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UNDERSTANDING SULFUR BASED REDOX BIOLOGY THROUGH ADVANCEMENTS in CHEMICAL BIOLOGY by THOMAS POOLE a Dissertation Submitted to T

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UNDERSTANDING SULFUR BASED REDOX BIOLOGY THROUGH ADVANCEMENTS IN CHEMICAL BIOLOGY BY THOMAS POOLE A Dissertation Submitted to the Graduate Faculty of WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Chemistry May 2017 Winston-Salem, North Carolina Approved By: S. Bruce King, Ph.D., Advisor Leslie B. Poole, Ph.D., Chair Patricia C. Dos Santos, Ph.D. Paul B. Jones, Ph.D. Mark E. Welker, Ph.D. ACKNOWLEDGEMENTS First and foremost I would like to thank professor S. Bruce King. His approach to science makes him an excellent role model that I strive to emulate. He is uniquely masterful at parsing science into important pieces and identifying the gaps and opportunities that others have missed. I thank him for the continued patience and invaluable insight as I’ve progressed through my graduate studies. This work would not have been possible without his insight and guiding suggestions. I would like to thank our collaborators who have proven invaluable in their support. Cristina Furdui has provided the knowledge, context, and biochemical support that allowed our work to be published in esteemed journals. Leslie Poole has provided the insight into redox biology and guidance towards appropriate experiments. I thank my committee members, Mark Welker and Patricia Dos Santos who have guided my graduate work from the beginning. Professor Dos Santos provided the biological perspective to evaluate my redox biology work. In addition, she helped guide the biochemical understanding required to legitimize my independent proposal. Professor Welker provided the scrutinizing eye for my early computational work and suggested validating the computational work with experimental data. Professor Paul Jones was kind enough to join my committee on late notice, and I look forward to his questions about radicals. Professor Leslie Poole will act as my committee chair and I thank her for taking the time out of her busy schedule to lead the assessment. I thank you all for your valuable time in reviewing my work. I thank all of my professors at Wake Forest. Professor Paul Jones helped hone my understanding of reactive intermediates and synthesis from a rudimentary understanding ii to a complex tool box capable of being applied throughout my work. Professor Amanda Jones bolstered my understanding of physical organic chemistry and provided the basis of knowledge (strain concepts) that lead to my first publication here. Professor Marcus Wright expanded my understanding of spectroscopy to the point where I feel confident in accurately assigning structure to complex molecules with a variety of techniques. In addition, Marcus has provided continued support and advice that has proven valuable both in science and in life. Professor Mark Welker opened my eyes to the possibilities of transition metal organic chemistry, and provided the background to understanding and successfully executing the ring closing metathesis shown in this work. I would like to thank all members of Team King, past and present. My lab mates throughout the years have provided invaluable insight, advice, and company when the going got tough. Zhengrui Miao and I started at the same time and he has been an excellent sounding board, and source of insightful alternative perspectives. Julie Reisz contributed heavily to my first publication here, and provided strong advice for how to navigate graduate school. Most importantly, I’d like to thank my family and friends. Mom and Dad, you have given me everything I need to succeed. My accomplishments would not be possible without you. You have raised me to be the scientist and person I am today. A sincere thanks to my girlfriend Kelsey Shore, I don't know how you’ve put up with me as I’ve written this dissertation. Thank you for the continued support and understanding. I’d also like to thank my bike people, you all keep my wheels rollin. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................ ii TABLE OF CONTENTS ................................................................................................... iv LIST OF FIGURES ........................................................................................................... vi LIST OF SCHEMES.......................................................................................................... ix ABBREVIATIONS .......................................................................................................... xii LIST OF STRUCTURES ................................................................................................ xiv ABSTRACT ...................................................................................................................... xv CHAPTER 1 SULFENIC ACIDS AND REDOX BIOLOGY .......................................... 1 1.1 An Introduction to Sulfenic Acids in Redox Biology .................................... 2 1.2 Disease ............................................................................................................ 7 1.3 Redox Balance and Signaling ....................................................................... 11 1.4 Cysteine and Sulfenic acids .......................................................................... 19 1.5 Synopsis ........................................................................................................ 32 CHAPTER 2 STRAINED CYCLOALKYNES AS NEW PROTEIN SULFENIC ACID TRAPS ......................................................................................................................... 34 2.1 Introduction .................................................................................................. 36 2.2 Evaluating the reactivity and selectivity of strained alkynes for sulfenic acids ............................................................................................................................ 37 2.3 Evaluating BCN and BCN-Bio in proteins .................................................. 45 2.4 Conclusion .................................................................................................... 62 2.5 Materials and Methods ................................................................................. 63 CHAPTER 3 OPTIMIZING ALKYNE BASED SULFENIC ACID TRAPS ................ 79 3.1 Reactions with thiols .................................................................................... 79 3.2 Persulfide Addition to Alkynes .................................................................... 84 3.3 Optimizing Strain and Electronics of Alkynes. ............................................ 86 3.4 Results .......................................................................................................... 92 iv 3.5 Discussion and Future Aims ....................................................................... 107 3.6 Methods Section ......................................................................................... 110 CONCLUSIONS AND FUTURE OUTLOOK .............................................................. 132 REFERENCES ............................................................................................................... 134 SCHOLASTIC VITA ..................................................................................................... 153 v LIST OF FIGURES Figure 1. Radical initiation, propagation, and termination. ................................................ 3 Figure 2. Lipid oxidation. ................................................................................................... 4 Figure 3. Oxidations of nitrogenous bases, ribose backbone, and amino acids .................. 5 Figure 4. Oxidation of guanine to 8-oxoguanine. ............................................................... 9 Figure 5. Oxidation of arachidonic acid by ROS or lipoxygenase. ................................. 10 Figure 6. Hypothetical transition state of AhpC ............................................................... 13 Figure 7. Peroxiredoxins are recycled by thioredoxins .................................................... 14 Figure 8. Peroxiredoxin hyperoxidation and resurrection ................................................ 16 Figure 9. Biological reactions and fates of sulfenic acids.. .............................................. 20 Figure 10. Oxidation states & nucleophilic/electrophilic reactions .................................. 22 Figure 11. Stable organic sulfenic acids ........................................................................... 22 Figure 12. AhpC with resolving and peroxidatic cysteine thiols ...................................... 24 Figure 13. Early dicarbonyl probes for sulfenic acids. ..................................................... 26 Figure 14. Commercially available 1,3 dicarbonyl probes. .............................................. 27 Figure 15. Properties of nucleophilic probes. ................................................................... 29 Figure 16. The electrophilic probe NBD-Cl ..................................................................... 31 Figure 17. Examples of sulfenic acid reacting with pi bonds. .......................................... 32 Figure 18. UV-Vis Kinetics; Fries Acid + BCN in acetonitrile. ...................................... 40 Figure 19. UV-Vis Kinetics; Fries Acid + BCN in 50:50 MeCN:H2O ............................. 41 Figure 20. UV-Vis Kinetics; Fries Acid + trans-cyclooctene (tCOT)
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