Towards Targeted Photodynamic Therapy: Synthesis and Characterization of Aziridine Aldehyde-Cyclized Cancer- Targeting Peptides and Bacteriochlorin Photosensitizers by Áron Roxin A thesis submitted in conformity with the requirements for the degree of Doctorate of Philosophy Graduate Department of Pharmaceutical Sciences University of Toronto © Copyright by Áron Roxin 2014 Towards Targeted Photodynamic Therapy: Synthesis and Characterization of Aziridine Aldehyde-Cyclized Cancer-Targeting Peptides and Bacteriochlorin Photosensitizers Áron Roxin Doctorate of Philosophy Graduate Department of Pharmaceutical Sciences University of Toronto 2014 Abstract This thesis presents the contributions we made towards achieving targeted photodynamic therapy (PDT) by synthesizing and characterizing new aziridine aldehyde-cyclized cancer-targeting peptides and bacteriochlorin photosensitizers (PSs). The new peptides are based on the integrin- targeting sequence, arginine-glycine-aspartic acid (RGD), and were cyclized by aziridine aldehyde-driven macrocyclization chemistry. We developed a versatile conjugation strategy, and created two useful RGD macrocycles that specifically bound to integrin receptors in vitro. Computer modeling and competitive binding studies found that this macrocyclization chemistry modulated the binding affinity of these peptides by tuning the geometry of peptides of different lengths. The new PSs described herein include bacteriochlorophyll derivatives that efficiently produced reactive oxygen species (ROS) upon illumination with red light. We discovered two simple structural modifications that enhanced the photogeneration of ROS, and suggest that future studies make use of these design parameters to create next generation bacteriochlorin PSs with superior photoactivity compared to known derivatives. In addition, we optimized a facile synthetic reaction for creating a new natural product analog with a distinct exocyclic F-ring, and ii found it was capable of PDT. Unlike some of its natural chlorin counterparts, our bacteriochlorin was not a potent antioxidant. Yet, we suggest that future efforts make use of our simple reaction to expand the library of F-ring containing bacteriochlorins to elucidate key structural modifications that can tune the PDT efficacy and antioxidant activity of this distinct class of bacteriochlorins. Finally, this thesis presents preliminary investigations that attempted to develop new cancer-targeting PDT agents. While the goal of achieving optimal cancer-targeted PDT was not accomplished in this work, it is envisioned that this thesis will provide useful data and insights that will contribute to the development of optimal aziridine aldehyde-cyclized peptide- bacteriochlorin PS conjugates for efficient in vivo cancer-specific PDT. iii Acknowledgments I would like to acknowledge several people who have helped me throughout my PhD experience at the University of Toronto. First and foremost, I thank my supervisor and mentor, Dr. Gang Zheng. His constant support, encouragement and guidance gave me a rich and rewarding graduate school experience. Working in Dr. Zheng‘s lab gave me the opportunity to pursue my research interests and allowed me to develop many useful skills. Thanks also to Dr. Zheng‘s senior scientist, Dr. Juan Chen. Dr. Chen taught me a lot about pharmaceutical research and always made time to discuss my experiments. I‘ll always look up to Dr. Zheng and Dr. Chen, and appreciate that they‘ve greatly helped me progress to the next level in my research career. I would also like to thank the members of my advisory committee, Dr. Andrei K. Yudin, Dr. David Jaffray and Dr. Raymond Reilly, for their valuable guidance. There are many people in Dr. Zheng‘s lab and close group of collaborators who I would like to thank for helping me and for enriching my work experience. Dr. Lili Ding taught me many important microbiology skills. Dr. Andrew Cao greatly contributed to my development as a chemist. Also, the other graduate students and postdocs in Dr. Zheng‘s lab; Dr. Jon Lovell, Ken Ng, Cheng Jin, Dr. Ben Scott, T.D. MacDonald, Ben Luby, Sophie Wang, Arash Farhadi, Elizabeth Huynh, Sarah Cui, Dr. Jiyun Shi and Dr. Neeshma Dave were all great colleagues and I will always treasure their friendship. I am very thankful for my loving partner, Dr. Katie Marshall. As a fellow scientist, she was always understanding and encouraging throughout my program. I am also grateful to my parents, Dr. George and Dr. Sara Roxin for their constant love and support. As I continue to pursue my career goals, I will forever be thankful to everyone who has made my experience as a PhD student at the University of Toronto a great one. iv Table of Contents Abstract ........................................................................................................................................... ii Acknowledgments .......................................................................................................................... iv Table of Contents ............................................................................................................................ v List of Tables ................................................................................................................................ xii List of Figures, Charts and Schemes ............................................................................................ xiv List of Abbreviations ................................................................................................................. xxvi List of Appendices ...................................................................................................................... xxx Chapter 1 ......................................................................................................................................... 1 Introduction to Cyclic Cancer-Targeting Peptides and Bacteriochlorin Photosensitizers .............. 1 Preamble ..................................................................................................................................... 1 Section 1. Flexible or fixed: a comparative review of linear and cyclic cancer-targeting peptides ............................................................................................................................... 2 Summary ............................................................................................................................. 2 Introduction ......................................................................................................................... 2 Geometry of linear and cyclic peptides .............................................................................. 3 Library screenings and structure-activity studies ............................................................... 7 Receptor subtype specificity of linear and cyclic peptides ................................................. 9 Stability of linear and cyclic peptides ............................................................................... 11 Unconjugated peptides as anti-cancer therapeutics .......................................................... 14 Cancer-targeting peptide conjugates ................................................................................. 16 Cell penetrating peptides ................................................................................................... 19 Limitations of peptide cyclization .................................................................................... 20 Unique aspects of peptide cyclization ............................................................................... 23 Conclusions ....................................................................................................................... 27 Section 2. Towards potent bacteriochlorin photosensitizers: insights into structural modifications with potential to enhance PDT-relevant photophysical properties ............ 28 v Summary ........................................................................................................................... 28 Introduction ....................................................................................................................... 28 Metal insertion .................................................................................................................. 31 The heavy atom effect ....................................................................................................... 36 The electron-donating meso 5-methoxy group ................................................................. 39 Modifications along the y-axis .......................................................................................... 43 Cationic substituents along the x-axis and the y-axis ....................................................... 47 Additional fused rings ....................................................................................................... 49 Photosensitizers capable of type 1 PDT ............................................................................ 51 Conclusions ....................................................................................................................... 55 Section 3. Towards Cancer-Targeted Photodynamic Therapy: Overarching Thesis Goals ..... 56 Aziridine aldehyde-cyclized integrin-targeting peptides .................................................. 56 Structural features for modulating the photoproperties of bacteriochlorin photosensitizers