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Sulfur and Selenium in Peptides and Proteins: Part I – Chemoselective Methods for Disulfide Bond Ormation,F Part Ii – Function of Selenium in Enzymes

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Sulfur and Selenium in Peptides and Proteins: Part I – Chemoselective Methods for Disulfide Bond Ormation,F Part Ii – Function of Selenium in Enzymes University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2020 Sulfur And Selenium In Peptides And Proteins: Part I – Chemoselective Methods For Disulfide Bond ormation,F Part Ii – Function Of Selenium In Enzymes. Emma Jean Ste.Marie University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Organic Chemistry Commons Recommended Citation Ste.Marie, Emma Jean, "Sulfur And Selenium In Peptides And Proteins: Part I – Chemoselective Methods For Disulfide Bond ormation,F Part Ii – Function Of Selenium In Enzymes." (2020). Graduate College Dissertations and Theses. 1270. https://scholarworks.uvm.edu/graddis/1270 This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. SULFUR AND SELENIUM IN PEPTIDES AND PROTEINS: PART I – CHEMOSELECTIVE METHODS FOR DISULFIDE BOND FORMATION, PART II – FUNCTION OF SELENIUM IN ENZYMES. A Dissertation Presented by Emma Jean Ste.Marie to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Chemistry August, 2020 Defense Date: June 4, 2020 Dissertation Examination Committee: Robert J. Hondal, Ph. D., Advisor Robert J. Kelm, Ph. D., Chairperson Matthew D. Liptak, Ph. D. Jose S. Madalengoitia, Ph. D. Cynthia J. Forehand, Ph. D., Dean of the Graduate College 1 ABSTRACT Selenocysteine (Sec), the 21st proteogenic amino acid, was first identified in 1976 by Thressa Stadtman. In proteins, Sec replaces the far more common sulfur-containing amino acid cysteine (Cys). A key question since Stadtman’s discovery is: Why does Sec replace Cys? This question is especially relevant since Cys-orthologs of Sec-enzymes catalyze the identical reaction with only slightly reduced efficiency, and incorporation of Sec into a protein is much more complicated and bioenergetically costly compared to Cys. The study of selenoproteins is very difficult because Sec is incorporated into proteins by recoding a UGA stop codon as a sense codon. Production of recombinant selenoproteins involves reconstituting the recoding machinery. Alternatively, selenoproteins can be produced using a combination of recombinant DNA technology and peptide synthesis. While the development of solid phase peptide synthesis has provided a synthetic route for studying Sec, the Sec side chain requires the use of sturdy protecting groups (PGs) during synthesis. Work herein first addresses complications associated with Sec and Cys PGs, which until now have required harsh conditions for removal. My work has developed facile new methods for the deprotection of Sec and Cys residues. For Sec, we found that the use of DTNP to remove various PGs with subsequent ascorbolysis results in a Sec-selenol. Likewise, we developed 2,2’-dipyridyl diselenide (PySeSePy) to deprotect Cys, which can be used with subsequent ascorbolysis to provide a Cys-thiol. Notably, we found the ascorbolysis step to be chemoselective; ascorbate can reduce a selenosulfide bond, but not a formed disulfide bond. We harnessed this chemoselectivity for the synthesis of peptides that contain multiple disulfide bonds, which we demonstrate by synthesizing guanylin and tachyplesin-1 using PySeSePy as a chemical tool. Another chemical tool that we utilize to explore selenoprotein chemistry is alpha- methyl selenocysteine (αMe)Sec. This unique amino acid has a methyl group in place of its α-H. We found that a peptide containing (αMe)Sec (compared to a Sec-peptide control), showed enhanced stability when incubated in oxygenated buffer for prolonged periods of time. We also utilized our (αMe)Sec-peptide as a glutathione peroxidase mimic to reduce peroxides, and postulate that this peptide could serve as a therapeutic in times of high oxidative stress. Finally, it is now commonly accepted in the field that selenoproteins evolved to resist oxidative stress. Herein, we expand this hypothesis: Sec replaces Cys in proteins to resist all types of electrophilic stress. We found that when Sec residues are alkylated by reactive biological electrophiles (such as acrolein), the formed adduct can be reversed. There are many potential mechanisms of reversal, but we provide evidence supporting a selenoxide elimination mechanism using a mutant form of thioredoxin reductase that contains (αMe)Sec in place of the native Sec residue at the C-terminal active site. Taken together, the works described in this dissertation expand the chemical toolbox for the study of Se-containing biomolecules and provides new hypotheses for the chemical role of Se in proteins. 0 CITATIONS Material from this dissertation has been published in the following form: Ste.Marie, E.J., & Hondal, R.J.. (2019). 2,2'-Dipyridyl diselenide: A chemoselective tool for cysteine deprotection and disulfide bond formation. Journal of Peptide Science, e3236. Wehrle, R.J.*, Ste.Marie, E.J.*, Hondal, R.J., & Masterson, D.S.. (2019). Synthesis of alpha‐methyl selenocysteine and its utilization as a glutathione peroxidase mimic. Journal of Peptide Science, 25(6), e3173. *these authors contributed equally to this work. Ste.Marie, E.J., & Hondal, R.J.. (2018). Reduction of cysteine‐S‐protecting groups by triisopropylsilane. Journal of Peptide Science, 24(11), e3130. Ste. Marie, E.J., Ruggles, E.L., & Hondal, R. J.. (2016). Removal of the 5‐nitro‐2‐ pyridine‐sulfenyl protecting group from selenocysteine and cysteine by ascorbolysis. Journal of Peptide Science, 22(9), 571-576. AND Material from this dissertation has been submitted for publication in ACS Biochemistry in the following form: Ste.Marie, E.J.*, Wehrle, R.J.*, Haupt, D.J., Wood, N., Previs, M.J., Masterson, D.S., & Hondal, R.J.. (2020). Incorporation of alpha-methyl selenocysteine into thioredoxin reductase confers resistance to (i) oxidative inactivation and (ii) alkylation by reactive biological electrophiles. Submitted to: Biochemistry. *these authors contributed equally to this work. Remington, J.M., Liao, C., Ste.Marie, E.J., Sharafi, M., Ferrell, J.B., Hondal, R.J., Wargo, M., Schneebeli, S.T., Li, J.. (2020). A molecular basis of antimicrobial peptide synergy. Submitted to: Chemical Communication. ii DEDICATION I dedicate this dissertation to my parents, Greg and Julie Ste.Marie. Without them, none of this would have been possible. They are the utmost example of dedication and perseverance. iii ACKNOWLEDGMENTS First and foremost, I would like to thank the members of my committee, Dr. Robert Kelm, Dr. Matthew Liptak, and Dr. Jose Madalengoitia. Each of you has expressed genuine interest in my progress throughout my time here. The insight, advice, and encouragement you have provided is greatly appreciated. Collectively, you are a brilliant, well-rounded committee who I had the great pleasure of working with. Thank you! I also want to express my deepest gratitude to my advisor, Dr. Robert Hondal. Your mentorship, guidance, and unique ideas have helped me grow as a researcher beyond what I thought possible. Your dedication to your craft is inspiring, and you have given me every tool I need to succeed going forward. Thank you for challenging me with the lofty task of completing the numerous projects in this dissertation, and for your support along the way. Thank you for trusting me to work autonomously while providing encouragement and direction any time I needed it. Your unwavering mentorship and friendship have undoubtably shaped me into the researcher, chemist, and teacher I am today. To my fellow graduate students, thank you for the friendship and support you have provided me; there simply are not words to describe the comradery among the UVM graduate students. I especially want to thank my cohort who completed every milestone with me and shared in the challenges of earning this degree; without each of you, this journey would have been much more dismal. There are several people who I would like to thank specifically: Jessica Bocanegra, Joey Campbell, Kevin Fischer, and Bobby Tracey. I will be forever grateful for the friendships I have formed with each of you. I would also like to thank all the previous and current members of the Hondal research group. You were the best kind of colleagues to work with. Thank you for making iv our lab environment friendly, inspiring, and overall pleasant, even during times when our science was not particularly cooperative. To my small army of undergraduates, especially Gracyn Mose, Linnea Saunders, and Kaelyn Jenny, thank you for trusting my advice and direction; the opportunity to provide you mentorship has been among my most rewarding experiences. Dr. Erik Ruggles, thank you for your guidance and hands-on training in the lab. The training and advice you provided me was vital for the completion of this dissertation, especially as a HPLC mechanic. Without the unwavering support of my family, I would not be where I am today. Greg and Julie (Mom and Dad), words cannot describe the gratitude I have for you. You are sine qua non in every one of my achievements. You’ve both been a constant source of support, love, and encouragement throughout this journey, and I am eternally grateful. I rely on you for so much, but you somehow make that burden seem light. I hope to someday, in some small way, find a way to repay you for the countless sacrifices you have always made for us. Anna, Manny, and Gina; thank you for providing me laughter and comradery throughout my journey. You have been my confidants and best friends. To my grandparents, Raymond, Janine, Paul, and Norma; thank you for your never-ending love, support, and cookies. You have been my greatest cheerleaders, and your confidence in me has never faded. I hope I can continue to make you all proud. Finally, I want to thank my fiancé Eli Barrett for being my constant through times of great changes.
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