Expanding the Toolbox: Exploring the Lewis Acid Assisted 19F/18F-Isotopic Exchange Radiochemistry of BODIPY Dyes, and DiPODS: Development of a Novel Bioconjugation Reagent. A Thesis Submitted to the College of Graduate & Postdoctoral Studies In Partial Fulfillment of the Requirements For the Degree of Master of Science In the Department of Chemistry University of Saskatchewan Saskatoon By Whitney Shannon © Copyright Whitney Shannon, March 1st, 2021. All rights reserved. Unless otherwise noted, copyright of the material in this thesis belongs to the author. Permission to Use In presenting this thesis in partial fulfillment of the requirements for a graduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professors who supervised this thesis work or, in their absences, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to make other use of material in this thesis in whole or in part should be addressed to: Head of the Department of Chemistry Dean of College of Graduate & Postdoctoral Studies 170 Thorvaldson Building, 110 Science Place 116 Thorvaldson Building, 110 Science Place University of Saskatchewan University of Saskatchewan Saskatoon, Saskatchewan S7N 5C9 Saskatoon, Saskatchewan S7N 5C9 Canada Canada i Abstract Radiochemical diagnostics and therapeutics benefit from pairing with a compliment second imaging modality. However, methods to radiolabel and link these molecules to targeting vectors are problematic. In this dissertation, I explored new radiosynthetic strategies for producing fluorophore-based bimodal agents and new bioconjugation reagents for antibody drug conjugation with improved in vivo stability. 18F-radiolabeled 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores are hypothetically ideal bimodal imaging candidates. Preliminary experiments focused on facilitating 19F/18F-transfluorination of Pseudomonas aeruginosa-selective BODIPY dye, CDy11, using Lewis acidic [18F]F-BODIPY labeling strategies described in literature. However, the conventionally applied tin(IV)-chloride (SnCl4) Lewis acid’s instability affords reaction yield variability, and further promoted degradation of the CDy11. Thus, research transitioned towards investigating an alternative Lewis acid. To our knowledge, we are the first to use hydrated 18 magnesium nitrate [Mg(NO3)2] as an air-stable substitute for promoting F-fluorination of commercially-available BODIPY dyes. Order of reagent addition significantly affected the 19F/18F-isotopic exchange reaction; with addition of azeotropically dried [18F]F-TBAF to a mixture of the commercial dye and Lewis acidic agent proving most effective. 19F/18F-transfluorination was achieved using both SnCl4 and Mg(NO3)2 to varying degrees. Increasing the equivalences of applied Mg(NO3)2 salt generally improved radiochemical yields. Bioconjugation chemistry enables the covalent attachment of molecules of interest to larger proteins such as antibodies. While conjugation with thiol groups (cysteine) in antibodies is common, conventional maleimide-based coupling agents are prone to in vivo hydrolysis. Price lab research efforts sought to develop radioimmunoconjugates that operate through two phenyloxadiazolyl methyl sulfone (DiPODS) functionality to form irreversible bonds with antibody cysteine pairs. My contribution was to assist in synthesizing several of the multistep synthesis products and apply spectroscopic methods towards analysis of the initial step’s product mixture, which was hypothesized to exist as a rotameric mixture. Several intermediate agents were ii successfully synthesized to produce the desired DIPODS end product. The three components of the 5-[[(1,1-dimethylethoxy)carbonyl]amino]-1,3-dimethyl ester (compound 1)product mixture were attributed to an inseparable mixture of rotamers, doubly-di-tert-butyl decarbonate (Boc)- protected derivative of the compound, and imidic acid tautomers using spectroscopic methods including variable temperature 1H NMR. Lastly, an additional chemical species was observed via 1H NMR as temperature was increased to 75 °C and attributed to a second set of product rotamers. Ultimately, this thesis served to improve future radiotherapeutic agents and radio-diagnostics through the investigation of alternative and reproducible [18F]F-BODIPY labeling strategies and radioimmunoconjugates of improved stability. iii Acknowledgements In an attempt to thank all those who have made this body of work possible, foremost gratitude must be given to my co-supervisors, Dr. Eric Price and Dr. Steven Siciliano. Thank you both for your guidance, support, patience, and for the privilege to learn from both of your expertise. Sincerest thanks to my laboratory colleagues for many laughs and lessons. Special thanks must be given to the staff of the Sylvia Fedoruk Canadian Centre for Nuclear Innovation for providing a safe and enthusiastic work environment, Kelly Christopher for bringing so much life and enthusiasm into any and all research spaces and to Dr. Elaheh Khozeimeh Sarbisheh for being both a role model and mentor. Thank you for believing in me and pushing me to strive for excellence. I would like to thank the Department of Chemistry, the College of Arts and Science, and the College of Graduate and Postdoctoral Studies at the University of Saskatchewan, my committee members as well as the funding agencies whom have contributed to the work performed during the course of my graduate studies; namely the NSERC CREATE SAFER program. Lastly, to my family, whom has always been my greatest source of love and inspiration, none of this would have been possible without you. Thank you for exemplifying what it means to be both hard working and caring. To my parents especially, the person I am today is credited to you and being your daughter is my greatest source of pride. Thanks Everyone, Whitney Shannon iv Table of Contents Permission to Use ........................................................................................................................... i Abstract .......................................................................................................................................... ii Acknowledgements ...................................................................................................................... iv Table of Contents .......................................................................................................................... v List of Tables ................................................................................................................................ xi List of Figures ............................................................................................................................. xiii List of Schemes ....................................................................................................................... xxxiii Glossary of Abbreviations and Symbols .............................................................................. xxxvi General Introduction .................................................................................................................... 1 Radiochemistry ....................................................................................................................... 1 1.1. Fluorine-18 ([18F]F-). ....................................................................................................... 3 1.1.1. Fluorine in Medicine. ................................................................................................ 3 1.1.2. Fluorine-18 Radioisotope.......................................................................................... 3 1.2. ( 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) BODIPY Dyes. ....................................... 6 1.2.1. Optical Imaging. ....................................................................................................... 7 1.2.2. Positron Emission Tomography (PET). .................................................................... 7 1.2.3. Optical/PET Dual-Modality. ..................................................................................... 9 1.3. [18F]-Labeling Strategies for BODIPY Dyes. .................................................................. 9 v 1.3.1. Brønsted-Lowry Acid/Base Theory .......................................................................... 9 1.3.2. Brønsted-Lowry Acid-Assisted 19F/18F-Isotopic Exchange.................................... 10 1.3.3. Lewis Acids/Base Theory. ...................................................................................... 11 1.3.4. Hard Soft Lewis Acid Base (HSAB) Theory & Lewis Acid Selection. ................. 12 1.3.5. Lewis Acid-Assisted 19F/18F-Isotopic Exchange. ................................................... 13 1.4. Antibody Drug Conjugates. ........................................................................................... 14 1.4.1. Cleavable and Non-Cleavable