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And Light-Directed Peptide Microarray Synthesis Development of Chip-Based Electrochemically- and Light-Directed Peptide Microarray Synthesis by Pallav Kumar A Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Approved October 2013 by the Graduate Supervisory Committee: Neal Woodbury, Chair James Allen Stephen Johnston ARIZONA STATE UNIVERSITY December 2013 ABSTRACT Peptide microarrays may prove to be a powerful tool for proteomics research and clinical diagnosis applications. Fodor et al. and Maurer et al. have shown proof-of- concept methods of light- and electrochemically-directed peptide microarray fabrication on glass and semiconductor microchips respectively. In this work, peptide microarray fabrication based on the abovementioned techniques were optimized. In addition, MALDI mass spectrometry based peptide synthesis characterization on semiconductor microchips was developed and novel applications of a CombiMatrix (CBMX) platform for electrochemically controlled synthesis were explored. We have investigated performance of 2-(2-nitrophenyl)propoxycarbonyl (NPPOC) derivatives as photo-labile protecting group. Specifically, influence of substituents on 4 and 5 positions of phenyl ring of NPPOC group on the rate of photolysis and the yield of the amine was investigated. The results indicated that substituents capable of forming a π-network with the nitro group enhanced the rate of photolysis and yield. Once such properly substituted NPPOC groups were used, the rate of photolysis/yield depended on the nature of protected amino group indicating that a different chemical step during the photocleavage process became the rate limiting step. We also focused on electrochemically-directed parallel synthesis of high-density peptide microarrays using the CBMX technology referred to above which uses electrochemically generated acids to perform patterned chemistry. Several issues related to peptide synthesis on the CBMX platform were studied and optimized, with emphasis i placed on the reactions of electro-generated acids during the deprotection step of peptide synthesis. We have developed a MALDI mass spectrometry based method to determine the chemical composition of microarray synthesis, directly on the feature. This method utilizes non-diffusional chemical cleavage from the surface, thereby making the chemical characterization of high-density microarray features simple, accurate, and amenable to high-throughput. CBMX Corp. has developed a microarray reader which is based on electro- chemical detection of redox chemical species. Several parameters of the instrument were studied and optimized and novel redox applications of peptide microarrays on CBMX platform were also investigated using the instrument. These include (i) a search of metal binding catalytic peptides to reduce overpotential associated with water oxidation reaction and (ii) an immobilization of peptide microarrays using electro-polymerized polypyrrole. ii I would like to dedicate this work to my family and friends. First, to my loving parents, Shri Digvijay Kumar Sharma and Smt. Savita Sharma. Their unconditional love and support is a great source of strength to me. To my brothers, Pankaj Sharma and Neeraj Sharma who make me very proud and always love and support me. I am indebted to Neeraj for his steadfast support and faith in my abilities. I could not have accomplished this degree without his support. I would also like to dedicate this work to my sisters-in- law Aparna Sharma and Priyanka Sharma who are the new pillars of our family and are shouldering the responsibilities with great love and dedication. To the wonderful kids of my family, Vedagya, Riddhima, and Nandini who are a great source of happiness in our lives. To my wonderful friends, Abhishek Roy, Loren Howell, and Kul Bhushan who always love and support me and I feel blessed to have them. iii ACKNOWLEDGEMENTS My heartfelt thanks to my supervisory committee, Prof. Dr. Neal Woodbury, Prof. Dr. James Allen, and Prof. Dr. Stephen Johnston, for giving me a second chance to complete my degree. I shall ever remain greatly indebted to the committee. I am very grateful to Dr. Neal Woodbury for his support throughout my academic studies, for introducing me to microarray area, for his expert advice, for the supervision of my thesis, and especially for allowing me to complete my degree. He has been a great mentor and dealt with me with great patience and perseverance. I greatly admire his dedication to science, kindness, acumen, energy, patience, perseverance, creativity, and management skills. I offer special thanks to Dr. James Allen and Dr. JoAnn Williams for their support and guidance during the DOE project. Their focus on minute details of the experiments and strategizing future experiments always amazed me. I will try to develop these qualities earnestly and apply in my future endeavors. I would also like to thank Dr. Stephen Johnston for his support and guidance. Like many researchers, I too benefitted a lot from his willingness to safely use several scientific instruments of his lab. Several interactions with him and talented researchers of his lab broadened my perspective about peptide microarray area. I am very thankful to Dr. Arman Ghodousi, Dr. Zhan-Gong Zhao, and Dr. Allan Scruggs with whom I worked very closely on microarray related projects. Dr. Ghodousi taught me several key aspects of organic synthesis while studying the efficiency of different photo-labile protecting groups. His support and several discussions that we had iv were very helpful. Dr. Zhao and I worked together to develop peptide microarray fabrication capability on CombiMatrix platform. His expert advice in designing experiments and analyzing results were very helpful and was a learning experience. Dr. Scruggs and I worked together in understanding the capabilities of PotentioSense unit, and developing a protocol for measuring the redox assays involving high currents/voltages. During this period the discussions that we had, and his logical reasoning about the experiments and protocol development were very useful. I would like to thank Dr. Karl Maurer, Mr. Sho Fuji, and Dr. Kia Peyvan of CombiMatrix for all the technology support and guidance they provided. I would like to thank Mr. John Lopez and Dr. Zachary Laughrey of Proteomics Center at Arizona State University, Dr. Dan Brune of School of Life Sciences at Arizona State University, and Dr. Randall Nelson of The Biodesign Institute at Arizona State University for helping with MALDI characterization of microarray synthesis. I would also like to thank Kevin Brown for integrating Peptide synthesizer and Electro-synthesis instruments. I am very thankful to Carole Flores for providing every possible professional assistance and my thanks also go out to Mikayla Madjidi for editing my dissertation. Finally, I want to thank the DOE for financial support. In addition, I also want to thank the past and present members of Woodbury lab for all the fun along the way: Jinglin, Su Lin, Sean, Crystal, Jason, Shervin Shariari, Tram Lyna Vu, and Julie Noh, Matt, Jie, Guon, Wei, Evaldas, Amy, Doug, Jo, Laimonas, Terri, Haiyu, Trent, Rashaad, Michaela and April. v TABLE OF CONTENTS Page LIST OF TABLES ................................................................................................................... x LIST OF FIGURES .......................................................................................................... xii LIST OF EQUATIONS .................................................................................................. xvii LIST OF SCHEMES...................................................................................................... xviii CHAPTER 1. GENERAL INTRODUCTION TO PEPTIDE MICROARRAYS ...................................... 1 Abstract .......................................................................................................................... 1 Introduction ................................................................................................................... 1 Microarray Technology ............................................................ 3 Peptides at Molecular Level ..................................................... 5 Peptide Synthesis ...................................................................... 5 Peptide Microarray Synthesis ................................................... 7 Project Organization ............................................................... 11 Note ......................................................................................... 23 References.................................................................................................................... 24 2. INVESTIGATION OF THE 2-(2-NITROPHENYL)PROPOXYCARBONYL PROTECTING GROUP SUBSTITUENT-EFFECT ON THE RATE OF PHOTOLYSIS AND YIELD OF AMINE ............................................................................ 30 Abstract ........................................................................................................................ 30 Introduction ................................................................................................................. 31 vi CHAPTER Page Results and Discussion .............................................................................................. 32 Materials and Methods .............................................................................................. 40 Conclusion ..................................................................................................................
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