Synthetic Developments for the Treatment of Organophosphorus Nerve Agent Poisoning DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Amneh Young Graduate Program in Chemistry The Ohio State University 2016 Dissertation Committee: Professor Christopher M. Hadad, Advisor Professor Jon R. Parquette Professor Thomas M. Magliery Copyrighted by Amneh Young 2016 Abstract Acetylcholinesterase (AChE) is a serine hydrolase responsible for the hydrolysis of the neurotransmitter acetylcholine. It hydrolyzes over 25,000 acetylcholine (ACh) molecules every second. Inhibition of AChE catalytic site results in muscle contractions, blurry vision, seizures, and respiratory failure. Organophosphorus (OP) compounds are potent inhibitors of the enzyme AChE. Once the OP enters the enzyme active site, it phosphylates a serine residue (Ser203) to give an irreversibly inhibited AChE that is incapable of hydrolyzing ACh. This leads to a buildup of ACh at cholinergic receptors and constant stimulation of nerve fibers. Reactivation of AChE can occur by hydrolysis of the phosphylated enzyme which is usually accomplished by use of a nucleophilic oxime, such as 2-PAM, often administered after OP exposure as a treatment. However, if reactivation does not occur, the phosphylated enzyme will then undergo a spontaneous dealkylation process (called aging) to give an aged enzyme which, to date, cannot be reactivated. This dissertation covers multiple strategies to combat OP poisoning. First is the design of a pre-treatment in the form of catalytic antibodies capable of hydrolyzing OPs before they can inhibit AChE. The potential for antibodies to catalyze hydrolysis reactions inspired several reports of abzyme neutralization of OP nerve agents. To produce the catalytic antibodies, our group designed and synthesized a small library of haptens that mimic the transition state of nerve agent hydrolysis. These haptens are then ii conjugated to an immunogenic protein and antibodies are raised that hopefully hydrolyze nerve agents before they can inhibit AChE. Ten haptens were synthesized and preliminary results indicate that these haptens either bind or hydrolyze our target OP nerve agent. The second approach is the use of a cyclic peptide to mimic the active site of known OP bioscavengers. A combinatorial approach was used to develop a library of peptides synthesized on a bead using common solid phase peptide chemistry. A novel screening approach will be discussed. The final strategy is the design of quinone methide precursors (QMPs) that will enter the active site of AChE and alkylate the “aged” enzyme. Once alkylated, AChE can be reactivated using a nucleophilic oxime (2-PAM). Our goal is to design QMPs which will bind to and selectively alkylate aged AChE. We designed four different families of QMPs that can be easily accessed via simple transformations of commercially available starting materials. An undergraduate organic chemistry lab experiment was designed to aid in the synthesis of the QMP library and produced over 100 QMPs in gram quantities. Approximately half of the QMPs react with model nucleophiles under physiological conditions. The reactivity of these QMPs with model phosphonylated peptides and aged AChE will be discussed. iii Dedication To my loving husband Philip iv Acknowledgments There are many people who have helped me grow and succeed, both on an academic and personal level. I must first thank the people who mentored me through my graduate career: Professor Chris Hadad for helping me achieve my goals and for the freedom to pursue my own projects, and Dr. James Stambuli for teaching me the skills I needed to succeed and giving me continued guidance to help me find my path. Dr. Will Henderson showed me the ropes when I first started graduate school and Dr. Matt Lauer continued my training-teaching me everything I know about Wittig reactions and softball. All the Stambuli group members made work truly enjoyable and intellectually stimulating. I would like to thank Tom Corrigan, Chi Le, Ben Garrett, Ryan McKenney, Dr. Jeremey Erb, Qinggeng Zhuang, and our many collaborators for making the Hapten project a success. Special thanks to Ryan McKenney for helping me turn my idea of cyclic peptides as OP scavengers into a reality. Professor Dehua Pei was instrumental in the design of that project and without his guidance our success would have been unlikely. There are so many people involved in the QMP project, many of which I haven’t worked with directly, but without whom the project would likely still be in infancy. There are countless undergraduate students, under the guidance of Dr. Chris Callam, who did the bulk of the synthesis. Qinggeng Zhuang, worked tirelessly on this project to identify hit compounds. v My graduate career followed an unconventional path and I would like to thank the people who helped me navigate those waters-I would not be where I am today without them. Ben Garrett and Luke Baldwin patiently counseled me over many months while I struggled to determine my best course of action. I am really glad I listened to both of you. Professor Chris Hadad and Dr. James Stambuli always had an open door and listened to me before doing everything in their capacity to help me accomplish my goals. I am lucky to have both of you on my side. Finally, I would like to thank my wonderful husband Philip for all the love, support, and happiness you bring to my life. I look forward to the years ahead and all the adventures we will have along the way. vi Vita 2011................................................................B.S. Chemistry and Biochemistry, The University of Michigan-Dearborn 2014................................................................M.S. Chemistry, The Ohio State University 2011 to present ..............................................Graduate Research Associate, Department of Chemistry and Biochemistry, The Ohio State University Publications 1. Palladium-Catalyzed Reactions of Enol Ethers: Access to Enals, Furans, and Dihydrofurans. Lauer, M.G.; Henderson, W.H.; Awad, A.; Stambuli, J.P. Org. Lett. 2012, 14, 6000 – 6003. 2. Dimeric FeFe-Hydrogenase Mimics Bearing Carboxylic Acids: Synthesis and Electrochemical Investigation. Garrett, B.R.; Awad, A.; He, M.; Click, K.; Durr, C.; Gallucci, J.; Hadad, C.M.; Wu, Y. Polyhedron 2016, 103, 21-27. vii Fields of Study Major Field: Chemistry viii Table of Contents Abstract ............................................................................................................................... ii Acknowledgments............................................................................................................... v Vita .................................................................................................................................... vii List of Schemes ................................................................................................................. xii List of Tables .................................................................................................................... xv List of Figures .................................................................................................................. xvi List of Abbreviations ....................................................................................................... xix CHAPTER 1: INTRODUCTION TO ORGANOPHOSPHORUS NERVE AGENTS AND AChE ......................................................................................................................... 1 1.1: Brief History of the Development and Use of Organophosphorus Nerve Agents ... 1 1.2: Use of OP Compounds as Pesticides ....................................................................... 4 1.3: Structure and Activity of Acetylcholinesterase........................................................ 6 1.4: Current Therapies for OP Inhibition of AChE ....................................................... 10 1.5: Statement of Purpose.............................................................................................. 15 CHAPTER 2: SYNTHESIS OF NOVEL PHOSPHORANE HAPTENS ........................ 16 ix 2.1: Introduction ............................................................................................................ 16 2.2: Division of Work Described in this Chapter .......................................................... 23 2.3: Hapten Design and Synthesis ................................................................................. 24 2.4: Results and Discussion ........................................................................................... 45 2.5: Conclusions ............................................................................................................ 54 CHAPTER 3: DEVELOPMENT OF CYCLIC PEPTIDES FOR VX HYDROLYSIS ... 56 3.1: Introduction ............................................................................................................ 56 3.2: Division of Work Described in this Chapter .......................................................... 62 3.3: Cyclic Peptide Design and Synthesis ..................................................................... 62 3.4: Model Phosphonate Design and Synthesis ............................................................ 67 3.5: Screening Protocols and Hit Identification ............................................................ 74 3.6: Future Directions ...................................................................................................