Mechanistic Studies on Memory of Chirality Alkylations of 1,4- Benzodiazepin-2-ones & Structural-based Design of Insecticidal AChE Inhibitors for Malaria Mosquito, Anopheles gambiae Danny Chung Hsu Dissertation submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry Dr. Paul R. Carlier, Chairman Dr. Felicia Etzkorn Dr. Paul A. Deck Dr. David G.I. Kingston Dr. James M. Tanko Dr. Larry T. Taylor September 18th, 2007 Blacksburg, Virginia Keywords: Memory of Chirality, Asymmetric Synthesis, 1,4-Benzodiazepin-2-one, Density Functional Theory, Enolate Chemistry, Freeze Frame Observation, Kinetics, Acetylcholinesterase, Free Cysteine Targeting, Sulfhydryl Reagents, Anopheles gambiae Copyright 2007, Danny Chung Hsu Mechanistic Studies on Memory of Chirality Alkylations of 1,4- Benzodiazepin-2-ones & Structural-based Design of Insecticidal AChE Inhibitors for Malaria Mosquito, Anopheles gambiae Danny Chung Hsu ABSTRACT Memory of chirality (MOC) is an emerging strategy for asymmetric synthesis which relies upon the intermediacy of transiently non-racemic reactive species. In these reactions the configuration of the sole stereogenic center of the enantiopure starting material is "memorized" by a chiral non-racemic conformation in the intermediate; trapping then captures the stereochemical information, and generates a new stereogenic center with high fidelity. We experimentally and computationally studied the highly retentive deprotonation/alkylations of 1,4-benzodiazepin-2-ones (BZDs) that rely upon this strategy. We captured a transiently non- racemic BZD enolate intermediate in enantiopure form, then released the enolate and observed its subsequent reaction. This approach allowed the first ever step-wise observation of the stereochemical course of such a MOC process. Approximately 2 million deaths are caused by malaria every year in the world. In total roughly 3.2 billion people are living under the risk of malaria transmission. Current use of anticholinesterase insecticides has been limited by their toxicity to human beings. A major African malaria insect vector, Anopheles gambiae (Ag), was targeted. Based on sequence alignment and homology models of AgAChE, a strategy of dual-site binding was adopted that targets Trp84 in the active site and Cys286 at the peripheral site. Selective AChE inhibitors have been designed and synthesized. Acknowledgements I would like to express my heartfelt gratitude to my advisor Dr. Paul R. Carlier for his guidance, encouragement, and continuous support. He has inspired and advised me to grow intellectually and technically in multidisciplinary research areas. I am very honored and deeply indebted to be his student. I am grateful to my advisory committee, Dr. Felicia Etzkorn, Dr. Paul A. Deck, Dr. David G. I. Kingston, Dr. James M. Tanko, and Dr. Larry T. Taylor for their advice, patience, and kindness. I am obliged to Dr. Jeff R. Bloomquist and Dr. Troy D. Anderson of the Department of Entomology for conducting AChE bioassay. I thank Dr. Eric A. Wong and Dr. Ranginee Choudhury of the Department of Animal and Poultry Science for preparing recombinant enzymes. I also thank Dr. Larry T. Taylor for his HPLC thermostat. I would like to thank the Carlier group members, past and present, for creating supportive and fun environment in our lab. Particularly I am indebted to Dr. Polo C.-H. Lam, Dr. Dawn M. Wong, and Dr. Hongwu Zhao for sharing their knowledge and expertise, and for valuable discussions. I thank Nipa Deora, Yiqun Zhang, Yang-Sheng Sun, Larry Williams, Dr. Ming Ma, Jason Harmon, and Joe DeGuzman, Josh Hartsel, Chris Monceaux for their friendship. I would also like to thank Mr. Tom Glass (NMR), Mr. Bill Bebout (Mass Spectroscopy), Mr. Kim Harich (Mass Spectroscopy), and Ms. Carla Slebodnick (X-ray) for their help and support. I acknowledge the financial support of the Department of Chemistry at Virginia Tech. I am grateful to my parents, grandma, and brother for their everlasting love and encouragement. Without their unconditional support I could not come this far. iii Dedication To my parents iv Chapter 1. Memory of Chirality (MOC)........................................................................................................ 1 1.1 Introduction ...................................................................................................................................... 1 1.2 Requirements for Memory of Chirality............................................................................................ 2 1.3 Dynamic Chirality ............................................................................................................................ 4 1.4 Memory of Chirality in Enolate Chemistry...................................................................................... 6 1.4.1 α-Alkylation of an Aspartic Acid Ester Enolate........................................................................... 6 1.4.2 Designed Asymmetric Alkylation of a Napthyl Ketone............................................................... 7 1.4.3 Enantioselective α-alkylation of Amino Acid Esters without External Chiral Sources. .............. 9 1.4.4 Enantioselective Synthesis of Aza-cyclic Amino Acids............................................................. 12 1.4.5 Proposed Mechanisms of Asymmetric Induction in Deprotonation/Alkylation of Amino Acid Esters ................................................................................................................................. 15 1.4.6 Other Cyclization Reactions Involving Axially Chiral Enolate Intermediates........................... 19 1.4.7 Enantioselective Synthesis of Quaternary 1,4-Benzodiazepin-2-ones………………………..23 1.5 Memory of Chirality in Radical Chemistry....................................................................................32 1.5.1 Retentive Benzylic Substitution Induced by Dynamic Planar Chirality .................................... 32 1.5.2 Retentive Radical Trapping Controlled by a Slow Ring Inversion ............................................ 34 1.5.3 Memory of Chirality in Radical Cyclization.............................................................................. 36 1.5.4 Memory of Chirality in the Cyclization of Photochemically-generated Diradicals................... 38 1.5.5 Diastereoselective Photocycloaddition Controlled by Crystallization………………………..40 1.6 Memory of Chirality Involving Carbocation Intermediates ........................................................... 42 1.7 Memory of Chirality in Gold(I)-Catalyzed Reactions ................................................................... 44 1.7.1 Enantioselective Gold(I)-Catalyzed Rautenstrauch Rearrangement………...…………………44 1.7.2 Memory of Chirality in the Gold(I)-Catalyzed Intramolecular Carboalkoxylation of Alkynes..46 1.8 Summary ........................................................................................................................................ 48 References for Chapter 1………………...…………...………………..…………………………………...49 Chapter 2. Mechanistic Studies on Memory of Chirality Alkylation of 1,4-Benzodiazepin-2-ones............ 54 2.1 Medicinal Importance of 1,4-Benzodiazepin-2-ones ........................................................................ 54 2.2 High Enantioselectivity and Retention of Configuration Resulted in Previous MOC Alkylations of 1,4-Benzodiazepin-2-ones ................................................................................................................... 56 2.3 Investigation of Ring Inversion Barrier of Glycine-derived 1,4-Benzodiazepin-2-ones .................. 57 2.3.1 Synthesis of Glycine-derived BZDs ......................................................................................... 57 2.3.2 Coalescence Temperature Measurements of Glycine-derived BZDs ....................................... 59 2.3.3 DFT Calculations of Ring Inversion Barrier of Glycine-derived BZDs .................................... 61 2.4 Enantioselective Alkylations of Alanine-derived BZD...................................................................... 64 2.4.1 Synthesis of Alanine-derived BZDs .......................................................................................... 64 2.4.2 Memory of Chirality Alkylations of Alanine-derived BZDs .................................................... 65 2.4.3 Characterization of BZD conformers........................................................................................ 68 2.5 Proposal of Two Stereochemical Controlled Mechanisms............................................................... 70 2.6 Possible Means to Discriminate between the Two Mechanisms..................................................... 71 2.6.1 Inspiration from HPLC and 2D-TLC........................................................................................ 71 2.6.2 Freeze Frame Observations after Cold Work-up ...................................................................... 74 2.7 DFT Calculations of the BZD Deprotonation Transition Structures................................................ 80 v 2.8 Kinetic and Computational Studies of the 1,4-Benzodiazepin-2-one Enolate Racemization Barrier ...................................................................................................................................................... 93 2.8.1 Kinetic Studies on BZD Enolate Racemization Barrier............................................................