A mechanistic investigation of the reactions of diselenides with biological oxidants Kelly Gardiner Faculty of Medicine (Sydney Medical School) The University of Sydney A thesis submitted to fulfil the requirements for the degree of Doctor of Philosophy 2019 Declaration This is to certify that, to the best of my knowledge, the content of this thesis is my own work. This thesis has not been submitted for any degree or other purpose. I certify that the intellectual content of this thesis is the product of my own work and that all the assistance received in preparing this thesis and sources have been acknowledged. Signed, Kelly Gardiner, BBiomedSc (Hons) ii Table of Contents Abstract ...................................................................................................................................ix Acknowledgements .................................................................................................................xi List of Abbreviations ............................................................................................................xii List of Figures ......................................................................................................................xvii List of Tables ........................................................................................................................xxv Publications Arising from this Thesis ..............................................................................xxvi 1. Introduction ........................................................................................................................1 1.1. The Immune Response and Inflammation .........................................................................2 1.2. Generation of Reactive Oxygen Species (ROS) ................................................................3 1 1.3. Singlet Oxygen ( O2) and Photo-Oxidation Reactions ......................................................5 1.4. Protein Peroxides ...............................................................................................................6 1.5. The Catalytic Mechanism of Myeloperoxidase (MPO) ....................................................9 1.6. Myeloperoxidase-Derived Oxidants in Disease ..............................................................10 1.7. Endogenous Protective Mechanisms against ROS-Mediated Damage ...........................12 1.8. Selenium and Sulfur in Human Health and Disease .......................................................13 1.8.1. Selenomethionine (SeMet) and Methionine (Met) ...........................................14 1.8.2. Selenocysteine (Sec) and Cysteine (Cys) .........................................................17 1.8.3. Catalytic Mechanisms of Selenoenzymes ........................................................19 1.8.3.1. Thioredoxin Reductase (TrxR) ..........................................................19 iii 1.8.3.2. Methionine Sulfoxide Reductase (Msr) ............................................20 1.8.3.3. Glutathione Peroxidase (GPx) ...........................................................22 1.9. Therapeutic Application of Exogenous Selenocompounds .............................................23 1.9.1. Diselenides .......................................................................................................26 1.9.1.1. Diphenyl Diselenide [Ph(Se)2] ..........................................................27 1.9.1.2. Selenocystamine (SeCA) ...................................................................28 1.9.1.3. 3,3’-Diselenodipropionic Acid (DSePA) ..........................................29 1.9.1.4. Other Diselenide Compounds ...........................................................29 1.10. Factors Influencing the Reactivity of Disulfides and Diselenides ................................32 1.11. Hypothesis and Aims ....................................................................................................33 2. Materials and Methods ....................................................................................................35 2.1. General Information ........................................................................................................36 2.2. Materials ..........................................................................................................................36 2.3. Preparation of Sodium Phosphate Buffer (NaH2PO4; 0.1 M, pH 7.4) ............................38 2.4. Determination of Commercial H2O2 and HOCl Stock Concentrations ...........................38 2.5. Preparation of Photochemically-Generated Hydroperoxides ..........................................39 2.6. General Protocol for Ferrous-Oxidation Xylenol Orange (FOX) Assay ........................39 2.7. Methodology for Ultra-Performance Liquid Chromotography (UPLC) .........................40 2.7.1. Preparation of UPLC Mobile Phase .................................................................41 2.7.2. UPLC Instrumentation .....................................................................................41 iv 2.7.3. Fmoc-SeCystine Sample Preparation and Analysis .........................................41 2.7.4. Automated Dilution of Oxidised Fmoc-SeCystine for UPLC Product Analysis ..........................................................................................................................42 2.8. Detection of Oxidant-Induced Diselenide Bond Cleavage by UV/vis Spectroscopy .....43 2.8.1. Determination of Rate Constants for Reaction of SeCA and DSePA with H2O2 by UV/vis Spectroscopy ..............................................................................................43 2.9. Characterisation of Diselenide Oxidation Products by LC/MS ......................................44 2.9.1. Mass Spectrometry Instrumentation .................................................................44 2.9.2. Direct Injection MS for Characterising Diselenides ........................................44 2.9.3. LC/MS Protocol for Characterisation of Fmoc-SeCystine Oxidation Products ..........................................................................................................................45 2.9.4. LC/MS Protocol for Characterisation of GSeSeG Oxidation Products ...........46 2.10. Determining the Rate Constants of Reaction for HOCl with Diselenides via a Competitive CBA Assay .............................................................................................47 2.10.1. Stopped-Flow to Evaluate the Rate Constant for Reaction of CBA with HOCl ......................................................................................................................................47 2.10.2. CBA Competition Assay for Determining the Rate Constant of Reaction of HOCl with Diselenides ................................................................................................48 2.11. Statistical Analysis ........................................................................................................48 3. Reactions of Diselenides with Hypochlorous Acid (HOCl) ..........................................50 3.1. Introduction .....................................................................................................................51 3.2. Aims ................................................................................................................................53 v 3.3. Results .............................................................................................................................53 3.3.1. Investigation of the Oxidation of Selenocystamine by HOCl using UV/vis Spectroscopy ...............................................................................................................53 3.3.2. Optimisation of UPLC Conditions to enable the Detection of Diselenides and their Oxidation Product(s) ...........................................................................................55 3.3.3. Examination of the Oxidation of Fmoc-Selenocystine by Hypochlorous Acid using UPLC .................................................................................................................71 3.3.4. Characterisation of the Products of Fmoc-SeCystine Oxidation by HOCl using LC/MS .........................................................................................................................76 3.3.5. Characterisation of the Products of Seleno-Diglutathione Oxidation by HOCl using LC/MS ...............................................................................................................88 3.3.6. Oxidation of Coumarin Boronic Acid by HOCl to Form Fluorescent COH ....................................................................................................................................100 3.3.7. Determination of Rate Constants for the Reaction of HOCl with Diselenides by Competition Kinetics using the CBA Assay .............................................................104 3.4. Discussion .....................................................................................................................110 3.5. Conclusions ...................................................................................................................117 4. Reactions of Diselenides With Hydrogen Peroxide (H2O2) ........................................118 4.1. Introduction ...................................................................................................................119 4.2. Aims ..............................................................................................................................120