Design and Synthesis of Caged Thiols for Development of Photo-Activatable Peptides, Inhibitors and Biomaterials A Dissertation SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Mohammad Mohsen Mahmoodi IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR THE GRADUATE DEGREE OF DOCTORAL PHYLOSOPHY Mark D. Distefano, Advisor June, 2017 © Mohammad Mohsen Mahmoodi 2017. i Acknowledgement I was 22 years old when I moved to US, a new environment and culture far away from my family, in order to pursue my graduate studies. Thus far, this has been the most challenging journey in my life full of numerous learning and growing experiences. I would have never imagined that I would grow this much academically and as a person. This could not have been done without the support and help of many people. First, I would like to extend my sincere thanks to my advisor Mark Distefano for giving me this great opportunity to be a part of his fantastic research group. He was a great mentor full of encouragement, patience, and support helping me overcome the obstacles and challenges I faced during my research. A big thank you goes to MDD group members past and present. Thank you for your help and support you have provided me all these years. As in particular, my old friend and teacher Dr. Mohammad Rashidian, for his help, support and guidance both in academic a personal life. Dr. John Dozier for his humorous and supportive personality and his help for reviewing my proposals. Dr. Dan Mullen for teaching me peptide synthesis. Dr. Charuta Palsuledesai for teaching me mammalian cell culture. I would also like to thank all of my collaborators: Professor David Bank and his group members for their help on two-photon laser. Professor Molly Shoichet and her lab members Stephanie Fisher and Dr. Roger Tam for their contribution. Dr. Guillermo Marques for his help on confocal microscopy experiments. My PhD experience was a full of challenges and disappointments. I was not able to overcome these difficulties without the support of my family back home and also my great friends in Minneapolis: Davood Taherinia, Fazel Zare Bidoky, Mohammad Danesh Yazdi, Hamidreza Badri, Shahrzad Zaferanloo, Mahdi Ahmadi, Mammad Nasiri, Sara Parhizgari, Reza Halvagar and Mohammad Amin Tadayon. I am very blessed and thankful to have you as my friends. Lastly, I must thank my mom and dad, and my sisters for their endless love, help and support. You have always been a great source of inspiration, motivation and encouragement for me. I love you and this thesis is all dedicated to you. ii To my parents, Mahdi and Aghdas Mahmoodi and my sisters, Narges and Najmeh My endless gratitude for I would never made it this far without you iii Table of Contents Preface .......................................................................................................................................... vi Introduction ................................................................................................................................ vii Table of Figures ........................................................................................................................... x Table of Schemes...................................................................................................................... xiv List of Tables .............................................................................................................................. xv 1 Photo-cleavable protecting groups for thiols: Synthesis, biological and biomaterial applications ............................................................................................................ 1 1.1 Introduction ..................................................................................................................... 1 1.2 Ortho-nitrobenzyl based caging groups ..................................................................... 3 1.3 Coumarin-based caging groups ................................................................................ 14 1.4 Nitrodibenzofuran Cages for Thiols .......................................................................... 23 1.5 p-Hydroxyphenacyl Cages for Thiols ....................................................................... 26 1.6 Benzoin and Benzoyl Cages for Thiols .................................................................... 30 1.7 Conclusion .................................................................................................................... 31 2 Nitrodibenzofuran: A One- and Two-Photon Sensitive Protecting Group That Is Superior to Brominated Hydroxycoumarin for Thiol Caging in Peptides ............. 35 2.1 Introduction ................................................................................................................... 35 2.2 Results and Discussion .............................................................................................. 39 2.2.1 Synthesis and Studies of the Photolysis of Bhc- Protected Cysteine- Containing Peptides. ........................................................................................................ 39 2.2.2 Use of Nitrodibenzofuran for Development of Caged Cysteine- Containing Peptides. ........................................................................................................ 49 2.2.3 Light Activation of a Caged Peptide inside Live Cells. ........................... 57 2.3 CONCLUSION ......................................................................................................... 63 2.4 Experimental Section ........................................................................................... 64 3 6-Bromo-7-hydroxy-3-methyl coumarin (mBhc) is an efficient multi-photon labile protecting group for thiol caging and three-dimensional chemical patterning 74 3.1 Introduction ................................................................................................................... 74 3.2 Results and Discussion .............................................................................................. 77 3.2.1 Design and synthesis of a coumarin-based caging group for efficient thiol protection .................................................................................................................. 77 3.2.2 Photo-physical properties of an mBhc protected thiol ........................... 85 3.2.3 One- and two-photon activation of protein prenylation .......................... 88 iv 3.2.4 Two-photon patterning using a mBhc-caged thiol .................................. 91 3.3 Conclusion .................................................................................................................... 94 3.4 Experimental Section .................................................................................................. 95 4 Development of caged farnesyltransferase inhibitor for photo-chemical modulation of Ras-localization ............................................................................................ 105 4.1 Introduction ................................................................................................................. 105 4.2 Result and Discussion .............................................................................................. 107 4.2.1 Synthesis and Photo-chemical Properties of Caged FTI ...................... 107 4.2.2 Photo-triggered Release of FTI Inside Cells and Modulation of Ras Localization ...................................................................................................................... 110 4.3 Conclusion .................................................................................................................. 113 4.4 Experimental Section ................................................................................................ 113 5 Synthesis of a nitrodibenzofuran-based caging group with red-shifted absorption ................................................................................................................................. 117 5.1 Results and discussion ............................................................................................. 118 5.1.1 Synthesis of 7-amino nitrodibenzofuran (AminoNDBF) ....................... 118 5.2 Conclusion and future directions............................................................................. 120 5.3 Experimental secion .................................................................................................. 120 6 References ........................................................................................................................ 124 v Preface Please note the following: Chapter 2: Mahmoodi, M. M.; Abate-Pella, D.; Pundsack; T.J., Palsuledesai C. C.; Goff, P. C., Blank D. A.; Distefano, M. D. “Nitrodibenzofuran: A One- and Two- Photon Sensitive Protecting Group That Is Superior to Brominated Hydroxycoumarin for Thiol Caging in Peptides” J. Am. Chem. Soc., 2016, 138 (18), pp 5848–5859. Chapter 3: Mahmoodi, M. M.; Fisher, S. A.; Tam, R. Y.; Goff, P. C.; Anderson, R.; Wissinger, J. E.; Blank, D. A.; Shoichet, M. S.; Distefano M. D. “6-bromo-7- hydroxy-3-methyl coumarin (mbhc): an efficient photo-cleavable protecting group useful for thiol caging in peptides and creation of three-dimensional chemical patterns in hydrogels” Org. Biomol. Chem., 2016, 14, 8289–8300. vi Introduction The ability of light to traverse various chemical and biological barriers and be modulated by time and amplitude makes light- regulated molecules unique tools