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Chemical Tools for Residue-Specific and Secondary Amine Selective Petasis (SASP) Bioconjugation of Peptides and Proteins

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Seton Hall University eRepository @ Seton Hall Seton Hall University Dissertations and Theses (ETDs) Seton Hall University Dissertations and Theses Spring 5-4-2020 Chemical tools for Residue-Specific and Secondary Amine Selective Petasis (SASP) bioconjugation of Peptides and Proteins Yonnette Sim [email protected] Follow this and additional works at: https://scholarship.shu.edu/dissertations Part of the Biochemistry Commons Recommended Citation Sim, Yonnette, "Chemical tools for Residue-Specific and Secondary Amine Selective Petasis (SASP) bioconjugation of Peptides and Proteins" (2020). Seton Hall University Dissertations and Theses (ETDs). 2776. https://scholarship.shu.edu/dissertations/2776 Chemical tools for Residue-Specific and Secondary Amine Selective Petasis (SASP) bioconjugation of Peptides and Proteins A dissertation submitted to Seton Hall University in partial fulfillment for the Doctor of Philosophy Degree By: Yonnette E. Sim May 2020 Department of Chemistry and Biochemistry Seton Hall University South Orange, NJ. 07079 USA © 2020 Yonnette E. Sim DocuSign Envelope ID: 9EF6018D-72DB-4316-B86E-3A103260E910 We certify that we have read this dissertation and in our opinion it is adequate in scientific scope and quality as dissertation for the degree of Doctor of Philosophy Dr. Gregory R. Wiedman Mentor Dr. Monika Raj Co-Mentor (No Longer SHU Faculty) Dr. Joseph Badillo Member of Dissertation Committee Dr. Stephen Kelty Department Chair Seton Hall University I dedicate this thesis to my husband, Ebo, my children Ebony and Ethan for their tremendous love and support & My late parents Rupert and Theresa for their love and wisdom. “The only limit to the height of your achievements is the reach of your dreams and your willingness to work for them” – Michelle Obama i ACKNOWLEDGEMENTS First I would like to express my appreciation and thanks to my research mentor, Dr. Monika Raj for accepting me in her group as a Masters student and for encouraging me to transfer to the Doctorate program. Thank you Dr. Raj for believing in me and for encouraging your students to work hard, support each other, give their best to whatever they do and to set high standards for themselves. Your passion and enthusiasm for research greatly impacted my success. I would like to thank Dr. Gregory R. Wiedman for serving as my mentor to complete my dissertation. To Dr. Cecilia Marzabadi – thank you for being my mentor, for all your support and guidance throughout my journey in graduate school. Dr. David Sabatino thank you for all your guidance and support and for always being there to answer the tough questions. I would like to express my gratitude to Dr. Monika Raj, Dr. Cecilia Marzabadi, Dr. Joseph Badillo, Dr. Cosimo Antonacci and Reverend Gerald Buonopane for their willingness to serve on my dissertation committee. Thanks to the department of Chemistry and Biochemistry for accepting me in the Doctorate program and to the staff and professors in the department for their support and teaching over the years. To my colleagues in Raj Group at Seton Hall University, Ryan D. Cohen, Hader Elashal, Neelam N. Lahankar, Tiauna S. Howard, Zilma Muneeswaran, Lyssa Buisserth and Heidi Elashal – thank you all for your support in and out of the lab. Our journey through this will always be remembered. I would also like to thank my labmates in McNulty labs 419 and 401 that helped me through this journey - especially Marius Pelmus, Mufeda Zhort, Abdul Azeez and Sunil Shah. Finally, I would like to thank my family for their support, especially my husband Ebo, who has been mom and dad to my children throughout this journey, for supporting me, encouraging me and kept me going during the tough times. I am especially grateful to my kids for being patient and for not making me feel like I was taking time away from them – Thank You! ii Abstract Chemical protein synthesis is an invaluable tool that enables the construction of novel protein design, incorporation of non-native functionalities, elucidation of structure-function relationships and enable a wide diversity of modifications (such as Post Translation Modification) to study protein functions. Native Chemical Ligation (NCL) in conjunction with Fmoc Solid Phase Peptide Synthesis (SPPS) is the most used method for synthesis of functional peptides and proteins. The synthesis relies on the reaction of a C-terminal peptide thioester and a N-terminal cysteine peptide. Fmoc SPPS of peptide thioester for chemical protein synthesis via NCL is a challenge. Methods that exist for the synthesis of peptide thioester by Fmoc SPPS require special resins, linkers, additional chemistry and is difficult and time consuming. Therefore, a simple and robust method to synthesize peptide thioester is highly desirable. Chapter two of this thesis describes the development of a versatile approach for direct synthesis of peptide thioesters from a solid support utilizing Fmoc chemistry. The method utilizes a cyclic urethane activation technique for the synthesis of peptide thioesters directly from solid support. The resulting thioester is stable and free of epimerization. The usefulness of this methodology was demonstrated by the synthesis of a 19 amino acid peptide thioester, which was utilized for the synthesis of a 29 amino acid long peptide derived from rabies virus glycoprotein (Rvg) using NCL. This accessible and robust Fmoc-based thioesterication technique provides a significant advance to chemical protein synthesis due to its uncomplicated nature, whereby eliminating special precautions and additional steps typically needed for synthesis of peptide thioesters. This approach can be used to incorporate post-translational modifications for the synthesis of complex post translational peptides and proteins in milligram quantities that can be used to study their structure and function. This modification approach can also be used for the synthesis of other complex protein modifications through residue specific N-terminal chemical modification by NCL. iii Chemical modification of protein is a critically important tool for various biological applications such as probing protein dynamics, elucidating protein structure and functions, enhancing protein stability in biological system and construction of protein-drug conjugates. Traditional modification techniques utilize non-specific lysine and cysteine conjugation strategies that produces a heterogenous mixture of conjugates. Over the last two decades there has been an increasing need to developed methods which would modify protein in a controlled manner. Chapter three describes the development of a novel site-specific Secondary Amine Selective Petasis (SASP) bioconjugation strategy using Petasis reaction to modify secondary amines and N- terminal proline. The SASP reaction has been shown to modify a wide variety of peptides and proteins with high selectivity for N-terminal proline. The resulting bioconjugate is highly stereoselective (de >99%) which can be useful in drug discovery. Also, the multicomponent nature of this conjugation technique enables dual labeling of complex proteins in one pot with various cargoes such as dye, biotin and alkynes. The applicability of the SASP bioconjugation technique was demonstrated on a variety of different peptides and proteins with various aldehyde and organoboronate derivatives. The chemo-, regio- and site-specific nature of this method will enable the construction of biomolecular hybrids that can be used to study protein functions, identify new drug targets, deliver potent therapeutics to cellular targets, and engineer new materials. This strategy also provides a powerful tool to study the function and dynamics of post-translational modification such as mono methylated lysine that regulates transcription factor function. iv Table of Contents Dedication i Acknowledgement ii Abstract iii Table of Contents v List of Tables vii List of Figures ix List of Schemes xii List of Spectra xiii Abbreviations and Symbols xv Chapter 1: General Introduction to Protein Chemistry, Chemical Synthesis and Site-Specific modification Strategies. 1.1 General Introduction of Peptide and Protein Structure…………………………………..1 1.2 Solid Phase Peptide Synthesis……………………………………………………….......3 1.3 Native Chemical Ligation……………………………………………………………...10 1.4 Introduction to Peptide and Protein Modification……………………………….……..11 1.4.1 Post Translational Modification…………………………………………….....11 1.4.2 Chemical Modification of native amino acids……………………………..….13 1.4.3 Incorporation and modification of non-natural amino acids………………..…18 1.4.4 Chemical modification of peptide and protein termini…………….………..…20 1.5 Applications of chemical medication……………………………………………….….24 1.6 Thesis aim………………………………………………………………………….......29 1.7 References…………………………………………………………………………...…31 v Chapter 2: Serine Promoted Synthesis of Peptide Thioester-Precursor on Solid Support for Native Chemical Ligation 2.1 Abstract………………………………………………………………………………....36 2.2 Chapter Objective………………………………………………….……………….…..37 2.3 Introduction…………………………………………………………….……….....…....38 2.4 Result and Discussion………………………………………………...……….….….…40 2.4.1 Synthesis of the cyclic urethane moiety…………………………….………....40 2.4.2 Optimization of the reaction conditions for the formation of cyclic urethane moiety on solid support…………………………………………………….….41 2.4.3 Exploring the reaction various peptides with different sequences……..….......42 2.4.4 Stability and epimerization studies during the formation of cyclic urethane moiety………………………………………………………………………….43
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