SYNTHESIS AND SCREENING OF SUPPORT-BOUND COMBINATORIAL CYCLIC PEPTIDE AND FREE C-TERMINAL PEPTIDE LIBRARIES DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Sang Hoon Joo, M. S. ***** The Ohio State University 2007 Dissertation Committee: Approved by Professor Dehua Pei, Advisor Professor Ross E. Dalbey _________________________________ Professor Thomas J. Magliery Advisor Graduate Program in Chemistry Abstract ABSTRACT One-bead one-compound (OBOC) peptide libraries have been useful tools in the biomedical sciences. However, OBOC peptide libraries usually display the N-termini of peptides on the surface as conventional solid phase peptide synthesis proceeds in the C to N direction. While large combinatorial libraries of cyclic peptides can be synthesized by the split-and-pool synthesis method, the sequence determination has been a challenge. Also, peptide libraries with free C-termini face the same problem as well as the difficulty of synthesis in the N to C direction. We report here the development of cyclic peptide libraries and C-terminal peptide libraries for high-throughput screening and sequencing. TentaGel microbeads (90 μm) were spatially segregated into outer and inner layers; cyclic peptides were displayed on the bead surface, whereas the inner core of each bead contained the corresponding linear encoding peptide. After screening of the cyclic peptide library, the identity of hit peptides was determined by sequencing the linear encoding peptides using a partial Edman degradation/mass spectrometry method. Using the same spatial segregation approach peptides were synthesized in the conventional C to N direction, with their C-termini attached to the support through an ester linkage on the bead surface but through an amide bond in the inner layer. The surface peptides were cyclized between N-terminal amine and a carboxyl group installed at a C-terminal linker sequence, while the internal peptides stayed in the linear form. Base hydrolysis of the ii ester linkage in the cyclic peptides exposed a free α-carboxyl group at the C-termini of the peptides attached to the resin via the N-termini. An inverted peptide library containing five random residues was synthesized and screened for binding to PDZ domains. The identity of the binding peptides was determined from the encoding peptides. Consensus recognition motifs were identified for the PDZ domains and representative peptides were individually synthesized and confirmed for binding to their cognate PDZ domains. These methods expanded the utility of OBOC peptide libraries by displaying peptides in different ways. iii Dedication To My Father in Heaven iv Acknowledgments ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Dehua Pei, for his support and encouragement. His dedication to the excellence in Science inspired me to continue this journey. I also thank Dr. Ming-Daw Tsai, my former advisor, for letting me work on diverse projects allowing me to experience the challenges and opportunities. In addition, I would like to thank the professors in the Biological Division, especially Drs. Ross Dalbey and Thomas Magliery for their kind support and inspiration. I would like to thank my labmates, former and current, Drs. Junguk Park, Yun Ling, Jinge Grace Zhu, Qing Xiao, Anne-Sophie Wavreille, Bhaskar Gopishetty, and, Mss. Jing Zhang, Yanyan Zhang, Pauline Tan, and, Messrs. Amit Thakkar, Mathieu Garaud, and particularly Tao Liu, for their stimulating discussions. I am indebted to Dr. Junan Li for his mentoring, and I am grateful to Drs. Deborah Parris for the help with baculovirus system, Michael Zhu and Charles Brooks for their collaborations and active discussions. I cannot thank enough my parents for their love and support. Finally, my special thanks go to my wife Sook Kyung for her understanding and support. v Vita VITA 1972. 4. 18. Born - Seoul, South Korea 1995. 2. B.S. Pharmacy, Seoul National University, Seoul, South Korea 2000. 8. M.S. Pharmacy, Seoul National University, Seoul, South Korea 2001-2007 Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS 1. You-Chin Lin, Mitchell B. Diccianni, Youngjin Kim, Hsin-Hung Lin, Chien- Hsin Lee, Ruey-Jen Lin, Sang Hoon Joo, Junan Li, An-Suei Yang, Huan- Hsien Kuo, Ming-Daw Tsai, Alice L. Yu. Human p16γ, a novel transcriptional variant of p16INK4A, co-expresses with p16INK4A in cancer cells and inhibits cell cycle progression. Oncogene (2007) in press. 2. Sang Hoon Joo, Qing Xiao, Yun Ling, Bhaskar Gopishetty, and Dehua Pei. High-Throughput Sequence Determination of Cyclic Peptide Library Members by Partial Edman Degradation/Mass Spectrometry. Journal of the American Chemical Society (2006) 128(39), 13000-13009. 3. Jeong-In Oh, Kwang-Hoon Chun, Sang-Hoon Joo, You-Take Oh, and Seung- Ki Lee. Caspase-3-dependent protein kinase C delta activity is required for the progression of Ginsenoside-Rh2-induced apoptosis in SK-HEP-1 cells. Cancer Letter (2005) 230(2), 228-238. vi 4. Junan Li, Peter Muscarella, Sang Hoon Joo, Thomas, J. Knobloch, W. Scott Melvin, Christopher, M. Weghorst, and Ming-Daw Tsai. Dissection of CDK4-Binding and Transactivation Activities of p34SEI-1 and Comparison between Functions of p34SEI-1 and p16INK4A.Biochemistry (2005) 44(40), 13246 – 13256. 5. Young-Mi Ham, Joon-Seok Choi, Kwang-Hoon Chun, Sang-Hoon Joo, and Seung-Ki Lee. The c-Jun N-terminal Kinase 1 Activity Is Differentially Regulated by Specific Mechanisms during Apoptosis. The Journal of Biological Chemistry (2003) 278(50), 50330-50337. 6. Junan Li, Sang Hoon Joo, and Ming-Daw Tsai. An NF-kappaB-specific inhibitor, IkappaBalpha, binds to and inhibits cyclin-dependent kinase 4. Biochemistry (2003) 42(46), 13476-13483. FIELDS OF STUDY Major Field: Chemistry vii TABLE OF CONTENTS P a g e Abstract............................................................................................................................... ii Dedication.......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita..................................................................................................................................... vi List of Tables ..................................................................................................................... xi List of Figures................................................................................................................... xii List of Abbreviations ....................................................................................................... xiv Chapter 1 Introduction ........................................................................................................ 1 1.1 Combinatorial Peptide Libraries......................................................................... 1 1.2 Cyclic Peptides.................................................................................................... 3 1.3 Biologically Active Cyclic Peptides ................................................................... 4 1.3.1 Tyrocidine and Gramicidin S...................................................................... 4 1.3.2 Cyclosporin A ............................................................................................. 9 1.3.3 RGD Peptide ............................................................................................. 10 1.3.4 RNA Binding Cyclic Peptides .................................................................. 11 1.4 Cyclic Peptides with Genetic Encoding............................................................ 12 1.4.1 Phage Display for Cyclic Peptides............................................................ 12 1.4.2 Intein-Mediated Cyclization ..................................................................... 13 1.4.3 mRNA Display Based Cyclic Peptides..................................................... 15 1.5 Synthetic Cyclic Peptide Libraries.................................................................... 19 1.5.1 Iterative Deconvolution ............................................................................ 20 1.5.2 Tandem Mass Spectrometry ..................................................................... 23 1.6 Peptide Libraries with Free C-Termini............................................................. 25 1.6.1 PDZ Domains............................................................................................ 26 1.6.2 14-3-3 Proteins.......................................................................................... 27 viii 1.7 Biological Libraries for Studying the Specificities of PDZ Domains .............. 28 1.7.1 Phage Display ........................................................................................... 28 1.7.2 lacI Repressor ........................................................................................... 29 1.7.3 Two-Hybrid System.................................................................................. 30 1.7.4 FRET Based Screening............................................................................. 31 1.8 Synthetic Libraries for Studying the Specificities of PDZ Domains................ 32 1.8.1 Solution Phase Screening Using Synthetic Peptide Library..................... 32 1.8.2 Inverted Peptides on Solid Support..........................................................