Investigation of C-H Bond Oxidation Reactions: I. Asymmetric Amination of C-H Bonds Using Ruthenium(II) Catalysts II. Investigation of Structural Determinants of Monoamine Oxidase B Open Access

Abstract

ABSTRACT
Investigation of C-H Bond Oxidation Reactions:
I. Asymmetric Amination of C-H Bonds Using Ruthenium(II) Catalysts
II. Investigation of Structural Determinants of Monoamine Oxidase B
By Erika M. Milczek

This dissertation focuses on exploiting catalytic processes for the oxidation of C-H bonds. Part I of this dissertation describes the development of an efficient asymmetric intramolecular C-H amination protocol. A series of chiral Ru(II)-pyridine bisoxazoline (pybox) complexes were synthesized, and their catalytic properties were investigated. These Ru(II)-pybox complexes catalyze the cyclization of a variety of aryl- and aliphatic-sulfamate ester (R-(CH2)3OSO-2NH2) in the presence of hypervalent iodide oxidants. The cyclized sulfamidates were formed in up to 94% conversion and up to 92%ee.

Part II of this dissertation focuses on amine oxidation by the flavoprotein, monoamine oxidase (MAO). MAO exists in two isoforms, MAO A and MAO B, which differ greatly in substrate specificity and tissue distribution although they demonstrate similar catalytic function. The investigations detailed in this dissertation focus on two goals: 1. understanding the structural features unique to MAO B; and 2. exploiting these properties for the design of MAO B specific inhibitors.

The major structural difference in MAO A and MAO B is that MAO A has a monopartite substrate cavity of 550 ų and MAO B is dipartite with a 290 ų entrance cavity and a 400 ų substrate cavity. Ile199 and Tyr326 function to separate these two cavities. To probe the function of these gating residues, Ile199Ala and Ile199Ala-Tyr326Ala mutant forms of MAO B were constructed, expressed in Pichia pastoris, and purified. Both mutants exhibit catalytic activities that are altered with increased Km values. These insights are applied to understand the molecular mode of inhibition of three distinct classes of MAO B specific inhibitors: 1. Mechanism based inhibition of MAO B by mofegiline; 2. Selective and reversible inhibition of MAO B by styrylisatin analogues; and 3. Allosteric mechanism of inhibition through imidazoline binding to the entrance cavity of MAO B.

Table of Contents

Table of Contents

Chapter 1 Introduction to C-H Bond Cleavage Reactions........... 1

References........... 4

Chapter 2 Asymmetric Amination of C-H Bonds Using Cationic Ruthenium(II)-pybox Catalysts..... 5

2.1 Background and Significance........... 5 2.1.1 Porphyrin Catalysts........... 7 2.1.2 Dirhodium(II) Tetracarboxylate Catalysts........... 8 2.2 Project Objectives........... 10 2.3 Results and Discussion........... 11 2.3.1 Catalyst Design........... 11

2.3.2 Surveying the reactivity of Catalyst 8 and Reaction Development........... 12

2.3.3 Novel Metal Complexes........... 18 2.4 Conclusions........... 23 2.5 Materials and Methods........... 24 2.6 References........... 38 Chapter 3 Monoamine Oxidase: Laboratory to Clinical..... 43 3.1 MAO as a Genetic Control for Behavior........... 45 3.2 Pharmacological Importance of MAO B........... 46 3.3 Pharmacological Importance of MAO A........... 48 3.4 Clinical Use of MAOIs........... 49

3.5 Understanding the Differences in MAO A and MAO B: A Molecular Look........... 50

3.6 Crystallographic Studies of MAO........... 51 3.7 Probing the Oligomeric States of MAO........... 54

3.8 Comparison of MAO A and MAO B Active Site Cavities........... 55

3.9 Inhibitor Design and Inhibitor Binding to MAO........... 58 3.10 Reactions Catalyzed by MAO........... 60 3.11 Mechanism of Amine Oxidation........... 63 3.11.1 Hydride Transfer Mechanism........... 64 3.11.2 Radical Mechanism........... 65 3.11.3. Deprotonation Mechanisms........... 66

3.11.3.1. Aminium Cation Radical Mechanism 66

3.11.3.2. Polar Nucleophillic Mechanism 69

3.11.4. Mechanism for Oxidation of Flavin by O2 ........... 70 3.12 Dissertation Objectives........... 71 3.12.1 Elucidating the Role of Ile199 and Tyr326........... 71

3.12.2 Investigation of the Molecular Mechanisms of MAO B Specific Inhibitors.... 72

3.12.2.1 Exploiting Substrate Reactivity Unique to MAO B 72

3.12.2.2 Rational Design of Inhibitors that Bridge the Entrance and Substrate cavities of MAO B..... 73

3.12.2.3 Targeting the Entrance Cavity of MAO B 74

3.13 References........... 75

Chapter 4 Development of MAO B Specific Inhibitors: From Investigation to Design..... 91

4.1 Mechanistic Studies of the Mechanism Based Inhibitor Mofegiline........... 91

4.1.1 Development of Mofegiline as an Inhibitor of MAO B........... 91

4.1.2 Results and Discussion........... 95

4.1.2.1 Inhibition of MAO B with Mofegiline 95

4.1.2.2 Structure of Mofegiline Inhibited MAO B 100

4.1.2.3 Mofegiline Interactions with Purified MAO A 102

4.1.2.4 Differential Inhibition Motifs with MAO A and MAO B 103

4.1.3 Conclusions........... 107 4.1.4 Materials and Methods........... 108 4.1.5 References........... 110

4.2 Development of Inhibitors that Bridge Both Active Site Cavities........... 115

4.2.1 Inhibitors that Bridge Both Cavities of MAO B........... 115 4.2.2 Results and Discussion........... 117

4.2.2.1 Design of Inhibitor Compounds 117

4.2.2.2 Biological Evaluation of Styrylisatin Analogues 119

4.2.2.3 Importance of Planarity in Designing MAO B Inhibitors 123

4.2.2.4 Differential Inhibition Motifs with MAO A and MAO B 123

4.2.3 Conclusions........... 125 4.2.4 Materials and Methods........... 125 4.2.5 References........... 126

4.3 Targeting the Entrance Cavity of MAO B as a Novel Site for Inhibition: Elucidation of the Imidazoline Binding Site........... 129

4.3.1 History of Imidazoline Ligands and MAO........... 129

4.3.1.1 Classification of IBS 130

4.3.1.2 Elucidation of the I2-IBS 130

4.3.1.3 Evidence for MAO B as I2-IBS 132

4.3.1.4 Tranylcypromine Potentiation 133

4.3.2 Results........... 136

4.3.2.1 Crystallization of MAO with 2-BFI 136

4.3.2.2 Competition Experiments with 2-BFI 140

4.3.2.3 Competition Experiments using Farnesylamine 144

4.3.2.4 Fluorescence Binding Assay 146

4.3.2.5 Thermal Denaturation Experiments 147

4.3.3 Discussion........... 149 4.3.4 Conclusions........... 152 4.3.5 Materials and Methods........... 153 4.3.6 References........... 160

Chapter 5 Elucidating the Role of the ‘Gating' Residues of Human Monoamine Oxidase B..... 168

5.1 Background and Significance........... 168

5.1.1 Identification of the ‘Gating' Functions of Ile199........... 169

5.1.2 Role of the Tyr326........... 171 5.1.3 Point Mutation of the Ile199 to Phe........... 172 5.1.4 Abolishing the ‘Gating' Mechanism of MAO B........... 173 5.1.5 Creating a Monopartite Active Site in MAO B........... 174 5.2 Inhibition Studies........... 174 5.2.1 Nonspecific Inhibitors........... 175 5.2.2 MAO B Specific Inhibitors........... 175 5.2.3 MAO ASpecific Inhibitors........... 176 5.2.4 Mechanism Based Inhibitors........... 177 5.2.5 Aminoindans: Small Reversible Inhibitors........... 178 5.3 Discussion........... 179 5.3.1 Binding Properties of the Ile199Ala Mutant........... 179

5.3.2 Binding Properties of the Ile199Ala-Tyr326Ala Mutant........... 180

5.4 Structural Studies........... 180 5.4.1 Ile199Ala Mutant Structural Studies........... 181 5.4.2 Ile199Ala-Tyr326Ala Mutant Structural Studies........... 182 5.5 Activity Studies of the Ile199Ala Mutant ........... 183

5.6 Activity Studies of the Ile199Ala-Tyr326Ala Mutant ........... 185

5.7 pH Studies........... 186 5.8 Mechanistic Studies........... 191 5.8.1 QSAR Effects: Ile199Ala Mutant........... 195 5.8.2 QSAR Effects: Ile199Ala-Tyr326Ala Mutant........... 197 5.9 Conclusions........... 200 5.10 Materials and Methods........... 201 5.11 References........... 204

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School	Laney Graduate School
Department	Chemistry
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Organic Chemistry, Biochemistry
Keyword	Monoamine Oxidase B C-H Oxidation
Committee Chair / Thesis Advisor	Edmondson, Dale E, Emory University
Committee Members	Liotta, Dennis C, Emory University Blakey, Simon, Emory University

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