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And Chemical-Activated Nucleosides and Unnatural Amino Acids. (Under the Direction of Dr ABSTRACT LIU, QINGYANG. Synthesis of Photo- and Chemical-Activated Nucleosides and Unnatural Amino Acids. (Under the direction of Dr. Alexander Deiters). Synthetic oligonucleotides coupled with photolabile caging groups have been developed to regulate a variety of biological processes in a spatial and temporal fashion. A UV-cleavable caging group was installed on deoxyadenosine and two morpholino oligonucleotide (MO) monomers of which the morpholino core synthesis was also investigated. The synthesis of a two-photon caging group was optimized and two chromophores with > 400 nm absorption maximum were applied to cage thymidine. These caged monomers can serve as light-triggers of oligonucleotide function upon incorporation. Two phosphine-labile azido thymidine derivatives were synthesized as orthogonal small molecule-triggers to the above light-triggers. Additionally, two coumarin linkers were synthesized, which can cyclize a linear MO so as to inactivate MO activity until > 400 nm light irradiation. These two linkers have been applied to the wavelength-selective regulation of zebrafish embryo development. An azide linker was also synthesized to control MOs using phosphines, as well as a UV-cleavable phosphoramidite to regulate DNA oligonucleotide activities. On the regulation of proteins, a two-photon caged lysine, four azido lysines and an azido tyrosine were synthesized to control protein function with either light or small molecules. The phosphine-induced cleavage of the azido groups were investigated on a coumarin reporter. A fluorescent lysine and an isotope labeled lysine were also synthesized as additional biophysical probes to label protein. These unnatural amino acids have been or will be incorporated into proteins through exogenous tRNA-aaRSs pairs. Synthesis of Photo- and Chemical-Activated Nucleosides and Unnatural Amino Acids by Qingyang Liu 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 DEDICATION 谨以此文致家严家慈，感谢他们的理解，鼓励和支持。 谨以此文悼念先师黄宪教授，感谢他教导我以务实、非功利的态度面对科研。 I dedicate this work to my parents for their understanding, encouragement and support. I dedicate this work in memory of Prof. Xian Huang for showing me the ethic of work. ii BIOGRAPHY Qingyang Liu was born on March 7th, 1986 in Hebei, China and grown up to be a big sports fan. She enjoys watching any kind of sports and ran track and played soccer till high school. After graduation from high school, she went to Zhejiang University in Hangzhou, China where she received her BS degree in Chemistry in 2009 and experienced a different culture in southern China. This developed her interest in organic chemistry working on the methodology of vinylidenecyclopropane under Prof. Xian Huang’s mentorship. In the summer of 2008, she came to North Carolina State University as an exchange student for two months and worked on the development of microwave assisted cyclotrimerization towards natural product synthesis. This experience motivated her to pursue a graduate degree in the US. She started her PhD training under the supervision of Dr. Alexander Deiters at NCSU where she enjoyed the fusion of western and eastern cultures. This fusion was well demonstrated in her wedding ceremony to Thomas K. Chen on June 15, 2013. With curiosity and excitement, she looks forward to explore wherever and whatever the Spirit will lead her. iii ACKNOWLEDGMENTS First of all, I want to thank my advisor, Dr. Alexander Deiters for his guidance in the past five years. He taught me to be confident as a non-native English speaker and encouraged me to practice my English. He also trained me to maintain good time management and to develop a good work ethic which will be beneficial to me even after graduation. I would like to thank Yan who has been such a great friend and mentor to me for all these years. Thanks to Doug, Jesse, and Harry for their kindness during my visit in 2008 which played an important part in my decision to join this lab. Thanks to Jeane, Meryl, Alex P., James, and Andrew for making our office such an enjoyable place and for helping me both in research and in improving my English; Laura and Kalyn for teaching me protein expression and about American culture; Rajendra, Matt and Subhas for sharing their expertise in chemistry. Thanks to Colleen, Jessica, Ji, and Jihe for staying in Raleigh with me after the lab moved and to Taylor for making the boring moments of life more lively. I also want to thank Robin for her help outside the lab and Dr. Melander for supervising me in the past year. I would like to thank my parents for their love and support. Knowing that I have a place to go back to no matter what happens has always been a comfort during hard times. I thank my husband and my in-law families who have treated me as a member of their own ever since we first met and give me a home in this unfamiliar country. Finally I want to thank the Shaws for being my family in Raleigh, and my church family at St. Giles who gives me lots of help and comfort. iv TABLE OF CONTENTS LIST OF FIGURES .............................................................................................................vii LIST OF SCHEMES ...........................................................................................................ix LIST OF ABBREVIATIONS .............................................................................................xiii CHAPTER 1: Regulation of Biological Activity with Oligonucleotides .........................1 1.1 Synthetic oligonucleotides in the regulation of biological processes ..................1 1.1.1 Transcriptional level of regulation ........................................................2 1.1.2 Translational level of regulation ...........................................................3 1.1.3 Protein level of regulation .....................................................................7 1.2 Oligonucleotide improvement through chemical modification ...........................8 1.3 Regulation of synthetic oligonucleotide activites with light ................................11 1.3.1 Evolution of caging groups ...................................................................11 1.3.2 Caging approaches to regulate oligonucleotide function ......................18 CHAPTER 2: Synthesis of Photocaged Nucleoside Phosphoramidites ..........................25 2.1 Synthesis of 6-nitropiperonylmethyl-caged deoxyadenosine phosphoramidite ..25 2.2 Development of caged nucleosides with red-shifted cleavage wavelengths .......28 2.2.1 Synthesis and photolysis of coumarin caged nucleosides ....................29 2.2.2 Synthesis and photolysis of 3-(2-propyl)-4-dimethylamino-4-nitro- biphenyl nucleosides ......................................................................................32 2.3 Development of small molecule-triggered nucleosides .......................................39 2.4 Conclusion ...........................................................................................................46 2.5 Experimental ........................................................................................................47 CHAPTER 3: Synthesis of Photocaged Morpholino Oligonucleotide Subunits ............82 3.1 Synthesis of NPOM-caged MO subunit ..............................................................84 3.1.1 Optimization of the NPOM-caged thymine-MO subunit synthesis ......84 3.1.2 Synthesis of the NPOM-caged guanine-MO subunit ...........................90 3.2 Synthesis of two-photon caged thymine-MO subunit .........................................92 3.3 Synthesis of the PNVOM MO-T .........................................................................95 3.4 Conclusion ...........................................................................................................96 3.5 Experimental ........................................................................................................97 CHAPTER 4: Synthesis of Photocleavable Oligonucleotide Linkers .............................120 4.1 Synthesis of o-nitrobenzyl linker for DNA oligonucleotides ..............................120 4.2 Synthesis of coumarin-based linkers for MOs .....................................................121 4.2.1 Synthesis of 7-diethylaminocoumarin linkers ......................................122 4.2.2 Development of coumarin linkers with red-shifted absorption maximums ......................................................................................................128 4.2.3 Synthesis of azidobenzyl MO linker .....................................................136 4.3 Conclusion ...........................................................................................................138 v 4.4 Experimental ........................................................................................................139 CHAPTER 5: Engineering Protein Function through Unnatural Amino Acid Mutagenesis ........................................................................................................................168 5.1 Post transcriptional modification of protein ........................................................168 5.2 Unnatural amino
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