687-2692. (12) Kempfle, J. S.; Nguyen, K.; Hamadani, C.; Koen, N.; Edge, A. S.; Kashemirov, B. A.; Jung, D. H.; McKenna, C. E. Bisphosphonate-Linked TrkB Agonist: Cochlea- Targeted Delivery of a Neurotrophic Agent as a Strategy for the Treatment of Hearing Loss. Bioconjug. Chem. 2018, 29 (4), 1240–1250. (13) Lee, H. B.; Zaccaro, M. C.; Pattarawarapan, M.; Roy, S.; Saragovi, H. U.; Burgess, K. Syntheses and Activities of New C10 β-Turn Peptidomimetics. J. Org. Chem. 2004, 69 (3), 701–713. (14) Zaccaro, M. C.; Lee, H. B.; Pattarawarapan, M.; Xia, Z.; Caron, A.; L’Heureux, P.- J.; Bengio, Y.; Burgess, K.; Saragovi, H. U. Selective Small Molecule Peptidomimetic Ligands of TrkC and TrkA Receptors Afford Discrete or Complete Neurotrophic Activities. Chem. Biol. 2005, 12 (9), 1015–1028 (15) Pattarawarapan, M.; Zaccaro, M. C.; Saragovi, U. H.; Burgess, K. New Templates for Syntheses of Ring-Fused, C10 β-Turn Peptidomimetics Leading to the First Reported Small-Molecule Mimic of Neurotrophin-3. J. Med. Chem. 2002, 45 (20), 4387–4390. (16) Reich, S.; Bleckman, T.; Kephart, S.; Romines, W.; Wallace, M. Indazole Compounds, Pharmaceutical Compositions, and Methods for Mediating or Inhibiting Cell Proliferation. U.S. patent 20060111322A1. January 18, 2000. (17) Kumar, A.; Ye, G.; Wang, Y.; Lin, X.; Sun, G.; Parang, K. Synthesis and Structure−Activity Relationships of Linear and Conformationally Constrained Peptide Analogues of CIYKYY as Src Tyrosine Kinase Inhibitors. J. Med. Chem. 2006, 49 (11), 3395–3401. (18) Liu, R.; Liu, M.; Hood, D.; Chen, C.-Y.; MacNevin, J. C.; Holten, D.; Lindsey, S. J. Chlorophyll-Inspired Red-Region Fluorophores: Building Block Synthesis and Studies in Aqueous Media. Molecules 2018, 23 (1), 1-30. 112 (19) Villaverde, G.; Baeza, A.; Melen, G. J.; Alfranca, A.; Ramirez, M.; Vallet-Regí, M. A New Targeting Agent for the Selective Drug Delivery of Nanocarriers for Treating Neuroblastoma. J. Mater. Chem. B 2015, 3 (24), 4831–4842. (20) Wang, Q., and Green, S. H. Functional role of neurotrophin-3 in synapse regeneration by spiral ganglion neurons on inner hair cells after excitotoxic trauma in vitro. J. Neurosci. 2011, 31, 7938−7949 (21) Newcomb, W. S.; Deegan, T. L.; Miller, W.; Porco, J. A. Analysis of 9- Fluorenylmethoxycarbonyl (FMOC) Loading of Solid-Phase Synthesis Resins by Gas Chromatography. Biotechnol. Bioeng. 2000, 61 (1), 55–60. 113 Appendix A. Chapter 2 Supporting Data Part I Figure A1- 1 H NMR (500 MHz, D 2 O, pH 10.3) of compound 1. W= acetone; X = HDO; Y, Z = Et 3 N. Figure A2- 19 F NMR (376 MHz, D 2 O, pH 10.0) of compound 1. O P OH HO P O OH O NO 2 F 114 Figure A3- 31 P NMR (162 MHz, D 2 O, pH 10.0) of compound 1. Figure A4- MS (ESI) [M-H] – of compound 1. O P OH O P O OH O NO 2 F 115 Figure A5- 1 H NMR (500 MHz, CD 3 OD) of (R)-isomer of compound 2. U = unidentified peaks; X = HDO; Y, Z = Et 3 N. Figure A6- 31 P NMR (202 MHz, CD 3 OD) of (R)-isomer of compound 2. 10, 14 1, 3 4 5 2 11, 13 12 7 6 8 9 O P (R) OH N H P O OH O NO 2 F 1 2 3 4 5 6 7 8 9 10 11 12 13 14 g b O P (R) OH N H P O OH O NO 2 F β γ 116 Figure A7- 19 F NMR (470 MHz, CD 3 OD) of (R)-isomer of compound 2. O P (R) OH N H P O OH O NO 2 F 117 Figure A8- MS (ESI) [M-H] – of (R)-isomer of compound 2. O P (R) OH N H P O O O NO 2 F 118 Figure A9- 1 H NMR (400 MHz, CD 3 OD) of (S)-isomer of compound 2. U = unidentified peaks; V= methanol; W= acetone; X = HDO; Y, Z = Et 3 N Figure A10- 19 F NMR (376 MHz, CD 3 OD) of (S)-isomer of compound 2. 9 8 6 7 5 2, 10, 14 12 11, 13 1 3, 4 O P (S) OH N H P O OH O NO 2 F 1 2 3 4 5 6 7 8 9 10 11 12 13 14 O P (S) OH N H P O OH O NO 2 F 119 Figure A11- 31 P NMR (162 MHz, CD 3 OD) of (S)-isomer of compound 2. b g O P (S) OH N H P O OH O NO 2 F β γ 120 Figure A12- MS (ESI) [M-H] – of (S)-isomer of compound 2. O P (S) OH N H P O O O NO 2 F β γ 121 Figure A13- 1 H NMR (400 MHz, CD 3 OD) of (R)-isomer of compound 3. U = unidentified peaks; V= methanol; W= acetone; X = HDO; Y, Z = Et 3 N. Figure A14- 19 F NMR (376 MHz, CD 3 OD) of (R)-isomer of compound 3. O P (R) OH HO P O OH O NO 2 F 122 Figure A15- 31 P NMR (162 MHz, D 2 O, pH 10.0) of (R)-isomer of compound 3. O P (R) OH HO P O OH O NO 2 F β γ 123 Figure A16- MS (ESI) [M-H] – of (R)-isomer of compound 3. O P (R) OH O P O OH O NO 2 F 124 Figure A17- 1 H NMR (500 MHz, D 2 O, pH 10.0) of (S)-isomer of compound 3. U = unidentified peaks; W= acetone; X = HDO; Y, Z = Et 3 N. Figure A18- 19 F NMR (470 MHz, D 2 O, pH 10.0) of (S)-isomer of compound 3. O P (S) OH HO P O OH O NO 2 F 125 Figure A19- 31 P NMR (202 MHz, D 2 O, pH 10.0) of (S)-isomer of compound 3. 126 Figure A20- MS (ESI) [M-H] – of (S)-isomer of compound 3. O P (S) OH O P O OH O NO 2 F 127 Figure A21- 1 H NMR (400 MHz, D 2 O, pH 10.0) of (R)-β,γ-CHF dCTP. V= methanol; W= acetone; X = HDO; Y, Z = Et 3 N. Figure A22- 19 F NMR (564 MHz, D 2 O, pH 10.0) of (R)-β,γ-CHF dCTP. O P (R) OH P O OH HO O P OH O O OH O N N NH 2 O F 128 Figure A23- 31 P NMR (243 MHz, D 2 O, pH 10.0) of (R)-β,γ-CHF dCTP. 129 Figure A24- MS (ESI) [M-H] – of (R)-β,γ-CHF dCTP. Figure A25- Preparative SAX HPLC (second pass) of (R)-β,γ-CHF dCTP. O P (R) OH P O OH HO O P O O O OH O N N NH 2 O F 130 Figure A26- 1 H NMR (400 MHz, D 2 O, pH 10.0) of (S)-β,γ-CHF dCTP. V= methanol; X = HDO; Y, Z = Et 3 N. Figure A27- 19 F NMR (564 MHz, D 2 O, pH 10.0) of (S)-β,γ-CHF dCTP. O P (S) OH P O OH HO O P OH O O OH O N N NH 2 O F 131 Figure A28- 31 P NMR (243 MHz, D 2 O, pH 10.0) of (S)-β,γ-CHF dCTP. 132 Figure A29- MS (ESI) [M-H] – of (S)-β,γ-CHF dCTP. Figure A30- Preparative SAX HPLC (second pass) of (S)-β,γ-CHF dCTP. O P (S) OH P O OH HO O P O O O OH O N N NH 2 O F 133 Part II Figure A31- 1 H NMR (500 MHz, CDCl 3 ) of compound 1. X=residual solvents. Previously published compound (ref 20 from Chapter 2). Figure A32- 31 P NMR (202 MHz, CDCl 3 ) of compound 1. X= impurity. Previously published compound (ref 20 from Chapter 2). 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 f1 (ppm) -100 0 100 200 300 400 500 600 700 800 900 1000 1100 6.19 4.04 1.00 2.08 0.95 3.14 1.36 1.38 1.39 4.23 4.24 4.26 4.27 4.30 7.27 7.37 7.37 7.38 7.39 7.44 7.45 7.46 7.50 7.51 7.70 7.70 7.70 7.70 7.71 7.80 7.82 7.84 O P O O C H 3 O C H 3 a b c d e f g X h d , e h a , b , c , f i i i h X g -30 -20 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 f1 (ppm) -200 -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 -6.21 x O P O O C H 3 O C H 3 134 Figure A33- 31 P NMR (202 MHz, CDCl 3 ) of compounds 2 and 3. X= impurity. Previously published compound (refs 20 and 21 from Chapter 2). Figure A34- 31 P NMR (202 MHz, CDCl 3 ) of compounds 4. X= impurity 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 f1 (ppm) 0 50 100 150 200 250 300 350 400 450 500 1.80 1.00 21.11 25.22 O H P O O C H 3 O C H 3 O H P O O C H 3 O C H 3 2 3 3 2 x 0 5 10 15 20 25 30 35 40 45 f1 (ppm) -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 1 . 0 0 22.17 O H O H P O P O O C H 3 O C H 3 O C H 3 O C H 3 x 4 135 Figure A35- 31 P NMR (202 MHz, CDCl 3 ) of compound 7. X= impurities 0 5 10 15 20 25 30 35 40 45 f1 (ppm) -20 0 20 40 60 80 100 120 140 160 180 200 220 240 1.00 21.81 x x O H O H P O O C H 3 O C H 3 x x 136 Figure A36- MS (ESI) [M-H] – of compound 7. [M-H] - 137 Figure A37- 1 H NMR (500 MHz, CD 3 OD) of compound 8. X,Y= residual solvents Figure A38- 31 P NMR (202 MHz, CD 3 OD) of compound 8. 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0 5 . 5 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0 8 . 5 f 1 ( p p m ) 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0 3 0 0 0 2 . 9 0 2 . 0 0 2 . 3 9 5 . 0 2 1 . 0 8 1 . 0 5 0 . 9 9 1 . 1 3 1 . 0 2 0 . 9 8 1 . 1 5 1 . 1 6 1 . 1 8 1 . 1 8 1 . 2 0 3 . 8 4 3 . 8 5 3 . 8 6 3 . 8 8 3 . 8 9 4 . 8 3 7 . 0 8 7 . 1 0 7 . 1 4 7 . 1 6 7 . 1 9 7 . 5 3 7 . 5 5 7 . 5 8 7 . 5 8 7 . 6 0 7 . 6 0 7 . 6 4 7 . 6 5 8 . 0 0 8 . 0 3 8 . 1 9 8 . 2 0 8 . 2 2 8 . 2 7 8 . 2 8 x x Y Y C D 3 O D O H O H P O O H O C H 3 a b c a b c N a p h t h a l e n e r i n g s - 4 0 - 3 0 - 2 0 - 1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 f 1 ( p p m ) - 1 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 8 . 7 2 O H O H P O O H O C H 3 138 Figure A39- MS (ESI) [M-H] – of compound 8. Docking experiments and analysis of the active site of the lyase domain Figure A40- Docking experiment and analysis of Pamoic acid (PA). [M-H] - [2M-2H] - C:\Xcalibur\data\Boris\CA-3-13b 3/13/2015 3:32:34 PM RT: 0.00 - 20.00 SM: 11B 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Time (min) 0 100000 200000 uAU 50 100 0 500000 1000000 uAU 3.73 12.40 12.77 12.90 13.56 3.72 12.40 9.38 2.86 6.13 6.93 NL: 1.31E6 Channel A UV CA-3-13b NL: 5.49E7 Base Peak m/z= 406.50- 407.50 MS CA-3-13b NL: 2.84E5 Total Scan PDA CA-3-13b CA-3-13b #878 RT: 12.89 AV: 1 NL: 5.21E7 T: - c ESI Full ms [200.00-1000.00] 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance 407.11 814.61 263.22 852.84 361.18 504.73 205.16 444.90 296.86 875.44 553.98 973.67 773.15 586.05 625.09 946.14 693.24 748.79 139 Figure A41- Docking experiment and analysis of a tyrosine derivative of PA. Figure A42- Docking experiment and analysis of a phenyl derivative of PA. 140 Figure A43- Docking experiment and analysis of a phenyl and tyrosine derivatives of PA. Figure A44- Docking experiment and analysis of a phenyl and n-butyl amide tyrosine derivatives of PA. 141 Appendix B. Chapter 3 Supporting Data Figure B1- 1 H NMR (400 MHz, D 2 O, pH 8.0) of β,γ-CCl 2 ATP. X= residual solvents Figure B2- 31 P NMR (162 MHz, D 2 O, pH 8.0) of β,γ-CCl 2 ATP. 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0 5 . 5 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0 8 . 5 9 . 0 f 1 ( p p m ) 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0 3 0 0 0 3 5 0 0 4 0 0 0 1 . 1 8 2 . 0 6 0 . 9 8 1 . 0 0 0 . 8 8 0 . 9 1 4 . 0 8 4 . 0 8 4 . 0 9 4 . 0 9 4 . 1 1 4 . 1 1 4 . 1 2 4 . 1 2 4 . 1 8 4 . 1 9 4 . 2 0 4 . 2 0 4 . 2 1 4 . 2 2 4 . 2 3 4 . 2 3 4 . 2 4 4 . 2 5 4 . 2 6 4 . 2 6 4 . 2 7 4 . 4 9 4 . 5 0 4 . 5 1 4 . 5 2 5 . 9 7 5 . 9 8 6 . 0 0 8 . 0 8 8 . 0 8 8 . 0 9 8 . 1 0 8 . 1 1 8 . 4 1 a , a ' O P O H P O O H O H C l O P O H O O O H O C l O H N N N N N H 2 b c d e f g g f e c a , a ' , b , d x x x x x - 3 0 - 2 5 - 2 0 - 1 5 - 1 0 - 5 0 5 1 0 1 5 2 0 2 5 3 0 f 1 ( p p m ) - 5 0 0 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0 3 0 0 0 3 5 0 0 4 0 0 0 4 5 0 0 5 0 0 0 5 5 0 0 6 0 0 0 6 5 0 0 7 0 0 0 0 . 9 5 1 . 0 0 1 . 0 0 - 1 0 . 9 5 - 1 0 . 7 6 1 . 3 5 1 . 4 5 1 . 5 4 1 . 6 5 7 . 8 5 7 . 8 8 7 . 9 7 O P O H P O O H O H C l O P O H O O O H O C l O H N N N N N H 2 β γ α γ β α 142 Figure B3- MS (ESI) [M-H] - of β,γ-CCl 2 ATP. [M-H] - [M+Na] - [M-H] 2- [M-H] - [M-2H+Na] - [M-H] 2- 143 Figure B4- 1 H NMR (500 MHz, D 2 O, pH 7.5) of β,γ-CCl 2 UTP. X= residual solvents Figure B5- 31 P NMR (202 MHz, D 2 O, pH 7.5) of β,γ-CCl 2 UTP. - 2 0 - 1 5 - 1 0 - 5 0 5 1 0 1 5 f 1 ( p p m ) - 4 0 - 2 0 0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 0 . 9 8 0 . 9 3 1 . 0 0 - 1 1 . 0 4 - 1 0 . 8 8 1 . 6 3 1 . 7 1 1 . 7 8 1 . 8 7 7 . 8 7 7 . 9 6 O P O H P O O H O H C l O P O H O O O H O C l O H N N H O O β β γ γ α α x x x 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0 5 . 5 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0 f 1 ( p p m ) 0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 . 5 8 1 . 8 1 1 . 0 9 1 . 1 3 1 . 0 0 1 . 0 5 4 . 1 4 4 . 2 0 4 . 2 1 4 . 2 2 4 . 2 3 4 . 2 4 4 . 2 5 4 . 2 5 4 . 2 6 4 . 3 5 4 . 3 6 4 . 3 7 5 . 7 9 5 . 8 0 5 . 9 0 5 . 9 0 5 . 9 1 5 . 9 1 7 . 7 6 7 . 7 8 O P O H P O O H O H C l O P O H O O O H O C l O H N N H O O g f a , a ' b c d e f g e d , c b , a , a ' 144 Figure B6- MS (ESI) [M-H] - of β,γ-CCl 2 UTP. [M-H] - [M-H] 2- 145 Figure B7- 1 H NMR (400 MHz, D 2 O, pH 8.0) of β,γ-CHCl ATP. X= residual solvents Figure B8- 1 H NMR (243 MHz, D2O, pH 10.0) of β,γ-CHCl ATP. 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 5 . 0 5 . 5 6 . 0 6 . 5 7 . 0 7 . 5 8 . 0 8 . 5 9 . 0 f 1 ( p p m ) - 5 0 0 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0 3 0 0 0 3 5 0 0 4 0 0 0 4 5 0 0 5 0 0 0 5 5 0 0 6 0 0 0 6 5 0 0 7 0 0 0 1 . 1 3 1 . 9 6 0 . 9 5 0 . 9 8 1 . 0 0 0 . 8 7 0 . 9 7 3 . 8 6 4 . 0 8 4 . 0 9 4 . 1 1 4 . 1 2 4 . 1 2 4 . 1 3 4 . 1 4 4 . 1 5 4 . 1 6 4 . 1 6 4 . 1 7 4 . 2 7 4 . 2 8 4 . 2 8 4 . 4 5 4 . 4 6 4 . 4 7 6 . 0 1 6 . 0 2 8 . 1 2 8 . 4 1 g f e c a , a ' , b , d x x x x h O P O H P O O H O H C l O P O H O O O H O O H N N N N N H 2 a , a ' b c d e f g h -22 -20 -18 -16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 f1 (ppm) 0 50 100 150 200 250 300 350 400 1 . 0 1 1 . 0 0 0 . 9 5 -11.02 -10.91 6.64 6.75 8.44 α β γ 146 Figure B9- 1 H NMR (600 MHz, D 2 O, pH 7.5) of β,γ-CHCl UTP. X= residual solvents Figure B10- 31 P NMR (243 MHz, D 2 O, pH 7.5) of β,γ-CHCl UTP. 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 f1 (ppm) -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 8 . 4 2 5 . 5 0 1 . 1 5 3 . 1 7 2 . 0 6 1 . 9 6 1 . 0 0 1.12 1.14 1.15 3.04 3.05 3.06 3.08 3.80 3.83 3.86 4.11 4.14 4.24 4.25 4.26 4.28 4.28 5.83 5.85 5.86 7.82 7.84 f e , g X X d c b a a ' h X X O P O H P O O H O H C l O P O H O O O H O O H N N H O O a , a ' b c d e f g h -15 -10 -5 0 5 10 15 20 f1 (ppm) -50 0 50 100 150 200 250 300 350 400 450 500 550 600 1 . 0 3 0 . 9 2 1 . 0 0 -11.27 -11.16 4.12 9.24 β α γ 147 Figure B11- MS (ESI) [M-H] - of β,γ-CHCl UTP. [ M - H ] - [ M - 2 H + N a ] - [ M - 3 H + 2 N a ] - [ M - H ] 2 - 148 Figure B12- Purity analysis of β,γ-CHCl UTP (HPLC, 254 nm, C18 analytical column, flow 3.5 mL/min, isocratic mode, buffer: 0.1 M TEAB, pH 7.4 buffer, 7.5% acetonitrile). Figure B13- 31 P NMR of compound 1 (162 MHz, D 2 O, pH 10.0). X= impurities - 2 5 - 2 0 - 1 5 - 1 0 - 5 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 f 1 ( p p m ) - 1 0 0 1 0 0 3 0 0 5 0 0 7 0 0 9 0 0 1 1 0 0 1 3 0 0 1 5 0 0 1 7 0 0 1 . 0 0 1 . 1 5 8 . 4 7 1 5 . 8 9 x x O H P O O H P C l O O O H N O 2 β γ γ β 149 Figure B14- Purification of individual diastereomers (R)- and (S)-2 (HPLC-C18, analytical column, 280 nm, flow 3 mL/min, buffer: 0.1 M TEAB, pH 8.5, 32% acetonitrile) Figure B15- 1 H NMR (400 MHz, CD 3 OD) of compound 2 - HPLC fast. X= residual solvents, tea= triethylamine 0 min 14 min 8.5 min 9.4 min P P O O OH O HO Cl NO 2 N H a b c d e 150 Figure B16- 31 P NMR (162 MHz, CD 3 OD) of compound 2 - HPLC fast. P P O O OH O HO Cl NO 2 N H 151 Figure B17- MS (ESI) [M-H] - of compound 2 - HPLC fast. [M-H] - fragmentation [M-2H+Na] - 152 Figure B18- 1 H NMR (400 MHz, CD 3 OD) of compound 2 - HPLC slow. X= residual solvents, tea= triethylamine P P O O OH O HO Cl NO 2 N H a b c d e 153 Figure B19- 1 H 2D NMR (400 MHz, CD 3 OD) of the aromatic area of compound 2 - HPLC slow. P P O O OH O HO Cl NO 2 N H a b c d e f g h i j k l f g h i l j k f g h i l j k 154 Figure B20- 31 P NMR (162 MHz, CD 3 OD) of compound 2 - HPLC slow. P P O O OH O HO Cl NO 2 N H 155 Figure B21- MS (ESI) [M-H] - of compound 2 - HPLC slow. [M-H] - fragmentation [M-2H+Na] - 156 Figure B22- 1 H NMR (400 MHz, D 2 O, pH 1.0) of compound 3 - HPLC fast. X= residual solvents Figure B23- 31 P NMR (243 MHz, D 2 O, pH 9.1) of compound 3 - HPLC fast. 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 f1 (ppm) 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 1 . 0 0 1 . 7 1 0 . 8 3 0 . 8 5 0 . 8 1 0 . 7 6 4.10 4.14 4.18 5.45 5.47 7.53 7.55 7.57 7.76 7.78 7.80 7.88 7.91 8.14 8.17 x x x x x O H P O O H P C l O O O H N + O O - a a b b c d e f f c e d -5 0 5 10 15 20 25 30 35 40 45 f1 (ppm) -4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 0 . 9 8 1 . 0 0 8.74 15.82 O H P O O H P C l O O O H N + O O - 157 Figure B24- 1 H NMR (400 MHz, D 2 O, pH 1.0) of compound 3 - HPLC slow. X= residual solvents Figure B25- 31 P NMR (162 MHz, D 2 O, pH 1.0) of compound 3 - HPLC slow. 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 f1 (ppm) -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 1 . 0 0 1 . 8 8 1 . 0 0 0 . 9 8 0 . 9 6 0 . 9 5 3.96 4.00 4.04 5.33 5.35 7.41 7.43 7.45 7.64 7.67 7.68 7.77 7.79 8.03 8.05 O H P O O H P C l O O O H N + O O - a b c d e f x x a b f c d e 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 f1 (ppm) 0 50 100 150 200 250 300 350 400 1 . 1 5 1 . 0 0 11.07 12.73 O H P O O H P C l O O O H N + O O - 158 Figure B26- MS (ESI) [M-H] - of compound 3 - HPLC slow. [M-H] - [M-2H+Na] - 159 Figure B27- 31 P NMR (162 MHz, D 2 O, pH 9.7) of compound 4 - HPLC fast. -25 -20 -15 -10 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 f1 (ppm) -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 0 . 9 8 1 . 0 3 1 . 0 0 -11.38 -11.22 1.89 1.91 2.05 2.08 11.66 11.68 O P O H P O O H O C l O P O H O O O H O N N H O O O H N + O O - α β γ γ β α 160 Figure B28- MS (ESI) [M-H] - of compound 4 - HPLC fast. [M-H] - [M-2H+Na] - [M-H] 2- [M-3H+2Na] - 161 Figure B29- 31 P NMR (162 MHz, D 2 O, pH 9.2) of compound 4 - HPLC slow. -20 -10 0 10 20 30 40 50 60 70 f1 (ppm) -40 0 40 80 120 160 200 240 280 320 360 -11.40 -11.24 1.88 2.06 11.57 O P O H P O O H O C l O P O H O O O H O N N H O O O H N + O O - α β γ γ β α 162 Figure B30- MS (ESI) [M-H] - of compound 4 - HPLC slow. [M-H] - [M-H] 2- [M-2H+Na] - [M-3H+2Na] - 163 Figure B31- 1 H NMR (400 MHz, D 2 O, pH 6.5) of β,γ-CHCl UTP – HPLC fast. X= residual solvents Figure B32- 31 P NMR (162 MHz, D 2 O, pH 6.5) of β,γ-CHCl UTP – HPLC fast. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 f1 (ppm) -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 8 . 7 1 4 . 7 2 1 . 0 5 2 . 3 6 1 . 6 1 1 . 6 1 1 . 0 0 g f , h a e d x x x x x X c b b ' x -15 -10 -5 0 5 10 15 20 25 30 35 40 f1 (ppm) -50 0 50 100 150 200 250 300 350 400 450 500 550 0 . 9 1 1 . 0 8 1 . 0 0 -10.83 -10.66 3.76 3.79 3.91 10.03 β α γ 164 Figure B33- MS (ESI) [M-H] - of β,γ-CHCl UTP – HPLC fast. Figure B34- Purity check of β,γ-CHCl UTP – HPLC fast. [ M - H ] - [ M - 2 H + N a ] - [ M - 3 H + 2 N a ] - [ M - H ] 2 - [ M - 2 H + N a ] 2 - [ M ] f r a g m e n t 7/28/2017 10:51:01 AM Page 1 / 1 C:\LabSolutions\Data\Carolina\UMP_CHCl_final_iso1-CAII172a_iso1-purity_test1.lcd Analysis Report Sample Name : UMP_CHCl_final_iso1-CAII172a_iso1-purity_test Sample ID : UMP_CHCl_final_iso1-CAII172a_is Data Filename : UMP_CHCl_final_iso1-CAII172a_iso1-purity_test1.lcd Method Filename : CA_semiprep.lcm Batch Filename : Vial # : 1-1 Sample Type : Unknown Injection Volume : 200 uL Date Acquired : 7/28/2017 10:30:18 AM Acquired by : mckennalab Date Processed : 7/28/2017 10:48:22 AM Processed by : mckennalab <Chromatogram> min mAU 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 0 5 10 PDA Multi 1 254nm,4nm 4.534 10.579 <Peak Table> PDA Ch1 254nm Peak# 1 2 Total Ret. Time 4.534 10.579 Area 1571 341414 342985 Height 124 13305 13429 Area% 0.458 99.542 100.000 <Sample Information> 165 Figure B35- 1 H NMR (400 MHz, D 2 O, pH 6.5) of β,γ-CHCl UTP – HPLC slow. Figure B36- 31 P NMR (162 MHz, D 2 O, pH 6.5) of β,γ-CHCl UTP – HPLC slow. 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 f1 (ppm) -20 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 1 3 . 2 7 6 . 6 2 1 . 0 4 3 . 1 8 2 . 2 2 1 . 9 7 1 . 0 0 g f , h X c b b ' e d a x x x x x -24 -20 -16 -12 -8 -4 0 4 8 12 16 20 24 f1 (ppm) -20 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 0 . 8 8 1 . 0 0 1 . 0 0 -10.78 -10.61 3.89 10.02 β α γ 166 Figure B37- MS (ESI) [M-H] - of β,γ-CHCl UTP – HPLC slow. [ M ] f r a g m e n t [ M - H ] - [ M - 2 H + N a ] - [ M - 3 H + 2 N a ] - [ M - H ] 2 - [ M - 2 H + N a ] 2 - 167 Figure B38- Purity check of β,γ-CHCl UTP – HPLC slow. 7/28/2017 11:14:39 AM Page 1 / 1 C:\LabSolutions\Data\Carolina\UMP_CHCl_final_iso2-CAII172b_iso2-purity_test1.lcd Analysis Report Sample Name : UMP_CHCl_final_iso2-CAII172b_iso2-purity_test Sample ID : UMP_CHCl_final_iso2-CAII172b_is Data Filename : UMP_CHCl_final_iso2-CAII172b_iso2-purity_test1.lcd Method Filename : CA_semiprep.lcm Batch Filename : Vial # : 1-1 Sample Type : Unknown Injection Volume : 200 uL Date Acquired : 7/28/2017 10:52:22 AM Acquired by : mckennalab Date Processed : 7/28/2017 11:10:37 AM Processed by : mckennalab <Chromatogram> min mAU 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 0.0 2.5 5.0 7.5 PDA Multi 1 254nm,4nm 3.001 10.759 <Peak Table> PDA Ch1 254nm Peak# 1 2 Total Ret. Time 3.001 10.759 Area 3458 199287 202746 Height 778 8553 9331 Area% 1.706 98.294 100.000 <Sample Information> 168 Appendix C. Chapter 4 Supporting Data Figure C1- 1 H NMR (400 MHz, CD 3 OD) of azide 2. X=residual solvents Figure C2- 1 H NMR (500 MHz, CD 3 OD) studies of the different chemical shift between the desired product 2 (P) and the starting material (SM) 4-(bromomethyl)-3-nitrobenzoic acid. X=residual solvents 169 Figure C3- 13 C NMR (101 MHz, CD 3 OD). DMF= dimethylformamide. Previously published compound (refs 13 and 15 from Chapter 4). 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 f1 (ppm) -20 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 30.34 35.71 47.73 51.36 125.59 130.43 131.83 134.05 135.86 147.69 163.37 165.40 D M F D M F C D 3 O D 1 4 B r - C H 2 ( S M ) 8 A r o m a t i c s 2 , 3 , 5 , 6 , 7 170 Figure C4- MS ESI [M-H] - for azide 2. [2M-H] - [M-H] - 171 Figure C5- 1 H NMR (600 MHz, D 2 O pH 10.7) of 3. X=residual solvents. Previously published compound (refs 13 and 15 from Chapter 4). Figure C6- MS ESI [M-H] - for compound 3. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 f1 (ppm) -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 2 . 1 3 0 . 9 7 1 . 0 0 0 . 6 5 3.91 7.51 7.52 7.98 7.99 8.34 a b c d H D O x x Scanned by CamScanner į į Ţ ą Ą ñ [M-H] - 172 Figure C7- ISCO chromatography of compound 4. Conditions: solvent A- Hexane, solvent B- ethyl acetate, solvent B4- methanol, flow 40 mL/min, silica column 40g, wavelength 1 (red)- 254nm, wavelength 2 (purple)- 280nm, ELSD detector (green), area collected with desired product (yellow section). Figure C8- 1 H NMR (500 MHz, d-acetone) of 4. X=residual solvents. Previously published compound (refs 13 and 15 from Chapter 4). Sample: caii244-d Rf+ Wednesday 17 January 2018 01:28PM Page 1 of 1 RediSep Column: Silica 40g SN: E041508D9F7AB Lot: 272123604W Flow Rate: 40 ml/min Equilibration Volume: 240.0 ml Initial Waste: 0.0 ml Air Purge: 1.0 min Peak Tube Volume: Max. Non-Peak Tube Volume: Max. Loading Type: Solid Wavelength 1 (red): 254nm Peak Width: 2 min Threshold: 0.20 AU Wavelength 2 (purple): 280nm Evaporative Light Scattering (green) Peak Width: 2 min Threshold: 0.05 v Spray Temperature: 30C Drift Temperature: 60C Run Notes: 0.0 5.0 10.0 15.0 20.0 0 10 20 30 40 50 60 70 80 90 100 0.00 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 0.00 0.00 0.25 0.50 0.75 1.00 ELS Absorbance Percent B 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 B4 Run Length 24.0 Min Duration %B Solvent A Solvent B 0.0 0.0 hexane ethyl acetate 1.0 0.0 hexane ethyl acetate 5.0 35.6 hexane ethyl acetate 3.9 35.6 hexane ethyl acetate 0.0 35.6 hexane ethyl acetate 2.3 52.0 hexane ethyl acetate 3.0 52.0 hexane ethyl acetate 0.0 52.0 hexane ethyl acetate 3.6 78.2 hexane ethyl acetate 0.2 100.0 hexane ethyl acetate ... ... ... ... Peak # Start Tube End Tube 1 A:41 A:42 2 A:43 A:44 3 A:45 A:46 4 A:47 A:47 5 A:48 A:52 6 A:53 A:57 7 A:58 A:60 8 A:61 A:61 9 A:62 A:63 ... ... ... 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 Rack A 18 mm x 150 mm Tubes 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 f1 (ppm) -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 2 . 9 1 2 . 0 0 1 . 9 5 1 . 8 8 4 . 2 2 2 . 0 4 3 . 8 8 1 . 9 4 0 . 7 7 2.05 2.29 3.69 7.12 7.14 7.20 7.21 7.23 7.30 7.31 7.33 7.42 7.43 7.55 7.57 8.35 8.37 8.38 8.39 8.48 8.49 d - a ce t o n e C H C l 3 x d e a b , c M t t a r o m a t i c s x 173 Figure C9- MS ESI [M-H] - for compound 4. [M-H] - [2M-2H+Na] - [3M-3H+2Na] - [M-H+2Na] - 174 Figure C10- 1 H NMR (600 MHz, CD 3 OD) of 5. Previously published compound (refs 13 and 15 from Chapter 4). Figure C11- MS ESI [M-H] - for compound 5. 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 f1 (ppm) -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 3 . 2 1 2 . 0 0 2 . 4 3 3 . 3 4 4 . 6 5 3 . 3 0 4 . 1 7 2.29 3.29 3.33 3.35 4.85 7.09 7.11 7.16 7.17 7.18 7.19 7.26 7.27 7.28 7.34 7.35 7.47 7.49 d e C D 3 O D C H 3 O H H 2 O A r o m a t i c s ( 1 7 H ) [M-H] - [2M-H] - [3M-H] - 175 Figure C12- 1 H NMR (400 MHz, CDCl 3 ) of benzoyl chloride 9. Previously published compound (ref 16 from Chapter 4). Figure C13- 1 H NMR (400 MHz, CDCl 3 ) studies of the different chemical shift between the desired product benzoyl chloride 9 (P) and the starting material 2-fluoro-5-nitrobenzoic acid (SM). 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8.0 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 9.0 9.1 9.2 9.3 9.4 f1 (ppm) 0 50 100 150 200 250 300 350 400 450 2 . 9 2 1 . 7 4 1 . 1 3 1 . 6 3 1 . 0 0 7.26 7.37 7.39 7.39 7.41 7.42 7.44 8.47 8.48 8.48 8.49 8.49 8.50 8.50 8.51 8.52 8.53 8.53 8.54 8.54 8.55 8.55 8.56 8.94 8.95 8.95 8.96 8.99 8.99 9.00 9.01 P P S M S M P + S M 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 f1 (ppm) -50 0 50 100 150 200 250 300 350 400 450 500 1 . 1 0 1 . 1 0 1 . 0 0 1.55 7.26 7.39 7.41 7.44 8.52 8.53 8.53 8.54 8.54 8.55 8.55 8.56 8.99 9.00 9.01 9.01 a b c C D C l 3 H 2 O 176 Figure C14- LCMS of 1Aa-NO 2 . MS spectrum (below) shows m/z values for the peak at 8.13 min. Previously published compound (ref 15 from Chapter 4). [M+H] + [2M+H] + [3M+H] + 177 Figure C15- Semi-preparative RP-C18 HPLC of 1Aa (t r = 25.9 min). Conditions: 3.5 mL/min, 254 nm, gradient mode, A/ 0.1% formic acid 5% acetonitrile in H 2 O and B/ 0.1% formic acid 10% acetonitrile in H 2 O: 0 to 20 min (A), 20 to 40 min (B). 3/14/2018 5:23:38 PM Page 1 / 2 C:\LabSolutions\Data\Carolina\CA_II_SP_NH2_March14_t4_r2.lcd Analysis Report Sample Name : CA_II_SP_NH2_March14_t4_r1 Sample ID : CA_II_SP_NH2_March14_t4_r1 Data Filename : CA_II_SP_NH2_March14_t4_r2.lcd Method Filename : CA_II_SP_NH2_MArch13_gradient.lcm Batch Filename : Vial # : 1-1 Sample Type : Unknown Injection Volume : 10 uL Date Acquired : 3/14/2018 11:40:44 AM Acquired by : mckennalab Date Processed : 3/14/2018 12:36:47 PM Processed by : mckennalab <Chromatogram> min mAU 0 10 20 30 40 50 0 500 1000 1500 2000 PDA Multi 1 254nm,4nm 3.556 3.954 4.271 4.492 5.897 7.747 9.299 9.960 10.688 12.116 20.148 25.981 26.300 26.954 27.239 27.748 28.800 30.531 31.968 32.585 33.646 39.049 40.230 47.728 <Peak Table> PDA Ch1 254nm Peak# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Ret. Time 3.556 3.954 4.271 4.492 5.897 7.747 9.299 9.960 10.688 12.116 20.148 25.981 26.300 26.954 27.239 27.748 28.800 30.531 Area 365767 1264802 808794 683955 64611 55834 43466 12981 211573 579101 456197 14464451 120375 317560 162513 153101 937960 1517405 Area% 1.463 5.060 3.236 2.736 0.259 0.223 0.174 0.052 0.846 2.317 1.825 57.872 0.482 1.271 0.650 0.613 3.753 6.071 <Sample Information> 178 Figure C16- LCMS of 1Aa. MS spectrum (below) shows m/z values for the 14.11 min peak. [M+H] + [M+TEA] + 179 Figure C17- 1 H NMR (400 MHz, D 2 O) of 1Aa. Previously published compound (ref 15 from Chapter 4). 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 f1 (ppm) -20 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 3 . 4 5 3 . 0 0 6 . 0 3 2 . 5 6 2 . 5 4 0 . 8 8 0 . 9 1 1 . 2 5 1 . 5 7 1 . 1 6 1 . 3 0 1 . 1 6 1 . 5 4 1 . 0 5 0.86 0.98 1.01 1.03 1.05 1.66 1.82 1.93 2.07 3.01 3.03 3.05 3.59 3.61 3.72 4.14 4.19 4.32 4.34 4.79 7.09 7.11 7.20 7.22 7.28 7.76 7.95 a T E A T E A D 2 O M e t h a n o l j n , o , p , k d , m , h , i , q b , c , e , f l x x 180 Figure C18- MS ESI [M-H] - for 1Aa-COOH (no Boc). Scanned by CamScanner [M-H] - 181 Figure C19- MS ESI [M-H] - for 1Aa-COOH. Scanned by CamScanner ļ ļ į ş Ùș ĵÈ î ļ ĺļĮįţį ļ £ ļįţå į [M-H] - 182 Figure C20- LCMS of compound 14 (entry 5 from Table 4.3). MS spectrum (below) shows m/z values for the 11.72 min peak. LCMS conditions: flow 1.0 mL/min, buffer A: 0.1% formic acid H 2 O, 10% acetonitrile, buffer B: 0.1% formic acid H 2 O, 75% acetonitrile, gradient method: 0-5 min 100% A; 5-7 min 0-40% of B; 7-15 min 40% B; 15-20 min 40- 100% of B and 20-25 min of 100% B. [M-H] - [2M-H] - 183 Figure C21- LCMS of compound 14 (entry 6 from Table 4.3). MS spectrum (below) shows m/z values for the 9.71 min peak. LCMS conditions: flow 1.0 mL/min, buffer A: 0.1% formic acid H 2 O, 10% acetonitrile, buffer B: 0.1% formic acid H 2 O, 75% acetonitrile, gradient method: 0-5 min 100% A; 5-7 min 0-40% of B; 7-15 min 40% B; 15-20 min 40- 100% of B and 20-25 min of 100% B. [M-H] - [2M-H] - 184 Figure C22- MS ESI [M-H] - of compound 14. Scanned by CamScanner [M-H] -

Abstract (if available)

Abstract DNA polymerase β (DNA pol β) is responsible for DNA repair mechanisms that are involved with gap-filling DNA synthesis in the single nucleotide base excision repair (BER) pathways. BER is very important in maintaining healthy cells because it removes the damaged base, avoiding further mutations. On another hand, RNA polymerases are involved in the transcription process, where a single-stranded mRNA is created from a dsDNA chain, in combination with a diverse number of general transcription factors. This process is crucial for cell growth and differentiation and therefore studying the mechanism of action of these enzymes is of extreme importance. The functionality and structure of both DNA pol β and RNA polymerase II (RNA pol II), as well as their fidelity mechanism and the use of these enzymes as potential cancer therapeutic targets, is explored in detail in the introductory chapter, Chapter 1. ❧ In a complementary manner, Chapter 2 is focused on the synthesis of β,γ-CHF dCTP probes for DNA pol β. A series of β,γ-CXY dNTP compounds have been extensively synthesized by the McKenna lab over the years in order to provide an accessible tool kit of dNTPs to study how DNA pol β cleaves the bisphosphonate moiety of the nucleotide depending on the different CXY derivatives they possess. The diastereomers of β,γ-CHF dCTP were synthesized, analyzed and both kinetically and structurally studied. When observing similar electronegative effects of CXY, it can be seen that the dihalo derivatives display a lower kₚₒₗ when compared with the monohalo and non-halo line (Figure 2.3). ❧ In addition, Chapter 2 further explores the use of small molecule inhibitors to target the lyase domain of DNA pol β. Previous studies have shown that pamoic acid has lyase inhibitory properties, therefore we aimed to synthesize modified pamoic acid derivatives, which possess a phosphorus moiety, and test their inhibitory effect. Compound 8 from Generation 2 (Scheme 2.4) was successfully synthesized and characterized and shown to inhibit the lyase domain of DNA pol β at a concentration of 500 μM (Figure 2.12). ❧ Moreover, the synthesis of β,γ-CXY UTP probes to study the mechanism and fidelity of RNA pol II is analyzed in Chapter 3. This unique enzyme possesses a well-organized network, using a crucially conserved motif called the trigger loop. β,γ-CCI₂ UTP, β,γ-CCI₂ ATP, β,γ-CHCl UTP and β,γ-CHCl ATP were synthesized and the individual diastereomers β,γ-CHCl-1 and β,γ-CHCl-2 UTP were successfully isolated (Scheme 3.2). According to in vitro transcription assays, β,γ-CCI₂ UTP showed a lower incorporation rate when compared with the fluorine derivative, but higher than he methylene version (Figure 3.6). The optimal coupling step conditions were also examined. ❧ Lastly, Chapter 4 is focused on the solid-phase synthesis of the bisphosphonate conjugate 1Aa to target Tropomyosin receptor kinase B and C (TrkB and TrkC) in the inner ear. Recently, our group has published preliminary data regarding the use of a bisphosphonate-linked TrkB agonist as a delivery method to reach the cochlea. 1Aa was synthesized over 12 steps, 7 of them being in solid-phase support with an overall yield of 16% (Scheme 4.1). 1Aa promotes spiral ganglion neurite outgrowth in vitro and also bolsters the regeneration of cochlear ribbon synapses in vitro (Figures 4.4 and 4.5). Further studies involve the synthesis of the bisphosphonate counterpart, RIS-1Aa, and its effect on the spiral ganglion neurite outgrowth and the regeneration of cochlear ribbon synapses.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Novel stereochemical probes for DNA polymerases: nucleoside triphosphate beta,gamma-CXY analogues

PDF

Design and synthesis of a series of methylenebisphosphonates: a nucleotide analogue toolkit to probe nucleic acid polymerase structure and function

PDF

Nucleotide analogs and molecular probes for LFER, time-resolved crystallography, FRET, and EPR studies of human DNA polymerase: probing mechanism, conformation and structure

PDF

Deoxynucleotide analog probes and model compounds for studying DNA polymerase structure and mechanism: synthesis and evaluation of alkyl-, azido-, and halomethylene bisphosphonate-substituted tri...

PDF

Deoxyribonucleoside triphosphate analogues for inhibition of therapeutically important enzymes

PDF

Nucleophilic fluorination of bisphosphonates and its application in PET imaging

PDF

Fluorinated probes of enzyme mechanisms

PDF

Expansion of deoxynucleotide analog probes for studying DNA polymerase mechanism: synthesis of novel beta, gamma-CXY deoxycytidine series

PDF

Studies of bisphosphate-conjugated fluorescent imaging compounds and 8-oxo-dGTP derivatives

PDF

I. Microwave-assisted synthesis of phosphonic acids; II. Design and synthesis of polymerase β lyase domain inhibitors

Asset Metadata

Creator Amador, Carolina D. (author)

Core Title Synthetic studies of chemical probes for i) DNA, ii) RNA polymerases and iii) tropomyosin receptor kinase

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 02/20/2019

Defense Date 02/16/2019

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag bisphosphonate,cancer,chemical probes,DNA,hearing loss,OAI-PMH Harvest,polymerases,RNA,triphosphate analogues,tropomyosin receptor kinase

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor McKenna, Charles E. (committee chair)

Creator Email carolina.d.amador@gmail.com,cdamador@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-125319

Unique identifier UC11675279

Identifier etd-AmadorCaro-7093.pdf (filename),usctheses-c89-125319 (legacy record id)

Legacy Identifier etd-AmadorCaro-7093.pdf

Dmrecord 125319

Document Type Dissertation

Format application/pdf (imt)

Rights Amador, Carolina D.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Repository Email cisadmin@lib.usc.edu