ABSTRACT TORRES, JESSICA. Synthesis of Unnatural Amino Acids for Protein Labeling and Activation. (Under the direction of Alexander Deiters). Site-specific incorporation of unnatural amino acids into proteins, both in vivo and in vitro, is a promising technology with tremendous potential to advance studies in protein structure and function. The technique allows the incorporation of a vast diversity of functional groups that extends beyond the conventional mutagenesis of the twenty common amino acids. The rapid development of orthogonal PylRS/PylT pairs has resulted in an increasing number of novel unnatural amino acids that can be site-specifically introduced into proteins. This dissertation presents the syntheses of several unnatural amino acids for use in protein labeling and activation. A bipyridine lysine was synthesized for the assembly of metal-binding proteins and its genetic encoding is shown. In addition, unnatural amino acids bearing reactive functionalities for bioconjugation reactions for the selective labeling of proteins were assembled. These include a variety of lysine analogs that can be applied to carbonyl/aminooxy condensations, thiol-ene reactions, and Diels-Alder cycloadditions. Moreover, bioorthogonal reaction partners such as aminooxy dyes, thiol, and tetrazine probes for subsequent protein labeling were prepared. For the photoregulation of protein activity in live cells, several caged tyrosine analogs were synthesized. The assembly of caged phosphoryl tyrosines to study tyrosine phosphorylation on proteins by light activation is presented; as well as, the synthesis of tyrosine derivatives bearing ortho-nitrobenzyl caging groups to improve decaging kinetics and bioavailability of photocaged tyrosines. In addition, an isotope labeled, photolabile tyrosine was synthesized as a biophysical probe to study protein structure and dynamics by infrared spectroscopy. Lastly, for the light-triggered regulation of oligonucleotides and gene expression, a caged thymidine phosphoramidite bearing a norbornene functional group was synthesized. This synthetic monomer can be incorporated into oligonucleotides and enable the dual functions of selectively modifying an oligonucleotide post-synthetically and have precise control over oligonucleotide activation by the use of light. Synthesis of Unnatural Amino Acids for Protein Labeling and Activation by Jessica Torres A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Chemistry Raleigh, North Carolina 2014 APPROVED BY: _______________________________ ______________________________ Dr. Alexander Deiters Dr. Christian Melander Committee Co-chair Committee Co-chair ________________________________ ________________________________ Dr. Daniel L. Comins Dr. Gavin Williams Committee Member Committee Member DEDICATION To my parents, who guided me to where I am today. DEDICATORIA A mis padres, quienes me encaminaron a donde estoy hoy. ii BIOGRAPHY Jessica Torres was born on January 20, 1988 to Jose and Gerlinde Torres in Boston, MA. Jessica has an older brother, Alexander, and older and younger sisters, Erika and Jomarie. Her parents moved to Villalba, PR where she was raised and graduated from Lysander Borrero Terry High School in 2005. She then moved to Cayey, PR to attend the University of Puerto Rico at Cayey, pursuing her Bachelors of Science degree in Chemistry. During this time she worked in the lab of Dr. Elba Reyes as an awardee of a BioMINDS scholarship by AMGEN; won three summer research internships, one at the University of Pittsburgh School of Pharmacy under the supervision of Dr. Wen Xie in 2007, and the other two at North Carolina State University with Dr. Alexander Deiters in 2008 and Dr. Christopher Gorman in 2009. After graduating in the top three of her class with magna cum laude in 2009, she moved to Raleigh, NC to continue her studies at North Carolina State University in the Ph.D. program in Chemistry. In 2010, Jessica received a National Science Foundation Graduate Research Fellowship and has worked under the supervision of Prof. Alexander Deiters on the synthesis of novel unnatural amino acids for the expression of proteins with new function. iii ACKNOWLEDGEMENTS I would first like to thank my parents for their love and support, for always believing in me and motivating me to chase my dreams. I would like to acknowledge my brother and sisters, you guys are my best friends for life and even in the distance I could always feel your love and support. I wish to thank Dr. Elba Reyes, Dr. Rene Rodriguez, Dr. Robert Ross, Dr. Maria Oliver-Hoyo, and Alison Wynn who believed in me and in one way or another contributed to my decision of pursuing my PhD. I would like to thank my friend Efrain Rivera Serrano for our journey together from Puerto Rico to Raleigh and helping me find a little of home in all of our talks! I wish to give very special thanks to my advisor Alexander Deiters for his guidance and high expectations. All that I have learned from him in the past five years have made me a better scientist. He has been very supportive and his enthusiasm in research was always a great motivation to keep me going on with my projects. I would like to thank to the Deiters lab past and present members that I have had the pleasure of working with. To Doug, for being an awesome mentor during my undergraduate research summer internship, an experience that contributed to my decision of joining the lab. Yan, Andrew, Meryl, Matt, Subhas, and Qingyang for being great hood neighbors, for their fun talks, and their knowledge in chemistry. To Rajendra, for his wise and long talks, and his impressive expertise in chemistry. Thanks to Hank, Alex P., Ji, and Jihe for their contributions on my projects, as well as James and Sander for their talks and knowledge in biology. I would like to thank Ana and Luis for being great summer students, and I wish good luck to Luis who is iv now joining the lab! Special thanks to the ladies, Colleen, Jeane, Laura, Jie, and Kalyn for the great moments we shared inside and outside of lab. I want to add a special shout-out for Colleen and Qingyang for their very appreciated company after the lab moved to Pittsburgh, to Sarah for helping me around with chemistry and lab cleanup, and to Taylor for being a great friend, keeping me sane, and teaching me some English. I would also like to thank Robin Tanner and Dr. Christian Melander for always being friendly faces on the fifth floor and checking up on those of us left at NCSU. Also thanks to Dr. Comins, Dr. Williams and Dr. Lalush for serving as committee members. I would like to acknowledge the Chin lab for research collaborations. And finally, I would like to acknowledge the National Science Foundation Graduate Research Fellowship for financial support. v TABLE OF CONTENTS LIST OF FIGURES ...............................................................................................................ix LIST OF SCHEMES..............................................................................................................iv LIST OF ABBREVIATIONS ............................................................................................. xvi CHAPTER 1: GENETIC CODE EXPANSION WITH UNNATURAL AMINO ACIDS ...1 1.1 Introduction ..........................................................................................................1 1.2 Protein modification via genetic encoding of unnatural amino acids ..................2 1.3 Genetic code expansion using orthogonal aminoacyl-tRNA synthetase/aminoacyl- tRNA pairs .................................................................................................................4 1.4 Pyrrolysyl-tRNA synthetase as a genetic code expansion tool ............................7 1.5 Summary ..............................................................................................................13 CHAPTER 2: SYNTHESIS AND GENETIC ENCODONG OF LYSINE ANALOGS FOR PROTEIN LABELING ..........................................................................................................15 2.1 Genetic code expansion with a bipyridine lysine ................................................15 2.1.1 Synthesis of a bipyridine lysine ........................................................................17 2.1.2 Genetic encoding of a bipyridine lysine ...........................................................19 2.2 Labeling of proteins via bioorthogonal reactions ................................................20 2.3 Genetic code expansion with unnatural amino acids for protein labeling via aldehyde/ketone condensations ..................................................................................23 2.3.1 Synthesis of ketone and diol functionalized unnatural amino acids, and hydroxylamine probes ................................................................................................26 vi 2.3.2 Genetic encoding of ketone and diol functionalized unnatural amino acids ....................................................................................................................................34 2.4 Genetic code expansion with alkene lysines for protein labeling ........................36 2.4.1 Synthesis of alkene lysines ...............................................................................37 2.4.2 Genetic encoding of alkene lysines ..................................................................40 2.4.3 Synthesis of alkene-reactive