Hybrid-Phase Native Chemical Ligation Approaches to Overcome the Limitations of Protein Total Synthesis DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ruixuan Ryan Yu Graduate Program in the Ohio State Biochemistry Program The Ohio State University 2016 Committee: Jennifer J. Ottesen – Advisor Michael G. Poirier Michael A. Freitas Dennis Bong Copyrighted by Ruixuan Ryan Yu 2016 Abstract Total protein synthesis allows the preparation of proteins with chemically diverse modifications. The numerous advantages of total synthesis are sometimes offset by some major limitations. Protein synthesis is a non-trivial task involving many chemical steps, and these steps increase with the size of the protein. Therefore, larger proteins are difficult to synthesize with high yield. We have developed a strategy which we term hybrid-phase native chemical ligation (NCL) to overcome some of the limitations of size and yield. Hybrid-phase NCL combines ligating peptides on a solid support (solid-phase NCL) and in solution (solution-phase NCL) to maximize synthetic yield. We have successfully used this method to synthesize triple-acetylated histone H4-K5ac,K12ac,K91ac and, for the first time, acetylated centromeric histone CENP-A-K124ac (CpA-K124ac). In order to improve the yield of CENP-A total synthesis, we have incorporated a convergent ligation element in our hybrid-phase strategy. This new approach reduced the number of purification steps, leading to a synthetic yield that was almost triple that of the original approach. Finally, we introduce the convergent solid-phase hybrid NCL approach that allows the preparation of a long peptide segment bearing a masked thioester on a solid support. ii Through a newly developed resin-anchoring strategy, cleavage of the product from solid- phase generated a ligation-compatible segment that could be used directly with no purification. This method has the potential to synthesize large proteins in good yield, effectively overcoming the size and yield limits of protein total synthesis. iii Dedication Alice and Owen. iv Acknowledgments I would like to thank all members of the Ottesen lab for their constant support, assistance, and guidance throughout my graduate career. I would like to thank Dr. Santosh Mahto for mentoring me during the first few years, and pushing me to continue his H4 total synthesis project. Thank you to C.J. Howard for advising me with his broad knowledge of biology. Thank you to Michael Cotten and Mallory Alexander for helping move my project forward. Thank you to Kurt Justus for assisting me with all the problems plaguing the histone peptides. Thank you to Dr. John Shimko for giving me his expert opinions on biochemistry, organic chemistry, and nerd culture. Thank you to Ziyong Hong for assisting me with all of the time-consuming experiments. My project would not have moved forward so efficiently without his assistance. Most of all, I thank my advisor Dr. Jennifer Ottesen for giving me unconditional support both inside and outside the lab. Her confidence in me did not change even during the year when I was starting to drift off from my research. She encouraged me to keep going even during the times when I was ready to give up. Her support even extended to my family, especially after my son was born. In addition, she used all of her available resources to support my decision to attend medical school after graduation. She is a mentor who truly cares about the success of her students. v Vita 2002-2006 ......................................................Adrian Wilcox High School 2010................................................................B.A. Molecular and Cell Biology, University of California, Berkeley 2010................................................................B.A. Practice of Art, University of California, Berkeley 2010-2016 ......................................................Graduate Teaching and Research Associate, The Ohio State University 2013................................................................M.S. Biochemistry, The Ohio State University Publications Ruixuan R. Yu, Santosh K. Mahto, Kurt Justus, Mallory Alexander, Cecil J. Howard, Jennifer J. Ottesen, “Hybrid phase ligation for efficient synthesis of histone proteins”. Organic and Biomolecular Chemistry, 2016, 14:2603-2607 Cecil J. Howard, Ruixuan R. Yu, Miranda L. Gardner, John C. Shimko, Jennifer J. Ottesen, “Chemical and Biological Tools for the preparation of modified histone proteins. Topics in Current Chemistry, 2015. 363:193-226 vi Fields of Study Major Field: The Ohio State Biochemistry Program vii Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments ............................................................................................................... v Vita ..................................................................................................................................... vi Publications ........................................................................................................................ vi Fields of Study .................................................................................................................. vii Table of Contents ............................................................................................................. viii List of Tables .................................................................................................................. xvii List of Figures ................................................................................................................ xviii List of Acronyms ............................................................................................................ xxii Chapter 1: Introduction ...................................................................................................... 1 Protein Total Synthesis ....................................................................................................... 1 Native Chemical Ligation ............................................................................................... 2 Applications of total synthesis ........................................................................................ 7 Histones............................................................................................................................. 10 Histone Post-Translational Modification ...................................................................... 13 viii Methods to Prepare Histone PTMs ............................................................................ 13 Genetic mimics .......................................................................................................... 14 Expanded genetic code .............................................................................................. 15 Dehydroalanine .......................................................................................................... 16 Chemical installation through cysteine ...................................................................... 17 Disulfide stapling ....................................................................................................... 18 Chemical Synthesis .................................................................................................... 20 Goals ................................................................................................................................. 20 Outline ........................................................................................................................... 22 Chapter 2: Solid-Phase Ligation vs. Hybrid-Phase Ligation of Histones ......................... 23 Introduction ....................................................................................................................... 23 Solution-Phase NCL ...................................................................................................... 23 Solid-Phase NCL ........................................................................................................... 25 Experimental Methods ...................................................................................................... 28 Materials ........................................................................................................................ 28 RP-HPLC ...................................................................................................................... 29 Mass spectrometry ......................................................................................................... 29 Solid-Phase Peptide Synthesis ...................................................................................... 30 Synthesis of 3-Fmoc-Dbz-OH ................................................................................... 30 ix Automated Solid-Phase Peptide Synthesis ................................................................ 30 Manual peptide synthesis ........................................................................................... 32 Manual synthesis of Dbz(Alloc) resin ....................................................................... 34 Loading the first amino acid on Dbz(Alloc)