Development of Bifunctional Alkylating Agents for Association and Migration along DNA By Mark A. Hutchinson A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland August 2016 © 2016 Mark A. Hutchinson All rights reserved Abstract Environmental toxins and a number of drugs have been shown to react with and cause damage to cellular components including DNA. Alkylation of DNA has been shown to result in mutations that may cause detrimental effects to the cell, including cancer. One class of DNA alkylating agents is quinone methides (QM). These compounds are highly electrophilic and are generated by a variety of anti-cancer compounds such as mitomycin C. In order to further understand their ability to alkylate DNA both their selectivity and mechanism of action must be studied. These intermediates have been shown to form from metabolism inside of cells and have been found to alkylate DNA in both an irreversible and reversible manner. The reversible DNA adducts may persistent enough to elicit a cellular response, but are difficult to observe for standard analysis. In order to study the QMs ability to alkylate DNA, a simple QM was used to observe reversible DNA adducts. These adducts could be irreversibly trapped through the use of bis[(trifluoroacetoxy)iodo]benzene (BTI). Once oxidized through the use of BTI, the reversible QM-DNA adducts could withstand lengthy analysis (>24 h) for detection by LC/MS analysis. Additionally, QMs have been synthesized as bifunctional alkylating agents capable of forming interstrand crosslinking within DNA (BisQM). Once crosslinked, BisQM is able to exploit the reversible nature of their DNA-adducts providing a potential to migrate along DNA. This has been shown through the use of a BisQM containing an acridine moiety for the association to DNA, however migration was relatively slow (~5% interstrand crosslinks (ISC) after 7 days)). By exchanging the DNA associating moiety ii from acridine to ammonium complexes, the molecule might be able to slide along the backbone of DNA and allow the QM to migrate rapidly. Several new BisQMs containing varying amounts of positive charges and electron density were synthesized. These compounds had a lower association to DNA than its BisQM acridine counterpart, but were able to generate ISC at a faster rate (~4 h). However, these compounds were unable to exhibit high ISC yields in strand transfer, exchange, or migration. Through additional modifications of BisQMs it is possible to increase the ability of the compounds to associate and migrate along DNA. Thesis Advisor: Dr. Steven E. Rokita Readers: Dr. Marc M. Greenberg, Dr. John P. Toscano iii Dedication: For my Grandparents: Stephen Donald and Mary Elizabeth Hutchinson iv Acknowledgements First and foremost, I would like to thank my advisor Dr. Rokita for the opportunity to work on this project. I could not have asked for a better mentor. You have always given me the support and motivation to further develop myself into a better scientist. You have always been there for discussion with an open mind for the freedom to advance my project and make it my own. I always left your office more confident in the progress and path that the project was heading, as well as in my own abilities. I cannot thank you enough for your continuing support and encouragement throughout these years. I hope to remain close as colleagues and friends. I would also like to thank my committee members Dr. Greenberg and Dr. Toscano for taking the time out of their busy schedules to accommodate me. Dr. Greenberg, thank you for allowing me to audit your nucleic acids course to improve my knowledge of chemistry as well as opening your lab doors for any questions I might have had in regards to troubleshooting PAGE analysis. I would also like to thank Dr. Toscano for assisting me in my job search with the letters of recommendations and allowing me to stop by your lab to talk science and football with Tyler. Also thank you for being so kind as to lend me several compounds here and there for small scale trail reactions. Thank you to all of the other Johns Hopkins professors who taught my classes and a special thank you to those that I had the privilege to TA for. I would like to specifically thank Dr. Cathy Moore and Joel Tang for their help with NMR experiments and for improving my knowledge of the instruments and their capabilities. Also, a special thank you to Dr. Phil Mortimer for running all of my FAB-HRMS samples and maintaining all v of the instrumentation to include the LC-MS and UPLC; these instruments were invaluable to my research. Additionally, thank you to the staff here at Johns Hopkins for always being available to take care of ordering, scheduling, printing, and being able to answer any question that I had. I’d also like to thank the staff for the happy hours and holiday parties; they were always a great treat to the end of a week in the lab. Thank you to all members of the Rokita Lab, both past and present. Everyone was a privilege to work with and was extremely friendly and welcoming from the day I joined. Especially thank you, Dr. Mike McCrane for showing me the ropes when I first joined the lab and allowing me to assist on his project which resulted in my first publication. My desk-mate Dr. Petrina Boucher for being there to talk to and listening when things weren’t working. Our discussions were very useful to my research. Dr. Abhishek Phatarphekar, thank you for making the lab fun to come to, especially during football season and for all of your assistance and comradery during the writing of our theses. A final thank you to Shane Byrne for saving me several trips into Baltimore by coming in at odd times to freeze time points for me; good luck with the rest of grad school. I’m happy to be handing the project over to you and I know that it is in good hands. I would like to thank my Mom and Dad for their continued support not only throughout graduate school but in everything that I’ve ever embarked upon. Thank you to my brother David for always being there when I needed a break from chemistry. Thank you to my sister Kelly and brother-in-law Jason for your assistance in preparing me for graduate school entrance exams while I was in Afghanistan, and for listening and reassuring me about my project and graduate school. Additionally I would like to thank vi my cousin Erin for being there for me while down in the DMV area, and my Aunt Theresa for all of her support, encouragement, and always being there to share a beer with. Lastly, thank you to all of the Marines and Sailors that I’ve had the honor of serving with. You are an inspiration for achieving all that I have. Semper Fidelis. Finally, thank you to my loving wife Alison. You have been my biggest supporter during my time in graduate school. Thank you for putting up with all of the complaining, unknown times I’d be home, and the early morning start to the work days. Words cannot explain how much you mean to me and I can never thank you enough for all you’ve done to help me pursue this endeavor. I love you and cannot wait to start the next chapter of our lives together. Tungsten and Bella, thank you for the unconditional love you gave me each and every day that I came home. You made even the worst days of failed experiments so much better. vii Table of Contents Abstract ............................................................................................................................... ii Dedication: ......................................................................................................................... iv Acknowledgements ............................................................................................................. v List of Figures .................................................................................................................... xi List of Schemes ................................................................................................................ xiv List of Tables ................................................................................................................... xvi List of Supporting Figures .............................................................................................. xvii List of Abbreviations ...................................................................................................... xxii Chapter 1: Introduction ..................................................................................................... 1 Chapter 1.1. Deoxyribonucleic Acid ............................................................................... 1 1.2. DNA Damage and Repair ........................................................................................ 3 1.2.1 DNA Damage ..................................................................................................... 3 1.2.1 DNA Repair ........................................................................................................ 4 1.3. DNA Alkylation ....................................................................................................... 5 1.3.1 Reversible and Non-Reversible Alkylation ........................................................ 5 1.3.2 Non-Reversible Alkylating Agent ...................................................................... 6 1.3.2 Reversible Alkylating Agent .............................................................................. 8 1.4. Quinone Methides .................................................................................................