Development of Bifunctional Alkylating Agents for Association and Migration along DNA

194 Pages·2016·7.47 MB·English

by  Mark Hutchinson

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
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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

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Dedication:
For my Grandparents:
Stephen Donald and Mary Elizabeth Hutchinson

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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
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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
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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.

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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 .................................................................................................. 10
1.4.1. Quinone Methides as Alkylating Agents ......................................................... 10
1.4.2. Quinone Methides and Migration .................................................................... 12
Chapter 2: Oxidative Trapping of a Model Quinone Methide on dA-N1 and Analysis of
Adducts in Single and Double Stranded DNA ................................................................. 17
2.1 Introduction ............................................................................................................. 17
2.2 Results and Discussion ............................................................................................ 18
2.2.1 Trapping and Characterization of dA-N1 ......................................................... 18
2.2.2 Deglycosylation of dG-N7 Adduct ................................................................... 25
2.2.3. Trapping Studies of o-QM with ssDNA and dsDNA ...................................... 27
2.3. Summary ................................................................................................................ 30
2.4. Materials and Methods ........................................................................................... 30
2.4.1. Materials .......................................................................................................... 30
2.4.2. Methods ........................................................................................................... 31
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2.4.2.2. Oxidative Trapping and Detection of MeQM-DNA Adducts in DNA .... 32
Chapter 3: Development of a New Quinone Methide Precursor for Association and
Migration along DNA. ...................................................................................................... 34
3.1. Introduction ............................................................................................................ 34
3.2. Results and Discussion ........................................................................................... 35
3.2.1. Synthesis of BisQMPN .................................................................................. 35
1
3.2.1.1. Tyramine as a Starting Material ................................................................ 35
3.2.1.2. 4-Hydroxyphenethyl Bromide .................................................................. 37
3.2.1.3. 3 (4-Hydroxyphenyl)propanoic acid ......................................................... 37
3.2.1.4. Acetylation of Benzylic TBDMS Alcohols .............................................. 39
3.2.1.5. Methylation of Tertiary Amine – (BisQMPN ) ....................................... 40
1
3.2.2. Synthesis of BisQMPN .................................................................................. 42
2
3.2.3. Synthesis of BisQMPN .................................................................................. 44
3
3.2.3.1. Triamination of Compound 5.................................................................... 44
3.2.4. Reaction of BisQMPN with DNA ................................................................. 47
x
3.2.4.1 Aggregation of DNA through Noncovalent Interactions with Amines ..... 47
3.2.4.2. Concentration Dependence of BisQMPN ............................................... 52
x
3.2.4.3. Time Dependence for the Formation of Interstrand Crosslinks ................ 53
3.2.4.4. Strand Transfer and Exchange of BisQMPN .......................................... 54
x
3.2.4.4. Migration of BisQMPN ....................................................................... 57
2&3
3.3. Summary ................................................................................................................ 59
3.4. Materials and Methods ........................................................................................... 60
Chapter 4: Development of an Electron Rich Bis-Quinone Methide for Association and
Migration along DNA. ...................................................................................................... 72
4.1. Introduction ............................................................................................................ 72
4.2. Results and Discussion ........................................................................................... 74
4.2.1. Synthesis of eBisQMPN ............................................................................ 74
2&3
4.2.2. Reaction of eBisQMPN with DNA ............................................................ 75
2&3
4.2.2.1. Concentration Dependence of BisQMPN ........................................... 76
2&3
4.2.2.2. Time Dependence for the Formation of Interstrand Crosslinks ................ 77
4.2.2.3. Quenching of eBisQMPN ................................................................... 78
2&3
4.2.2.4. Strand Transfer and Exchange of eBisQMPN .................................... 79
2&3
4.2.2.5. Migration of eBisQMPN ..................................................................... 81
2&3
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4.2.2.6. Over Alkylation of ssDNA ....................................................................... 82
4.3. Summary ................................................................................................................ 84
4.4 Materials and Methods ............................................................................................ 85
4.4.1 Materials ........................................................................................................... 85
4.4.2 Methods ............................................................................................................ 85
Chapter 5: Synthesis of Di-Quinone Methides................................................................ 95
5.1 Introduction: ............................................................................................................ 95
5.2 Results and Discussion ............................................................................................ 98
5.2.1 Synthesis of Di-QMPN ................................................................................... 98
x
5.2.1.1 Hydroxymethylation .................................................................................. 98
5.2.1.2 TBDMS Protection .................................................................................... 99
5.2.1.3 Amination and Coupling of DiQMN Precursors ................................... 100
x
5.2.1.4 Benzylic TBDMS Cleavage ..................................................................... 101
5.2.1.5 Acetylation ............................................................................................... 102
5.2.1.6 Methylation of Amines ............................................................................ 103
5.2 Summary ............................................................................................................... 104
5.3 Materials and Methods .......................................................................................... 105
5.3.1 Materials ......................................................................................................... 105
5.3.2 Methods .......................................................................................................... 105
Chapter 6: Conclusions ................................................................................................. 112
References ....................................................................................................................... 116
Appendix A: Supporting Information for Chapter 2 ..................................................... 121
Appendix B: Supporting Information for Chapter 3 ..................................................... 124
Appendix C: Supporting Information for Chapter 4 ..................................................... 145
Appendix D: Supporting Information for Chapter 5 ..................................................... 157
Curriculum Vitae ............................................................................................................ 168

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