A Mechanistic Investigation of the Reactions of Diselenides with Biological Oxidants

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)

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

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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

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

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

4.3. Results ...... 121

4.3.1. Oxidation of Selenocystamine by Hydrogen Peroxide ...... 121

4.3.2. Calculation of Rate Constants for the Reaction of Diselenides with Hydrogen

Peroxide ...... 123

4.3.3. Characterisation of the Products of Selenocystamine Oxidation by Hydrogen Peroxide using MS ...... 128

4.3.4. Quantification of the Oxidation of Fmoc-Selenocystine by Hydrogen Peroxide using UPLC ...... 132

4.3.5. Characterisation of the Products of Fmoc-Selenocystine Oxidation by Hydrogen Peroxide using LC/MS ...... 139

4.3.6. Characterisation of the Products of Seleno-Diglutathione Oxidation by Hydrogen Peroxide using LC/MS ...... 146

4.4. Discussion ...... 150

4.5. Conclusions ...... 152

5. Reactions of Diselenides with Peroxides ...... 153

5.1. Introduction ...... 154

5.2. Aims ...... 155

5.3. Results ...... 156

5.3.1. SeCA Oxidation by Reagent Peroxides ...... 156

5.3.2. SeCA Oxidation by Photochemically-Generated Peroxides ...... 162

5.3.3. Diselenide Oxidation by Photochemically-Generated TrpOOH ...... 168

5.3.4. The Products of Diselenide Oxidation are Consistent between Various Oxidant Species ...... 172

5.4. Discussion ...... 175

5.5. Conclusions ...... 179

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6. Discussion and Future Research ...... 180

6.1. General Overview of Oxidant Generation and Oxidative Stress ...... 181

6.2. Oxidation of Key Sulfur-Containing Amino Acid Residues ...... 182

6.3. Endogenous Antioxidant Defence Mechanisms ...... 183

6.4. Selenium Compounds as Exogenous Antioxidants ...... 186

6.4.1. Diselenide Species ...... 186

6.5. Kinetics and Products of the Oxidation of Selenium Compounds ...... 188

6.5.1. Oxidation of Diselenides by Hydrogen Peroxide ...... 189

6.5.2. Oxidation of Diselenides by Reagent and Photochemically-Generated Peroxides ...... 190

6.5.3. Oxidation of Diselenides by Hypochlorous Acid ...... 192

6.6. Comparison of the Reactions of Sulfur- and Selenium-Containing Compounds with Various Biological Oxidants ...... 195

6.6.1. Disulfides and Diselenides ...... 197

6.6.2. Thiols and Selenols ...... 199

6.6.3. Synthetic Antioxidants ...... 199

6.7. Future Directions of Research ...... 200

6.8. Conclusions ...... 202

References ...... 204

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Abstract

Activated neutrophils release myeloperoxidase (MPO) which generates antimicrobial reactive oxygen species (ROS) including hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and 1 singlet oxygen ( O2). Inappropriate or excess formation of these species or failure of protective mechanisms can lead to localised oxidative stress and host tissue damage, particularly to proteins. Sulfur-containing residues are particularly susceptible to oxidation due to rapid rates of reaction with ROS and this damage has been implicated in a wide variety of inflammatory conditions including atherosclerosis, neurodegenerative diseases and some forms of cancer.

Antioxidants react with oxidants or their precursors to protect against and repair oxidative damage within biological systems and include both enzymatic [e.g. glutathione peroxidase (GPx)] and non-enzymatic [glutathione (GSH)] species, many of which rely upon reactive sulfur or selenium centres to facilitate their activity. The selenium atom is more nucleophilic than sulfur, allowing selenocompounds [e.g. selenomethionine (SeMet) and selenocysteine (Sec)] to display higher rate constants than the analogous sulfur species (Met and Cys, respectively). Thus, selenium-containing species may effectively minimise biological damage via the rapid removal of oxidants and subsequent rapid reduction of oxidised compounds.

The reactions of HOCl with thiol (RSH, e.g. Cys) and thioether (RSR’, e.g. Met) groups are extremely rapid (k = 107 – 108 M-1 s-1), generating disulfides (RSSR) and sulfoxides [RS(=O)R’], respectively, and further irreversible products at high excesses of oxidant. -1 -1 Oxidation of thiol-containing proteins by H2O2 is much slower (k ~ 1 – 3 M s ) and initially yields the sulfenic acid (RSOH), with subsequent reactions forming the sulfinic acid (RSO2H) or disulfide. Oxidation of non-thiol-containing residues (e.g. Trp, Tyr, His), by radical or non- 1 radical (e.g. O2) reactions, generates hydroperoxides (ROOH) as a major product. Further reactions of hydroperoxides can perpetuate damage to other biomolecules, causing DNA fragmentation and inactivation of thiol-dependent enzymes.

Selenols (RSeH, e.g. Sec) and selenoethers (RSeR’, e.g. SeMet) typically react more rapidly than their respective sulfur analogues, thiols and thioethers. Peroxynitrous acid (ONOOH) and hypothiocyanous acid (HOSCN) oxidise SeMet ca. 10-fold faster (k ~ 103 M-1 s-1) than Met (k ~ 102 M-1 s-1). Similarly, HOSCN reacts with Sec ca. 15-fold faster than with Cys (k ~ 106 and k ~ 104 M-1 s-1, respectively). However, selenols are highly unstable and undergo rapid auto- oxidation, making both research and therapeutic application problematic. Therefore, this thesis

ix investigates the reactions of diselenides (RSeSeR) as shelf-stable alternatives to selenols, which can be reduced to the more reactive selenol form in situ.

There has been extensive research into the oxidation of disulfide compounds by HOCl (k ~ 105 M-1 s-1), which reveals thiosulfinates [RSS(=O)R’; disulfide S-oxides] and thiosulfonates

[RSS(=O)2R’] to be the major initial products, with secondary structure playing a key role in determining disulfide reactivity, yet data on diselenide oxidation is scarce. In this thesis, several aliphatic and aromatic low-molecular-mass diselenides have been investigated [selenocystamine (SeCA), 3,3’-diselenodipropionic acid (DSePA), 2,2’-dipyridyl diselenide (DPDSe) and dipyrimidin-2-yl diselenide (DPMSe)], as well as fluorescently-tagged selenocystine (Fmoc-SeCystine; the diselenide of Sec) and the selenium analogue of oxidised GSH [seleno-diglutathione (GSeSeG)]. Rate constants were determined for the reactions of 1 many of these diselenides with HOCl, H2O2 and O2-generated amino acid/peptide hydroperoxides and the products characterised by LC/MS.

Oxidation of low-molecular-mass diselenides by HOCl yielded rate constants in the range of 103 – 105 M-1 s-1, with the aliphatic compounds reacting more rapidly than the aromatic derivatives. Reaction with H2O2 was much slower, with rate constants determined for SeCA (k = 9.2 x 10-4 M-1 s-1) and DSePA (k = 1.5 x 10-2 M-1 s-1). Also, SeCA reacted at similar rates with various hydroperoxides (k ~ 10-2 M-1 s-1), whereas DSePA was oxidised by Trp hydroperoxide (TrpOOH) ca. 50 – 150-fold faster than the other diselenides investigated. Taken together, these data reveal that the secondary structure and ionisation/protonation state of the terminal groups of a diselenide affect the kinetics of the reactions of oxidants with the diselenide moiety. The favourable positioning of nearby heteroatoms (e.g. N, O) is postulated to elevate the lone-pair energy level of the reactive selenium centre and increase reactivity.

Product characterisation studies were performed for SeCA, Fmoc-SeCystine and GSeSeG with various oxidants (HOCl, H2O2, TrpOOH), which revealed the major product in each case to be the seleninic acid (RSeO2H). Oxidation is postulated to occur via initial formation of a transient intermediate [RSeSe(=O)R’ or RSeSe(=O)2R’] and subsequent diselenide bond cleavage to yield the seleninic acid, as per the typical oxidative pathway of disulfide species. However, the reaction pathways for these reactions have yet to be elucidated.

The studies reported in this thesis demonstrate the ability of diselenide compounds to act as competitive scavengers of biological oxidants, providing a basis for future research to investigate their potential in vivo application to mitigate oxidative stress-induced pathologies.

Acknowledgements

I would firstly like to thank my supervisors, Dr. David Pattison and Prof. Michael Davies, for your ongoing guidance, support and encouragement throughout my candidature. Your generosity of spirit in sharing your knowledge, time and expertise has been invaluable to the completion of this project and has helped to make me a better scientist.

I would also like to thank Dr. Luke Carroll for your help both in and out of the lab, for being so reliable and for always patiently answering my many questions. Thank you for your support and your friendship during my candidature and for being someone to look up to.

Thank you to Prof. Paul Pilowsky for your pastoral support and unwavering belief in me and for the very kind (and very unique) pep-talks. Your encouragement has been truly appreciated, particularly towards the end of my candidature.

To the Free Radical Group (HRI) and Protein Oxidation Group (University of Copenhagen), who have all been wonderful colleagues; thank you for fostering such positive, successful and encouraging work environments. Being a part of each team has been an honour. In particular, I’d like to thank Pam and Adrian for being my coffee buddies and my work family.

Finally, to my parents, Margaret and Peter, and my brother, Scott - I am so grateful for the unyielding love and support that you have shown me during my candidature as well as my life outside of it. I am truly grateful to be surrounded by such wonderful family and friends. Thank you all for your encouragement from beginning to end and for getting me through the tougher times with laughter and good company.

List of Abbreviations

This thesis employs the “three-letter system” for abbreviating amino acids, as described by the IUPAC-IUB Joint Commission on Biomedical Nomenclature.

ACN Acetonitrile

CAD Coronary artery disease

CBA Coumarin boronic acid

CHP Cumene hydroperoxide

CK Creatine kinase

3-ClTyr 3-Chlorotyrosine

COH 7-Hydroxycoumarin

CVD Cardiovascular disease

DHA Dehydroalanine

DHS Dihydroxy-1-selenolane

DMSO Dimethyl sulfoxide

DOPA 3,4’-Dihydroxyphenylalanine

DPDSe 2,2’-Dipyridl diselenide

DPMSe Dipyrimidin-2-yl diselenide

DSePA 3,3’-Diselenodipropionic acid

DTDPA 3,3’-Dithiodipropionic acid

DTT Diothiothreitol

Dox Doxorubicin

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(EbSe)2 Ebselen diselenide

EFsec Sec-specific elongation factor

EPO Eosinophil peroxidase

Fmoc 1-(9-Fluorenyl)methyl chloroformate

FOX Ferrous-oxidation xylenol orange

GAPDH Glyceraldehyde-3-phosphate dehydrogenase

GPx Glutathione peroxidase

GR Glutathione reductase

GSA Glutathione sulfonamide

GSeA Selenium analogue of GSA

GSeH Selenoglutathione

GSeSeG Seleno-diglutathione (Glutathione diselenide)

GSH Glutathione (L-ɣ-glutamyl-L-cysteinyl-glycine)

GSSG Glutathione disulfide (Oxidised glutathione)

HCl Hydrochloric acid

H2O2 Hydrogen peroxide

HOBr Hypobromous acid

HOCl Hypochlorous acid

HOHICA 3a-hydroxy-6-oxo-2,3,3a,6,7,7a-hexahydro-1H-indol-2- carboxylic acid

HOI Hypoiodous acid

HOSCN Hypothiocyanous acid

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HSV-2 Herpes simplex virus 2

LPO Lactoperoxidase

LysTrp H-Lys-Trp-OH acetate salt

LysTrpLys H-Lys-Trp-Lys-OH acetate salt

MetO Methionine sulfoxide

MetO2 Methionine sulfone

MPO Myeloperoxidase

Msr Methionine sulfoxide reductase

NADPH Nicotinamide adenine dinucleotide phosphate

NaOAc Sodium acetate trihydrate

NaOH Sodium hydroxide

NCE Normalised collision energy

NEM N-Ethylmaleimide

•NO Nitric oxide

• NO2 Nitrogen dioxide

NOS Nitric oxide synthase

NOX NADPH oxidase

NPC Nutritional Prevention of Cancer trial

1 O2 Singlet oxygen

•- O2 Superoxide anion

•OH Hydroxyl radical

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ONOOH/ONOO- Peroxynitrous acid/Peroxynitrite oxLDL Oxidised low-density lipoprotein

PDA Photodiode array

(PhSe)2 Diphenyl diselenide

RB Rose Bengal

RNHCl Chloramine (Monochloramine)

ROS Reactive oxygen species

SBP Sec binding protein

Sec Selenocysteine

SeCA Selenocystamine

SECIS Selenocysteine insertion sequence

SeCystine Selenocystine

SELECT Selenium and Vitamin E Cancer Prevention Trial

SeMet Selenomethionine

SeMetO Selenomethionine selenoxide

SeMSC Methyl-selenocysteine

SIM Selective ion monitoring

SOD Superoxide dismutase tBuOOH tert-Butyl hydroperoxide

TFA Trifluoroacetic acid

TGR Thioredoxin glutathione reductase

THF Tetrahydrofuran

TrpLys H-Trp-Lys-OH formiate salt

Trx/TrxS2 Thioredoxin/Oxidised thioredoxin

TrxR Thioredoxin reductase

XO Xylenol orange

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List of Figures

1 Figure 1.1: Reaction pathways of Trp via UV absorption or O2-mediated oxidation ...... 7

1 Figure 1.2: Reaction pathways of Tyr via UV absorption or O2-mediated oxidation ...... 8

1 Figure 1.3: Reaction pathways of O2-mediated oxidation of His ...... 8

Figure 1.4: Catalytic cycle of MPO ...... 10

Figure 1.5: Catalytic cycle of TrxR ...... 20

Figure 1.6: Catalytic cycle of Sec-containing Msr enzymes (e.g. MsrB1) ...... 21

Figure 1.7: Traditional catalytic cycle of glutathione peroxidase (GPx) ...... 23

Figure 1.8: Chemical structure and catalytic cycle of ebselen ...... 24

Figure 1.9: Proposed GPx-like catalytic cycle of GSeSeG ...... 30

Figure 3.1: Structures of the diselenides investigated in this Chapter ...... 52

Figure 3.2: Oxidation of SeCA (1 mM) by HOCl (0 to 4 mM); A) t = 0 min, B) t = 24 h ...... 54

Figure 3.3: UPLC chromatograms of SeCA (0 – 200 µM) obtained with different columns; A) Shim-Pack XR-ODS, B) Hichrom Ultrasphere 5 ODS, C) Beckman Coulter ODS...... 56

Figure 3.4: UPLC chromatogram of SeCA (0 – 200 µM) ...... 57

Figure 3.5: UPLC chromatogram of SeCA (0 – 200 µM) ...... 58

Figure 3.6: UPLC chromatogram of SeCA (0 – 200 µM) ...... 59

Figure 3.7: UPLC chromatograms of; A) SeCA (0 – 200 µM) and B) DSePA (0 – 200 µM) ...... 60

Figure 3.8: UPLC chromatogram of SeCA (0 – 200 µM) ...... 61

Figure 3.9: UPLC chromatogram of SeCA Standards (50 – 500 µM) ...... 62

Figure 3.10: UPLC chromatogram of Fmoc-SeCystine (100 µM) at various MeOH

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percentages (20 – 100%) ...... 65

Figure 3.11: UPLC chromatograms of; A) Fmoc-SeCystine standards (0 – 200 µM), B) Fmoc-SeCystine (100 µM) with HOCl (0, 50, 100, 200 µM) ...... 66

Figure 3.12: UPLC chromatogram of Fmoc-SeCystine (100 µM) with traces showing different initial buffer B concentrations (20 – 75%) ...... 67

Figure 3.15: Representative UPLC chromatograms of Fmoc-SeCystine (100 µM) oxidation by HOCl (0 to 400 µM). A) Loss in Fmoc-SeCystine, B) Formation of Fmoc- SeCystine Products ...... 72

Figure 3.16: Oxidative loss of Fmoc-SeCystine (100 µM) with HOCl (0 to 200 µM) ...... 73

Figure 3.17: Half maximal inhibitory concentration (IC50) plot of Fmoc-SeCystine (100 µM) loss with HOCl treatment (0 to 400 µM) ...... 74

Figure 3.18: Formation of product peaks upon oxidation of Fmoc-SeCystine (100 µM) by HOCl (0 to 200 µM). A) Product 1, B) Product 2 ...... 75

Figure 3.19: MS Spectra of Fmoc-SeCystine; A) Experimental direct injection, B) Computational simulation ...... 77

Figure 3.20: Representative full-scan LC/MS chromatogram of Fmoc-SeCystine (100 µM) in the absence (A) or presence (B) of HOCl (100 µM) ...... 78

Figure 3.21: MS spectra of the major chromatogram peaks of Fmoc-SeCystine (100 µM) in the absence or presence of HOCl (100 µM), which eluted at: A) 33.3 min, B) 35.3 min, C) 38.9 min ...... 79

Figure 3.22: MS spectra of the major chromatogram peaks of Fmoc-SeCystine (100 µM)

oxidised by HOCl (100 µM), which eluted at: A) 28.0 min (RSeO2H), B) 46.4

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min [Fmoc-SeCystine + H]+, and C) 46.4 min [2Fmoc-SeCystine + H]+ ...... 80

Figure 3.23: MS spectrum of the seleninic acid (RSeO2H) product formed by oxidation of Fmoc-SeCystine (100 µM) by HOCl (100 µM) ...... 81

Figure 3.24: Proposed structure of the seleninic acid (RSeO2H) product formed by Fmoc- SeCystine oxidation ...... 82

Figure 3.25: Representative UV/vis traces (λ = 265 nm) of Fmoc-SeCystine (100 µM) with increasing concentrations of HOCl: A) 0 µM, B) 25 µM, C) 100 µM, D) 200 µM ...... 82

Figure 3.26: MS spectra corresponding to the UV/vis peaks (λ = 265 nm) eluting at; A) 35.3 min, B) 40.1 min ...... 83

Figure 3.27: LC/MS extracted ion chromatograms of the oxidation of Fmoc-SeCystine (100 µM) by HOCl (0 to 400 µM); A) Fmoc-SeCystine loss, B) Product formation

(RSeO2H), and the corresponding MS spectra of these peaks, C) Fmoc-

SeCystine, D) RSeO2H ...... 84

Figure 3.28: Plots of; A) Fmoc-SeCystine loss, and B) RSeO2H formation against increasing concentrations of HOCl (0 – 400 µM) ...... 85

Figure 3.29: MS/MS spectra generated from fragmentation of the major ions of; A) Fmoc-

SeCystine (m/z 780.9 ± 7.5) and B) RSeO2H (m/z 424.0 ± 7.5) ...... 87

Figure 3.30: Full scan MS chromatogram (m/z 100 – 1500) of GSeSeG (100 µM) oxidised by HOCl (100 µM), MS spectra (A, B) and simulated spectra (C, D). MS

spectra are of peaks eluting at; A) 5.5 min (GSeO2H), B) 24.0 min (GSeSeG);

Simulated spectra are of; C) GSeO2H, D) GSeSeG ...... 89

Figure 3.31: Structures of GSeSeG and GSeO2H ...... 90

Figure 3.32: LC/MS extracted ion chromatograms (A, B) and full scan MS spectra (C, D) of the oxidation of GSeSeG (100 µM) by HOCl (0 – 400 µM); A) GSeSeG

loss, B) GSeO2H formation, C) GSeSeG MS spectrum, D) GSeO2H MS spectrum ...... 91

Figure 3.33: Representative full scan MS chromatogram (m/z 100 – 1500) of the chromatogram peak eluting at 8.8 min, formed in the presence of GSeSeG

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(100 µM) and HOCl (400 µM) ...... 93

Figure 3.34: Proposed pathway for the formation of 5-hydroxybutyrolactam from oxidation of GSSG by HOCl ...... 93

Figure 3.35: Plots of; A) GSeSeG loss, and B) GSeO2H formation against increasing concentrations of HOCl (0 – 200 µM) ...... 95

Figure 3.36: Half maximal inhibitory concentration (IC50) plot of GSeSeG (100 µM) loss with HOCl (0 – 400 µM) treatment ...... 96

Figure 3.37: Representative MS/MS analysis (m/z 708.4 ± 7.5) of GSeSeG (100 µM) oxidised by HOCl (100 µM); A) Full scan MS/MS chromatogram (m/z 100 – 1500), B) Spectrum of peak eluting at 22.4 min, C) Spectrum of peak eluting at 34.5 min ...... 98

Figure 3.38: Representative MS/MS analysis (m/z 387.8 ± 7.5) of GSeSeG (100 µM) oxidised by HOCl (100 µM); A) Full scan MS/MS chromatogram (m/z 100 – 1500), B) Spectrum of peak eluting at 5.5 min, C) Spectrum of peak eluting at 20.5 min ...... 99

Figure 3.39: Oxidation of Coumarin Boronic Acid (CBA) to 7-Hydroxycoumarin (COH) by biological oxidants ...... 100

Figure 3.40: COH fluorescence (350 – 450 nm) at 15 min following the oxidation of CBA (25 µM) by HOCl (0 to 25 µM) ...... 101

Figure 3.41: Example of a kinetic trace obtained by stopped-flow (50 µM HOCl + 300 µM CBA), showing COH fluorescence (350 – 450 nm) over 1 s post-mixing ...102

Figure 3.42: Plot of kobs against CBA concentration for the oxidation of CBA (250 - 500 µM) by HOCl (50 µM) ...... 103

Figure 3.43: Light-induced oxidation of CBA (500 µM), observed as formation of fluorescent COH ...... 104

Figure 3.45: Loss in final COH fluorescence (350 – 450 nm; at 30 min) following oxidation

of CBA (25 µM) by HOCl (25 µM) in the presence of diselenide; A) DSePA (0 to 100 µM), B) SeCA (0 to 100 µM), C) DPDSe (0 to 100 µM), D) DPMSe (0 to 200 µM) ...... 107

Figure 3.46: Competition kinetics plots from experimentally-generated COH fluorescence data: A) DSePA (0 to 25 µM), B) SeCA (0 to 20 µM), C) DPDSe (0 to 25 µM), D) DPMSe (0 to 200 µM) ...... 109

Figure 3.47: Proposed structure of GSeA ...... 113

Figure 3.48: Theoretical MS spectra of; A) GSeO2H, and B) GSeA ...... 113

Figure 4.1: UV/visible spectrum showing absorbance of untreated SeCA (1 mM) over 7 days ...... 121

Figure 4.2: UV/vis spectra of SeCA (1 mM) oxidation by H2O2 (10 mM) over 7 days...122

Figure 4.3: Oxidation of diselenides (1 mM) by H2O2 (20 mM) over time; A) SeCA, B) DSePA ...... 124

Figure 4.4: Diselenide (1 mM) absorbance loss over time (16.5 h) with H2O2 (20 mM); A) SeCA, B) DSePA ...... 125

Figure 4.5: Pseudo first-order kinetic plots for the loss of diselenides (1 mM) with

increasing concentrations of H2O2 (10 – 50 mM); A) SeCA, B) DSePA .....126

Figure 4.6: Oxidation of GSeSeG (1 mM) by H2O2 (50 mM) over time (16.5 hrs) ...... 127

Figure 4.8: Computational simulation of MS Spectra of SeCA, and the respective di- and

tri-oxygenated species, RSeO2H and RSeO3H ...... 131

Figure 4.9: Proposed structures of the products of SeCA oxidation by H2O2 ...... 131

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Figure 4.13: Oxidation of Fmoc-SeCystine (100 µM) by H2O2 (0 to 100 mM) over time (24 h), with automated dilution (1 in 40) prior to analysis. A) Fmoc-SeCystine Loss (% Control), B) Formation of Fmoc-SeCystine Product 1, C) Formation of Fmoc-SeCystine Product 2 ...... 138

Figure 4.14: Representative LC/MS data of Fmoc-SeCystine (100 µM) treated with H2O2 (500 µM), 24 h post-oxidant addition; A) Full scan chromatogram (m/z 150 – 2000), B) MS spectrum of peak eluting at 46.4 min (Fmoc-SeCystine) ...... 140

Figure 4.15: Representative UV/vis traces (λ = 265 nm) showing Fmoc-SeCystine (100

µM) oxidation by increasing concentrations of H2O2; A) 0 mM, B) 10 mM, C) 100 mM ...... 141

Figure 4.16: Representative MS spectra generated from the UV/vis (λ = 265 nm) chromatogram peaks eluting at; A) 27.9 min, B) 46.1 min, C) 37.6 min, D) 39.7 min ...... 142

Figure 4.17: Representative LC/MS extracted ion chromatograms showing the oxidation of

Fmoc-SeCystine (100 µM) by H2O2 (0 – 100 mM); A) Fmoc-SeCystine loss at

t = 0 min (m/z 780.4 ± 7.5), B) RSeO2H formation at t = 24 h (m/z 424.0 ± 7.5), and MS spectra of these peaks; C) Fmoc-SeCystine (46.1 min), D)

RSeO2H (27.9 min) ...... 143

Figure 4.18: MS/MS analysis of Fmoc-SeCystine (m/z 780.4 ± 7.5); A) MS/MS chromatogram, B) MS/MS spectrum of peak at 46.1 min ...... 144

Figure 4.19: MS/MS analysis of RSeO2H (m/z 424.0 ± 7.5); A) Extracted ion

chromatogram, B) MS/MS spectrum of peak at 27.9 min (RSeO2H), C) MS/MS spectrum of peak at 37.6 min (contaminant) ...... 145

Figure 4.20: Representative LC/MS data of GSeSeG (100 µM) oxidation by H2O2 (0 or 1

xxii

mM); A) Full scan MS chromatogram (m/z 100 - 1500), B) MS spectrum of peak at 31.9 min (GSeSeG) ...... 147

Figure 4.21: Representative LC/MS data of the oxidation of GSeSeG (100 µM) by H2O2 (0 or 1 mM) ...... 149

Figure 5.1: Structures of amino acids (Trp, Tyr, His) and peptides (LysTrp, TrpLys, LysTrpLys) investigated in this Chapter ...... 155

3 Figure 5.2: Absorbance of the Fe -XO complex generated by reagent peroxides (H2O2 and CHP, 0 to 25 µM; tBuOOH, 0 to 12.5 µM) measured using the FOX assay, as

equivalents of H2O2 ...... 157

Figure 5.3: Intrinsic decay of reagent peroxides (H2O2, tBuOOH and CHP; 25 mM) up to 7 days ...... 158

Figure 5.4: Extent of peroxide loss (H2O2, tBuOOH, CHP; initially 25 mM) over time in

the absence or presence of SeCA (0 to 10 mM): A) H2O2 (48 h), B) tBuOOH (7 days), C) CHP (7 days) ...... 159

Figure 5.5: Observed loss (kobs) of peroxides (25 mM) with increasing concentrations of

SeCA (0 to 10 mM); A) H2O2, B) tBuOOH, C) CHP ...... 161

Figure 5.6: Absorbance values of hydroperoxides expressed as equivalents of H2O2 ....163

Figure 5.7: Intrinsic decay of photochemically-generated peroxides over time (48 h) ...164

Figure 5.10: Loss of photochemically-generated TrpOOH (ca. 500 µM) with increasing concentrations of diselenides; A) DSePA (0 to 1 mM), B) DPMSe (0 to 10 mM), C) DPDSe (0 to 3 mM) ...... 169

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Figure 5.11: Plots of kobs against concentration of diselenide compounds upon reaction with TrpOOH: A) DSePA (0 to 1 mM), B) DPMSe (0 to 10 mM), C) DPDSe (0 to 3 mM) ...... 171

Figure 5.12: UPLC Chromatograms of; A) Blank and Control samples, B) TrpOOH Control over time (t = 0 and 72 h) ...... 173

Figure 5.13: UPLC chromatogram of Fmoc-SeCystine (500 µM in 50% MeOH) at decreasing injection volumes (10, 8, 6, 4, 2 µL) ...... 174

Figure 5.14: UPLC chromatogram showing oxidation of Fmoc-SeCystine (500 or 100 µM)

by TrpOOH (ca. 500 µM) and H2O2 (1 mM) ...... 175

Figure 6.1: Mechanisms of action and kinetics of Cys- vs Sec-dependent enzymes ...... 185

Figure 6.2: Thiol/disulfide and selenol/diselenide exchange reactions ...... 197

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List of Tables

Table 3.1: Summary of UPLC conditions employed during optimisation of diselenide detection ...... 63

Table 3.2: Abundance of naturally-occurring selenium isotopes ...... 76

Table 3.3: Absolute rate constants (k) for the reaction of various diselenides with HOCl ...... 108

Table 3.4: Absolute (k) or second-order (k2) rate constants for the reaction of diselenides and disulfides with HOCl ...... 116

Table 4.1: Reported rate constants of reaction (k) of H2O2 with biological substrates ..120

Table 4.2: Absolute rate constants (k) for reaction of H2O2 with diselenides ...... 128

Table 5.1: Absolute rate constants (k) for the reaction of SeCA with reagent peroxides ...... 162

Table 5.2: Observed rate constants (kobs) for the intrinsic decay of photochemically- generated peroxides ...... 164

Table 5.3: Absolute rate constants (k) for the reaction of SeCA with photochemically- generated peroxides ...... 166

Table 5.4: Absolute rate constants (k) for the reaction of various diselenides with photochemically-generated TrpOOH ...... 170

Table 5.5: Absolute rate constants (k) for the reaction of various diselenides with biological and model oxidants ...... 176

Table 6.1: Absolute rate constants (k) for the reaction of various diselenides with biological oxidants ...... 188

Table 6.2: Absolute rate constants (k) for the reaction of SeCA with various peroxides ...... 189

Table 6.3: Comparison of rate constants (k) for the reactions of sulfur compounds (and some of their selenium analogues) with biological oxidants ...... 196

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Publications Arising from this Thesis

Conference Presentations

Kelly A. Gardiner, Michael J. Davies & David I. Pattison (2017) Characterising the reactions of diselenides with reactive oxidants generated during chronic inflammation, Poster Presentation, Sydney Cardiovascular Symposium: Big Data and the Future of Cardiology, Garvan Institute of Medical Research, Darlinghurst, Australia, 7 – 8 Dec 2017

Kelly A. Gardiner, Michael J. Davies & David I. Pattison (2016) Characterising the reactions of diselenides with reactive oxidants generated during chronic inflammation, Poster Presentation, 23rd Annual Meeting of the Societies for Redox Biology and Medicine & Society for Free Radical Research International 18th Biennial Congress, San Francisco, California, USA, 16 – 19 Nov 2016

Kelly A. Gardiner, Michael J. Davies & David I. Pattison (2015), Characterising the reactions of diselenides with reactive oxidants generated during chronic inflammation, Poster Presentation, 23rd Annual Meeting of the Societies for Free Radical Research of Australasia and Japan, Christchurch, New Zealand, 7 – 10 Dec 2015

Kelly A. Gardiner, Michael J. Davies & David I. Pattison (2014), Mechanisms of damage to low-molecular-mass selenoethers and diselenides induced by inflammation-related oxidants, Poster Presentation, 22nd Annual Meeting of the Societies for Free Radical Research of Australasia, Melbourne, Australia, 3 – 6 Dec 2014

Kelly A. Gardiner, David I. Pattison, David van Reyk & Michael J. Davies (2013), Mechanisms of damage to selenium-containing amino acids induced by reactive oxidants that are generated during chronic inflammation, Poster Presentation, 21st Annual Meeting of the Societies for Free Radical Research of Australasia and Japan, Sydney, Australia, 12 – 14 Sept 2013

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Chapter 1

Introduction

1.1. The Immune Response and Inflammation

In the event of mechanical injury or pathogenic invasion, the human immune system is evolutionarily designed to employ a cascade of processes aimed at returning host tissue to its homeostatic state.1 The efficacy of the immune response is dependent upon its ability to initiate a response that is both rapid and specific to the inflammatory trigger, which facilitates the clearance of potentially harmful agents whilst minimising damage to host tissue.2, 3

The process of inflammation involves the recruitment and activation of leukocytes (‘white blood cells’), the mobile mediatory components of the immune system.4 Leukocytes infiltrate inflammatory sites by migrating along a chemical gradient of chemotactic factors such as pro- inflammatory cytokines, which are released from bacteria, damage-activated macrophages, mast cells and endothelial cells, and also arise from activation of the complement system.3 The most abundant leukocyte and first cell line to be recruited to the injury site is the neutrophil, which accounts for ca. 60 per cent of total circulating leukocytes under physiological conditions.4, 5

Neutrophils are granulocytic cells which act via phagocytosis, ingesting pathogenic material and releasing antimicrobial agents including multiple enzymes and reactive oxygen species (ROS) into the phagolysosome of the cell.6, 7 One of the major enzymes released upon neutrophil degranulation is the haem enzyme myeloperoxidase (MPO), which is the most abundant protein in the neutrophil, constituting approximately 5 per cent of the cells’ total dry mass.7-9 Other members of the haem peroxidase family include lactoperoxidase (LPO), an antimicrobial protein found in milk, saliva, tears and airway secretions, and eosinophil peroxidase (EPO), expressed in eosinophils.10, 11

MPO-mediated reactions are responsible for the generation of many of the potent antimicrobial ROS that neutrophils produce.12 It is well established that various ROS play an important role in the immune response and inflammation, owing to their ability to destroy invading pathogens6, 13 and tumour cells.14, 15 However, MPO-derived ROS can also have deleterious effects on host tissue, causing damage via the induction of localised oxidative stress.16, 17 Oxidative stress is a state wherein excess or inappropriate ROS are produced or ROS- scavenging or -degrading mechanisms are inadequate, favouring the balance of oxidants over antioxidants.18, 19 ROS-induced oxidative stress is believed to play a pivotal role in the onset and progression of inflammatory pathologies including some cancers14, 20, 21, neurodegenerative diseases17 and atherosclerosis.22, 23

1.2. Generation of Reactive Oxygen Species (ROS)

ROS are any chemically reactive species containing oxygen, encompassing both free radical (one electron) and non-radical (two electron) oxidants.19 Free radical species are typically short-lived molecules with an unpaired valence electron, such as the superoxide radical anion •- • 19 (O2 ) and the hydroxyl radical ( OH). Non-radical ROS are oxidising compounds such as 1 24 hypochlorous acid (HOCl), hydrogen peroxide (H2O2) and singlet oxygen ( O2). Inappropriate or excess formation of ROS has been associated with the onset of inflammatory pathologies due to the capacity of ROS to oxidise biological substrates,25 with the species of oxidant influencing the type and severity of damage.6

Immune response activation is concomitant with the production of ROS via a process termed the respiratory (or oxidative) burst.24 Neutrophil activation upon exposure to pathogenic stimuli incites an oxygen-dependent pathway characterised by an increased initial consumption of 6, 24, 26 molecular oxygen (O2) by the cell. This O2 acts as a substrate for a membrane-bound enzyme complex, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), •- 14, 16, 20 which catalyses the production of O2 and H2O2. The NOX family of enzymes occur widely in many animal and plant tissues, where they generate ROS in order to carry out numerous biological functions including immune defence and antimicrobial actions, cellular proliferation and differentiation, vascular regeneration and redox regulation.6, 10

•- Through the action of NOX-2, a neutrophil-specific NOX enzyme, O2 is converted to O2 within the phagosomal space of neutrophils at a rate of up to 5 mM per second.6, 24 The majority •- of O2 is converted to H2O2 by either spontaneous dismutation or enzymatic reaction using 6, 24 6 superoxide dismutase (SOD). H2O2 is a relatively weak oxidant in itself, but the MPO- - - catalysed reaction of H2O2 with halide (chloride, Cl ; and bromide, Br ) and pseudo-halide (thiocyanate, SCN-) ions facilitates the formation of more potent oxidants, namely, the hypohalous acids (HOX); that is, hypochlorous (X = Cl), hypobromous (X = Br) and hypothiocyanous acids (X = SCN), respectively.7, 9 The predominant MPO-derived products at physiological plasma ion concentrations are around 50 per cent HOSCN and 45 per cent HOCl (Reaction 1):27, 28

푀푃푂 − + 퐻2푂2 + 퐶푙 + 퐻 → 퐻푂퐶푙 + 퐻2푂 (1)

Another considerable source of exogenous ROS originates from ultraviolet (UV) radiation,

1 29 which can generate both free radicals on susceptible biological substrates and O2. Singlet oxygen is the first excited state of molecular oxygen, with both electrons in the same orbital 30 1 and having antiparallel spin. It was proposed in 1972 that O2 may be produced by the respiratory burst of neutrophils during inflammation or infection.31 No incontrovertible 1 evidence was found in support of this hypothesis until a decade later when the first O2 emission spectrum was reported to be generated from a biological enzymatic system, leading to the 1 32-34 suggestion that MPO-derived HOCl can form O2 via Reaction 2:

− − 1 푂퐶푙 + 퐻2푂2 → 퐶푙 + 퐻2푂 + 푂2 (2)

•- Singlet oxygen is a potent oxidant that is much more reactive than O2 , is readily formed and 29, 35 1 has a relatively long lifetime within the range of microseconds. The lifetime of O2 and its potential to perpetuate secondary damage within a protein means that significant damage is 1 29 possible despite low concentrations of O2 being produced. As such, in conjunction with the 1 hypohalous acids, O2 constitutes a potentially significant pathway for the accumulation of 1 19, 29, 36, 37 oxidative damage, particularly as a major target of O2 is proteins.

Nitric oxide (•NO) is a free radical generated by nitric oxide synthases (NOS), which reacts •- - with O2 at diffusion-controlled rates to form peroxynitrite (ONOO ), a peroxy acid with strong 38 oxidising and nitrating properties. The peroxynitrite anion has a pKa value of 6.8 so that under physiological conditions, both ONOO- and peroxynitrous acid (ONOOH) will be present, with the exact ratio being strongly pH-dependent.38 The labile O-O bond of ONOOH is • • homolytically cleaved to form OH and nitrogen dioxide ( NO2), which are potent oxidising and nitrating species, respectively.38 Much of the in vivo biological activity of ONOOH is due to its nucleophilicity, which facilitates the rapid one- and two-electron oxidation of biological substrates, including sulfur-containing thiols (RSH) and thioethers (RSR’).38 ONOOH and the secondary radicals that arise from it also target lipids, proteins, carbohydrates and nucleic acids, making this species a potent cytotoxic effector against invading pathogens, as well as an endogenous toxicant, causing ONOOH to be implicated in human pathologies including motor neuron death, neurodegeneration and cardiovascular disease (CVD).38, 39

An additional common source of endogenous ROS is normal aerobic cellular respiration, wherein mitochondria facilitate multiple sequential one-electron reductions of molecular oxygen for energy metabolism.19 However, exogenous factors also represent a source of ROS; for example, particulates from fuel combustion, dust and fibres such as asbestos, ozone and

4 drug metabolism.40

1 1.3. Singlet Oxygen ( O2) and Photo-Oxidation Reactions

1 1 In addition to the generation of O2 by MPO-derived HOCl (Reaction 2), O2 may be formed via alternative mechanisms, such as by decomposition of lipid hydroperoxides41 and •- 42 theoretically by oxidation of O2 by MPO, although this latter reaction has not yet been 6 1 verified experimentally. O2 can also be produced in biological systems via energy transfer from a sensitiser (such as various dyes and porphyrins)43 to molecular oxygen.29 Peroxyl • 1 44, 45 radicals (ROO ) also represent a considerable source of O2.

Photo-oxidation reactions, which employ a photosensitiser, were amongst the earliest indicators that neutral oxygen molecules may be activated via a light-dependent mechanism36 1 and O2 was proved to be an intermediate species in both chemical and photo-oxidation 46-48 1 reactions. Methods of experimentally producing and detecting O2 were subsequently developed49 with the intention of applying this oxidant in a beneficial and therapeutic approach; 1 for example, utilisation of O2 in the photodynamic therapy of superficial human tumours to effectively eliminate localised skin cancers.50

Photo-oxidation of proteins can occur via several pathways. Exposure to visible light or UV radiation in the presence of suitable chromophores (e.g. certain amino acids and side-chain residues) can induce damage to proteins, either directly or indirectly.37 The initial energy absorption is performed by either a protein or its side chains (direct), or by a sensitising compound (indirect). Direct absorption (referred to as a Type I mechanism) is chiefly pertinent to Trp, His, Tyr, Phe, Met, Cys and cystine (disulfide) residues, which are the major chromophoric amino acids present in proteins.29, 51 Direct absorption of UV radiation by these residues facilitates photo-ionisation reactions, which result in the generation of excited state •- 37, 51 species (singlets or triplets) and radicals (e.g. O2 ).

1 Indirect protein oxidation results from the formation and ensuing reactions of O2 (Type II processes).51 Initial energy absorption by endogenous (e.g. porphyrins, vitamins) or exogenous 1 (e.g. drugs, dyes) sensitisers results in energy transfer to molecular oxygen, forming O2, which 37 1 then reacts with target molecules to form the longer-lived triplet state. O2 is known to react with Trp, Tyr, His, Met and Cys more rapidly than with most other cellular targets at physiological pH values (k ~ 107 M-1 s-1).51, 52 The specific configuration and location of the reactive residue within the protein is also known to affect the rate constant for reaction with

1 53, 54 1 O2. Direct reaction of O2 with these susceptible residues forms protein peroxides, which can decay to give radical species (alkoxyl, peroxyl and carbon-centred), or react directly with other molecules, thereby facilitating the perpetuation of oxidative damage in biological 55, 56 1 systems, even after cessation of photolysis (‘dark reactions’). Subsequently, O2 and its photo-products have been implicated in the pathogenesis of cataract formation,52 sunburn and skin cancer.29

1.4. Protein Peroxides

Protein peroxides (ROOR’), which include reactive hydro- (R’ = H) and endo-peroxides (R’ = any heterocylic ring), are major products of protein photo-oxidation.57 These intermediates are 1 55 formed in high yields on amino acids and proteins by reactive oxidants, including O2, and also by oxygen-derived radicals, activated neutrophils and peroxynitrite.51 Hydroperoxides can undergo rapid reaction with reducing substrates such as protein and low-molecular-mass thiols, concomitant with the oxidation of the thiol group, resulting in perturbation of cellular signalling and redox homeostasis.37, 57

Due to their rapid decomposition and further reactions, direct detection of protein peroxides in vivo is difficult, but indirect evidence, i.e. the detection of decay products (e.g. alcohols) is compelling.57 Furthermore, hydroxylated amino acids [e.g. 3,4’-dihydroxyphenylalanine (DOPA), dityrosine] have been detected in both normal and pathological samples such as human lens cataracts and atherosclerotic lesions.52, 57 The relative abundance of these markers in pathological samples has provided sound evidence indicating the role of peroxide-derived •OH radicals in the progression of these conditions.57-59

Trp and Tyr undergo UV light-induced electron transfer with suitable substrates (e.g. disulfides such as cystine) to give radical ions (cations e.g. Trp+•/Tyr+• and anions e.g. RSSR-•) which can • • deprotonate to the neutral indolyl/tyrosyl radical (Trp /Tyr ), which then react with O2 (albeit slowly) to give a peroxyl radical (ROO•; Figure 1.1).29 Trp is the strongest UV chromophore, but its potential for oxidative damage is limited by its relatively low abundance within proteins.37 Two of the major end products of Trp photooxidation (N-formylkynurenine and kynurenine) are more effective photosensitising agents than the parent amino acid and can thus produce further reactive species and exacerbate photo-oxidation through perpetuation of secondary damage (e.g. through ring-closure reactions), particularly within proteins that are exposed to UV radiation.37 Evidence suggests that Trp• and ROO• radicals can be repaired by

6 low-molecular-mass antioxidants including ascorbate and certain thiol-containing species.37

1 Figure 1.1: Reaction pathways of Trp via UV absorption or O2-mediated oxidation. R represents the ɑ-amino acid moiety [CH(COOH)NH2]. Image adapted from Pattison, Suryo Rahmanto & Davies (2012)37

1 Oxidation of free Tyr by O2 primarily forms unstable endoperoxides, which rapidly decompose via ring-opening pathways to form the hydroperoxide and cyclised products (Figure 1.2).29, 55 However, ring-closure of Tyr is less favourable when the residue is bound in proteins, allowing other reactions to take place which result in the direct formation of the hydroperoxide and its subsequent decomposition to the corresponding alcohol.51, 57 This reaction may be 29, 51 1 central to the formation of protein cross-links and aggregation. O2-mediated oxidation of protein-bound His residues is believed to proceed via initial endoperoxide formation and subsequent radical and ring-opening reactions (Figure 1.3).29 Further reactions of these products generate protein cross-links akin to the reactions of protein-bound Tyr.51

1 Figure 1.2: Reaction pathways of Tyr via UV absorption or O2-mediated oxidation. R represents the ɑ-amino acid moiety [CH(COOH)NH2]. HOHICA is 3a-hydroxy-6-oxo- 2,3,3a,6,7,7a-hexahydro-1H-indol-2-carboxylic acid. Image adapted from Pattison, Suryo Rahmanto & Davies (2012)37

1 Figure 1.3: Reaction pathways of O2-mediated oxidation of His. R represents the ɑ- amino acid moiety [CH(COOH)NH2]. Image adapted from Pattison, Suryo Rahmanto & Davies (2012)37

27, 60 1 Cys and Met are particularly susceptible to oxidative insult. Free Met reacts with O2 via + - the formation of a zwitterionic species (R2S -OO ), which then reacts with a second molecule of Met to generate two moles of methionine sulfoxide (MetO).29 Free Cys reacts rapidly with 1 51 O2 to produce the disulfide product, cystine. Cys and Met residues in proteins react with 1 photochemically-generated O2 at significant rates at physiological pH, becoming oxidised to the respective disulfide and sulfoxide and causing inactivation of Cys-dependent enzymes.29, 30 1 No biomarkers specific for O2-mediated protein oxidation have yet been established because the products generated are not unique to the oxidant (e.g. MetO).61

1.5. The Catalytic Mechanism of Myeloperoxidase (MPO)

Myeloperoxidase is a haem peroxidase that activates H2O2 to oxidise organic substrates to free radicals, cycling through three redox intermediates in the process; the native ferric haem iron 11 species, and the high-valent iron species, Compound I and Compound II (Figure 1.4). H2O2 reacts with the active site haem groups of native MPO to generate Compound I, which is a short-lived and strongly oxidising intermediate comprising an oxy-ferryl (FeIV-O) haem centre and porphyrin radical; Compound I can be reduced to native MPO via the peroxidase cycle, which proceeds via the reduction of Compound I in 2 one-electron steps via formation of the haem-centred intermediate, Compound II.62, 63 Alternatively, Compound I can react via the halogenation cycle, where it undergoes a two-electron reaction with a halide ion (Cl-, Br-, SCN- ) to yield the corresponding hypohalous acid (HOCl, HOBr, HOSCN) and the native ferric form of the enzyme.64, 65 Reaction of Compound I is rapid and it is the slower reaction of Compound II which limits the rate of enzyme turnover.64

•- MPO can also exhibit SOD activity through the reaction of the native enzyme with O2 to form Compound III, an oxy-haem complex which is then recycled to the native form via further •- 64 reaction with O2 to give H2O2 and molecular oxygen.

Figure 1.4: Catalytic cycle of MPO. Native MPO reacts with H2O2 to form Compound I. a) the halogenation cycle: Compound I oxidises halide ions in a two-electron step to form hypohalites. b) the peroxidase cycle: Compounds I and II oxidise organic substrates (RH) to radicals (R•) by removal of an electron, and oxidise superoxide to molecular oxygen. c) Ferric MPO dismutates superoxide to form H2O2 and molecular oxygen via a Compound III intermediate. The redox states of the haem moiety are given for each form of MPO. Image adapted from Winterbourn, Kettle and Hampton (2016).64

MPO is unique among the mammalian peroxidases in that is has a sufficiently high reduction potential (1.16 V, compared to 1.10 V and 1.09 V for EPO and LPO, respectively) to oxidise chloride ions, which are the major physiological substrate of the enzyme, to generate HOCl; by doing so, MPO plays a key role in host defence by generating antimicrobial ROS to combat pathogenic invasion.64, 66

1.6. Myeloperoxidase-Derived Oxidants in Disease

MPO-derived oxidants play a key role in the immune response, destroying pathogens and tumour cells.6, 13-15 However, in the event of increased oxidant generation or decreased antioxidant activity (or both), oxidants can adversely affect host tissue via induction of oxidative stress, resulting in the initiation and progression of many inflammatory pathologies, including atherosclerosis, arthritis, asthma, certain cancers and neurodegenerative conditions (e.g. Alzheimer’s and Parkinson’s diseases), among many others.66, 67

1 HOCl and O2 are known to react with a vast array of biomolecules, including lipids, DNA, cholesterol, amino acids and proteins.51, 60 HOCl also rapidly oxidises amino groups and unsaturated fatty acids,16, 60 bleaches haem proteins27 and damages elements of the electron transport chain.16, 68 Kinetic data are consistent with proteins and their side-chains being the major oxidative targets in biological systems, with the fastest known rate constants reported for the reaction of HOCl with sulfur moieties, such as the thiols (RSH) in Cys side chains (k = 3.1 x 108 M-1 s-1) and thioethers (RSR’) in Met side chains (k = 3.4 x 107 M-1 s-1).9, 27, 60, 69, 70 Oxidation of protein-bound thiol groups results in the formation of intramolecular disulfide bonds (RSSR) and further products [e.g. sulfinic (RSO2H) and sulfonic (RSO3H) acids], thereby inactivating Cys-dependent enzymes.25, 71 Oxidation of thioethers in Met residues results in formation of the sulfoxide [RS(=O)R’; MetO] and further oxidation by high excesses 72, 73 of oxidant yields the subsequent sulfone [RS(=O)2R’; MetO2). Protein oxidation results in aggregation, fragmentation and eventual unfolding of the protein, causing inactivation of thiol- dependent enzymes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH).37, 51, 74

The reaction of HOCl (or HOBr) with amines results in generation of organic chloramines (RNHCl; or bromamines, RNHBr). These chlorinating species are less reactive than their precursor, HOCl, reacting up to 104 times slower, but demonstrate similar selectivity, being able to induce oxidative damage on proteins, low pKa thiols and DNA, inactivating intracellular enzymes and inducing cell death.15, 24, 66, 75 Chloramines, particularly those formed on His residues and nucleotide bases, act as intermediates in the propagation of HOCl-induced damage by slowly chlorinating Tyr residues and selectively oxidising thiols over Met residues.66

HOSCN, despite being a major MPO-derived oxidant, is less reactive than HOCl as it has a lower standard reduction potential (0.56 V vs 1.28 V, respectively)76 and is more specific, reacting rapidly only with low-molecular-mass and protein-bound thiols.70, 77 The specificity of HOSCN makes it the less proficient of the two oxidants in causing irreversible damage to host tissue under inflammatory conditions.27, 78 In addition, the production of HOSCN may act as a regulatory mechanism for competitively removing H2O2, thereby modulating the formation of harmful HOCl.70

H2O2 and other peroxides (such as those formed on proteins, lipids and peptides) react slowly with thiols and thioethers via two-electron oxidation reactions, and react rapidly with redox- active metal ions (Fe2+, Cu2+) via one-electron reactions, to generate radicals (e.g. •OH).79 The reaction of these radicals with biological targets via hydrogen atom abstraction, addition or

11 electron transfer yields carbon-centred radicals (R•) and peroxyl radicals (ROO•), which can 79 perpetuate oxidative damage within biological systems. Reaction of H2O2 with thiols is slow, yielding the initial sulfenic acid (RSOH) which can slowly be oxidised to the sulfinic acid 80 (RSO2H). Reaction of RSOH with a second thiol group generates the disulfide.

MPO-derived oxidants, principally HOCl, have been associated with cardiovascular diseases.69 This is evidenced by clinical studies which have detected MPO in atherosclerotic lesions and have revealed a strong correlation between blood levels of MPO and the incidence of disease in humans; thus, MPO is regarded as an independent risk factor for coronary artery disease (CAD).66, 67, 81 Detection of the protein-derived biomarker of HOCl-mediated damage, namely 3-chloro-tyrosine (3-ClTyr), as well as modification of lipids and DNA in tissue samples,27 has given further evidence for the role of MPO-derived HOCl in the aetiology of disease.12, 72, 81, 82

1.7. Endogenous Protective Mechanisms against ROS-Mediated Damage

Physiological systems possess antioxidant mechanisms to protect against and repair oxidative damage.40 An antioxidant is any compound that is able to remove, or to delay or prevent the formation of, an oxidant.83 These include enzymes that remove oxidants or their precursors [catalase, SOD and GPx], repair enzymes [sulfiredoxins, methionine sulfoxide reductase (Msr)] and enzymes that eliminate irreparably damaged substrates (proteases, DNA repair enzymes, phospholipases).40 Antioxidant mechanisms also include non-enzymatic processes such as low-molecular-mass oxidant scavengers, namely glutathione (GSH), ascorbate and vitamin E.40 Many of these mechanisms rely upon an active sulfur or selenium centre at the active site of a protein to carry out their antioxidant activity.

Glutathione (GSH; L-ɣ-glutamyl-L-cysteinyl-glycine) is an ubiquitous low-molecular-mass antioxidant involved in nutrient metabolism, with a high cellular concentration in the range of 0.1 to 10 mM under physiological conditions.84, 85 It is the most important known redox-active thiol because it acts as a co-factor for many critical antioxidant enzymes, including GPx.84 In addition, the chemical structure of GSH makes it resistant to proteolysis.84 ROS-mediated depletion of low-molecular-mass thiol antioxidants such as GSH has negative ramifications for the activity of the key antioxidant enzyme, glutathione peroxidase (GPx), which utilises GSH 37 as a co-factor to detoxify H2O2.

1 The thiol moiety of GSH is a significant target for in vivo oxidation by HOCl and O2. Oxidation of GSH results in the formation of glutathione disulfide (GSSG) and other products

12 such as glutathione sulfonamide (GSA);9, 60, 71, 86 the former is reducible by the action of NADPH-dependent enzymes (e.g. glutathione reductase, GR), allowing GSH to funcation as a recyclable antioxidant defence system against HOCl-induced damage.40 The formation of GSA may constitute a pathway to oxidative stress, as this product irreversibly consumes GSH upon reaction with HOCl, indicating that this species could be used as a sensitive biomarker in detecting HOCl-induced damage.87, 88 The accumulation of GSSG may also potentially contribute to oxidative stress by preventing GSH recycling.85, 89

If the oxidative challenge presented by ROS is greater than the capacity of antioxidants to repair this damage, the depletion of antioxidants such as GSH can contribute to or exacerbate oxidative stress.90 Antioxidant depletion has thus been associated with myocardial ischemia and reperfusion injury, aging and Alzheimer’s disease.84

In recent years, organoselenium compounds have received attention as potential antioxidants due to increasing evidence of their beneficial role in the prevention and treatment of oxidative stress-associated pathologies, including atherosclerosis and cancer progression.91-93

1.8. Selenium and Sulfur in Human Health and Disease

Selenium (Se) is an essential trace element required for many physiological processes.40 It plays a role in thyroid hormone metabolism, in the immune system and in both male and female reproduction.92, 94 Low selenium status has been associated with poor immune function, neurodegenerative diseases and cardiomyopathy (Keshan disease), which result from decreased activity of selenium-dependent enzymes, including GPx.92, 94 Excess of the element, although less common than deficiency, results in selenosis (selenium toxicity), which manifests as hair and nail loss (keratosis), dermatitis and rickets.92

The main dietary form of selenium is the amino acid selenomethionine (SeMet),95 the analogue of Met wherein a selenoether group (RSeR’) replaces the thioether group (RSR’).96, 97 However, most of the benefits to human health that derive from elemental selenium are due to its presence in selenoproteins as the genetically-encoded amino acid selenocysteine (Sec), the analogue of Cys in which a selenol group (RSeH) replaces the thiol group (RSH).98-100 Selenium and sulfur are in the same Group of elements as oxygen (Group 16, the chalcogens) and thereby demonstrate similar electron configuration, each having six valence electrons.101 This accounts for the similarity between the elements in terms of oxidation states, electronegativity and ionic radii.98, 101-103 These shared characteristics are reflected in their

13 chemical behaviour, with selenium-containing compounds exhibiting close analogy to the corresponding sulfur compounds.95

Within the microenvironment of a protein, the chemical differences between selenium and sulfur become significant, altering the potential biochemical interactions of the resultant compounds.102 The larger atomic radius and longer bond length of selenium relative to sulfur suggest that in structurally analogous compounds, the species containing selenium would be more polarised and more nucleophilic and electronegative.102 Also, the lower di-atomic bond strength of selenium indicates that cleavage of the diselenide bond (Se-Se) is more favourable than that of the disulfide bond (S-S).98 Cumulatively, these properties allow selenium species to be fundamentally more reactive, and consequently more susceptible to oxidation, than the corresponding sulfur analogues.89, 102, 104-107

The atomic properties of selenium also allow selenols to have a lower pKa value (5.3) than thiols (8.3). 102, 106, 107 This causes selenols (unlike thiols) to be almost completely ionised at physiological pH 7.4, making them more reactive under the mildly acidic conditions that 102, 107 predominate within inflammatory microenvironments. The lower pKa of selenols is crucial for the potential therapeutic application of selenium compounds, because the antioxidant function of these species typically rely upon reactions which require the generation of a reactive selenol intermediate.40

1.8.1. Selenomethionine (SeMet) and Methionine (Met)

Met plays an important biological role in the initiation of protein synthesis, structure, redox regulation and antioxidant defence.108, 109 Unlike Cys, which can be synthesised by the body, 84, 110 1 Met is an essential amino acid. Oxidants including HOCl, H2O2 and O2 oxidise Met to MetO by addition of oxygen to its sulfur atom,111 introducing a chiral centre at the sulfur moiety, resulting in formation of two stereoisomers, termed R- and S-MetO.108, 112, 113 The oxidation and reduction cycle of Met has been proposed to regulate the biological activity of some proteins.114 It has been suggested that oxidation of Met to MetO will increase the surface hydrophobicity of the molecule by perturbing the 3-dimensional folding conformation and exposing residues previously buried within the hydrophobic core, consistent with Met oxidation increasing the surface hydrophobicity of proteins.108, 111 It has also been suggested that Met residues present on the surface of a protein may be positioned to intercept ROS and undergo preferential oxidation in order to prevent damage to other residues that are critical to

14 protein function, e.g. reactive Cys residues at the active site of enzymes.108, 115 Some experimental evidence supports this hypothesis, as glutamine synthetase from E. coli was found to undergo oxidation of eight of its sixteen surface-exposed Met residues before losing its catalytic activity.116

MetO requires reduction by methionine sulfoxide reductase enzymes (Msr), which are part of a family of oxidoreductases, i.e. enzymes which catalyse reduction-oxidation (i.e. redox) reactions.117 Msr enzymes contain either a catalytic Cys or Sec residue at the active site, which is oxidised by MetO and requires regeneration [e.g. by thioredoxin (Trx)] before the next catalytic cycle can proceed.113, 117 The S- and R-stereoisomers of MetO each require a different Msr for reduction, termed MsrA and MsrB, respectively, which have little structural homology other than exhibiting a mirror-like relationship in the structure of their active sites to account for their specificity in substrate.108, 113 MsrAs reduce both free and protein-bound MetO, whereas MsrBs demonstrate a high specificity for protein-bound MetO, which constitutes around 99 % of total MetO.113 The Msr-mediated oxidation and reduction cycle of Met is postulated to be an important endogenous antioxidant mechanism in the protection and repair of oxidatively damaged proteins, to scavenge biological oxidants and increase the resistance of a protein to oxidative stress.84, 109, 110, 112

Further oxidation of MetO to MetO2 causes the side chain to become more rigid and increase in size, polarity and charge. These structural changes can result in loss of protein functionality and subsequent oxidative stress, which has been associated with the progression of aging.110, 114, 117, 118 MetO2 is biologically stable, so cannot be enzymatically reduced in the same way as MetO, thus, accumulation of this product is indicative of a significant level of oxidative stress.84, 110, 111 Met over-oxidation has been implicated in the progression of neurodegeneration and the resulting development of Parkinson’s119 and Alzheimer’s120 diseases.

The bioavailability of selenocompounds is generally fairly high and all dietary selenium species are readily absorbed, although organic forms (e.g. SeMet and Sec) are retained more 2- 2- 92, 121 efficiently than inorganic forms [e.g. selenate (SeO4 ) and selenite (SeO3 )]. Initial uptake of SeMet is very high, particularly in the exocrine tissue of the pancreas due to rapid synthesis of proteins in this tissue.97, 122, 123 The majority is incorporated into proteins, where it is stored in tissues for further metabolism into more reactive forms of selenium.97, 124 SeMet is rapidly absorbed and incorporated via the same pathway (and at the same rate) as Met, allowing it to replace the sulfur species in proteins and act as a natural amino acid without any prior

15 metabolic alterations.97, 122, 125

The random incorporation of SeMet via the Met pathway allows the selenium derivative to undergo the same reactions and perform the same function as Met in the protection and antioxidant repair of proteins.97 However, the greater intrinsic reactivity of the selenium atom causes SeMet to be oxidised by ROS much more rapidly than Met with the same oxidants (ONOOH, HOSCN and HOCl), the primary product being the analogous mono-oxygenated species, selenomethionine selenoxide (SeMetO).19, 85, 89 If a system contains both amino acids, SeMet is preferentially oxidised, as evidenced by the reaction of ONOOH with SeMet occurring at a rate 10 to 1000 times faster than with Met.89 Whilst MetO can be reduced only by Msr enzymes, the selenoxide is able to be non-enzymatically reduced by GSH to regenerate SeMet (and form GSSG; Reaction 3).85, 89

푆푒푀푒푡푂 + 2퐺푆퐻 → 푆푒푀푒푡 + 퐺푆푆퐺 + 퐻2푂 (3)

The preferential oxidation of SeMet and the rapid non-enzymatic recycling of the selenoxide may act as a biological mechanism for the protection of Met (and other susceptible amino acids, e.g. Cys, which displays comparable rate constants),79 from irreversible over-oxidation, as well as a mechanism for scavenging biological oxidants.85, 89, 104 In this way, SeMet might protect against inflammatory ROS, preventing DNA strand-breaks and thiol, lipid and protein oxidation.89, 104 Other antioxidants such as ascorbate can also reduce selenoxides, but this particular reaction is much slower than that of GSH.85

The rapid absorption and incorporation of SeMet has encouraged the supposition that it may represent an appropriate form of selenium supplementation for deficient populations to improve patient outcomes in cases of cancer or diabetes.123, 126 Clinical trials examining the effects of selenium supplementation have revealed a U-shaped dose-response relationship between selenium intake and diabetes and cancer risk, where both deficiency and excesses of selenium can contribute to the pathogenesis of disease.94, 127 The Selenium and Vitamin E Cancer Prevention Trial (SELECT) reported that oral supplementation of selenium (as 200 µg SeMet/day) or vitamin E (400 IU/day), alone or in combination, did not prevent prostate cancer in relatively healthy men.128 In the SELECT study, median baseline serum selenium was increased from 135 µg L-1 to 250 µg L-1 following SeMet supplementation, which is postulated to be above the beneficial concentration range, as participants demonstrated a non-significant increased incidence of diabetes.128

This result is consistent with the results of the Nutritional Prevention of Cancer (NPC) trial, where selenium supplementation (as selenised yeast, 200 µg/day) non-significantly decreased total cancer and prostate cancer incidence only in participants whose baseline plasma selenium concentrations were in the lowest two tertiles (< 121.6 ng L-1), whereas those in the highest tertile demonstrated an elevated total cancer incidence and an almost 3-fold increase in incidence of diabetes.129 These studies concluded that the benefits to human health that may be gained from selenium supplementation rely on factors that affect plasma/serum selenium levels and selenoenzyme activity, with disease incidence increasing both below and above the physiological range which is optimal for the activity of some or all selenoproteins, highlighting the need for future research.127

Methyl-selenocysteine (SeMSC; C4H9NO2Se) is another selenium species present in selenium- enriched yeast, which (unlike SeMet) is not incorporated into proteins, thus remaining available for incorporation into selenoproteins.130 A separate report indicated that SeMSC was a more efficacious anti-cancer agent than SeMet,131 demonstrating that a more comprehensive mechanistic understanding of the nature of selenium compounds, their bioavailability and antioxidant activity is required in order to develop more successful future clinical trials of disease prevention by supplementation.132

In addition to providing direct antioxidant properties, SeMet can also act as a source of selenium for the synthesis of antioxidant and reparative selenoproteins (i.e. Sec-containing proteins).40, 95 Some of the established roles of selenoproteins include redox homeostasis, thyroid hormone metabolism,94 antioxidant defence, embryonic muscle development and immune response augmentation.133

1.8.2. Selenocysteine (Sec) and Cysteine (Cys)

Sec is the twenty-first naturally-occurring genetically-encoded amino acid.98 It is actively and preferentially incorporated into the active site of specific selenoproteins, including GPx, thioredoxin reductase (TrxR) and some Msr enzymes (e.g. MsrB1).30, 133 In the event of dietary selenium deficiency, the synthesis of Sec-containing selenoproteins (particularly GPx and TrxR) is prioritised so as to maintain enzyme activity.94, 98, 133 The synthesis and incorporation of Sec residues is more energetically expensive to the cell than Cys incorporation, and requires unique synthesis machinery.98, 133 Sec insertion into the polypeptide chain involves the recoding of a stop codon (UGA) as a sense codon.105 This mechanism requires the use of cis- and trans-

17 active factors, including a structure termed the SECIS (Sec insertion sequence) element in the mRNA.134 In eukaryotic mRNA, the SECIS element is found in the 3’-untranslated region, whereas in prokaryotic mRNA it is located in the open reading frame immediately downstream from the UGA-selenocysteine codon.134 This disparity results in mechanistic differences in Sec incorporation between species.134 The trans-factors essential for Sec incorporation also include SectRNA([Ser]Sec) which possesses a unique mechanism of biosynthesis, as well as a Sec- specific elongation factor (EFsec) and Sec binding protein (SBP2).134 This complex Sec- insertion machinery is energetically costly and unique, suggesting that Sec is essential for human health and fulfils a role which cannot be achieved by Cys.98, 105, 106, 134

It has been proposed that the incorporation of selenium (Sec) to replace sulfur (Cys) in the active site of enzymes to form selenoproteins (e.g. GPx, TrxR, MsrB1) occurs for one of two reasons; firstly, that it is an ‘evolutionary relic’, dating back to anaerobic cellular origins,105 which is consistent with the presence of the Sec codon in both prokaryotes and eukaryotes, indicating that it predates the separation of the lineages.135 The second line of thought is that selenols are more reactive than thiols, so replacement of Cys with Sec at enzyme active sites increases the kinetic and catalytic efficiency of the enzyme.99, 105, 135 This has been validated by experiments showing that substitution of the active site Sec in selenoenzymes with a Cys residue decreases enzyme activity.136-139 Thus, Kim et al. (2006) created by site-directed mutagenesis a Cys-containing variant of Sec-MsrA from C. reinhardtii cDNA, and a Sec- containing variant of a native mammalian Cys-MsrA, then expressed these enzymes in mammalian cells for comparison of their activity in the presence of low-molecular-mass thiol reductants [Trx or dithiothreitol (DTT)].136 In a DTT-dependent reaction, the native Sec-MsrA was 10-fold more reactive than its Cys-mutant, and 50-fold more reactive than the native Cys- MsrA.136 The introduction of Sec into the native Cys-MsrA also increased the activity of this enzyme for the DTT-dependent reaction.136 Similarly, Sec-substitution of the active site catalytic Cys in plant phospholipid hydroperoxidase GPx resulted in a 4-fold increase in catalytic activity compared to the wild-type Cys-enzyme.140 Also, mutation of the selenol group of Sec-TrxR to a thiol caused the mutant Cys-TrxR to suffer a 10- to 100-fold decrease in rate constant relative to the wild-type enzyme upon reaction with Trx.138, 141 Cumulatively, these data imply that the selenium moiety provides a functional advantage to the catalytic activity of selenoproteins.

In addition to providing possible catalytic advantages, it has been suggested that the incorporation of Sec residues into selenoenzymes (GPx, TrxR, some Msrs) provides the

18 enzyme with a greater inherent ability to resist oxidative inactivation.106, 141, 142 At physiological pH, the selenol of the Sec residue is ionised to the selenate form (RSe-), which is particularly susceptible to oxidation, potentially leading to aggregation, fragmentation and inactivation of Sec-enzymes, concomitant with the detoxification of the oxidising species.30, 143, 144 However, the major products of Sec oxidation [selenenic (RSeOH) and seleninic (RSeO2H) acids], which are analogous to those formed with Cys, are rapidly reduced to regenerate the enzymatically- active selenol form (e.g. using low-molecular-mass thiols such as GSH) due to the highly electrophilic nature of the oxidised selenium atom; a process which is performed much less efficiently by the corresponding sulfur species.102, 105, 106 The enhanced catalytic activity of Sec- enzymes coupled with the more favourable reduction to the selenol allows for both the rapid turnover of substrate and rapid enzyme regeneration, enhancing the ROS-scavenging efficiency of Sec-enzymes relative to their Cys analogues.100

The products of Sec oxidation in vivo remain largely uncharacterised but it is generally assumed that the mechanisms, and hence the products, of selenocompound oxidation are analogous to those of the corresponding sulfur compounds.27, 86 However, it has been reported that treatment of GPx1 with H2O2 oxidised the active site Sec residue to dehydroalanaine (DHA) by elimination of the Se moiety, as determined by mass spectrometry (MS),143 as 27, 30, 145 opposed to the expected selenonic acid (RSeO3H) predicted by thiol chemistry. It was proposed that the oxidative environment promoted the conversion of the selenol to RSeO2H 143 and subsequent loss of selenium as H2SeO2. These experimental data suggest that oxidation of selenocompounds may proceed via alternative mechanisms, thus generating alternative products, to those predicted by sulfur chemistry, contrary to what has been previously believed.86, 145 Therefore, the mechanisms of selenocompound oxidation and the nature of the resulting products require further investigation.

1.8.3. Catalytic Mechanisms of Selenoenzymes

1.8.3.1. Thioredoxin Reductase (TrxR)

Thioredoxin reductases (TrxR) are Sec-containing enzymes which maintain redox balance within cells by catalysing the reduction of disulfide bonds in a broad range of substrates, 40, 146 concomitantly reducing H2O2 and other peroxides, HOCl and HOSCN. The action of TrxR relies on thioredoxin (Trx), a small redox-active protein with a dual thiol moiety, which supplies electrons to reduce the disulfide substrate, itself becoming oxidised to a disulfide

147 (TrxS2) in the process. TrxS2 is then reduced by TrxR via oxidation of the active site Sec residue to form a selenenylsulfide intermediate (RSeSR), which is then reduced to regenerate the selenol using electrons supplied by NADPH (Figure 1.5).148-150

Figure 1.5: Catalytic cycle of TrxR. Image adapted from Holmgren and Lu (2010).150

There are three isoforms of TrxR; TrxR1, TrxR2 and TGR (thioredoxin glutathione reductase), each of which are wholly dependent upon selenium for reducing TrxS2, thereby supporting and maintaining cellular proliferation and redox-regulated signalling pathways.144 As the Trx system (Trx/TrxR/NADPH) reduces disulfide bonds, a number of coagulation factors are substrates for this system. Inhibition of the Trx system has been shown to result in platelet aggregation and to be of relevance in vascular occlusion and thrombogenesis.144

The active site Sec residue is also commonly credited for the broad substrate specificity of TrxR, as the efficacy of this system is lost when Sec is mutated to Cys.40 However, this has been countered by experimental evidence implying that non-Sec-dependent regions of the enzyme are capable of equally rapid reduction of certain substrates.142 Therefore, it has been suggested that the benefits of Sec incorporation into TrxR are not necessarily due to its increased catalytic activity, but are due to it bestowing greater substrate specificity and providing a greater range of environmental conditions in which it remains efficacious.149 Furthermore, a direct comparison of the abilities of mammalian Sec-TrxR and a Cys-analogue • to resist oxidation was undertaken using a variety of oxidants including H2O2, OH, ONOOH, HOCl and HOBr, wherein the Sec enzyme was demonstrably superior to the Cys enzyme in terms of resisting overoxidation.141 Based on this evidence, Sec was proposed to be evolutionarily favoured based on its ability to resist oxidation, to be rapidly reduced, and to retain functionality at the lower pH ranges that occur at inflammatory foci.141

1.8.3.2. Methionine Sulfoxide Reductase (Msr)

The reaction mechanism of Cys- and Sec-Msrs are generally accepted to proceed via the initial formation of a sulfenic (RSOH) or selenenic (RSeOH) acid intermediate, respectively,

20 concomitant with reduction of the sulfoxide in MetO to Met (Figure 1.6).113, 118, 136 The RSOH/RSeOH is then reduced by an adjacent intramolecular Cys residue (the evolutionarily- conserved resolving Cys), resulting in a RSSR/RSeSR bond and the liberation of water.113, 118, 136 Finally, the RSSR/RSeSR species is reduced by a reductant, typically Trx in vivo or DTT 118, 136 110, 118, in vitro, to regenerate the active site RSH/RSeH of the enzyme and produce TrxS2. 119 The resolving Cys is necessary for the Trx-dependent reduction of the RSeSR intermediate in Sec-containing MsrB1.118 Cys-to-Sec mutant forms of MsrB2 and 3 are therefore inactive in the presence of the physiological reductant, Trx.118

Figure 1.6: Catalytic cycle of Sec-containing Msr enzymes (e.g. MsrB1). Image adapted from Kim and Gladyshev (2005).118

The presence of Sec in the active site of MsrB1 is therefore proposed to be “a compromise” between an increased rate of MetO reduction and the ability of the enzyme to be reduced by its natural reductant, Trx.118 Sec oxidoreductases are typically ca. 100- to 1000-fold more reactive than the corresponding Cys mutants, but some Cys-enzymes are equally as efficient catalysts as the native Sec-enzymes.118 These data suggest that incorporation of Sec into TrxR increases the range of potential substrates as well as allowing flexibility in microenvironmental conditions required for activity, thereby greatly increasing the potential protection against oxidative stress.118, 149 MsrB1 seems to be the most sensitive protein to minor changes in the amount of dietary selenium and may therefore be used as an indicator of selenium status in humans.95, 100

1.8.3.3. Glutathione Peroxidase (GPx)

Glutathione peroxidases are responsible for the reduction of organic hydroperoxides, ONOOH and H2O2 in both intra- and extra-cellular compartments, typically using GSH as the reductant.95, 147 There are eight currently identified GPx enzymes, some of which contain active site Sec residues (GPx1-4 and 6) and others that alternatively contain Cys (GPx 5, 7 and 8).95, 147 Each GPx isoform is tissue and site-specific; for example, Sec-GPx1 is cytosolic and mitochondrial, whereas Sec-GPx3 is present in plasma and Cys-GPx5 is secreted by the epididymis in the male reproductive system.147

The mechanisms by which GPx exert their antioxidant function varies between Sec- and Cys- forms.147 For a typical Cys-GPx, the RSH at the active site is oxidised to RSOH, which reacts with a second Cys thiol to form an intramolecular disulfide, which is crucial in completion of the catalytic cycle.86, 147 This disulfide is then reduced back to two active thiols by Trx, yielding 147 147 TrxS2. Cys-GPx7 and 8 undergo alternative reaction pathways.

In contrast, during the catalytic cycle of Sec-GPx (Figure 1.7), in the initial reaction (e.g. with - 147 H2O2), the selenol (as RSe ) is oxidised to RSeOH. Subsequently, GSH forms a RSeSR bond with the RSeOH, with the oxygen atom eliminated as water.86, 147 A second equivalent of GSH then reduces the RSeSR bond by thiol-disulfide exchange to form GSSG and regenerate the active selenol.147, 151 Interconversion between these three species has been demonstrated experimentally using 1,4-dithiols as reductants in place of GSH.152 This mechanism is also assumed to be valid for GPx3 and 4, which allows GPx to maintain activity under oxidative conditions.147 However, GPxs represent significant biological targets due to their abundance 1 and reactivity and can be inactivated by O2 and H2O2 via over-oxidation of the active site Sec residue.30, 143

The direct therapeutic potential of GPx is limited by its instability, poor availability and high molecular mass.153 There has been increasing interest in the development and application of low-molecular-mass selenocompounds that are able to mimic the peroxidase activity of GPx, such as ebselen, an organic aromatic selenide.154 It is through their ability to mimic GPx activity that synthetic low-molecular-mass selenium species may act therapeutically, although it is possible that other mechanisms are also occurring.153

Figure 1.7: Traditional catalytic cycle of glutathione peroxidase (GPx); a) the peroxidatic cycle: the selenol (as a selenolate ion) is oxidised to selenenic acid by a hydroperoxide, b) the reductive cycle: GSH forms a selenenylsulfide with the selenenic acid, with oxygen eliminated as water, and a second equivalent of GSH reduces the selenenylsulfide to release GSSG and regenerate the active selenol of native GPx. Image adapted from Brigelius-Flohe & Maiorino (2013).147

1.9. Therapeutic Application of Exogenous Selenocompounds

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one; Figure 1.8) is a synthetic, electrophilic organoselenium compound which demonstrates in vitro mimicry of GPx activity (e.g. H2O2 and hydroperoxide reduction), to a greater extent than its sulfur analogue.154, 155 Ebselen has been shown to possess antioxidant capabilities and an ability to modulate the immune system, and has been proposed as a treatment for inflammatory diseases including atherosclerosis, arthritis and asthma.154 Like other selenocompounds, ebselen is able to protect against ONOOH-induced DNA strand breaks.104, 156 It achieves this through a mode of action similar to that of GPx, wherein the oxidation state of the active site selenium moiety is altered, being oxidised to selenenic acid, which is reduced by GSH (or other thiols)157 through formation of a selenenylsulfide intermediate (Ebs-SeSG; Figure 1.8).154, 158 Ebselen is also an excellent substrate for the TrxR system, a factor which contributes to its pharmacological activity.159 Upon administration, ebselen is transported through the bloodstream bound to the cysteine residue of albumin, where it (or its metabolites) localises in the endoplasmic reticulum (ER).154 Here, ebselen is able to modulate the function of the ER and mitochondria, which may represent the basis of its pharmacological utility.154

Figure 1.8: Chemical structure and catalytic cycle of ebselen. Image adapted from Nogueira and Rocha (2011).157

Ebselen has been proposed as a therapeutic agent for multiple diseases and has been the subject of several clinical trials.160 It has been shown to reduce the incidence and severity of brain damage (measured as the area of low-density brain tissue) when administered as water- suspended granules to patients recently suffering subarachnoid haemorrhage.160 The administration of ebselen to patients suffering acute ischemic stroke significantly improved the outcome of the treatment group, provided that treatment was administered within 24 hours of the ischemic event.161 Experimental evidence suggests that ebselen acts as a neuroprotective agent through the modulation of lipid peroxidation and ROS generation events that are concomitant with apoptotic brain injury.160, 161 Animal studies in a rodent model established good oral absorption and brain-permeability of the drug, as well as its free-radical scavenging ability.162 Experimental data obtained using mice have also been suggestive of therapeutic potential in the treatment of diabetic complications by minimising the inflammatory insult, thus, balancing the loss of GPx activity.163 This model of advanced oxidative stress demonstrated the potential benefits to atherosclerotic outcome of administering ebselen in a diabetic setting.163

Despite the therapeutic potential of ebselen as a GPx mimetic, its utilisation (and that of similar

24 compounds) in vivo may be limited due to low water solubility.147 Furthermore, under certain conditions ebselen is known to cause cellular toxicity that is associated with thiol oxidation, causing depletion and inactivation of thiol-dependent enzymes (e.g. NO synthases, lactate dehydrogenase) and sulfhydryl-dependent enzymes (enzymes that use thiols as electron- donors), and inducing mitochondrial dysfunction, resulting in membrane disruption and perturbation of calcium homeostasis.154, 157, 164 High concentrations of the drug have also been associated with necrotic cell death, induction of DNA damage and apoptosis.154 Thus, ebselen (and other organoselenium compounds) can have both pharmacologic and toxic effects which will affect in vivo outcomes, and as such, these compounds must be investigated in detail to determine their suitability as candidates for therapeutic application.

Dihydroxy-1-selenolane (DHS) is a water-soluble cyclic monoselenide which demonstrates GPx-like activity, acting to accelerate the healing of stomach ulcers in mice by modulating anti-inflammatory pathways.165-167 DHS was shown to inhibit lipid peroxidation and to catalyse the oxidative folding of denatured proteins in cell-free systems.168-172 Furthermore, pre- treatment of mice by intraperitoneal injection of DHS (2 mg kg-1 bodyweight) improved survival rates by 40% following gamma-irradiation by inducing GPx activity in certain tissues, thus decreasing the resulting oxidative damage (e.g. lipid peroxidation and DNA damage).165

Other seleno-species have also been investigated as antioxidants. A method for the synthesis of selenium- and sulfur-substituted carbohydrates has been developed and in vitro studies showed that these compounds exhibited a dose-dependent prevention of HOCl-mediated oxidation of Met residues in human plasma proteins.173 The selenium sugar derivatives were considerably more effective than the sulfur analogues, with near complete Met protection achieved with 500 μM of the selenocompound (compared to 1 mM of the sulfur derivative).173 These seleno-sugars are potent water-soluble scavengers of HOCl, which provided significant protection at low concentrations without inducing toxicity; this is important as most organic selenides shown to exhibit GPx-like activity are limited by a lack of solubility in aqueous solutions and potential toxicity.173 Further study showed that these selenium sugars afforded protection to amino acids (Trp, Tyr, His, Lys and Met) against HOCl- and ONOOH-mediated protein damage, with rate constants of reaction in the range of k ~ 108 M-1 s1 with HOCl and k ~ 103 M-1 s1 for ONOOH.39, 174 The selenium sugars exhibited ca. 10-fold increased reactivity relative to the sulfur compounds with both HOCl and ONOOH.39, 174 The seleno-sugars were oxidised stoichiometrically to the corresponding selenoxides, which can be non-enzymatically reduced by low-molecular-mass thiols, such as GSH.174 As the precise secondary molecular

25 structure of the selenium sugars seems to have little effect on their antioxidant capacity, there exists a range of potential applications for these compounds to combat oxidative stress- associated pathologies.174 The effectiveness of these compounds requires further study, including investigation of their metabolism, bioavailability and toxicity.174 The capacity of 1 these seleno-sugars to scavenge O2 also warrants investigation.

1.9.1. Diselenides

The highly-reactive selenol group at the active site of GPx facilitates the antioxidant action of the enzyme, but the instability of low-molecular-mass selenols and their rapid auto-oxidation confounds both mechanistic research and therapeutic application.79 However, diselenide compounds (RSeSeR’) may act as shelf-stable alternatives to selenols which are more therapeutically viable and might be reduced to the more reactive selenol form in vivo.102

Unlike the analogous diselenides, the oxidation of disulfides is well-characterised. The one- and two-electron oxidation of disulfides typically occurs at slower rates than for the corresponding thiols and thioethers due to their partially-oxidised state, initially giving rise to the mono- and di-oxygenated species [the thiosulfinate (RSS(=O)R’) and thiosulfonate

(RSS(=O)2R’)], which react further by subsequent cleavage of the disulfide bond to form the 79, 86, 175, 176 sulfonic acid (RSO3H). Thiosulfinates can also undergo reaction with GSH to yield mixed disulfides and sulfenic acids, with subsequent reduction to the native disulfide, thereby representing a catalytic oxidant-scavenging pathway.176

Kinetic data suggest that disulfides can react rapidly with certain ROS, depending upon both the oxidant species and the conformation of the disulfide, with favourable electrostatic interaction arising from neighbouring heteroatoms acting to stabilise the incipient positive charge on the sulfur reaction centre.176 The ionisation state of the disulfide is also important in determining its reactivity with oxidants, with favourable interactions of the positively-charged sulfur centre with lone pairs of electrons being strongly pH-dependent and sensitive to the microenvironment of the target disulfide.176 Certain protein-bound disulfides show enhanced reactivity, as do five-membered ring structures when compared to six-membered rings and aliphatic disulfides.176 The structural and functional significance of the disulfide bond within proteins means that oxidation plays a major role in the aggregation, fragmentation or unfolding of proteins and the ultimate loss in functionality of disulfide-containing proteins.66, 177

The diselenide bond is weaker than the disulfide bond, so that oxidation is more

26 thermodynamically and kinetically favourable.102 Few kinetic data have been reported for the direct reaction of diselenides with two-electron oxidants, but certain diselenides have been reported to lessen the effects of inflammatory pathologies;91 for example, selenocystamine (SeCA) when applied to cardiovascular stents has been shown to aid vascular integration by 178 anti-inflammatory means; diphenyl diselenide [(PhSe)2] has been shown to minimise inflammatory characteristics in mice with gastric ulcers;179 and pre-treatment with 3,3’- diselenodipropionic acid (DSePA) was shown to reduce gamma radiation-induced inflammation in mice.180

1.9.1.1. Diphenyl Diselenide [Ph(Se)2]

Diphenyl diselenide has gained increasing attention over recent years due to its reported ability to mimic the antioxidant activity of GPx, and is assumed to act via a classical GPx cycle.91, 179, 181-183 When administered orally to rats, (PhSe)2 exhibited an anti-ulcer effect which was attributed to a weak peroxidase activity.179 It was also found to protect macrophages (murine J774A.1 cells) against oxidised low density lipoprotein (oxLDL)-initiated atherogenic signalling by inhibiting the initiation of ROS-induced inflammatory pathways, demonstrating a potential to positively influence the incidence of cardiovascular events.91 This effect was further investigated using modified derivatives of (PhSe)2 which contained additional terminal 182 methoxy (-OCH3) or chlorine (-Cl) groups. Both compounds exhibited a greater effect than 2+ (PhSe)2 in reducing the extent of Cu -induced oxidation of LDL, and also reduced the uptake of oxLDL by macrophages.182 The chlorinated derivative reacted with GSH to produce an intermediate capable of interaction with H2O2, allowing for the detoxification of peroxides, whilst the methoxy compound did not.182 Furthermore, both diselenide derivatives were able to act as substrates for hepatic mammalian TrxR, indicating that they may constitute an antioxidant system via interaction with this key enzyme.182

The mechanisms by which (PhSe)2 exerts its action have not yet been elucidated, but it is widely supposed that GPx-like activity is fundamental to the efficacy of the compound, suggesting that GSH (or an equivalent reductant) is required.153, 184, 185 The GPx-like peroxidase activity of (PhSe)2 was maximal at physiological pH 7.4, was diminished in basic conditions and was non-existent under acidic conditions; however, antioxidant activity was maintained under each setting, indicating that the antioxidant action of (PhSe)2 may result from a 181 mechanism not related to its peroxidase activity. Physicochemical profiling of (PhSe)2

27 revealed the compound to have low aqueous solubility, poor absorption and high affinity for binding to plasma proteins (predominantly albumin), presumably by oxidation of Cys residues 186 and formation of a selenenylsulfide. No degradation products or metabolites of (PhSe)2 were detected by LC/MS, indicating that the compound is biologically stable, but was able to cross the blood-brain barrier and rapidly induce seizures, causing neurological damage in rodent models.186

Studies aimed to improve the pharmacokinetics of (PhSe)2 via loading the compound into nanocapsules, which has the potential to increase bioavailability, decrease toxicity, and physically protect labile drugs, improving the overall pharmacokinetics of the compound.187

Both free and nano-encapsulated (PhSe)2 was administered to mice by intragastric gavage (50 mg/kg bodyweight), and the encapsulated (PhSe)2 displayed increased excretion in the urine and decreased excretion in the faeces relative to the free compound, indicative of increased intestinal absorption of the encapsulated drug, and both forms were still able to penetrate the 187 blood-brain barrier. The low water solubility and high lipophilicity of (PhSe)2 benefited this application because it allowed the compound to bind efficiently to the structure of the 187 nanocapsule, preventing toxicity by protecting plasma proteins against free (PhSe)2.

1.9.1.2. Selenocystamine (SeCA)

Selenocystamine [SeCA, (SeCH2CH2NH2)2] is a diselenide compound with terminal amine groups which has been shown to exhibit GPx-like activity.188, 189 An investigation by Weng et al. (2011) utilised this diselenide as a ‘bioactive material’ coated onto cardiovascular stents.178 When SeCA-coated stents were implemented in an animal model (implantation into the femoral artery of dogs), the treatment resulted in a decrease in platelet aggregation and lowered the incidence of neointimal hyperplasia relative to uncoated stents, thus having significant implications for the potential treatment of cardiovascular disease.178 The authors suggest that SeCA is a preferable biomaterial to GPx as it is a smaller molecule, limiting steric hindrance, and it is apparently more resistant to enzymatic degradation in vivo, whereas GPx undergoes a significant loss in activity upon implantation.153, 178 Follow-up reports detailed a novel method for the layer-by-layer production of vascular grafts which used a SeCA coating for the catalytic in vitro generation of •NO to aid in the improvement of haemocompatability of vascular stents.190 These reports have significant implications for the treatment of CVD and improvement of cardiovascular outcomes.178, 190

1.9.1.3. 3,3’-Diselenodipropionic Acid (DSePA)

3,3’-Diselenodipropionic acid [DSePA; (SeCH2CH2COOH)2] is a selenocystine derivative which is both stable and water-soluble.191 Pre-treatment of RBCs with DSePA reduced the GSH depletion that follows oxidative insult, and the compound displayed no cytotoxicity to normal or tumour cells.191 Further experiments exposed mice to gamma radiation to induce an apoptotic response and the oxidative damage attributable to this acute radiation insult was nullified in the intestine by pre-treatment of the mice with DSePA at a dose of 2 mg/kg/day, five consecutive days prior to irradiation.192 Thus, DSePA has been proposed as a model for the development and synthesis of more potent selenocompounds for use as therapeutic agents.191-193 Research has revealed that certain aliphatic (non-aromatic) selenoethers and diselenides can show higher GPx activity than ebselen, depending on the substitution on the aliphatic chain.194, 195 Knowledge of the chemistry of these selenocompounds will prove vital in the research and development of compounds that can show GPx-activity for antioxidant function in the treatment of inflammatory pathologies.194

1.9.1.4. Other Diselenide Compounds

Other selenocompounds with GPx activity have been investigated, including seleno- diglutathione (GSeSeG; the Se-analogue of GSSG), which show promise for further research and development.196 GSeSeG has found application in the efficient and rapid oxidative folding of proteins for the production of redox buffers for medical and industrial use, as it demonstrates increased reactivity relative to the disulfide derivative (GSSG) that is traditionally used for generation of redox buffers.197, 198 Selenoglutathione (GSeH), the analogue of GSH with Sec in place of Cys, has been investigated as a radical scavenger.90 GSeSeG displays GPx-like activity, and can be reduced to GSeH by NADPH in the presence of glutathione reductase 197 (GR). In the presence of excess GSH, reaction of a 3:1 ratio of H2O2 to GSeSeG yielded 2 molecules of the seleninic acid (GSeO2H) via formation of a transient selenenic acid (GSeOH) intermediate (Reaction 4), concomitant with the generation of selenenylsulfide species (GSeSG) (Reaction 5):199

퐺푆푒푂2퐻 + 2퐺푆퐻 → 퐺푆푒푂퐻 + 퐺푆푆퐺 + 퐻2푂 (4)

퐺푆푒푂퐻 + 퐺푆퐻 → 퐺푆푒푆퐺 + 퐻2푂 (5)

In the absence of GSH, it is suggested that GSeOH would react with GSSG to produce GSeSG and GSOH.199 Reaction of the GSeSG species with GSH did not generate GSeH, but reaction with DTT generated the parent diselenide by thiol exchange, suggesting that the di-thiol with a higher reducing potential and less steric bulk than the mono-thiol (GSH) could cleave the Se- S bond to form GSe-, which subsequently reacted with GSeSG to yield GSeSeG.199 The GPx- like catalytic activity of GSeSeG is summarised in Figure 1.9.

Figure 1.9: Proposed GPx-like catalytic cycle of GSeSeG. Image adapted from Shimodaira et al. (2017).199

The sulfur atom of GSeSG is subject to interaction with nearby heteroatoms (e.g. N, O) which favours attack on the S atom by a thiol substrate to cleave the Se-S bond, resulting in formation of a disulfide and selenolate, although attack at the Se atom is intrinsically more favourable.200 These data suggest that the secondary structure of the diselenide and other species present in the reaction mixture have the potential to modify the speed of the reaction and, potentially, the products formed, thereby affecting the antioxidant capacity of selenocompounds.

Several studies have focused on the pharmacology of selenium compounds by introducing or altering the functional groups of diselenides in order to investigate the effect of secondary structure on antioxidant activity.201 Diaryl diselenides were synthesised by augmenting the aromatic ring of (PhSe)2 with different substituent groups (CF3, F, Cl, OCH3), or by introduction of a nitrogen atom into both aromatic rings [to generate 2,2’-dipyridyl diselenide

(DPDSe; C10H8N2Se2)], and the antioxidant ability of these compounds was found to be dependent upon chemical structure.201 DPDSe exhibited the highest in vitro antioxidant activity of the compounds investigated with respect to protein and lipid peroxidation, with this attributed to the generation of radicals on the N atoms creating a favourable electronic environment for scavenging of radicals.201 The diaryl diselenides with substituted -Cl and -

OCH3 groups showed greater inhibition of copper-induced LDL oxidation than (PhSe)2, and reduced the cellular uptake of oxLDL by macrophages, indicating an anti-atherogenic effect in a murine model.91, 182 Furthermore, these species were proposed to act as substrates for TrxR, thereby potentially participating in the catalytic detoxification of H2O2 and other organic peroxides.182

Another avenue for the biomedical application of diselenide compounds has arisen through the development and fabrication of polymers for use as chemical vehicles for drug delivery.180, 202 In one such study by Cao et al. (2014), a diselenide bond-containing monomer and a positively charged monomer were copolymerised, and made soluble through termination of the active end groups.180 A ‘hydrogel’ was then formed from these two species which consisted of long nanofibres fashioned by physical cross-linking of the diselenide polymer with a peptide building block.180 Exposure to low-dose gamma radiation caused the nanofibres to fragment as a result of diselenide bond cleavage, causing the polymer to transition from a gel to a solution state.180 Diselenide cleavage was facilitated by ROS generated via irradiation of water molecules in the solution, resulting in the disintegration of the polymeric structure.180 A disulfide-containing analogue of the polymer remained intact under gamma-radiation doses up to 10-fold higher, verifying that the diselenide bond is more susceptible to gamma-irradiation- generated ROS than the disulfide bond.180 This polymer could be fashioned as a stable vehicle for the delivery of sensitive or hydrophobic drugs, which could then be physically manipulated via the targeted application of low-dose radiation, causing the fragmentation of the diselenide structure and release of the pre-loaded therapeutic agents.180

This was investigated by loading of the anti-tumour chemotherapy drug Doxorubicin (Dox) into diselenide polymer micelles and release of the drug via targeted radiation.180 At a relatively low dose of 5 Gy (close to the radiation dose a patient would receive in a day of chemotherapy treatment), 45 per cent of the Dox was released from the micelles, which had partially disassociated via absorption of ROS.180 This combination therapy would allow the drug to be applied directly at the site of treatment and lower the necessary total radiation dose to the patient, minimising the detrimental side-effects of chemotherapy treatment (myelosuppression,

31 alopecia, etc.).180

1.10. Factors Influencing the Reactivity of Disulfides and Diselenides

The selenium atom is known to interact with nearby heteroatoms (atoms other than carbon and hydrogen; e.g. S, N, O)203 and the increased GPx activity of diselenide derivatives is attributed to these beneficial non-covalent interactions.204 Organoselenium compounds capable of reproducing the catalytic activity of GPx whilst simultaneously acting as a substrate for TrxR stand in good stead to operate as strong antioxidant drugs by replicating the endogenous antioxidant machinery.205 Nascimento et al (2014) reported on the efficient synthesis of new nitrogen-containing diselenides that interacted with selenium in this way to improve the antioxidant activity of the resulting compounds.205 Various diselenides were synthesised and their GPx activity assayed before assessment of their ability to inhibit lipid peroxidation and act as a substrate for TrxR, using ebselen as a positive control.205 The studies showed that factors including chain size, steric hinderance and strength of the Se-heteroatom interaction are of paramount importance in the diselenides’ mechanisms of action.205 For example, aromatisation of the side chain restricts the potential reactions by forcing the Se and N atoms into close proximity for interaction.205 Each of the diselenides synthesised were capable of inhibiting lipid peroxidation, some of which were more effective than ebselen, particularly the aromatic derivatives; however, the precise mechanism of action is yet to be elucidated.205 Ebselen, its diselenide and various other diselenide compounds act as substrates for mammalian TrxR,159 and aromatisation of the compounds was found to be detrimental to diselenide functionality in terms of TrxR affinity.205 Therefore, the aromatic nature of the secondary structure of a diselenide has a significant impact on its antioxidant activity, depending upon the mechanism by which it exerts this antioxidant action.

Given that the GPx activity of selenocystine depends upon one- and two-electron reduction potential,206 derivatives of selenocystine were synthesised and tested in terms of their respective GPx activity relative to that of the parent compound, and this activity was correlated to the redox properties of each compound.194 The GPx activity of the selenocompounds investigated was in the order of selenocystine ~ SeCA > DSePA, with selenocystine and SeCA exhibiting the highest GPx activity due to their ability to easily undergo one-electron reduction.194 With selenocystine and SeCA, the Se-Se bond acts as an electron recipient due to the (-I) effect on the bond, so these compounds react readily with the reducing radical (in this

•- 194 • case, CO2 ). Conversely, DSePA is readily oxidised (by the peroxyl radical, CCl3O2 ) as a result of its carboxylic acid substitution creating a (+I) effect on the Se-Se bond, thus increasing electron density and causing the compound to act as an electron donor.194 Ergo, the diselenides that more readily undergo reduction show the most efficient GPx activity.194

Nitrogen-containing diselenide compounds (e.g. selenocystine and SeCA) exhibit strong antioxidant abilities that are typically superior to ebselen, and may represent therapeutic options against oxidative stress-associated diseases through their biological activities and diverse mechanisms of action.205 However, further evaluation of their chemistry is required and study of these compounds will aid in the determination of the important intramolecular interactions that affect GPx-mimicking ability and subsequent antioxidant ability of novel diselenide compounds.205

1.11. Hypothesis and Aims

The ability of selenocompounds to rapidly undergo both oxidation and reduction represents a catalytic mechanism for the scavenging of MPO-derived ROS, affording protection to the biologically active sulfur species, which are highly susceptible to oxidative insult. Oxidised selenium species are also resistant to further oxidation to the biologically stable tri-oxygenated state, maximising the potential for facile recycling of ROS. Evidence strongly suggests an antioxidant role of selenium species and a favourable effect on inflammatory disease outcomes, implying a potentially therapeutic application of selenocompounds. Whilst the rate constants and products of reaction are well characterised for the sulfur compounds, less literature exists detailing the reactions of the analogous selenium species, particularly diselenides.

It is therefore hypothesised that diselenide species may act as efficient scavengers of biological oxidants, with faster rate constants of reaction than those reported for the corresponding disulfides and may therefore act to modulate the onset and progression of inflammation- associated pathologies.

The aims addressed within this thesis are;

1) To examine the mechanisms by which low-molecular-mass diselenides scavenge HOCl

and H2O2 2) To determine rate constants for, and characterise the products generated by, these reactions 3) To investigate the removal of model peptide peroxides by low-molecular-mass diselenides 4) To compare the reactions of diselenide compounds to those reported for the analogous sulfur-containing species

Chapter 2

Materials and Methods

2.1. General Information

This Chapter details the Materials and Methods used in completion of this thesis.

All buffers, solutions and samples, unless otherwise stated, were prepared using Nanopure water filtered through a four-stage Milli-Q System (Millipore Waters, Lane Cove, NSW,

Australia), hereafter referred to as H2O. Where indicated, samples were prepared in sodium phosphate buffer (0.1 M, pH 7.4, preparation detailed below), which shall be referred to as simply ‘buffer’. The pH of solutions was determined using a MeterLab PHM220 pH meter with pHC2401 probe (Radiometer Analytical, Villeurbanne Cedex, France). Where necessary, pH was adjusted using solutions of NaOH or HCl (0.1 M, Sigma Aldrich, Castle Hill, NSW,

Australia) diluted in H2O.

All buffers, solvents and mobile phases (used in UPLC and LC/MS) were prepared as per their respective methods and vacuum filtered before use through a 0.2 µm hydrophilic polypropylene membrane filter (Pall Life Sciences, Kent Town, SA, Australia). Where required, samples were centrifuged using a Model 5215R Refrigerated Micro Centrifuge (Eppendorf, Hamburg, Germany).

2.2. Materials

Reagent Supplier Acetic acid (glacial) ≥ 99.8% Sigma Aldrich (Castle Hill, NSW, Australia) Acetonitrile (ACN), LiChrosolv® for Merck Millipore (Kilsyth, VIC, Australia) liquid chromatography ≥ 99.9% Catalase, from bovine liver Sigma Aldrich (Castle Hill, NSW, Australia) Chelex® 100 Resin Bio-Rad Laboratories (Gladesville, NSW, Australia) Coumarin boronic acid (CBA) BioNordika (Herlev, Denmark) Cumene hydroperoxide (CHP), 80% Sigma Aldrich (Castle Hill, NSW, Australia) solution Dimethyl sulfoxide (DMSO) Merck (Darmstadt, Germany) 2,2’-Dipyridyl diselenide (DPDSe) Carbosynth (Berkshire, United Kingdom) Dipyrimidin-2-yl diselenide (DPMSe) Carbosynth (Berkshire, United Kingdom) 3,3’-Diselenodipropionic acid (DSePA) Custom Synthesis by Prof. K. Indira

Priyadarsini (Bhabha Atomic Research Centre, Mumbai, India) Fmoc-L-Selenocystine (Fmoc-SeCystine) Custom Synthesis by Dr. Lara Malins and Prof. Richard Payne (School of Chemistry, University of Sydney, Australia) L-Seleno-diglutathione (GSeSeG) Custom Synthesis by Dr. Lara Malins and Prof. Richard Payne (School of Chemistry, University of Sydney, Australia) L-Histidine monochloride monohydrate Sigma Aldrich (Castle Hill, NSW, Australia) (His) Hydrochloric acid (HCl), 37% solution Sigma Aldrich (Castle Hill, NSW, Australia)

Hydrogen peroxide (H2O2), 30% Merck Millipore (Kilsyth, VIC, Australia) Suprapur® Iron (II) sulfate heptahydrate Sigma Aldrich (Castle Hill, NSW, Australia)

(FeSO4•7H2O) ReagentPlus® ≥ 99% H-Lys-Trp-Lys-OH acetate salt Bachem (Bubendorf, Switzerland) (LysTrpLys) H-Lys-Trp-OH acetate salt (LysTrp) Bachem (Bubendorf, Switzerland) Methanol (MeOH) LiChrosolv® for Merck Millipore (Kilsyth, VIC, Australia) liquid chromatography ≥ 99.8% 2-Propanol, UPLC ACS Thermo Fisher Scientific (Scoresby, VIC, Australia) Rose Bengal sodium salt (RB) Sigma Aldrich (Castle Hill, NSW, Australia) Selenocystamine dihydrochloride (SeCA, Sigma Aldrich (Castle Hill, NSW, Australia)

C4H12N2Se2•2HCl) Sodium acetate trihydrate Sigma Aldrich (Castle Hill, NSW, Australia)

(C2H3NaO2•3H2O) Sodium hydroxide (NaOH) pellets ≥ 97% Sigma Aldrich (Castle Hill, NSW, Australia) Sodium hypochlorite (HOCl), 12.5% w/v Ajax Finechem (Taren Point, NSW, Australia) solution Sodium phosphate monobasic Sigma Aldrich (Castle Hill, NSW, Australia) monohydrate (H2NaO4P•H2O)

Sulfuric acid (H2SO4) (95-98%, ≈ 18 M) Ajax Finechem (Taren Point, NSW, Australia)

37 tert-Butyl hydroperoxide (tBuOOH), Sigma Aldrich (Castle Hill, NSW, Australia) Luperox® TBH70X solution 70 % wt % in H2O Tetrahydrofuran (THF) dried, Merck Millipore (Kilsyth, VIC, Australia) SeccoSolv® ≥ 99.9% Trifluoroacetic acid (TFA) ReagentPlus® Sigma Aldrich (Castle Hill, NSW, Australia) 99% H-Trp-Lys-OH formiate salt (TrpLys) Bachem (Bubendorf, Switzerland) L-Tryptophan (Trp) Sigma Aldrich (Castle Hill, NSW, Australia) L-Tyrosine (Tyr) Sigma Aldrich (Castle Hill, NSW, Australia) Xylenol orange tetrasodium salt (XO) Sigma Aldrich (Castle Hill, NSW, Australia)

2.3. Preparation of Sodium Phosphate Buffer (NaH2PO4; 0.1 M, pH 7.4)

Sodium phosphate buffer (0.1 M, pH 7.4) was prepared as previously described.57 Monobasic and dibasic orthophosphate solutions (0.1 M) were prepared by dissolution of sodium phosphate dibasic dodecahydrate (25.06 g) into 700 mL H2O and dissolution of sodium hydrogen orthophosphate monohydrate (4.138 g) into 300 mL H2O. Both solutions were treated with washed Chelex resin to remove potential interference from trace transition metal ions. After 1 h of stirring, the Chelex resin was removed from each solution by vacuum filtration. Whilst stirring, the filtered monobasic solution was added into the filtered dibasic solution until a pH of 7.4 was reached. This final buffer solution (0.1 M, pH 7.4) is stable and was stored at 4 ˚C when not in use.

2.4. Determination of Commercial H2O2 and HOCl Stock Concentrations

Concentrations of commercial stock solutions of H2O2 (30% v/v) and HOCl (12.5% w/v) were determined by spectrophotometric assay, measuring the optical absorbance of each solution and calculating concentration using their respective molar absorption coefficients (ɛ).

-1 -1 57, 207 The stock concentration of H2O2 was determined using ɛ = 39.4 M cm at 240 nm.

Immediately prior to experimental use, H2O2 was prepared by several dilution steps to ensure accuracy, into either buffer or H2O, in accordance with the methods detailed below.

The concentration of stock HOCl was determined by measuring the optical absorbance of

- -1 -1 solution at pH 12, achieved by dilution into NaOH (0.1 M), using ɛ of OCl = 350 M cm at 292 nm.208

2.5. Preparation of Photochemically-Generated Hydroperoxides

Amino acids (Trp, Tyr, His) and peptides (LysTrp, TrpLys, LysTrpLys) were prepared to 2.5 mM in 10 mL H2O and corrected to pH 7.2 – 7.4. To these solutions, Rose Bengal (RB) was added (100 µL of 1 mM; 10 µM final concentration). Samples were immediately placed on ice and exposed to high-intensity visible light, 5 cm from the outlet of a Slide Projector (Leica Pradovit P150, Leica Camera AG, Germany) through a 345-nm cut-off filter (UQG Optics, Cambridge, England). Under these conditions, light intensity was measured to be ca. 9500 lux using a light meter (Black-Comet-C-50, StellarNet Inc., Tampa, FL, USA). Samples were continuously bubbled with compressed air (Coregas, Yennora, NSW, Australia) for 30 min 1 under these conditions, resulting in the well-documented generation of singlet oxygen ( O2) and subsequent formation of hydroperoxide/s on the amino acid/peptide residue.55, 56, 61 Immediately after cessation of photolysis, catalase (10 µL of 1 mg mL-1, ≈ 65 U mL-1) was added and the sample was incubated in the dark at room temperature for 10 min; this step was performed to eliminate any H2O2 produced during photolysis. Each photochemically-generated hydroperoxide sample was generated in this way immediately prior to use; any further treatment is described in the respective methods, below.

2.6. General Protocol for Ferrous-Oxidation Xylenol Orange (FOX) Assay

These experiments aimed to assess the ability of low-molecular-mass diselenides to scavenge reagent peroxides (ROOR) and photochemically-generated amino acid- and peptide- hydroperoxides (ROOH, generation detailed in Section 2.5.). Diselenides at varying dosages were added to the peroxides and the FOX assay was employed to monitor the loss in absorbance over time at 560 nm as an indirect measure of peroxide loss.

The FOX assay relies upon a peroxide species oxidising the Fe2+ ions in the reagent to Fe3+, which then reacts with the xylenol orange (XO) dye to form a purple-blue complex, detectable by measuring absorbance of a sample at 560 nm. This assay provides an indirect measure of total peroxide concentration within a sample.209, 210 The following outline is general for each FOX assay undertaken; the peroxide samples undergoing analysis are specific to each experiment and their preparation and treatment are detailed in the ensuing sections of this

Chapter.

Initially, 250 mL of a H2SO4 (2.5 M) solution was prepared in H2O, ensuring that the acid was diluted into the H2O and not the converse due to the highly exothermic nature of the reaction. This acid is stable indefinitely at room temperature. A solution of acidified iron (II) sulfate heptahydrate (FeSO4•7H2O, 25 mM) was prepared by dissolving 0.0695 g of this compound into 10 mL of the diluted H2SO4 (2.5 M). XO solution (10 mM) was prepared by dissolving

0.1521 g of xylenol orange tetrasodium salt into 20 mL H2O. Both the acidified iron (II) sulfate and XO solutions are stable for several months when kept at 4 ˚C, with the XO also requiring storage in the dark.

The FOX assay working solution was prepared on the day of use by combining solutions in this order; one part acidified iron (II) sulfate (25 mM), two parts H2O and two parts XO (10 mM). The working solution was then passed through a 0.45 µm PVDF membrane syringe filter (Grace Davison Discovery Sciences, MD, USA) and kept on ice in the dark until needed.

The FOX assay was performed by adding 10 µL of working solution into each well of a 96- well polystyrene clear-bottom cell culture plate (Corning Inc., VIC, Australia) before adding 200 µL of the peroxide sample, ensuring that good mixing took place. The plate was then incubated at room temperature in the dark for 30 min prior to reading the absorbance at 560 nm on a SpectraMax M2e Plate Reader (Bio-Strategy Ltd., VIC, Australia). The analysis software used was that provided by the manufacturer (SoftMax Pro Analytical Software, version 5.3; MDS Analytical Technologies, CA, USA). For the FOX assay, all samples were prepared in H2O.

As this assay does not account for the species of peroxide present, where necessary, peroxides were expressed as H2O2 equivalents as determined by a concomitant FOX assay of the peroxide in comparison to a standard curve of a known range of H2O2 concentrations.

2.7. Methodology for Ultra-Performance Liquid Chromatography (UPLC)

These experiments aimed to determine the mechanisms by which diselenides were oxidised in the presence of biological oxidants, and to determine if different oxidants generated the same product/s. Initial experiments that aimed to visualise SeCA and DSePA by UPLC were unsuccessful, and so, an alternative diselenide was chosen for study; Fmoc-Selenocystine (Fmoc-SeCystine) is a diselenide of two Sec residues, wherein each Sec moiety is tagged with

40 a fluorescent Fmoc (fluorenylmethyloxycarbonyl chloride) group. This section details the optimised UPLC protocol for the detection of Fmoc-SeCystine and its oxidation products, which are formed upon reaction with HOCl, H2O2 and TrpOOH. Every sample analysed by UPLC was prepared as detailed in their respective methods and then filtered through 0.2 µm NanoSep centrifugal devices (Bio-Strategy Ltd., VIC, Australia), before being transferred to UPLC vials with 200 µL glass inserts and caps with silicone membranes (Thermo Fisher Scientific, VIC, Australia). Samples were then kept in the autosampler unit at 10 ˚C prior to analysis.

2.7.1. Preparation of UPLC Mobile Phase

Initial preparation of 1 M sodium acetate trihydrate (NaOAc, pH 5.0) was achieved by dissolving 136.08 g into 900 mL H2O and adjusting the pH value to 5.0 with glacial acetic acid

(ca. 29 mL) before addition of H2O to reach a final volume of 1 L. The pH of this solution was critical for the separation of amino acids by retention time.57

Buffers A and B were prepared as follows, making each up to a volume of 2 L and vacuum filtering through a 0.2 µm hydrophilic membrane prior to use. Buffer A contained 20% MeOH,

2.5% tetrahydrofuran (THF), 72.5% H2O and 5% NaOAc (1 M, pH 5.0) and Buffer B contained

80% MeOH, 2.5% THF, 12.5% H2O and 5% NaOAc (1 M, pH 5.0).

2.7.2. UPLC Instrumentation

The experiments were performed using a Shimadzu Nexera X2 UPLC System (Shimadzu, South Rydalmere, NSW, Australia), equipped with degassing unit (DGU-20A 5R), binary liquid chromatograph pumps (LC-30AD), autosampler (SIL-30AC), communications bus module (CBM-20A), column oven (CTO-20A), photodiode array (PDA) detector (SPD- M20A) and fluorescence detector (RF-20A X5). The software used for analysis was that provided by the manufacturer (LabSolutions, version 5.57; Shimadzu).

2.7.3. Fmoc-SeCystine Sample Preparation and Analysis

Due to the hydrophobic nature of Fmoc-SeCystine, the compound precipitates in buffer and therefore requires dilution into an organic solvent. The batch of Fmoc-SeCystine was initially tested for fluorescence by diluting 0.1 mg mL-1 of the compound into 100% MeOH and reading

fluorescence intensity on a LS50B Fluorimeter (Perkin Elmer, Waltham, MA, USA) with λex

= 265 nm and λem = 275 to 500 nm, comparing this to a MeOH control. A significant fluorescence peak at 310 nm showed that the Fmoc group gave sufficient fluorescence emission to undergo analysis via UPLC fluorescence detection.69

The final solvent concentration of Fmoc-SeCystine in all experiments was 50% MeOH and 50% buffer, unless otherwise stated. This ratio allows for sufficient dilution of the compound whilst allowing samples to retain some pH stability and biological relevance owing to the presence of the buffer. Furthermore, this dilution ratio also resulted in the largest and most consistent peaks when analysed by UPLC fluorescence detection, when compared over a range of MeOH concentrations (100% to 20%).

Quantification of Fmoc-SeCystine and its oxidation products was performed on a Shimadzu Nexera X2 UPLC system (Shimazu, South Rydalmere, NSW, Australia) and samples separated on a Shim-Pack XR-ODS UPLC column (2.2 µm, 100 x 4.6 mm) fitted with a KrudKatcher Ultra guard column (Phenomenex, Lane Cove, NSW, Australia) at 40 ˚C and a solvent flow rate of 1.2 mL min-1 over 30 min. The mobile phase comprised of a binary gradient of an aqueous component, A [20% MeOH, 2.5% THF, 72.5% H2O and 5% NaOAc (1 M, pH 5.0)] and an organic component, B [80% MeOH, 2.5% THF, 12.5% H2O and 5% NaOAc (1 M, pH 5.0)], prepared as detailed in Section 2.7.1. The solvent gradient was programmed as follows: a steady increase in B from 10% at 0 min to 87.5% at 26 min, increasing to 100% B over 0.5 min and a wash with 100% B for 1.5 min, before returning to initial conditions of 10% B for 2 min of re-equilibration preceding the next sample injection. The sample volume injected onto the column during analysis was 10 µL unless otherwise indicated in the individual experimental protocols. Fluorescence detection was optimised for the Fmoc groups (λex = 265 nm, λem = 310 nm).69 Analysis conditions were kept consistent for each Fmoc-SeCystine separation experiment.

2.7.4. Automated Dilution of Oxidised Fmoc-SeCystine for UPLC Product Analysis

The oxidation of Fmoc-SeCystine by H2O2 resulted in the formation of both a primary and secondary oxidation product. However, the fluorescence of the former was so intense as to exceed the limits of detection, resulting in an underestimation of the amount of product formed over longer time-periods. Therefore, follow-up experiments were performed with the addition

42 of an automated dilution step immediately prior to analysis. UPLC vials of filtered diluent [50% MeOH in buffer (0.1 M, pH 7.4)] and oxidised Fmoc-SeCystine samples were prepared and placed in the autosampler unit at 10 ˚C to await analysis. A pre-treatment method was programmed to aspirate either 5 or 10 µL of each Fmoc-SeCystine sample and mix it into the prepared diluent immediately prior to injection onto the column (final dilution 1 in 40 or 1 in 20, respectively). UPLC analysis was subsequently performed over time as before, via the protocol detailed in Section 2.7.3.

2.8. Detection of Oxidant-Induced Diselenide Bond Cleavage by UV/vis Spectroscopy

For all UV/vis spectroscopy experiments, conditions were kept consistent; all stocks and samples were prepared and diluted in buffer, oxidant was added to the diselenide immediately prior to analysis and all samples underwent analysis in 0.6 mL quartz cuvettes. Data was acquired at various time-points (t) over a wavelength range of 200 to 400 nm using a UV-2550 Spectrophotometer (Shimadzu, South Rydalmere, NSW, Australia) and the analysis software provided by the manufacturer (UVProbe, version 2.33). Both cells of the spectrophotometer were zeroed against buffer prior to analysis and each sample was read against a buffer blank. The cell temperature was set to 22 ˚C using the thermostat block for all experiments.

2.8.1. Determination of Rate Constants for Reaction of SeCA and DSePA with

H2O2 by UV/vis Spectroscopy

These experiments aimed to observe the spectral changes in SeCA and DSePA caused by

HOCl- and H2O2-induced oxidation using UV/vis spectroscopy. Pseudo-first-order rate constants (k1) for the reaction of the diselenides with H2O2 were obtained from analysis of spectral data over time. Any observed loss in the diselenide bond peak (maximum at ca. 300 nm) was interpreted as oxidative cleavage of the diselenide bond.

Spectral data for each diselenide was analysed using ProKIV Software (version 1.0.1.4, Applied Photophysics, Surrey, England) by first plotting absorbance loss over time for the wavelengths corresponding to the peak of the diselenide (295 to 315 nm, in 5 nm steps) and then fitting non-linear one-phase decay curves to these plots, from which an observed rate constant (kobs) at each of these wavelengths was obtained. The five resulting kobs values were averaged to give a single kobs representing the rate of absorbance loss for the whole diselenide

43 peak (295 to 315 nm). The data for each experimental triplicate at each H2O2 concentration was analysed as such, resulting in a total of three kobs values at each of five H2O2 concentrations.

These kobs values were plotted against their respective molar concentrations of H2O2, and the gradient of the resultant linear plot taken as the k1 for the reaction of each diselenide with the oxidant. The error was calculated as 95% confidence interval (CI).

2.9. Characterisation of Diselenide Oxidation Products by LC/MS

These experiments aimed to characterise the products formed by oxidation of SeCA, Fmoc- SeCystine and GSeSeG. The samples were introduced to the mass spectrometer using either direct injection or chromatographic separation by HPLC prior to analysis. The respective methodologies are detailed below.

2.9.1. Mass Spectrometry Instrumentation

Samples were analysed using a Finnigan LCQ Deca XP Max Plus ion trap mass spectrometer (MS) fitted with an ESI source. The MS was coupled to an HPLC system comprising of a Finnigan Surveyor MS Pump and a Finnigan Surveyor Autosampler, with degassing unit and column oven (Thermo Fisher Scientific, Scoresby, VIC, Australia). The system was controlled by a personal computer with Xcalibur and QualBrowser software (versions 1.4, ThermoElectron, IL, USA) for spectra collection and analysis.

Where chromatographic separation was performed prior to analysis, samples were prepared via their respective methods, before being filtered through 0.2 µm NanoSep centrifugal filters (Bio- Strategy Ltd., VIC, Australia) and transferred to UPLC vials with 200 µL glass inserts and caps with silicone membranes (Thermo Fisher Scientific, VIC, Australia).

2.9.2. Direct Injection MS for Characterising Diselenides

In order to observe the diselenide pattern of Fmoc-SeCystine and to examine the oxidation of

SeCA by H2O2, samples were introduced to the MS by direct injection from a syringe pump. Initially, a solution of 1% formic acid (v/v) in MeOH was prepared, which was used to dilute and acidify solutions before injection, promoting ionisation for more effective analysis.

Samples containing SeCA (1 mM) and H2O2 (1 or 10 mM) were prepared in H2O and first diluted 5-fold into H2O and then into an equal volume of the 1% formic acid solution. Samples

44 containing Fmoc-SeCystine (100 µM) were prepared in 50% MeOH before dilution into an equal volume of 1% formic acid solution. Samples were infused directly onto the MS source at a flow rate of 5 µL min-1, with analysis performed in positive-ion mode. The instrument parameters were: electrospray needle (4.5 kV), nitrogen as the sheath gas (held at 14 units), helium as the collision gas, and the capillary was maintained at a temperature of 275 ˚C. MS/MS studies were performed with a normalised collision energy (NCE) of 20%. The mass values were scanned over the range of m/z 100 to 400 for SeCA and m/z 150 to 2000 for Fmoc- SeCystine and spectra were generated from an average of 100 scans.

2.9.3. LC/MS Protocol for Characterisation of Fmoc-SeCystine Oxidation Products

For these experiments, chromatographic separation was performed using a Shim-Pack XR- ODS UPLC column (100 x 4.6 mm, 2.2 µm particle size, 12 nm pore size; Shimadzu, South Rydalmere, NSW, Australia) fitted with a KrudKatcher Ultra guard column (Phenomenex,

NSW, Australia). Buffer A (0.1% TFA in H2O) and Buffer B (0.1% TFA in ACN) were each prepared to 1 L and vacuum filtered prior to use. A binary elution method was used for Fmoc- SeCystine detection, comprising Buffer A and Buffer B with a total flow rate of 400 µL min-1 for 85 min. Buffer B increased steadily from 0% to 100% from t = 0 to 60 min, where it remained at 100% for 10 min to flush the column before returning to 0% at t = 72 min for re- equilibration until 85 min, in preparation for the following sample. For the first 5 and final 10 min of each run, the eluent was diverted to waste rather than to the mass spectrometer to prevent infusion of salts into the MS.

Samples were prepared to contain Fmoc-SeCystine (100 µM) and either HOCl (0 to 400 µM) or H2O2 (0 to 100 mM) in 50% MeOH/50% buffer. Immediately prior to analysis, each sample was diluted 5-fold into water, then filtered and transferred to UPLC vials. These were placed in the autosampler unit at 10 ˚C prior to injection of 20 µL onto the column, whilst the column oven was maintained at 30 ˚C. Analysis of HOCl samples was performed immediately after preparation, and analysis of H2O2 samples was performed at t = 0 and 24 h. All analyses were performed in positive ion-mode with the electrospray needle at 4.5 kV, capillary temperature of 250 ˚C and nitrogen sheath gas at 70 units. A full scan was performed from m/z 150 to 2000, as well as MS/MS scans of peaks at m/z 780.9 and 424.0, each at 28% normalised collision energy (NCE) and an isolation width of m/z ± 7.5. UV/vis analysis was performed concurrently

45 at 265 nm to detect Fmoc derivatives69 using a Perkin Elmer 785A UV/vis detector unit.

2.9.4. LC/MS Protocol for Characterisation of GSeSeG Oxidation Products

For these experiments, the liquid chromatography component of LC/MS analysis was modified from a previous method, comprising of a binary elution gradient consisting of Buffer A [H2O with 0.5% formic acid (v/v)] and Buffer B [a 50/50 solution of propan-2-ol and ACN, with 0.1% formic acid (v/v)], with a total flow rate of 200 µL min-1 for 55 min per run.211 The stationary phase was a Hypercarb UPLC Column (100 x 2.1 mm, 5 µm particle diameter) fitted with Uniguard guard column (Thermo Fisher Scientific, VIC, Australia). The column was initially equilibrated in 100% A for 10 min, decreasing to 80% between 10 and 30 min, and then to 0% between 30 and 35 min. The flow rates were kept stable for 5 min, followed by a rapid change from 100% B to 100% A between 40 and 45 min, remaining at 100% A for a further 10 min in order to re-equilibrate the column before the next sample injection. For the first 5 and final 20 min of each run, the eluent was diverted via the waste valve to prevent salts from being injected into the MS.

Samples were prepared in H2O containing GSeSeG (100 µM) and either HOCl (0 to 400 µM) or H2O2 (0 or 1 mM). Samples containing H2O2 were incubated in the dark at room temperature for 3 days before analysis. All samples were filtered through 0.2 µm centrifugal filters and transferred to UPLC vials for analysis. Vials were kept in the autosampler unit at 10 ˚C prior to injection of 20 µL of sample onto the column, which was maintained at 30 ˚C. Analysis was performed in positive ion mode with the electrospray needle at 4.5 kV, capillary temperature of 275 ˚C and nitrogen sheath gas set to 45 units, as previously described.87 Scan events were selected to identify the selenium-containing analogue of the well-characterised sulfur species, glutathione sulfonamide.211 As such, samples were monitored via a full MS scan from m/z 100 to 1500, as well as MS/MS scan events for m/z 708.9 to detect GSeSeG, and m/z 387.8, which corresponded to the mass for both the selenium analogues of glutathione sulfonamide, GSA

(GSeA, 384.2 g/mol) and the sulfinic acid derivative (GSeO2H, 386.2 g/mol) which have similar molecular masses. Each scan was performed with 28% NCA and isolation widths optimised for each compound. UV/vis analysis was performed simultaneously at 210 nm to detect any peptide bonds present.212

2.10. Determining the Rate Constants of Reaction for HOCl with Diselenides via a Competitive CBA Assay

Coumarin boronic acid (CBA) is a profluorescent compound which reacts with H2O2, ONOOH 213 and HOCl, to form a fluorescent product (7-hydroxycoumarin, COH, λmax = 370 nm), the detection and quantification of which provides a means of determining rate constants for these 214 -1 -1 reactions. CBA oxidation is known to occur slowly with H2O2 (1.5 M s ) and rapidly with ONOOH (1.1 x 106 M-1 s-1).213 Stopped-flow methodology has been employed herein to determine a rate constant for the reaction of CBA with HOCl. Subsequently, a competition kinetics assay was used to calculate the rate constants of reaction for HOCl with various diselenides in the presence of CBA as a competitive substrate. The methods for achieving these aims are detailed below.

2.10.1. Stopped-Flow to Evaluate the Rate Constant for Reaction of CBA with HOCl

Stopped-flow methodology was used to evaluate the rate constant of reaction between CBA and HOCl. The stopped-flow system (Applied Photophysics, Surrey, England) was made up of the following components; the light source was a 150 W Xenon lamp (Osram, GmbH, Munich,

Germany), which was held under an N2 atmosphere (Pharmaline, Strandmøllen, Denmark) to prevent formation of ozone. The monochromator (4.55 nm/mm bandpass) was set to a slit width of 1 mm and fluorescence detection was performed by a R6095 fluorescence photomultiplier, with voltage set to 310 V. The excitation wavelength was set to 332 nm, and emission was measured from 350 to 450 nm, which corresponds to the fluorescence maxima of COH at 370 nm.213 The stopped-flow system was linked to a personal computer with a Pro-Data electronics unit, running Pro-Data SX and Pro-Data Viewer software (Applied Photophysics, version 2.5.1883.0) for data acquisition and analysis.

CBA (5 mM) was prepared in 20% DMSO/80% buffer and diluted into buffer immediately prior to injection to achieve concentrations from 250 to 500 µM in 50 µM steps. HOCl (50 µM) was prepared in buffer by serial dilution to ensure accuracy. CBA and HOCl solutions were placed in the syringes of the stopped-flow system before rapid mixing (< 2 ms) with a pneumatic ram. For each sample, fluorescence data was acquired over 1 s for five replicates, then these traces were averaged. The experiment was performed in triplicate. The resulting

47 plots of average fluorescence over time were fitted with exponential curves, yielding a kobs value for each concentration of CBA. The rate of reaction of CBA with HOCl was determined from the gradient of this plot.

CBA (500 µM in 20% DMSO/80% buffer) was also analysed by stopped-flow to detect auto- oxidation under experimental conditions, under the same analysis conditions as described above. The sample was mixed with buffer in lieu of HOCl, and fluorescence (λex = 332 nm, λem = 350 – 450 nm) was measured every 1 s for 200 s. Analysis was performed five times per sample in three independent experiments. A linear regression was applied, representing the auto-oxidation of CBA (by measuring formation of COH) under experimental conditions over time.

2.10.2. CBA Competition Assay for Determining the Rate Constant for Reaction of HOCl with Diselenides

The HOCl-induced oxidation of CBA was monitored by measuring the formation of the fluorescent product, COH, using a SpectraMax i3 Plate Reader with SoftMax Pro Software version 6.4 (Molecular Devices, CA, USA). The excitation wavelength was set to 350 nm and fluorescence detection was performed at various time points in kinetic mode from 350 to 450 nm, which corresponds to the fluorescence maxima of COH (370 nm).213

Initially, the HOCl-induced oxidation of CBA (25 µM) was confirmed to be linearly dose- dependent within the experimental range of oxidant concentration (0 – 50 µM). Subsequently, CBA (25 µM) was oxidised by HOCl (25 µM) in the presence of increasing concentrations of various diselenides (SeCA, DSePA, DPDSe or DPMSe; 0 to 200 µM) to analyse the competitive inhibition of CBA oxidation in the presence of these compounds. Stock solutions of CBA and diselenide were prepared in 50% MeOH and 50% H2O, whereas HOCl was prepared in H2O only. HOCl was added immediately prior to the first fluorescence reading, ensuring that the final solvent concentration of each sample was equal to 50% MeOH and 50%

H2O. All samples were prepared in duplicate in Greiner Bio-One black polystyrene 96-well UV plates (Sigma, Brøndby, Denmark), and all experiments were performed in triplicate.

2.11. Statistical Analysis

Unless stated otherwise, all statistical analyses were performed using GraphPad Prism, version

7.02 (GraphPad Software, CA, USA). Data are typically expressed as mean ± 1 standard deviation, with p < 0.05 taken as a statistically significant result from at least three independent experimental replicates. Further statistical tests, where relevant, are described in each Figure legend.

Chapter 3

Reactions of Diselenides with Hypochlorous Acid (HOCl)

3.1. Introduction

Reactive oxygen species (ROS), including hypochlorous acid (HOCl), hydrogen peroxide 1 (H2O2) and singlet oxygen ( O2), are generated via multiple pathways in biological systems. The controlled production of biological oxidants is a crucial antimicrobial process during the immune system response to invading pathogens. However, excess or inappropriate generation of these oxidants can cause host tissue damage, which has been implicated in the pathogenesis of multiple inflammatory pathologies. HOCl is the major halogenating oxidant formed under physiological conditions, with proteins as key targets of oxidation due to their abundance within cells, plasma and most tissues.29, 66, 215 Previous studies have reported that sulfur- containing species such as thiols (RSH) and thioethers (RSR’) (e.g. in the amino acids Cys and Met, respectively) react rapidly with HOCl with rate constants in the range of 107 – 108 M-1 s- 1, making these some of the fastest known reactions in biological systems.69, 81 Thiols and thioethers are oxidised by HOCl to form disulfide bonds (RSSR’) and sulfoxides [RS(=O)R’], respectively.60, 70 Reaction with disulfides (e.g. cystine) can also occur, which can affect the stability of protein structures, causing a loss of function and activity.176

Cys residues present in the ubiquitous tripeptide antioxidant glutathione (GSH) react rapidly with HOCl to form the major oxidation product, glutathione disulfide (GSSG), which may be reduced by enzymatic processes back to GSH. However, under certain conditions, an additional product may be formed from the oxidation of GSH by HOCl (and to a lesser extent with other oxidants), i.e. glutathione sulfonamide (GSA), which is believed to be irreducible.216 GSA is a stable cyclic species which has been detected within physiological systems, and as such, is a potential candidate for use as biomarkers of HOCl-induced oxidative stress in vivo.87, 88, 217 For example, recent studies have reported the use of GSA as a biomarker of HOCl-induced oxidative stress in children with cystic fibrosis (CF).218

There are extensive kinetic data reported in the literature detailing the biological reactions of

HOCl, which reveal that it also reacts rapidly with amines (RNH2), unsaturated fatty acids, haem proteins and DNA,16, 27, 60 contributing to the pathogenesis of many inflammatory pathologies including atherosclerosis, neurodegenerative conditions and some forms of cancer.8, 66 Reaction of HOCl with amines can result in the formation of organic chloramines (RNHCl), which are less reactive and more stable oxidants than HOCl. The generation of chloramines can result in the propagation of HOCl-mediated oxidative damage.15, 24

The oxidation of diselenides is both thermodynamically and kinetically more favourable than

51 oxidation of the analogous disulfides,219 allowing diselenides to potentially be more effective scavengers of biological oxidants than the corresponding sulfur compounds and hence, effectively mitigate oxidative stress-induced pathologies. As such, several low-molecular-mass diselenides have been investigated to determine their efficacy in the detoxification of HOCl, by determining rate constants for, and characterising the products generated by, these reactions. The diselenides investigated in the studies reported in this Chapter were; selenocystamine (SeCA), 3,3’-diselenodipropionic acid (DSePA), 1-(9-fluorenyl)methyl chloroformate-tagged selenocystine (Fmoc-SeCystine), seleno-diglutathione (GSeSeG), 2’-dipyridyl diselenide (DPDSe) and dipyrimidin-2-yl diselenide (DPMSe), the structures of which are shown in Figure 3.1.

Figure 3.1: Structures of the diselenides investigated in this Chapter. The compounds shown are: selenocystamine (SeCA), 3,3’-diselenodipropionic acid (DSePA), selenocystine tagged with 1-(9-fluorenyl)methyl chloroformate (Fmoc-SeCystine), 2,2’-dipyridyl diselenide (DPDSe), dipyrimidin-2-yl diselenide (DPMSe) and seleno-diglutathione (GSeSeG).

3.2. Aims

Given the potential antioxidant capacity of selenium species, the experiments reported in this Chapter aimed to examine the mechanisms by which HOCl oxidises low-molecular-mass diselenides. Rate constants have been determined for these reactions by UV/vis spectroscopy, and the products characterised by LC/MS. UPLC has been used to quantify the oxidant dose- dependence of reactions.

3.3. Results

3.3.1. Investigation of the Oxidation of Selenocystamine by HOCl using UV/vis Spectroscopy

These experiments investigated the mechanisms by which HOCl oxidises SeCA by observing the relationship between diselenide bond loss (observed as a loss of absorbance at 305 nm)220 and HOCl concentration. Samples were prepared in buffer (0.1 M, pH 7.4) containing SeCA (1 mM) with increasing concentrations of HOCl (0 to 4 mM) added immediately prior to analysis by UV/vis spectroscopy (200 – 400 nm) as detailed in Section 2.8. Samples were prepared in duplicate and kept at 22 ˚C in the dark between analyses at t = 0, 1, 3 and 24 h, and at 4 days. A pH of 7.4 was recorded for each sample prior to analysis and no pH change was observed over the experimental time course. Data from a single representative experiment are shown in Figure 3.2, comparing the traces obtained immediately after HOCl addition (A, t = 0 min) to those obtained after 24 h of sample incubation (B, t = 24 h). The time between sample mixing and the first analysis was ca. 5 s, and each consecutive trace recorded over ca. 10 s.

- At physiological pH of 7.4, HOCl is present as a mixture of OCl and HOCl (pKa = 7.582 at 20 208 - -1 -1 ˚C). HOCl and OCl have molar absorption coefficients of ɛ235 = 100 M cm and ɛ292 = 350 M-1 cm-1, respectively, therefore the absorption band of OCl- has the potential to interfere with the diselenide peak at 305 nm when present in high concentrations.208, 221 However, the relative - -1 -1 absorbance of OCl relative to SeCA (SeCA has ɛ243 = 7510 M cm ) at the experimental concentrations employed is minimal and thus, does not generate significant absorbance when compared to that of the diselenide, except where the HOCl is in high excess.222

Figure 3.2: Oxidation of SeCA (1 mM) by HOCl (0 to 4 mM); A) t = 0 min, B) t = 24 h. Representative data of a single independent experiment, performed in duplicate, with HOCl added immediately prior to analysis by UV/vis spectroscopy (ca. 5 s). Initial pH was 7.4 and no change was observed over the experimental time course.

In the presence of amine groups such as those on SeCA, HOCl is known to form chloramines (RNHCl), which are oxidants that are less reactive than their precursor, HOCl, but can be crucial intermediates in the propagation of HOCl-mediated damage.223, 224 These relatively long-lived organic chloramines form on the -NH2 groups of SeCA and remain attached to the molecule, generating absorbance at ca. 260 nm,225, 226 thus, the formation of chloramines may introduce spectral interference, causing an underestimation of the total extent of diselenide bond loss. Chloramines degrade slowly over time, the effect of which is apparent in Figure 3.2, B, where the chloramines have degraded during the 24-hour incubation period, as evidenced

54 by a decrease in absorbance at ca. 260 nm, revealing a more pronounced decrease in absorbance at 305 nm. The higher absorbance values at ca. 260 nm in the presence of 4 mM HOCl is likely due to the higher concentrations of both the OCl- and organic chloramines at this concentration.208

The reaction of SeCA and HOCl is very rapid, as evidenced by the loss in diselenide bond absorbance immediately following oxidant addition (305 nm; Figure 3.2, A), concomitant with formation of products that absorb at shorter wavelengths (< 240 nm). The presence of an isosbestic point at ca. 280 nm suggests the formation of a single primary oxidation product, which might be the oxygenated mono-selenium species. Whilst the HOCl dose-dependence of the reaction is apparent, the rapidity of reaction and the spectral interference introduced by chloramine formation prevents the calculation of rate constants from these studies.

Subsequent experiments aimed to investigate this reaction in greater detail by monitoring the reaction of SeCA with increasing concentrations of HOCl by separation and quantification of the reaction components using ultra-high-performance liquid chromatography (UPLC).

3.3.2. Optimisation of UPLC Conditions to enable the Detection of Diselenides and their Oxidation Product(s)

The oxidation of SeCA by HOCl had been shown by UV/vis spectroscopy to result in loss of the diselenide bond, but products were not able to be accurately detected. Therefore, initial experiments aimed to establish a UPLC protocol by which the HOCl-induced loss of SeCA and formation of oxidation product/s could be monitored. A stock solution of SeCA (10 mM) was prepared in buffer (0.1 M, pH 7.4) and diluted into the mobile phase to obtain a range of standards (0 to 200 µM), which were then filtered by centrifugation and analysed by UPLC as per Section 2.7., using a Shimadzu Nexera X2 UPLC system (Shimadzu, South Rydalmere, NSW, Australia). The mobile phase used in these preliminary experiments was a filtered solution of 1% ACN in H2O adjusted to pH 2.5 with TFA. The initial UPLC method employed an isocratic flow with a rate of 1 mL min-1 over a run time of 12 min per sample. For each analysis, 10 µL of sample was injected onto the stationary phase (column) and the PDA detector monitored the absorbance at 230 nm (close to the absorbance maximum of SeCA at 243 nm).222 This Section details the process by which the final optimised UPLC conditions were established, by systematic deviation from these initial conditions.

Three columns were employed under these initial analysis conditions; a Shim-Pack XR-ODS

UPLC column (2.2 µm, 100 x 4.6 mm), an Hichrom Ultrasphere 5 ODS HPLC column (5 µm, 250 x 4.6 mm) and a Beckman Coulter ODS HPLC column (5 µm, 250 x 4.6 mm). Analysis using each column yielded a peak close to the solvent front which was attributed to SeCA as it increased in a dose-dependent manner with the SeCA concentration. The retention time for this peak ranged between 1 and 2.7 min (Figure 3.3).

Figure 3.3: UPLC chromatograms of SeCA (0 – 200 µM) obtained with different columns; A) Shim-Pack XR-ODS, B) Hichrom Ultrasphere 5 ODS, C) Beckman Coulter ODS. Mobile phase was 1% ACN in H2O, adjusted to pH 2.5 with TFA, with a flow rate of 1 mL min-1 over 12 min runs and 10 µL sample injection volume. PDA detector was set to 230 nm.

These columns showed that the diselenide is capable of detection by UPLC, but the conditions employed were not suitable for accurate quantification. Therefore, subsequent optimisation attempts aimed to increase the retention time of SeCA to minimise interference from the buffer components on the solvent front and allow for diselenide peak integration and analysis.

The stationary phase was changed to a Zorbax 300-SCX HPLC column (5 µm, 250 x 4.5 mm) and analysis repeated under the original experimental conditions, resulting in a negative peak at 2.4 min followed by the primary SeCA peak at 2.8 min. This peak was affected by an increase in retention time as SeCA concentrations increased (Figure 3.4). The co-elution of the diselenide with the solvent front is impractical for quantification.

Figure 3.4: UPLC chromatogram of SeCA (0 – 200 µM). Zorbax 300-SCX Column. Mobile -1 phase was 1% ACN in H2O, adjusted to pH 2.5 with TFA, with a flow rate of 1 mL min .

Using this same column and changing the mobile phase to buffer (0.1 M, pH 6.5) eliminated the negative peak and increased the retention of SeCA to 3.0 min (Figure 3.5). However, this also introduced a secondary peak which co-eluted with that of the diselenide, preventing accurate peak integration. This is presumed to be a component of the mobile phase interacting with the diselenide, as it also demonstrated diselenide dose-dependence. The secondary peak effect observed in these (and subsequent) traces, is believed to be a result of column deterioration, as most initial optimisation experiments utilised a column which was several years old and well-used. Following completion of optimisation experiments, a newer column was implemented, and the secondary peak effect was no longer observed; this is detailed in subsequent sections.

Figure 3.5: UPLC chromatogram of SeCA (0 – 200 µM). Zorbax 300-SCX Column. Mobile phase was buffer (0.1 M, pH 6.5), with a flow rate of 1 mL min-1.

The flow rate was then increased from 1 to 3 mL min-1 to increase system pressure in an attempt to force the diselenide into the packing material of the column for longer retention (Figure 3.6). Instead, a small peak eluted on the solvent front at 1.0 min which displayed no diselenide dose- dependence, indicating that this peak corresponded to a component of the mobile phase. The diselenide is believed to have been flushed from the column at the solvent front, as no dose- dependent peak attributed to SeCA was detected. Increasing the flow rate also introduced significant turbulence into the baseline signal of all samples.

Figure 3.6: UPLC chromatogram of SeCA (0 – 200 µM). Zorbax 300-SCX Column. Mobile phase was buffer (0.1 M, pH 6.5), with a flow rate of 3 mL min-1.

The flow rate was returned to 1 mL min-1 and, as per the recommendation of the column supplier, the buffer (0.1 M) was adjusted from pH 6.5 to pH 5.0. However, use of this mobile phase did not alter the retention time of the diselenide peak, nor did it eliminate the secondary peak eluting alongside SeCA (Figure 3.7, A). In addition, standards of DSePA (0 to 200 µM) were prepared and analysed in the same way as SeCA using the adjusted buffer (0.1 M, pH 5.0) as mobile phase (Figure 3.7, B). Whilst the absorbance intensity of DSePA was ca. 5-fold higher than that of the SeCA, the retention time was identical, and the secondary peak remained present, preventing accurate diselenide peak integration and analysis.

Figure 3.7: UPLC chromatograms of; A) SeCA (0 – 200 µM) and B) DSePA (0 – 200 µM). Zorbax 300-SCX Column. Mobile phase was buffer (0.1 M, pH 5.0), with a flow rate of 1 mL min-1.

The stationary phase was then changed to a Zorbax SB-C18 HPLC column (5 µm, 250 x 4.6 mm) and various analysis conditions were tested. Initially, an isocratic flow of 1 mL min-1 was employed using a mobile phase of 1% ACN in H2O with TFA (pH 2.5). Using this column, SeCA standards eluted in a sharp diselenide dose-dependent peak at 3.4 min, slightly separated from the solvent front (Figure 3.8).

Figure 3.8: UPLC chromatogram of SeCA (0 – 200 µM). Zorbax SB-C18 Column. Mobile -1 phase was 1% ACN in H2O, adjusted to pH 2.5 with TFA, with a flow rate of 1 mL min .

A binary buffer gradient was then introduced comprising an aqueous buffer (H2O with 0.01 or

0.1% TFA) and an organic buffer (ACN with 0 or 0.01% TFA), with the aim of increasing SeCA retention time on the column. Various buffer ratios were investigated, as well as the initial percentage of organic buffer and the rate of increase in the organic buffer component over time. The total SeCA concentration was increased from 200 to 500 µM to produce larger peaks, and the total run time was lengthened from 12 to 20 min. SeCA standards (0, 50, 100, 200, 300, 400, 500 µM) were analysed by UPLC using a binary buffer gradient of 0.1% TFA in H2O as the aqueous phase and 100% ACN as the organic phase. The gradual increase in the organic component over time caused the SeCA standards to elute in a small, wide peak at 4.8 min, which increased in intensity with SeCA concentration (Figure 3.9). This peak displayed

61 two intensity maxima, with the lower SeCA concentrations appearing to shift towards the later- eluting peak segment. The cause of this effect is unknown and makes integration unfavourable.

Figure 3.9: UPLC chromatogram of SeCA Standards (50 – 500 µM). Zorbax SB-C18 Column. A binary buffer gradient was employed using the mobile phases; A) 0.1% TFA in -1 H2O, and B) 100% ACN, with a total flow rate of 1 mL min .

All UPLC experiments up to this point aimed to determine optimal conditions for the detection and quantification of diselenides (SeCA and DSePA). The buffers and columns used during this stage of method development are summarised in Table 3.1.

Table 3.1: Summary of UPLC conditions employed during optimisation of diselenide detection

Diselenide Column Buffer(s) Shim-Pack XR-ODS (2.2 µm, 100 x 4.6 mm) Hichrom Ultrasphere 5 ODS 1% ACN in H2O with TFA (pH 2.5) (5 µm, 250 x 4.6 mm) Beckman Coulter ODS (5 µm, 250 x 4.6 mm)

1% ACN in H2O with TFA (pH 2.5) SeCA (0 – 500 µM) Zorbax 300-SCX 0.1 M buffer (pH 6.5)a (5 µm, 250 x 4.5 mm) 0.1 M buffer (pH 5.0)

1% ACN in H2O with TFA (pH 2.5) b A: 0.01% TFA in H2O Zorbax SB-C18 B: 0.01% TFA in ACN (5 µm, 250 x 4.6 mm) b A: 0.1% TFA in H2O B: 100% ACN Zorbax 300-SCX DSePA (0 – 200 µM) 0.1 M buffer (pH 5.0) (5 µm, 250 x 4.6 mm) aflow rates of 1 and 3 mL min-1 were each investigated; bbinary buffer gradients: column and buffers remained the same, but various gradients and buffer percentages were investigated.

Any products of diselenide oxidation would be expected to elute at earlier retention times than the reduced diselenide due to increased polarity owing to the addition of oxygen. As a result of this, any oxidation product/s of SeCA were postulated to elute at the solvent front, which would prevent integration and subsequent quantification, rendering this UPLC method impractical for diselenide product detection. Therefore, an alternative diselenide was chosen to undergo investigation into the mechanisms of diselenide oxidation by biological oxidants. The fluorescently-tagged diselenide Fmoc-SeCystine was employed for this purpose. The Fmoc groups were postulated to increase the retention time of the bound diselenide due to the steric bulk and hydrophobic nature of the aromatic structure, as well as providing a means by which to detect the diselenide and its products by fluorescence emission.69

Due to the hydrophobic nature of Fmoc-SeCystine, all Fmoc-SeCystine stocks were prepared in MeOH to facilitate dissolution of the compound. Oxidant stocks were prepared in buffer (0.1 M, pH 7.4) and mixed with Fmoc-SeCystine stock solutions to give a final concentration of 50% MeOH for all analytical samples, unless stated otherwise. This solvent composition had been found to facilitate Fmoc-SeCystine dissolution, whilst retaining some physiological relevance and pH stability owing to the presence of buffer. The fluorescence intensity of the Fmoc-SeCystine was measured, as per Section 2.7.3., which confirmed the strong fluorescence of the Fmoc groups, showing that the compound could be detected by UPLC.69

Fmoc-SeCystine was then investigated by UPLC using a protocol reported in the literature for the detection of Fmoc-Met.174 The initial UPLC parameters employed were detailed in the report by Storkey et al. (2012),174 with all deviations from this method outlined below. The final protocol that was developed from this method is detailed in Chapter 2, Section 2.7.3.

Fmoc-SeCystine standards (0 – 200 µM) were prepared in different MeOH to buffer (0.1 M, pH 7.4) ratios (final MeOH concentration = 20 – 80%) and analysed using these UPLC conditions to determine the effect of MeOH concentration on Fmoc-SeCystine elution time and peak intensity. It was found that the MeOH percentage did not affect the elution time of the Fmoc-SeCystine standards (all 4.6 min), but each sample containing 50% MeOH (final concentration) consistently generated the largest peaks at each Fmoc-SeCystine concentration employed. An example of this is given in Figure 3.10, where Fmoc-SeCystine concentration is 100 µM and the MeOH solvent concentration varies.

Figure 3.10: UPLC chromatogram of Fmoc-SeCystine (100 µM) at various MeOH percentages (20 – 100%). Fmoc-SeCystine was prepared in MeOH and diluted into different MeOH-to-buffer ratios. UPLC analysis used a Shim-Pack XR-ODS column, a binary buffer gradient with increasing organic component over time, with a total flow rate of 1.2 mL min-1.

Each of the samples analysed displayed a large, wide peak between 7.4 to 9.8 min, which corresponds to the end of the wash phase and beginning of the re-equilibration phase of the solvent gradient. The presence and intensity of this peak were not affected by altering the MeOH-to-buffer ratio, nor the concentration of Fmoc-SeCystine, and it was present in both blank (100% MeOH) and control (100 µM Fmoc-SeCystine) samples (not shown). The lack of diselenide dose-dependence implies that it is not a component of the Fmoc-SeCystine. These data indicate that the peak corresponds to a constituent of the solvents as they are flushed from the column. It is likely an organic species as it elutes when the organic buffer component is highest. The peak was deemed to not impact the result of the experiment as it did not interfere with the species under investigation and was therefore discounted in all subsequent experiments.

These experiments had served to determine the elution time of Fmoc-SeCystine (4.6 min) under these conditions, as well as the optimal MeOH concentration (50%) at which peak intensity was maximised. Therefore, all subsequent Fmoc-SeCystine samples were prepared to a final solvent concentration of 50% MeOH and 50% buffer (0.1 M, pH 7.4).

Further experiments aimed to investigate the elution times of any HOCl-induced oxidation products of Fmoc-SeCystine under these conditions. Standards of Fmoc-SeCystine (0 – 200 µM) and samples containing both Fmoc-SeCystine (100 µM) and HOCl (0, 50, 100 and 200 µM) were each prepared in 50% MeOH, and were analysed concurrently under the UPLC conditions described by Storkey et al. (2012),174 which was amended as follows to increase the

65 length of each run from 12 to 30 min; the buffer gradient was programmed to increase from 75% to 87.5% B from 0 to 26 min, followed by an increase to 100% B over the next 0.5 min, staying at 100% B for 1.5 min to flush the column, before returning to 75% B over 0.1 min, then 1.9 min of re-equilibration prior to the next sample injection. Representative chromatograms of the Fmoc-SeCystine standards (0 – 200 µM) and Fmoc-SeCystine (100 µM) oxidised by HOCl (0 – 200 µM) under these conditions are shown in Figure 3.11, A and B, respectively.

Figure 3.11: UPLC chromatograms of; A) Fmoc-SeCystine standards (0 – 200 µM), B) Fmoc-SeCystine (100 µM) with HOCl (0, 50, 100, 200 µM). Shim-Pack XR-ODS column with a binary buffer gradient (30 min runs) with increasing organic buffer percentage over time (B = 75% at t = 0 min), total flow rate of 1.2 mL min-1. The intensity of the fluorescent product relative to the parent diselenide is evident by comparing the scale on the vertical axes.

The slower rise in the organic component of the buffer allowed the Fmoc-SeCystine to remain on the column for longer, increasing the retention time of the diselenide from 4.6 to 5.7 min (Figure 3.11, A). The addition of HOCl resulted in the formation of a highly-fluorescent peak eluting on the solvent front at 2.0 min, which was generated in a HOCl dose-dependent manner (Figure 3.11, B). This was postulated to be a product of the oxidation of Fmoc-SeCystine by HOCl. This peak emitted fluorescence at an intensity that saturated the detector, having reached the upper limits of detection, causing the product peaks to display flattened tops and preventing an accurate calculation of peak area by integration. Both the standard and oxidised Fmoc- SeCystine samples displayed a smaller secondary peak eluting immediately subsequent to the major peak, believed to be result of column deterioration.

The UPLC protocol was again amended, the buffer gradient program being altered in an attempt to further increase the elution time of Fmoc-SeCystine, and also of the product peak. Several experiments were performed where the initial percentage of buffer B was set to a value between 20 – 75%. The initial concentration of buffer B remained constant from 0 to 5 min, followed by an increase to 87.5% B over the next 21 min, then a rapid increase to 100% B over 0.1 min where it remained at 100% for 2 min to wash the column, and finally a drop to the initial concentration over 0.1 min where the concentration remained constant for 1.8 min of re- equilibration prior to injection of the next sample. Two samples were prepared in 50% MeOH; one containing untreated Fmoc-SeCystine (100 µM) and the other containing equimolar concentrations of Fmoc-SeCystine and HOCl (each 100 µM), which were analysed using these amended UPLC conditions with different initial concentrations of buffer B. The chromatogram traces of the untreated Fmoc-SeCystine (100 µM) at the various initial buffer B percentages are shown in Figure 3.12.

Figure 3.12: UPLC chromatogram of Fmoc-SeCystine (100 µM) with traces showing different initial buffer B concentrations (20 – 75%). Data obtained using a Shim-Pack XR- ODS column with a binary buffer gradient over 30 min runs with increasing organic buffer B percentage over time, total flow rate of 1.2 mL min-1.

Decreasing the initial percentage of buffer B increased the rate of buffer B rise over time, causing a significant increase in the retention time of Fmoc-SeCystine. Ensuing experiments examined the elution times of both the parent diselenide and its oxidation product/s under various initial buffer B percentages, to further optimise their detection. An untreated Fmoc- SeCystine standard (100 µM) and an oxidised sample of Fmoc-SeCystine and HOCl (each 100 µM) were analysed under the UPLC conditions described above, with the gradient program amended to begin rising at 8 min, rather than at 5 min. The programming for the washing and

67 re-equilibration phases remained unchanged. Four methods were employed under these conditions, where only the initial percentage of buffer B was changed (40, 45, 50 and 55% B at t = 0 min), which altered the rate of organic buffer rise over time. The chromatograms in Figure 3.13 show the traces of both untreated (A) and HOCl-treated Fmoc-SeCystine (B), respectively, analysed using the various initial buffer B percentages undergoing investigation.

Figure 3.13: UPLC chromatograms generated using different initial buffer B concentrations (40 – 55% at t = 0 min). A) Untreated Fmoc-SeCystine (100 µM), B) Fmoc- SeCystine (100 µM) with HOCl (100 µM). Data acquired using a Shim-Pack XR-ODS column with a binary buffer gradient where the organic buffer percentage increases over time from 8 min onward, total flow rate of 1.2 mL min-1 for 30 min per sample.

Consistent with previous experiments, decreasing the initial percentage of buffer B resulted in a dramatic increase in the retention time of Fmoc-SeCystine (Figure 3.13, A). Furthermore, delaying the rise of the buffer gradient to begin at 8 min caused a slight increase in retention time of the diselenide, relative to previous experiments where the buffer began increasing from 5 min (see Figure 3.12). In addition to increasing the retention time of Fmoc-SeCystine, decreasing the initial buffer B percentage resulted in a concomitant increase in retention time of the peak corresponding to the observed product of HOCl-induced Fmoc-SeCystine oxidation

(Figure 3.13, B). It is unclear if the accompanying loss in product peak intensity is a result of the different buffer conditions or is due to the product decaying over the experimental time- course. Despite both the diselenide and oxidant being present at a 1:1 molar ratio, the fluorescence of the product is disproportionate to that of the parent diselenide, being so high as to reach the limits of detection, as observed by the product peaks showing flat tops. These experiments had determined that decreasing the initial percentage of the organic component of the binary buffer gradient (buffer B), resulted in a significant increase in the retention time of Fmoc-SeCystine and of the primary stable oxidation product observed under these conditions.

All subsequent experiments used a newer Shim-Pack XR-ODS column to improve the retention and elution of samples, to generate sharper peaks and allow quantitative analysis. This caused Fmoc-SeCystine (100 µM) to elute at 25.8 min and addition of increasing concentrations of HOCl (0 – 100 µM) resulted in the dose-dependent formation of a product which eluted at 11.2 min (Figure 3.14). This, and all subsequent UPLC experiments used for the detection of Fmoc- SeCystine and its product(s), used the protocol detailed in Section 2.7.

Figure 3.14: Representative UPLC chromatogram of Fmoc-SeCystine (100 µM) oxidation by HOCl (0, 10, 25, 50, 75, 100 µM); A) Full chromatogram, B) Inset of primary product peak (11.2 min), C) Inset of Fmoc-SeCystine peak (25.8 min). Data obtained using a newer Shim-Pack XR-ODS column with the final optimised protocol conditions, and a 2 µL injection volume for all samples. Baseline is a blank [50% MeOH in buffer (0.1 M, pH 7.4)].

Cumulatively, these experiments established an optimised protocol for the separation and detection of Fmoc-SeCystine and its primary stable oxidation product. This optimised protocol was then employed to examine the reaction pathway of Fmoc-SeCystine oxidation by HOCl

(and H2O2 in Chapter 4), to quantify parent diselenide loss and concomitant product formation.

3.3.3. Examination of the Oxidation of Fmoc-Selenocystine by Hypochlorous Acid using UPLC

The reaction of Fmoc-SeCystine with HOCl was analysed via the UPLC method developed herein (as detailed in Section 2.7.3.) to quantify diselenide loss and product formation. Preliminary analysis of samples containing Fmoc-SeCystine (100 µM) revealed that the diselenide eluted at 25.8 min under these conditions (Figure 3.15, A). Samples containing Fmoc-SeCystine (100 µM) were then prepared with increasing concentrations of HOCl (0 to 400 µM) added immediately prior to sample filtration and analysis (Figure 3.15, B).

Analysis of the resulting chromatograms revealed that the oxidation of Fmoc-SeCystine was dependent upon the dose of HOCl, and that this reaction occurred rapidly, having generated product in the brief time between oxidant addition and sample analysis (ca. 3 min). This is consistent with the UV/vis spectroscopy data described earlier (Section 3.1.1.), which demonstrated the rapid oxidation of SeCA by HOCl, suggesting that HOCl reacts with diselenides with very high rate constants.

Upon oxidation by HOCl, two additional peaks were observed which were not present in the non-oxidised control samples (Figure 3.15, B). A peak with high intensity was detected at 11.2 min (termed ‘Product 1’) which saturated the fluorescence detector under these conditions, and a much less intense fluorescent peak at 6.2 min (termed ‘Product 2’). The formation of both of these peaks was HOCl dose-dependent, contrary to UV/vis spectroscopy data above, where oxidation of SeCA by HOCl formed an isosbestic point, indicating the generation of a single primary oxidation product. This result indicates that the nature of the diselenide plays a key role in determining the formation of oxidation products generated by the same oxidant.

Figure 3.15: Representative UPLC chromatograms of Fmoc-SeCystine (100 µM) oxidation by HOCl (0 to 400 µM). A) Loss of Fmoc-SeCystine, B) Formation of Fmoc- SeCystine Products. Data obtained using a Shim-Pack XR-ODS column, a binary buffer gradient (B = 10% at t = 0 min) with organic component increasing over time, and a total flow -1 rate 1.2 mL min for 30 min. Fluorescence detection was optimised for the Fmoc group (λex = 265 nm, λem = 310 nm). Several intermediate HOCl concentrations have been removed for visual clarity.

The fluorescence yields are significantly higher for Product 1 relative to the parent compound. It is postulated that the two linked Fmoc groups in the parent diselenide interact to cause internal fluorescence quenching, with the oxidised diselenide having a much higher fluorescence as it only has a single Fmoc group, and thus, no internal fluorescence quenching. This provides insight into the possible structure of the oxidation product, which is examined in detail in subsequent Sections.

The chromatogram peak areas of the parent Fmoc-SeCystine and its two resulting products were then integrated for quantitative analysis. No Fmoc-SeCystine peak was detected when the

HOCl concentration was 400 µM, so this data point was excluded from analysis. A linear regression curve was fitted to the data (Figure 3.16). It was found that the loss in Fmoc- SeCystine (100 µM) became significant at equimolar concentrations of HOCl, measured as a loss in the percentage of diselenide peak area relative to the non-oxidised control. Upon addition of 150 µM HOCl, ca. 50% of the Fmoc-SeCystine fluorescence had been lost (i.e. 50 µM), indicating that the reaction has a stoichiometry of 3 moles of HOCl reacting with every 1 mole of Fmoc-SeCystine.

Figure 3.16: Oxidative loss of Fmoc-SeCystine (100 µM) with HOCl (0 to 200 µM). Average of 4 independent experimental replicates, error bars are ± 1 SD. Data point where HOCl = 400 µM was removed from linear analysis. The loss in Fmoc-SeCystine is relative to untreated Fmoc-SeCystine, as a percentage of integrated peak area. Significance was determined by one-way ANOVA with Dunnett’s post-hoc test for multiple comparisons. **P < 0.005, ***P < 0.0001. Dotted line shows the linear fit for the data with equation y = -0.3691x + 104.

It is possible that not all the HOCl is reacting exclusively with the diselenide bond; there may be reaction with the oxidised product itself to form further species. Previous studies have reported that HOCl does not interact with the Fmoc moiety in the case of Fmoc-Met (5 µM) at low HOCl concentrations (up to 2 µM).69 Due to the inherent reactivity of the diselenide bond, it is presumed to be the major target of oxidative attack on the Fmoc-SeCystine within these experiments, with the Fmoc groups remaining intact at the oxidant concentrations used.

The loss in Fmoc-SeCystine (as % of the untreated control) was plotted against the log of the

HOCl concentration to determine the half maximal inhibitory concentration (IC50) of the reaction (Figure 3.17). The plot yielded a log(IC50) value of 2.1, thus, the IC50 value was

73 calculated to equal 140 µM HOCl. This signifies that loss of 50 µM Fmoc-SeCystine occurs with ca. 140 µM HOCl and hence, a stoichiometry of ca. 3:1.

Figure 3.17: Half maximal inhibitory concentration (IC50) plot of Fmoc-SeCystine (100 µM) loss with HOCl treatment (0 to 400 µM). Fmoc-SeCystine is expressed as % peak area of the untreated control (100 µM) and HOCl concentration is converted to a logarithmic scale. Average of 4 independent experimental replicates, error bars show ± 1 SD.

The peaks corresponding to Product 1 and Product 2 were integrated to give the peak areas, with these then plotted against the concentration of HOCl and the data fitted with linear regression curves (Figure 3.18). Product 1 was formed in a linear, oxidant dose-dependent manner from 0 to 200 µM HOCl (R2 = 0.9999, Figure 3.18, A) but displayed a plateau at the highest HOCl concentration (400 µM) due to depletion of the Fmoc-SeCystine parent.

The generation of Product 2 occurs only at higher HOCl concentrations (≥ 75 µM, Figure 3.18, B). This may indicate that Product 2 is an over-oxidised species that is secondary to the formation of Product 1, and that its generation may contribute to the plateau effect demonstrated for Product 1 at high oxidant excesses. Between 75 – 200 µM HOCl, the formation of Product 2 appears linear (R2 = 0.9923, Figure 3.18, B), but this trend cannot be applied to all the data. At 400 µM HOCl, the integrated peak area of Product 2 is ca. 103-fold higher than that at 200 µM HOCl, further supporting the hypothesis that Product 2 is a secondary species generated via the over-oxidation of Product 1 at higher excesses of HOCl.

Figure 3.18: Formation of product peaks upon oxidation of Fmoc-SeCystine (100 µM) by HOCl (0 to 200 µM). A) Product 1, B) Product 2. Data are average of at least 3 independent experiments, error bars are ± 1 SD. Product 1 is linear from 0 to 200 µM HOCl (R2 = 0.9999). For Product 2, a linear regression was applied only to [HOCl] = 75 – 200 µM (R2 = 0.9923).

These experiments have established that Fmoc-SeCystine is oxidised by HOCl in a linear dose- dependent manner up to a two-fold excess of oxidant over diselenide, concomitant with the formation of two major stable fluorescent products. The primary product is initially formed in a linear dose-dependent manner before giving a plateau value, whereas the secondary product is only observed as the HOCl concentration approaches a 1-to-1 ratio of oxidant to diselenide, or higher. From these data, it is inferred that excess HOCl reacts with the primary product to drive the formation of the over-oxidised secondary product. Subsequent experiments aimed to characterise these products using liquid chromatography coupled to mass spectrometry (LC/MS).

3.3.4. Characterisation of the Products of Fmoc-SeCystine Oxidation by HOCl using LC/MS

The relative abundance of the six naturally-occurring isotopes of elemental selenium (Table 3.2) results in a distinctive pattern upon detection of the charged ions by MS, the observation of which can be used to confirm that either a single selenium atom or a diselenide bond is present within the species being investigated.227

Table 3.2: Abundance of naturally-occurring selenium isotopes

Se isotope % abundance Se74 0.9 Se76 9.4 Se77 7.6 Se78 23.8 Se80 49.6 Se82 8.7

Initial experiments examined the MS spectrum of untreated Fmoc-SeCystine to confirm the identity of the parent diselenide, as detailed in Section 2.9.2. A sample of Fmoc-SeCystine (100

µM) was prepared in MeOH then diluted into an equal volume of 1% formic acid in H2O solution (v/v) to give a final MeOH concentration of 50%, before being introduced by direct injection into the MS source at a rate of 5 µL min-1. A blank sample (50% MeOH, 50% formic acid solution) was analysed concurrently. Each sample was injected into the MS three times and spectra were generated for each injection from the average of 100 scans of mass-to-charge ratio (m/z) values in the range of m/z 150 to 2000. The average MS spectrum of three independent infusions of Fmoc-SeCystine (778.6 g/mol, final concentration = 50 µM) is shown in Figure 3.19, A, which displays a typical diselenide isotope pattern and a peak maximum at m/z 780.9. This is identical to the simulated MS spectrum of Fmoc-SeCystine (Figure 3.19, B), obtained by computational simulation using the chemical formula of Fmoc-SeCystine

[(C18H16O4NSe)2; Xcalibur and QualBrowser software, v 1.4., ThermoElectron, IL, USA], confirming the presence of Fmoc-SeCystine in the standard. This was not detected in the blank, where peak intensity at m/z 780.9 was negligible.

Figure 3.19: MS Spectra of Fmoc-SeCystine; A) Experimental direct injection, B) Computational simulation. A) representative data from the average of three direct infusions. ‘Blank’ is MeOH and ‘Standard’ is 100 µM Fmoc-SeCystine in MeOH, with both samples diluted immediately prior to injection into an equal volume of 1% formic acid (v/v) in H2O. B) Spectrum obtained by computational simulation using the chemical formula of Fmoc- SeCystine [(C18H16O4NSe)2, Xcalibur and QualBrowser software (v 1.4.)]. This isotope pattern is characteristic of a diselenide species.

Following the detection of Fmoc-SeCystine by direct injection MS, subsequent experiments were performed to determine the products of Fmoc-SeCystine oxidation by HOCl via LC/MS. Samples were prepared containing Fmoc-SeCystine (100 µM) and HOCl (0 to 400 µM) in a final solvent concentration of 50% MeOH and 50% H2O, along with a blank (50% MeOH in

H2O). Samples were diluted 5-fold into H2O and analysed immediately. A LC/MS protocol with a binary elution gradient was employed over 85 min, where the organic component increased steadily over time, with concomitant UV/vis analysis at 265 nm to detect Fmoc derivatives69 and MS/MS analysis of the major ion peaks. Representative full scan MS chromatograms from a single LC/MS experiment are given in Figure 3.20, which show untreated Fmoc-SeCystine (100 µM, A) and an oxidised sample containing equimolar concentrations of Fmoc-SeCystine and HOCl (100 µM, B). The chromatogram peaks at 33.3 and 38.9 min were present at a similar intensity in both the untreated and treated samples, whereas the addition of HOCl resulted in a significant decrease in the peak at 46.4 min, as well as formation of two small peaks which eluted at 28.0 and 35.3 min.

Figure 3.20: Representative full-scan LC/MS chromatogram of Fmoc-SeCystine (100 µM) in the absence (A) or presence (B) of HOCl (100 µM). Chromatogram data were obtained from a single MS scan (m/z 150 – 2000).

MS spectra were then generated for each chromatogram peak in order to characterise the species giving rise to each peak, based upon the isotopic pattern and mass of the major ions in each spectrum. The peak eluting at 33.3 min displayed significant peak intensity relative to the other compounds, but the major ion in the resulting spectrum was of low intensity, with a single selenium isotope pattern and m/z 478.1 (Figure 3.21, A). Due to the presence of this species in both the untreated and treated samples and the weakness of the resulting mass spectrum, it is suggested to be an unknown contaminant arising from the starting material, and not a product of the reaction of Fmoc-SeCystine with HOCl; therefore, this material was not investigated further.

Figure 3.21: MS spectra of the major chromatogram peaks of Fmoc-SeCystine (100 µM) in the absence or presence of HOCl (100 µM), which eluted at: A) 33.3 min, B) 35.3 min, C) 38.9 min. Data from a single representative experiment, with normalised peak intensity.

The mass spectra of the peaks eluting at 35.3 and 38.9 min are given in Figure 3.21, B and C, respectively, which display no apparent major peaks, nor a selenium or diselenide isotope pattern, as well as very low intensity across all mass ranges scanned. Thus, these peaks were deemed to be contaminants arising from sample preparation and analysis and were not investigated further.

The chromatogram peak eluting at 46.4 min was found to be Fmoc-SeCystine, as evidenced by the typical diselenide isotope pattern and major ion at m/z 780.9 (Figure 3.22, B), which is consistent with the data shown in Figure 3.19.

Figure 3.22: MS spectra of the major chromatogram peaks of Fmoc-SeCystine (100 µM) oxidised by HOCl (100 µM), which eluted at: A) 28.0 min (RSeO2H), B) 46.4 min [Fmoc- SeCystine + H]+, and C) 46.4 min [2Fmoc-SeCystine + H]+. Data from a single representative experiment, with normalised peak intensity.

This co-eluted with a species exhibiting a peak maximum at m/z 1561.0 (Figure 3.22, C), the mass and isotope pattern of which is suggestive of a singly-charged aggregate of two molecules of Fmoc-SeCystine within the MS (i.e. [2Fmoc-SeCystine + H+]). This species likely results from the use of acidic buffers to promote ionisation and does not represent a species that is present in samples at neutral pH.

The compound eluting at 28.0 min generated a MS spectrum with an isotope pattern typical of a species containing a single selenium atom and a peak maximum at m/z 424.0 (Figure 3.22, A), which was confirmed by computational simulation to be consistent with formation of the di-oxygenated seleninic acid derivative (RSeO2H) of oxidised Fmoc-SeCystine (Figure 3.23).

Figure 3.23: MS spectrum of the seleninic acid (RSeO2H) product formed by oxidation of Fmoc-SeCystine (100 µM) by HOCl (100 µM). Spectrum obtained by computational simulation using the chemical formula of the compound (C18H17O6NSe, Xcalibur and QualBrowser software, v 1.4.). The isotope pattern is typical of a species with a single selenium atom.

Formation of this product indicates that Fmoc-SeCystine oxidation occurs by addition of two oxygen and one hydrogen atom, preceding cleavage of the diselenide bond. The postulated structure of the seleninic acid derivative and its mechanism of formation are shown in Figure 3.24. Selective ion monitoring (SIM) was also performed to detect the possible formation of the selenenic (RSeOH; 408.3 g/mol, scanned m/z 410.0 ± 7.5) and selenonic acids (RSeO3H; 438.3 g/mol, scanned m/z 440.0 ± 7.5), but these species were not detected under the experimental conditions employed.

Figure 3.24: Proposed structure of the seleninic acid (RSeO2H) product formed by Fmoc- SeCystine oxidation. The single Se isotope pattern and m/z 424.0 are consistent with this product being the seleninic acid derivative, formed by addition of two oxygen atoms and a hydrogen atom, and cleavage of the diselenide bond.

In conjunction with full scan MS analysis, UV/vis detection was performed at 265 nm to identify the presence of Fmoc derivatives within the chromatogram peaks of both the parent and product compounds. Representative UV/vis data from a single experiment (of the three independent experiments carried out) are given in Figure 3.25, where Fmoc-SeCystine (100 µM) was oxidised by increasing concentrations of HOCl (0 – 200 µM).

Figure 3.25: Representative UV/vis traces (λ = 265 nm) of Fmoc-SeCystine (100 µM) with increasing concentrations of HOCl: A) 0 µM, B) 25 µM, C) 100 µM, D) 200 µM. Data obtained from a single experimental replicate in conjunction with LC/MS data, under the same conditions.

Fmoc absorbs strongly at 265 nm,69 so detection by UV/vis at this wavelength provided additional evidence to confirm which species contained at least one Fmoc group. The RSeO2H product peak at 28.0 min was shown to increase in intensity with increasing HOCl concentration, with a concomitant decrease in the intensity of the Fmoc-SeCystine peak at 46.4 min, which is indicative of a HOCl dose-dependent loss of the diselenide.

The peaks that increased in intensity at 35.3 min and 40.1 min were not observed in the absence of HOCl and had not previously been observed by inspection of the total ion chromatograms. Therefore, the MS spectra of these species were analysed to determine the composition of these Fmoc-containing compounds (Figure 3.26, A and B, respectively).

Figure 3.26: MS spectra corresponding to the UV/vis peaks (λ = 265 nm) eluting at; A) 35.3 min, B) 40.1 min. Representative data from a single experiment, with full MS scan (m/z 150 – 2000), cropped > 500 as no peaks above this mass were distinguishable from baseline intensity. No Se or SeSe isotope patterns were observed in either spectrum, and maximum peak intensity was relatively low.

The maximum peak intensity in both spectra was very low relative to the Fmoc-SeCystine and the RSeO2H product, and no diselenide or mono-selenium isotope pattern was observed. Therefore, these two peaks were postulated to be Fmoc-containing contaminants arising from sample preparation and MS analysis and were not analysed further.

Full MS scans were performed (m/z 150 – 2000) on samples containing Fmoc-SeCystine (100 µM) and increasing concentrations of HOCl (0 – 400 µM) and from these data, extracted ion chromatograms were generated for the mass ranges corresponding to Fmoc-SeCystine (m/z 780.9 ± 7.5; Figure 3.27, A) and to the major stable oxidation product, the seleninic acid derivative (RSeO2H, m/z 424.0 ± 7.5; Figure 3.27, B). The isolation widths were set so as to accommodate the entirety of the diselenide or selenium isotope pattern, which span across the major ion m/z ± 7.5. If the isolation width was narrower, the isotope patterns would not be detected, which are important for species identification.

Figure 3.27: LC/MS extracted ion chromatograms of the oxidation of Fmoc-SeCystine (100 µM) by HOCl (0 to 400 µM); A) Fmoc-SeCystine loss, B) Product formation (RSeO2H), and the corresponding MS spectra of these peaks, C) Fmoc-SeCystine, D) RSeO2H. Data shown are taken from the full MS scan (m/z 150 – 2000) with mass ranges selected for; A) Fmoc-SeCystine (m/z 780.9 ± 7.5) and B) the seleninic acid derivative (RSeO2H, m/z 424.0 ± 7.5), and spectra are taken from these peaks.

The extracted ion chromatograms revealed the loss of Fmoc-SeCystine and generation of the seleninic acid to be oxidant dose-dependent, with the resulting MS spectra confirming the identity of these compounds (Figure 3.27, C and D, respectively).

The extracted ion chromatograms of Fmoc-SeCystine and RSeO2H were integrated to find the area under the curve (between 46.0 – 47.0 min and 27.7 – 29.0 min, respectively), which was then plotted against HOCl concentration and fitted with a linear regression to quantify the oxidant dose-dependence of the reaction (Figure 3.28). The background peak area (i.e. peak area of the blank; 50% MeOH in H2O) was subtracted from the total peak area of each sample prior to integration.

Figure 3.28: Plots of; A) Fmoc-SeCystine loss, and B) RSeO2H formation against increasing concentrations of HOCl (0 – 400 µM). Data are the average of three independent experiments, where error is ± SEM. Fmoc-SeCystine data is expressed as a percentage of the peak area of the control sample (i.e. 100 µM Fmoc-SeCystine in the absence of HOCl), minus the background area (i.e. peak area of the blank). A linear trend is fitted from 0 – 200 µM HOCl only, as the diselenide is depleted at the higher HOCl concentrations.

Analysis revealed that Fmoc-SeCystine was lost in a linear manner up to two-fold excesses of oxidant (R2 = 0.9668), becoming significant at equimolar concentrations of diselenide to oxidant (100 µM) as determined by one-way ANOVA with Dunnett’s post-hoc test for multiple comparisons. Fmoc-SeCystine was depleted at 400 µM as evidenced by the area of the Fmoc- SeCystine being indistinguishable from baseline at this concentration (see Figure 3.27, A). Following the addition of 100 µM HOCl, approximately 40% of the Fmoc-SeCystine remained (i.e. 40 µM; Figure 3.28, A), providing an estimate of the stoichiometry of the reaction to be ca. 2.5 moles of HOCl reacting with every 1 mole of Fmoc-SeCystine. This is similar to UPLC data above (Figure 3.16), which estimated a stoichiometry of 3 moles of HOCl reacting with each mole of Fmoc-SeCystine.

The generation of the RSeO2H derivative of Fmoc-SeCystine was found to proceed in a linear HOCl dose-dependent manner (R2 = 0.9981) up to the highest experimental concentration of HOCl employed (400 µM; Figure 3.28, B). This is in contrast to the UPLC data, above, which displayed a plateau effect of primary product formation when HOCl is at a four-fold excess over Fmoc-SeCystine. Cumulatively, these data indicate that Fmoc-SeCystine undergoes HOCl dose-dependent diselenide bond oxidation via addition of two oxygen atoms (and one hydrogen atom) to form the major stable product, the seleninic acid (RSeO2H).

For each LC/MS experiment, concomitant MS/MS scans were performed at m/z 780.9 and 424.0 with an isolation width of m/z ± 7.5, which correspond to the major ions of Fmoc-

SeCystine and the RSeO2H derivative, respectively (Figure 3.29). The MS/MS spectrum of the Fmoc-SeCystine peak (m/z 780.9 ± 7.5) yielded a fragment ion with a maximum at m/z 556.9 and a typical diselenide isotope pattern (Figure 3.29, A). This loss of 224.0 mass units is consistent with the loss of a single Fmoc group (223.3 g/mol) from the Fmoc-SeCystine molecule within the MS.

MS/MS of the primary product (m/z 424.0 ± 7.5) resulted in two peaks, each with a single selenium isotope pattern, at m/z 405.7 and 273.0 (Figure 3.29, B), representing losses of fragments weighing 18.3 and 151.0 mass units, respectively. The loss of 18 units corresponds to a loss of H2O from the carboxylic acid function but does not give insight into the resulting structure of the product. The loss of 151 units is likely indicative of fragmentation of the Fmoc group, as the mass is consistent with the loss of the aromatic moieties from the dehydrated

RSeO2H molecule (m/z 405.7), but the structure of this product has not been elucidated.

Figure 3.29: MS/MS spectra generated from fragmentation of the major ions of; A) Fmoc-SeCystine (m/z 780.9 ± 7.5) and B) RSeO2H (m/z 424.0 ± 7.5). MS/MS analysis performed in conjunction with LC/MS, with NCE 28%.

SIM analysis was performed at m/z 796.9 and 812.9, which correspond to the addition of one and two oxygen atoms, respectively, to the intact Fmoc-SeCystine molecule. These masses were selected to detect the formation of transient oxidised diselenide species which may precede diselenide bond cleavage, as predicted by the analogous sulfur chemistry for which these species have been characterised [e.g. thiosulfinates, RSS(=O)R’ and thiosulfonates, 176, 228 RSS(=O)2R’]. No peaks were observed in the resulting spectra at these mass values. The lack of oxygenated Fmoc-SeCystine in the intact diselenide form indicates that these species, if formed (as predicted by sulfur chemistry), may undergo rapid fragmentation of the diselenide

87 bond following oxidation, or that these intermediate species are transient and undergo rapid further reaction.

These experiments have shown that the oxidation of Fmoc-SeCystine by HOCl is oxidant dose- dependent and results in the formation of a primary stable product, which was characterised to be the seleninic acid derivative (RSeO2H). This is evidenced by the product spectra displaying an isotope pattern typical of a species containing a single selenium atom, as well as the mass of the species being consistent with the seleninic acid (422.3 g/mol). It is proposed that the product is formed via oxidation of the diselenide bond by formation of a transient mono- or di- oxygenated species and rapid secondary reaction, ultimately resulting in cleavage of the diselenide bond, as per the reaction pathway of the corresponding disulfide, in which oxygen addition precedes disulfide cleavage.175, 176, 228 Subsequent experiments investigated the oxidation products of another diselenide, seleno-diglutathione (GSeSeG).

3.3.5. Characterisation of the Products of Seleno-Diglutathione Oxidation by HOCl using LC/MS

These experiments aimed to characterise the products generated by the HOCl-induced oxidation of GSeSeG via LC/MS analysis as detailed in Section 2.9.4. Samples were prepared in H2O containing GSeSeG (100 µM) with increasing concentrations of HOCl (0 – 400 µM).

These were diluted 5-fold into H2O, filtered and transferred to UPLC vials for immediate analysis, due to the rapidity of reaction. A blank sample (H2O) was analysed concurrently. A binary elution gradient was employed with a gradual increase in the organic buffer component over 55 min for each run. A full MS scan (m/z 100 – 1500) was performed for each sample, as well as MS/MS scans of the major ion peaks, as discussed below.

A representative full scan MS chromatogram (m/z 100 – 1500) of a sample containing untreated GSeSeG (100 µM) and a sample with equimolar concentrations of GSeSeG and HOCl (100 µM) is shown in Figure 3.30, along with the corresponding MS spectra of each major chromatogram peak. A peak eluted at 24.0 min, the spectrum of which displayed a major ion with a diselenide isotope pattern and peak maximum at m/z 708.4, consistent with the presence of parent GSeSeG in the samples (Figure 3.30, B). This was confirmed by computational simulation of the spectrum of GSeSeG using the molecular formula of the compound, i.e. + [(C10H16O6N3Se)2 + H] (Figure 3.30, D).

Figure 3.30: Full scan MS chromatogram (m/z 100 – 1500) of GSeSeG (100 µM) oxidised by HOCl (100 µM), MS spectra (A, B) and simulated spectra (C, D). MS spectra are of peaks eluting at; A) 5.5 min (GSeO2H), B) 24.0 min (GSeSeG); Simulated spectra are of; C) GSeO2H, D) GSeSeG. Chromatogram data were obtained in full MS scan mode (m/z 100 – 1500) monitoring total ion count (TIC). ‘GSeSeG Standard’ is untreated GSeSeG (100 µM) and ‘Oxidised GSeSeG’ contains GSeSeG and HOCl at equimolar concentrations (100 µM). Chromatograms were cropped above 28 min to remove background interference from wash- phase ion monitoring, and MS spectra were cropped to show only the major ion peak(s) detected. Simulated spectra obtained by computational simulation using the chemical formulae of the compounds [Xcalibur and QualBrowser software, v 1.4].

The addition of HOCl resulted in the generation of an additional peak at 5.5 min. Analysis of the resulting MS spectrum of this peak revealed a major ion at m/z 387.8 and a minor ion at m/z 369.9, each displaying an isotope pattern typical of species containing a single selenium atom (Figure 3.30, A). The identity of the major product was determined by computational simulation using the experimentally elucidated mass of the compound (m/z 387.8) to be the + seleninic acid derivative of GSeSeG (GSeO2H, [C10H17O8N3Se + H] ; Figure 3.30, C). The smaller ion peak (m/z 369.9; Figure 3.30, A) is suggested to result from a loss of 17.9 mass units from the GSeO2H species (m/z 387.8), corresponding to the loss of H2O from the molecule, presumably from a carboxylic acid functional group.

These data indicate that GSeSeG oxidation is likely to proceed via addition of 2 oxygen atoms and 1 hydrogen atom, resulting in cleavage of the diselenide bond. The proposed reaction pathway for the oxidation of GSeSeG by HOCl and structure of the major stable product is given in Figure 3.31.

Figure 3.31: Structures of GSeSeG and GSeO2H. The proposed structure of GSeO2H has been elucidated via the LC/MS experiments performed in this Chapter, using the m/z of the compound and predicted chemical formula.

The stoichiometry of this reaction was investigated by analysing the oxidation of GSeSeG (100 µM) in the presence of increasing concentrations of HOCl (0 – 400 µM). To reduce the signal- to-noise ratio of the full-scan MS data, extracted ion chromatograms were generated from these samples with masses selectively investigated which corresponded to the major ions of GSeSeG

(m/z 708.4 ± 7.5) and to the primary stable oxidation product (GSeO2H, m/z 388 ± 7.5). The

90 resulting chromatograms demonstrated an oxidant dose-dependent loss of GSeSeG (24.0 min,

Figure 3.32, A) and concomitant formation of GSeO2H (5.5 min, Figure 3.32, B). The GSeSeG peak demonstrated significant retention shift between analytical samples, eluting between 20.6 and 24.7 min across the experiments. This is likely to be due to slow column deterioration (or possibly degradation of buffer components), causing altered retention of the later-eluting materials. The shift was observed to be independent of HOCl concentration, and therefore deemed inconsequential to the outcome of the experiments. Therefore, to improve the visual clarity of Figure 3.32, A, the GSeSeG peaks have been superimposed over the elution time of the GSeSeG standard (i.e. 24.0 min). The product peak demonstrated no such retention shift, and therefore Figure 3.32, B, reflects the true elution times obtained during analysis.

Figure 3.32: LC/MS extracted ion chromatograms (A, B) and full scan MS spectra (C, D) of the oxidation of GSeSeG (100 µM) by HOCl (0 – 400 µM); A) GSeSeG loss, B) GSeO2H formation, C) GSeSeG MS spectrum, D) GSeO2H MS spectrum. Masses were monitored in full MS mode (m/z 100 – 1500) with extracted ion chromatograms generated for; A) GSeSeG (m/z 708.4 ± 7.5), B) GSeO2H (m/z 387.8 ± 7.5). C and D are representative spectra of the major ion peaks of A and B, respectively, where [GSeSeG] = 100 µM and [HOCl] = 200 µM.

The GSeSeG peak (24.0 min) yielded a MS spectrum with two major charged ions; one with a diselenide pattern at m/z 707.9 and one with half the spacing and half the mass at m/z 355.0 (Figure 3.32, C). This suggests the presence of both singly (m/z 707.9) and doubly charged (m/z 355.0) GSeSeG in the untreated sample. The double charge affects the m/z of the species, causing the spacing of the typical diselenide isotope pattern to be halved. This is a result of acidic buffers promoting ionisation of the species for MS detection and does not reflect the charge on the parent molecule when present in neutral solution. The charge of the molecule is postulated to have no effect on oxidative product formation, as both charged species were lost in a HOCl-dose dependent manner, concomitant with the formation of the product.

The formation of the primary stable product (5.5 min) was found to occur with increasing HOCl concentrations up to a two-fold excess of oxidant (200 µM), above which the GSeSeG is depleted. The product elutes from the column very rapidly, implying a high polarity of the molecule. The spectrum of this product (Figure 3.32, D) displays two major ions at m/z 387.8 and 369.9, each with single selenium isotope patterns. This is consistent with the generation of

GSeO2H, and the subsequent loss of H2O from the molecule. Analysis of the GSeO2H product spectra also revealed a low-intensity peak at m/z 775.7, which is double the mass of the m/z 387.8 peak and is therefore postulated to correspond to an aggregate of two molecules of + GSeO2H, i.e. [2GSeO2H + H] . No evidence of a doubly charged GSeO2H product was observed, which would be expected to have a compressed selenium isotope pattern at m/z 194.

Addition of a four-fold excess of HOCl resulted in a decrease in the intensity of the primary product peak at 5.5 min and concomitant formation of a small additional peak at 8.8 min, generated only at the highest oxidant concentration employed (Figure 3.32, B). Analysis of this peak yielded a MS spectrum with a peak at m/z 342.8 and a smaller peak at m/z 324.8, both with a single selenium isotope pattern (Figure 3.33), indicating the preservation of the selenium atom within these species. This loss of 45 and 63 mass units, respectively, from the GSeO2H molecule is consistent with the initial loss of a carboxylic acid functional group, followed by a further loss of H2O, which is consistent with fragmentation of the molecule at high molar excesses of oxidant. The loss of 45 mass units had previously been observed during the oxidation of GSSG by HOCl, which irreversibly converts the ɣ-glutamyl residues to a five- membered cyclic ring (5-hydroxybutyrolactam; Figure 3.34).229

Figure 3.33: Representative full scan MS chromatogram (m/z 100 – 1500) of the chromatogram peak eluting at 8.8 min, formed in the presence of GSeSeG (100 µM) and HOCl (400 µM). Data cropped to show the major ion peaks detected within the full MS scan range.

Figure 3.34: Proposed pathway for the formation of 5-hydroxybutyrolactam from oxidation of GSSG by HOCl. Image adapted from Yuan et al. (2009)229

The extracted ion chromatograms of GSeSeG (Figure 3.32, A) and the major stable oxidation product, GSeO2H (Figure 3.32, B), were integrated to find the peak area, which was then plotted against HOCl concentration and fitted with linear regression curves to quantify the oxidant dose-dependence of the reaction (Figure 3.35). Prior to analysis, the peak area of the blank sample (H2O) was subtracted to eliminate background intensity, and GSeSeG was normalised to give values as a percentage of the control (i.e. untreated 100 µM GSeSeG). GSeSeG (100 µM) was lost in a linear manner up to two-fold excess of oxidant (Figure 3.35, A; R2 = 0.9729), becoming significant at 400 µM HOCl, as determined by one-way ANOVA with Dunnett’s multiple comparisons test. After addition of 200 µM HOCl, GSeSeG loss was ca. 50 µM, suggesting that the reaction proceeds with a stoichiometry of 4 moles of HOCl reacting with each mole of GSeSeG. This is consistent with the di-oxygenation of both Se atoms of the GSeSeG molecule and cleavage of the diselenide bond to form 2 moles of

GSeO2H.

2 The generation of the GSeO2H derivative was found to proceed in a linear manner (R = 0.9943) up to a two-fold excess of HOCl (Figure 3.35, B). At the highest experimental oxidant concentration employed (400 µM), GSeSeG was depleted from the sample and the formation of GSeO2H demonstrated a plateau effect, therefore this point was excluded from linear analysis. The depletion of GSeSeG at 400 µM HOCl and the stoichiometry of the reaction suggest the di-oxygenation of both Se atoms in the parent diselenide at this oxidant concentration, which is consistent with the formation of GSeO2H.

Figure 3.35: Plots of; A) GSeSeG loss, and B) GSeO2H formation against increasing concentrations of HOCl (0 – 200 µM). Data are the average of three independent experiments, minus background (blank sample) peak area. Error bars show ± SEM. GSeSeG data are expressed as percentage of the control (i.e. 100 µM GSeSeG in the absence of oxidant). A linear trend is fitted from 0 – 200 µM only, as GSeSeG is depleted and product formation reaches a plateau above this concentration.

The loss of GSeSeG (as a percentage of the control) was plotted against the log of the HOCl concentration (Figure 3.36) to give a log(IC50) value of 2.3, yielding an IC50 value of 180 µM HOCl, indicating that GSeSeG loss reaches 50% when HOCl concentration is equal to ca. 180 µM.

Figure 3.36: Half maximal inhibitory concentration (IC50) plot of GSeSeG (100 µM) loss with HOCl (0 – 400 µM) treatment. Data are the average of three independent experiments and error bars are ± SEM. GSeSeG is expressed as a percentage of the peak area of the untreated control, minus background (blank sample) peak area.

Extracted ion chromatograms were also generated at masses corresponding to reduced GSeSeG

(GSeH, m/z 356.3) and to the tri-oxygenated selenonic acid derivative (GSeO3H, m/z 404.0), but these species were not detected during analysis. GSeH is a possible intermediate in the oxidation reaction but is too transient for detection due to the known reactivity of selenols with oxygen. GSeO3H was not detected, nor were any forms of oxidised GSeSeG with the diselenide intact [e.g. GSe(=O)2SeG], indicating that these species (if formed during the oxidation of GSeSeG) are transient, as was the case for Fmoc-SeCystine, above.

MS/MS scans were performed concurrently on the major ion peaks at m/z 708.4 (GSeSeG) and at m/z 387.8 (GSeO2H) with an isolation width of ± 7.5 to accommodate the spread of the diselenide or selenium isotope pattern. MS/MS analysis of GSeSeG (m/z 708.4 ± 7.5) yielded a chromatogram with two major peaks eluting at 22.4 min and 34.5 min (Figure 3.37, A). The major fragments of the peak at 22.4 min occurred at m/z 578.8, 449.9 and 688.8 (Figure 3.37, B), and the peak eluting at 24.5 min yielded major fragment ions with m/z 588.9 and 567.1 (Figure 3.37, C), none of which displayed isotope patterns to indicate the presence of selenium.

MS/MS analysis of the GSeO2H peak (m/z 387.8 ± 7.5) resulted in a chromatogram with major peaks eluting at 5.5 and 20.5 min (Figure 3.38, A). MS/MS spectra of the peak at 5.5 min gave ions with m/z 256.0 and 367.7 (Figure 3.38, B). The peak at 20.5 min (Figure 3.38, C) did not yield any usable data.

None of the MS/MS spectra generated by the analysis of GSeSeG or GSeO2H demonstrated a

96 mono-selenium or diselenide isotope pattern. Therefore, the spectral peaks detected during analysis are postulated to arise as a result of fragmentation of GSeSeG and GSeO2H due to the high collision energy employed during MS/MS analysis, but the structures of these species were not characterised. These compounds are not observed by full scan MS analysis under the experimental oxidation conditions employed, and as such, are not representative of the oxidation pathway of the GSeSeG molecule and are not analysed in greater detail.

These data cumulatively suggest that the oxidation of GSeSeG by HOCl proceeds in an oxidant-dose dependent manner with the primary stable oxidation product, characterised by

LC/MS, being the seleninic acid derivative of GSeSeG (GSeO2H). This is evidenced by MS spectra displaying a single selenium isotope pattern, and confirmed by computational simulation using the chemical formula of the species, which has a mass of 386.2 g/mol. It is postulated that this major product is formed by the addition of two oxygen and one hydrogen atom to the diselenide, resulting in cleavage of the diselenide bond and formation of the di- oxygenated species.

The ensuing experiments aimed to further examine the oxidation of diselenides by HOCl using a competition kinetics approach to determine rate constants for these reactions.

Figure 3.37: Representative MS/MS analysis (m/z 708.4 ± 7.5) of GSeSeG (100 µM) oxidised by HOCl (100 µM); A) Full scan chromatogram (m/z 100 – 1500), B) Spectrum of peak eluting at 22.4 min, C) Spectrum of peak eluting at 34.5 min. MS/MS analysis performed concurrently with LC/MS, with NCE 28%.

Figure 3.38: Representative MS/MS analysis (m/z 387.8 ± 7.5) of GSeSeG (100 µM) oxidised by HOCl (100 µM); A) Full scan chromatogram (m/z 100 – 1500), B) Spectrum of peak eluting at 5.5 min, C) Spectrum of peak eluting at 20.5 min. MS/MS analysis performed concurrently with LC/MS, with NCE 28%.

3.3.6. Oxidation of Coumarin Boronic Acid by HOCl to Form Fluorescent COH

One method by which the rate constants for diselenide oxidation by HOCl can be investigated is via competitive kinetics using coumarin boronic acid (CBA) as the probe. CBA is a profluorescent compound which has gained recent interest due to its use as a probe for the detection and quantification of various biological oxidants, including peroxynitrous acid (ONOOH), 214 HOCl and H2O2 as well as amino acid and protein hydroperoxides. CBA can be oxidised to form a fluorescent product, 7-hydroxycoumarin (COH), with rate constants, k = 1.5 ± 0.2 M-1 -1 6 -1 -1 230 s for H2O2 and k = (1.1 ± 0.2) x 10 M s for ONOOH (Figure 3.39). A rate constant for the reaction of CBA with HOCl has not previously been reported. In this study, stopped-flow was used to determine a rate constant for the reaction of CBA with HOCl, with this value then used to calculate rate constants for reaction various diselenides with HOCl via a competition kinetics approach.

Figure 3.39: Oxidation of Coumarin Boronic Acid (CBA) to 7-Hydroxycoumarin (COH) - by biological oxidants. Rate constants (k) are given for H2O2 and ONOO . Image adapted from Zielonka et al. (2010)213

Initially, CBA (25 µM in 50% MeOH) was oxidised by HOCl (0 to 50 µM in H2O) and shown 214 to form COH (λem = 370 nm), the fluorescence of which was measured (350 – 450 nm) every 30 s over 15 min, in triplicate over 3 independent experiments, as detailed in Section 2.10.2. The average COH fluorescence at 15 min was plotted against HOCl concentration and fitted with a linear regression (Figure 3.40). The formation of COH was found to proceed in a HOCl dose-dependent trend up to 25 µM of oxidant, where the CBA and HOCl are equimolar. This concentration range was then used in further experiments. The point at 50 µM HOCl, although fitting the linear trend, was removed from analysis as it was above stoichiometric levels and multiple oxidations of CBA could occur.

100

Figure 3.40: COH fluorescence (350 – 450 nm) at 15 min following the oxidation of CBA (25 µM) by HOCl (0 to 25 µM). Data show average of 3 independent experiments in triplicate, error bars show ± 1 SD. Linearity confirms that formation of COH from oxidation of CBA is HOCl dose-dependent within the experimental concentration range.

Given the rapid and linear dose-dependent oxidation of CBA by HOCl, the pseudo first-order rate constant for this reaction was then determined using stopped flow, where increasing concentrations of CBA (250 – 500 µM, in 50 µM steps) were added to HOCl (50 µM, see Section 2.10.1.). For each sample, fluorescence was measured over 1 s five consecutive times with λex = 332 nm over the range of λem = 350 – 450 nm, corresponding to the fluorescence maxima of COH at 370 nm.214 An example of one such kinetic trace is given in Figure 3.41 ([HOCl] = 50 µM, [CBA] = 300 µM) which shows the rapid exponential increase in COH fluorescence following mixing. For each replicate at all CBA concentrations, five consecutive traces were averaged and fitted with a single exponential curve from 0.003 to 1 s, with the first few microseconds discarded due to the dead time of the system. From each exponential curve, a kobs value was obtained.

101

Figure 3.41: Example of a kinetic trace obtained by stopped-flow (50 µM HOCl + 300 µM CBA), showing COH fluorescence (350 – 450 nm) over 1 s post-mixing. Representative data shown from a single experiment, with 5 successive traces. This experiment was independently performed three times, each with five traces per [CBA]. These traces were averaged and fitted with a single exponential curve over the range of 0.003 to 1 s, from which a kobs value was obtained.

As the formation of COH is proportional to the loss in CBA, these kobs values can be used to determine the rate of reaction between CBA with HOCl by plotting them against CBA concentration (Figure 3.42). The gradient of this plot yields the absolute rate constant for the 4 -1 -1 reaction of CBA with HOCl, kCBA+HOCl = (4.2 ± 0.1) x 10 M s , where the error value is expressed as 95% confidence intervals (C.I.). The y-intercept of this plot also provides an estimate of the rate constant for the auto-oxidation of CBA in the absence of HOCl, the extent of which was investigated concurrently.

102

Figure 3.42: Plot of kobs against CBA concentration for the oxidation of CBA (250 - 500 µM) by HOCl (50 µM). Error bars are ± 1 SD. Data are average from three independent experiments, performed in triplicate at each CBA concentration, obtained from the fit of a single exponential curve of average fluorescence increase (350 – 450 nm) over time (0.003 to 1 s). Gradient yields k1 for reaction of CBA and HOCl; y-intercept is an approximation of k for CBA auto-oxidation.

In conjunction with these experiments, CBA (500 µM) was analysed via stopped-flow (as above, see Section 2.10.1.) to detect background auto-oxidation induced by the high-intensity light of the xenon lamp in the stopped-flow apparatus. CBA was mixed with buffer in lieu of

HOCl, and fluorescence (λex = 332 nm, λem = 350 – 450 nm) was measured every 1 s for 200 s. Analysis was performed five consecutive times for each sample over three independent experiments. The data was fitted with a linear regression, which represents the auto-oxidative formation of COH under the experimental conditions employed over time (Figure 3.43).

It was determined from the line of best fit that the fluorescence resulting from CBA auto- oxidation was 9 x 10-4 units s-1, whereas in the presence of HOCl, COH fluorescence reached a plateau in under 0.5 s and was of much higher value (e.g., see Figure 3.41), indicating that all CBA had been oxidised in this time frame and that auto-oxidation of CBA was a relatively minor process. Therefore, although CBA does slowly auto-oxidise to COH under the stopped- flow conditions, the amount of background fluorescence produced in this way was deemed to be insignificant and did not substantially contribute to the overall fluorescence over the 1 s time frame of the CBA/HOCl reaction. Care was still taken in experiments to limit light exposure prior to reaction to minimise background CBA auto-oxidation and to ensure the initial

103 concentration of CBA present in solution was accurate.

If time had permitted, the photo-oxidation of additional CBA concentrations would have been investigated across the experimental range (250 – 500 µM). Only the highest concentration of CBA (500 µM) was selected for these experiments because it would provide the highest level of photo-oxidation and was therefore a valid indicator of any fluorescence interference caused by CBA photo-oxidation during the calculation of the CBA/HOCl rate constant.

Figure 3.43: Light-induced oxidation of CBA (500 µM) over time, observed as formation of fluorescent COH. Data are average of 3 independent experiments, each with 5 consecutive traces. λex = 332 nm, λem = 350 – 450 nm. Grey line is raw data and black dotted line is linear regression. Auto-fluorescence of CBA resulting from experimental conditions was determined to be insignificant relative to the 1 s time frame of the CBA/HOCl reaction.

3.3.7. Determination of Rate Constants for the Reaction of HOCl with Diselenides by Competition Kinetics using the CBA Assay

Having determined the rate constant for reaction of HOCl with CBA, the diselenide concentration-dependent inhibition of COH formation was analysed. A competition kinetics approach was employed to calculate rate constants for the reaction of HOCl (25 µM) with diselenides (SeCA, DSePA, DPDSe and DPMSe; 0 – 200 µM). CBA (25 µM) was mixed with increasing concentrations of each diselenide, and then HOCl was added immediately prior to analysis and COH fluorescence measured (350 – 450 nm) every 60 s for 30 min, as detailed in

Section 2.10.2. (Figure 3.44). The fluorescence of the solvent solution (50% MeOH in H2O), CBA control (25 µM) and diselenide controls (25 µM CBA + 25 µM diselenide) were negligible (not shown). In the presence of DSePA, SeCA and DPDSe concentrations > 25 µM,

104 the fluorescence yield was very low and hence the data attained with these concentrations was not used.

Increasing the concentration of each diselenide modulates the amount of CBA that is oxidised by HOCl, in turn, decreasing the yield of its fluorescent oxidation product, COH. With each of the diselenides (DSePA, SeCA, DPDSe and DPMSe; Figure 3.44, A, B, C and D, respectively), a dose-dependent trend is apparent in the yield of COH (as measured by fluorescence). Higher concentrations of DPMSe are required to modulate COH formation (Figure 3.44, D) compared to the other diselenides, indicating that the reaction between DPMSe and HOCl is slower than that of the other diselenides.

For each diselenide, the average fluorescence at 30 min was plotted against diselenide concentration (Figure 3.45). For DSePA, SeCA and DPDSe (Figure 3.45, A, B and C, respectively), the decrease in final COH fluorescence (350 – 450 nm) became significant (P < 0.05) at 5 µM diselenide, whereas with DPMSe (Figure 3.45, D), significant loss in COH fluorescence was only achieved at 100 µM, as determined by one-way ANOVA and Dunnett’s post-hoc test for multiple comparisons. DSePA, SeCA and DPDSe data were each fitted with a non-linear one-phase decay curve, demonstrating the exponential loss in fluorescence resulting from diselenide addition. Conversely, the DPMSe-induced loss in COH fluorescence appeared non-exponential and was therefore fitted with a linear regression curve (R2 = 0.9402). This final fluorescence data was then used to calculate rate constants for the reaction of each diselenide with HOCl, as a competitive reaction with CBA.

Figures 3.44 and 3.45 display variation in the y-intercepts (i.e. initial fluorescence) between each diselenide species. This is believed to occur because the data points at t = 0 min are not a true measure of the initial fluorescence as the reaction between HOCl and CBA is so rapid as to be complete prior to the first fluorescence reading. Also, it is possible that the higher fluorescence exhibited by DPMSe (Figure 3.44, D) may be due to the pyrimidyl groups of the diselenide contributing to the fluorescence signal231.

105

Figure 3.44: Oxidation of CBA (25 µM) by HOCl (25 µM) in the presence of increasing concentrations of diselenide; A) DSePA (0 to 25 µM), B) SeCA (0 to 25 µM), C) DPDSe (0 to 25 µM), D) DPMSe (0 to 200 µM). Data are the average of at least 4 independent experimental replicates, performed in duplicate. COH fluorescence corresponds to CBA oxidation. COH fluorescence measured at λex = 332 nm, λem = 350 – 450 nm, lines show average fluorescence.

106

Figure 3.45: Loss in final COH fluorescence (350 – 450 nm; at 30 min) following oxidation of CBA (25 µM) by HOCl (25 µM) in the presence of diselenide; A) DSePA (0 to 100 µM), B) SeCA (0 to 100 µM), C) DPDSe (0 to 100 µM), D) DPMSe (0 to 200 µM). Data are averages of at least 4 independent experimental replicates, performed in duplicate. Error bars show ± 1 SD. DSePA, SeCA and DPDSe are fitted with exponential curves, and fluorescence loss was significant at 5 µM diselenide. The data for DPDSe was fitted with a linear regression curve, and fluorescence loss was significant at 100 µM (R2 = 0.9402).

107

By analysing the decrease in COH fluorescence with increasing diselenide dosages and using the known rate constant of the CBA/HOCl reaction (see above), the rate constant for the reaction of HOCl with each diselenide can be calculated using a modified competitive kinetics equation, as described elsewhere (equation 1):174, 232

푌푚푎푥[퐶퐵퐴] = 푘푆푒푆푒 ∗ [푆푒푆푒](푚표푙푎푟) + [퐶퐵퐴] (1) 푌푆푒푆푒 푘퐶퐵퐴+퐻푂퐶푙

Equation 1 is in the linear form of y = mx + b, where; Ymax is the COH fluorescence measured -5 in the absence of diselenide, [CBA] = 2.5 x 10 M, YSeSe is the final COH fluorescence (at 30 4 -1 -1 min) measured for each diselenide at each concentration, kCBA+HOCl = 4.2 x 10 M s , [SeSe] -6 -4 = 5 x 10 to 2.0 x 10 M and kSeSe is the unknown rate of reaction between each diselenide and HOCl.

By plotting y against x in equation 1, i.e. Ymax[CBA]/YSeSe against [SeSe] for each diselenide, the gradient of this plot (m = kSeSe/kCBA+HOCl) can be rearranged to calculate kSeSe, i.e. kSeSe = m 4 -1 -1 * kCBA+HOCl = m * 4.2 x 10 M s . The linear plots of equation 1 for each diselenide are displayed in Figure 3.46, from which the gradients were obtained.

The plots for DSePA, SeCA, DPDSe and DPMSe were linear and yielded gradients of m = 4.166, m = 7.414, m = 1.629 and m = 0.09217, respectively. These values yielded k values for the reaction of HOCl with each diselenide, summarised in Table 3.3, with error value expressed as 95% CI. These values demonstrate that the structure of the diselenide plays an important role in determining the rate constant for oxidation by HOCl.

Table 3.3: Absolute rate constants (k) for the reaction of various diselenides with HOCl

-1 -1 Diselenide k /M s DSePA (1.8 ± 0.2) x 105 SeCA (3.1 ± 0.6) x 105 DPDSe (6.9 ± 1.2) x 104 DPMSe (3.9 ± 0.8) x 103

108

Figure 3.46: Competition kinetics plots from COH fluorescence data for various diselenides: A) DSePA (0 to 25 µM), B) SeCA (0 to 20 µM), C) DPDSe (0 to 25 µM), D) DPMSe (0 to 200 µM). CBA (25 µM) was oxidised by HOCl (25 µM) in the presence and absence of increasing diselenide concentrations to form fluorescent COH. Resulting fluorescence data (λex = 332, λem = 350 – 450 nm) was used to generate the y-value at each concentration of diselenide. The gradient of each plot was used to calculate a rate constant of reaction between each diselenide and HOCl, based on a competition kinetics equation described previously,232 i.e. equation 1, above.

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3.4. Discussion

The experiments reported in this Chapter investigated the reactions of various low-molecular- mass diselenides with HOCl by determining rate constants for, and characterising the products generated from, these reactions. The rapid rates of reaction and generation of products that are analogous to those characterised for the corresponding sulfur species may predicate the use of these diselenides as putative antioxidants in mitigating oxidative stress-associated pathologies. The reaction of HOCl with low-molecular-mass selenium species is reported to be more rapid than reaction with the corresponding sulfur compounds, potentially making selenium species more efficient scavengers of MPO-derived oxidants.102

The reaction of HOCl with amine groups, such as those on free amino acids and amino acid side chains (e.g. His, Lys), are known to form chloramines (RNHCl) with rate constants k = ~ 105 M-1 s-1 for alpha amino acids.75 Chloramines have the ability to react in an oxidative manner similar to that of HOCl with rate constants with thiols and thioethers in the range of 102 – 103 M-1 s-1, which can lead to the inactivation of thiol-dependent enzymes such as GAPDH and creatine kinase (CK), and induce apoptotic cell death.223, 233, 234 His-chloramines are less selective than other chloramines and react only 5-30 times slower than HOCl itself,226 consistent with data for other cyclic chloramines such as those on pyrimidine bases.224 The reduced reactivity relative to HOCl allows chloramines to diffuse away from their initial sites of formation, thereby propagating oxidative stress.234, 235 The oxidation of the amine groups of

SeCA [(NH2CH2CH2Se)2] by HOCl was shown to produce chloramines, as evidenced by an elevated absorbance at ca. 260 nm,21, 22 which degraded slowly over the experimental time course (24 h) to reveal a pronounced loss of diselenide absorbance at 305 nm. The rapidity of diselenide bond oxidation and HOCl dose-dependence of this reaction was evident immediately following oxidant addition, concomitant with the formation of products which absorb at lower wavelengths (< 240 nm). However, spectral interference caused by the presence of SeCA- chloramines prevented the calculation of a rate constant for this reaction via UV/vis spectroscopy.

Following unsuccessful attempts to examine the reactions of SeCA and DSePA with HOCl by UPLC, an alternative diselenide (Fmoc-SeCystine) was oxidised by increasing concentrations of HOCl, and a UPLC protocol was optimised for the separation and detection of the reaction components. The loss of Fmoc-SeCystine and generation of two major stable fluorescent oxidation products (‘Product 1’ and ‘Product 2’) were quantified. Product 1 was formed in a

110 linear HOCl-dose dependent manner up to a two-fold excess of oxidant, above which, formation displayed a plateau effect, presumably due to Fmoc-SeCystine depletion. The reaction is rapid, having reached completion within the time between oxidant addition and initial analysis. This is consistent with the high reactivity of HOCl with low-molecular-mass sulfur species (thiols, thioethers) having k = 3.8 x 107 M-1 s-1 for Met and 3.0 x 108 M-1 s-1 for Cys residues.69, 71, 81 Furthermore, a study of the oxidation of Fmoc-Met by HOCl reported a rate constant for this reaction to be k = (1.5 ± 0.2) x 108 M-1 s-1 at pH 7.4 and 22 ˚C, as determined by UPLC in conjunction with a competition kinetics approach.69

The generation of Product 2 occurred only at higher HOCl concentrations (≥ 75 µM), indicating that formation of this species is likely to be secondary to the formation of Product 1 and may contribute to the plateau effect of Product 1 generation at higher HOCl doses. These data suggest that excess HOCl reacts with the primary product to drive the formation of the over- oxidised secondary product. The reaction was found to have a stoichiometry of ca. 3 moles of HOCl reacting with every 1 mole of Fmoc-SeCystine. This may indicate that not all the HOCl is reacting exclusively with the diselenide bond; there may be reaction with the oxidised product itself to form further species.

The parent diselenide exhibited a lower fluorescence yield than its primary oxidation product. It is postulated that the two linked Fmoc groups of the parent interact to cause internal fluorescence quenching, whereas the single Fmoc group of the oxidised product exhibits much higher fluorescence due to a lack of internal quenching. This implies that diselenide bond oxidation may result in diselenide cleavage.

The data obtained indicate that HOCl does not react with the Fmoc groups of the diselenide to a significant degree at the experimental oxidant concentrations used because the fluorescence signal remained intact. It has been reported elsewhere that the Fmoc groups of a fluorescently- tagged thioether (Fmoc-Met; 5 µM) do not undergo oxidation by HOCl at low doses (2 µM),69 but neither the reaction of the Fmoc groups with HOCl nor the effect of oxidation on the fluorescence signal of the compound were examined in these experiments. However, the inherent reactivity of the diselenide bond makes it the most likely site of oxidative attack.

The products of Fmoc-SeCystine and GSeSeG oxidation by HOCl have been characterised using LC/MS. It has been shown that the major product that can be detected is the di- oxygenated seleninic acid derivative (RSeO2H/GSeO2H). No evidence was obtained for the formation of mono- or tri-oxygenated species, i.e. the selenenic or selenonic acid derivatives,

111 respectively (RSeOH/GSeOH or RSeO3H/GSeO3H). This contrasts to the oxidative pathway characterised for sulfur compounds, which have been shown to be readily oxidised initially to the corresponding sulfenic acid (RSOH) and sulfinic acids (RSO2H) with further reaction 79, 176 causing over-oxidation to the sulfonic acid (RSO3H) at high excesses of oxidant.

This difference is likely to result from the inherent physicochemical differences that exist between sulfur and selenium. Compared to their sulfur analogues, selenocompounds have a greater nucleophilicity, electronegativity and a lower pKa of selenols (RSeH, 5.2) relative to 102, 106 thiols (RSH, 8.3). Thus, whilst RSO2H is readily oxidised to RSO3H, RSeO2H appears 106 to be resistant to further oxidation. This may account for the lack of RSeO3H detected during the HOCl-induced oxidation of Fmoc-SeCystine and GSeSeG. These characteristics allow selenium-dependent enzymes such as GPx, TrxR and some Msrs to maintain redox homeostasis and remove species such hydroperoxides, hypohalous acids and other strong oxidants, without themselves becoming over-oxidised and thereby inactivated.100, 105

Whilst attempts to examine the reactions of SeCA and DSePA with HOCl via UPLC were unsuccessful, this may be worth re-visiting in the future because investigation of SeCA oxidation products could be compared to the products of Fmoc-SeCystine oxidation which were elucidated herein.

GSH is a key intracellular antioxidant which reacts rapidly with HOCl to form oxidised glutathione (GSSG). GSSG may be reduced by enzymatic processes (e.g. glutathione reductase, GR) to yield parent GSH. However, under certain conditions an additional product may be formed from the oxidation of GSH by HOCl, i.e. glutathione sulfonamide (GSA). GSA is a stable, cyclic and irreversible oxidation product formed with HOCl, but not by other 87 hypohalous acids [HOBr, HOSCN, hypoiodous acid (HOI)] or H2O2. It’s detection within physiological systems predicates its potential use as a biomarker of HOCl-induced oxidative stress in vivo.87, 88, 217 Equimolar concentrations of GSH and HOCl (100 mM) have been shown 217 to produce GSA without significant generation of GSO3H or GSO2SG. Whilst the formation of GSA has typically been observed in the HOCl-induced oxidation of GSH,87, 88, 217 reports also suggest its formation from GSSG.229 Upon oxidation of GSeSeG by HOCl, no evidence of the analogous product (GSeA; Figure 3.47) was detected, even at high excesses of oxidant.

112

Figure 3.47: Proposed structure of GSeA. GSeA is the selenium analogue of the sulfur- containing GSA, the structure of which has previously been elucidated elsewhere88

Although the calculated mass of GSeA (384.2 g/mol) is close to that of GSeO2H (386.2 g/mol), and the presence of a single Se atom in both species results in a characteristic selenium isotope pattern in each MS spectra, these two compounds can be easily distinguished by their respective m/z values (GSeO2H m/z 387.1; GSeA m/z 385.1) as determined by computation simulation using the masses of compounds (Figure 3.48). The exact mass of the GSeO2H confirmed its formulation as the primary stable product of GSeSeG oxidation by HOCl, whereas no GSeA was detected.

Figure 3.48: Theoretical MS spectra of; A) GSeO2H, and B) GSeA. Spectra obtained by computational simulation using the chemical formulae of the compounds [Xcalibur and QualBrowser software, v 1.4., ThermoElectron, IL, USA].

The lack of GSeA formation may imply that the mechanism of HOCl-induced oxidation of the diselenide differs from that of the corresponding sulfur compound. This may be due to the longer covalent bond length of the selenium atom relative to sulfur,102 which would be expected to introduce instability into the nine-membered ring structure, particularly at the N-SeO2 bond, 217 102 analogous to the N-SO2 bond of GSA. The 1.2-fold larger atomic radius of Se relative to S may also result in a decreased stability of the GSeA molecule by hindering the molecule from

113 adopting a compact structure, as is the case for GSA.217 The greater electronegativity and nucleophilicity of the diselenide relative to the disulfide may also play a role in the lack of detectable GSeA, due to the enhanced reactivity that results from the substitution of the selenium atom.98, 102 Any GSeA, if produced, may undergo rapid ring-opening and further oxidation, thus, preventing its detection by the LC/MS methodology employed. These data imply that whilst there may be similarities in the mechanisms and subsequent products of oxidation between the analogous selenium and sulfur compounds, the chemistry is not superimposable. This is consistent with literature data which show variation for compounds which are similar except for the substitution of the sulfur moiety for selenium. For example, the selenocysteine (Sec) residue of GPx1 is oxidised by H2O2 to form DHA, whereas formation of the analogous product is atypical with Cys residues, likely due to the relative strength of the C-S bond relative to the C-Se bond.143 Also, the one-electron oxidation of Met and SeMet each form radical-cations, but the substitution of Se enhances the stabilisation of the SeMet radical, enhancing its lifetime 300-fold over that of the S species and altering its further reaction pathways.79 Therefore, it is important to take into account the physicochemical differences between elemental sulfur and selenium when investigating the analogous species, particularly with respect to pH and pKa, atomic bond strength, atomic radius and the different interactions with nearby heteroatoms.102, 205

The oxidation of GSeSeG by HOCl was found to result in the formation of the seleninic acid derivative (GSeO2H) as the primary stable oxidation product. Contrary to the sulfur-containing species, GSSG, which has been shown to form the sulfonic acid (GSO3H) at greater than 87 equimolar ratios of HOCl, no evidence of the analogous tri-oxygenated species (GSeO3H) was detected with GSeSeG, even at high excesses of oxidant. This variance is likely due to the resistance of RSeO2H to further oxidation to RSeO3H, which contrasts to the rapid and 106 irreversible over-oxidation of RSO2H to RSO3H. This resistance to over-oxidation is the proposed rationale behind the incorporation of selenium as opposed to sulfur into some redox active enzymes (e.g. TrxR, GPx, MsrB1).105, 106, 141

Whilst the reactions of thiols and thioethers are well-characterised, comparatively less literature exists detailing the reaction pathways of disulfide oxidation, with limited reports suggesting the formation of thiosulfinates [RSS(=O)R’, disulfide S-oxides] as the major initial oxidation products, with further reaction resulting in the generation of the corresponding sulfonic acid and disulfide bond cleavage.175, 228 The analogous selenium species [RSeSe(=O)R’] were not detected on oxidation of Fmoc-SeCystine or GSeSeG by HOCl. This

114 is possibly due to such species being highly reactive intermediates in the formation of RSeO2H. A second-order rate constant for the reaction of GSSG with HOCl has recently been reported, 5 -1 -1 176 with k2 = (1.4 ± 0.3) x 10 M s at 10 ˚C. A rate constant for the reaction of GSeSeG with HOCl was not obtained within this study due to a lack of sufficient amounts of the compound but is worth re-visiting in future experiments.

Stopped-flow was employed to elucidate the rate of reaction between CBA and HOCl for the first time, yielding k = (4.2 ± 0.1) x 104 M-1 s-1, which is ca. 26 times slower than rate reported for the CBA/ONOO- reaction [k = (1.1 ± 0.2) x 106 M-1 s-1], but 4 orders of magnitude faster

-1 -1 213 than the rate of the CBA/H2O2 reaction (k = 1.5 M s ). This CBA/HOCl rate constant was used to calculate the rate constants for reaction between HOCl and diselenides using a modified competition kinetics approach.174 Using this approach, HOCl was found to react with DSePA, SeCA, DPDSe and DPMSe with k = (1.8 ± 0.2) x 105, (3.1 ± 0.6) x 105, (6.9 ± 1.2) x 104 and (3.9 ± 0.8) x 103 M-1 s-1, respectively. The variation in these values shows that the diselenide structure plays a key role in determining its susceptibility to reaction with biological oxidants.

Rate constants have been reported for the sulfur-containing analogues of SeCA and DSePA, i.e. cystamine [(NH2CH2CH2S)2] and 3,3’-dithiodipropionic acid [DTDPA;

(HOOCCH2CH2S)2], respectively, with the values for cystamine and DTDPA being k2 = (2.1 ± 0.8) x 105 M-1 s-1 and (1.0 ± 0.1) x 105 M-1 s-1, respectively, as measured by stopped-flow analysis at 10 ˚C.176 These values are of similar magnitude as the rate constants for the selenium compounds. Considerable variation in the apparent k2 value of both low-molecular-mass and protein-bound disulfides arises from interaction between the reaction centre and adjacent heteroatoms, which is strongly affected by the nature and ionisation state of the functional groups to which the disulfide is bound.176 It has been suggested that for acyclic species such as cystamine and DTDPA (and their selenium analogues, SeCA and DSePA) that the interaction between neighbouring electron-donor groups acts to stabilise the sulfur (or selenium) centre as it becomes positively charged during reaction, resulting in enhanced reactivity.176 These interactions are strongly pH-dependent and rely upon the protonation/deprotonation equilibria of both the oxidant and the target disulfide, highlighting the importance of the microenvironment in determining the reactivity of an acyclic disulfide.176 This elevated reactivity may clarify why the rates of reaction of the low-molecular-mass diselenides reported herein do not vary greatly from those of the corresponding sulfur compounds (summarised in Table 3.4.), despite the typically greater reactivity of selenium species. It is important to note that pH-dependence studies were not performed in this project due to time constraints but

115 would be very interesting and relevant for future research, as the electrochemical interaction between various groups within both diselenide and oxidant are strongly dependent upon microenvironmental factors, such as pH.

Table 3.4: Rate constants (k) for the reaction of diselenides and disulfides with HOCl

Structure k /M-1 s-1 (X = Se) k /M-1 s-1 (X = S)

5 a 5 b (HOOCCH2CH2X)2 (1.8 ± 0.2) x 10 (1.0 ± 0.1) x 10

5 a 5 b (NH2CH2CH2X)2 (3.1 ± 0.6) x 10 (2.1 ± 0.8) x 10

4 a (C5H4NX)2 (6.9 ± 1.2) x 10 ND 3 a (C4H3N2X)2 (3.9 ± 0.8) x 10 ND

a Determined in this thesis; b Karimi, M. et al (2016)176

These rate constants demonstrate that the addition of low concentrations of diselenides may be sufficient to scavenge biological oxidants in a competitive way, indicating that such compounds may prove useful in the prevention or treatment of oxidative stress-associated pathologies. However, the efficacy of diselenides as scavengers of biological oxidants depends strongly upon the secondary structure of the diselenide. It has been found that the interaction of the selenium moiety with adjacent heteroatoms such as N, O or S, within the same compound can increase the GPx-like antioxidant activity as a result of non-covalent interactions which act to stabilise and increase the electrophilicity of the selenium atom.203, 204

GPx-like activity is typically defined as the capability to catalyse the reduction of H2O2 and organic hydroperoxides to water or the corresponding alcohol, using GSH as a reductant. The selenol moiety (RSeH) becomes oxidised to the selenenic (RSeOH) or seleninic (RSeO2H) acid, which is reduced by GSH to the active parent selenol via a selenenylsulfide intermediate (RSe-SG), with the concurrent oxidation of GSH to GSSG.154 This implies that the efficacy of these selenocompounds as antioxidants may lie in their ability to be readily oxidised and readily reduced, thus driving the scavenging of biological oxidants through rapid recycling. Whilst the rate constants of reaction with HOCl elucidated for the diselenides in this Chapter do not vary greatly from those reported for the corresponding sulfur compound, these experiments were performed in the absence of a reductant, so data on the reduction and recycling of the parent selenium species remains to be determined; these may be faster for the selenium species compared to the sulfur analogues.

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3.5. Conclusions

Low-molecular-mass diselenides have been shown to react rapidly with HOCl, with reaction postulated to occur via oxygen addition and subsequent diselenide bond cleavage to form a major stable product, the seleninic acid (RSeO2H). The mono- and tri-oxygenated species

(RSeOH and RSeO3H) were not detected, implying that the oxidative reaction pathways of selenium compounds diverge from those of the analogous sulfur species, for which the analogous mono- and tri-oxygenated products (RSOH and RSO3H) have been characterised.

However, it is possible that RSeOH and RSeO3H are less stable than the sulfur analogues, which may account for why these species were not detected, rather than their lack of formation.

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Chapter 4

Reactions of Diselenides with Hydrogen Peroxide (H2O2)

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4.1. Introduction

H2O2 is a well-documented biological oxidant, produced as a by-product of aerobic cellular 80 •- respiration, excesses of which can induce oxidative stress. It is produced via superoxide (O2 ) generation and dismutation and functions as a secondary messenger in vivo.236 In the presence of some metal ions the decomposition of H2O2 is rapid with the O-O bond undergoing cleavage to give rise to the hydroxyl radical (HO•) and hydroxide anion (HO-), which is partially 80 • responsible for much of the strong oxidising action of H2O2. HO is one of the strongest known oxidants and reacts with rate constants close to the diffusion-controlled limit.80

Whilst H2O2 is a strong two-electron oxidant, high activation energy barriers result in slow rates of reaction with many biological substrates. It is a weak one-electron oxidant, rendering 80 H2O2 reactions to be kinetically, rather than thermodynamically, driven. H2O2 reacts primarily with transition metal centres, selenoproteins and select thiol proteins (e.g. catalase, GPxs, peroxiredoxins) and also haem groups. The reaction is faster with the thiolate ion (RS-) rather than the neutral thiol (RSH), so thiols with a low pKa are typically more reactive at 80 physiological pH 7.4. Rate constants have been reported for the reaction of H2O2 with low- molecular-mass thiols and thiol proteins; GSH (k = 0.89 M-1 s-1), cysteine (k = 2.9 M-1 s-1) and select peroxiredoxins (k = 1 – 4 x 107 M-1 s-1).237, 238 Initial oxidation of the thiolate yields the sulfenic acid (RSOH), which typically have lower pKa values than the parent thiol, and subsequent reaction with thiols forms the disulfide (RSSR). Sulfenic acids are also oxidised by

H2O2 to generate the sulfinic acid (RSO2H), but rates of reaction are typically ca. 1000-fold slower than for the parent thiol.80

As discussed in Chapter 1, host cells have multiple antioxidant defences against H2O2, including catalase, GPxs and peroxiredoxins. Catalase detoxifies H2O2 by converting it to oxygen and water.207 GPxs have an active site Sec (or Cys) which reacts rapidly with peroxides to form the selenenic (or sulfenic) acid, and a subsequent selenenylsulfide (RSeSR) (or disulfide) intermediate which is reduced to the active parent form using GSH as a reductant.239

H2O2 also reacts with the haem peroxidases MPO, eosinophil peroxidase (EPO) and lactoperoxidase (LPO) with k values of 1.4 x 107 M-1 s-1, 4.3 x 107 M-1 s-1 and 1.1 x 107 M-1 s- 1 66 , resulting in the reduction of H2O2 to water. Some of the reported rate constants for reaction of H2O2 with biological substrates are summarised in Table 4.1.

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Table 4.1: Reported rate constants of reaction (k) of H2O2 with biological substrates

-1 -1 Reactant k /M s GSH 0.89 a Cysteine 2.9 a Peroxiredoxins 1 – 4 x 107 a Catalase 2.4 x 107 b MPO 1.4 x 107 c EPO 4.3 x 107 c LPO 1.1 x 107 c a Winterbourn (2013),80 measured at pH 7.4 – 7.6 and 37 or 20 – 25 ˚C b Greenfield and Price (1954),240 measured at pH 6.8 and 29 ˚C c Davies et al (2008),66 measured at pH 7.0 and 15 ˚C

The greater size, nucleophilicity and electrophilicity of the selenium atom relative to sulfur confers selenocompounds with increased reactivity and an enhanced ability to resist over- oxidation (and hence, inactivation), allowing the more rapid turnover of oxidants (including

H2O2), making them potentially more efficacious ROS-scavengers to mitigate oxidative stress- associated pathologies.102, 105, 106, 141 Little kinetic data exist in the literature on the reaction of selenols (e.g. Sec) due to the rapid auto-oxidation of these species upon exposure to O2, thus, complicating experimental investigation.79 Some reports exist detailing the kinetics of selenoether (e.g. SeMet) and diselenide oxidation,219, 222 but the information is often incomplete [reviewed by Carroll, Davies and Pattison (2015)].79

4.2. Aims

The experiments reported in this Chapter aimed to examine the mechanisms by which H2O2 oxidises low-molecular-mass diselenides (see Chapter 3, Figure 3.1), to determine rate constants for these reactions and characterise the resulting products, using UV/vis spectroscopy and LC/MS.

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4.3. Results

4.3.1. Oxidation of Selenocystamine by Hydrogen Peroxide

Whilst the oxidation of SeCA by HOCl proved to be too rapid to calculate a rate constant for this reaction via UV/vis spectroscopy (see Chapter 3, Section 3.3.1.), the reactions of diselenides with H2O2 are likely to be much slower, potentially allowing rate constants to be calculated via this method. Initial experiments were carried out to examine the stability of the diselenide bond of untreated SeCA over time. Duplicate samples of SeCA (1 mM) were prepared in buffer (0.1 M, pH 7.4) and analysed by UV/vis spectrometry (200 to 400 nm, as detailed in Section 2.8.) at various time points over 7 days (t = 0, 1, 2, 4 and 7 days) to monitor 220 any changes to the wavelength characteristic of the diselenide bond (λmax = 305 nm). Samples were maintained at room temperature (22˚C) in the dark and acquisition of UV spectra was repeated in duplicate over three independent experiments. This control sample showed no significant change in absorbance profile at 305 nm over the time-period employed (Figure 4.1), indicating that the untreated diselenide remains stable in the absence of oxidant.

Figure 4.1: UV/visible spectrum showing absorbance of untreated SeCA (1 mM) over 7 days. SeCA was prepared in buffer (0.1 M, pH 7.4). Representative data from a single experiment are shown with each line representing the average of two duplicate samples.

121

A slight increase in the absorbance over time at 250 nm may be an indication of a slow reaction involving SeCA, possibly reflecting a slow degradation of the compound. This absorbance increase did not affect the diselenide region and was therefore not investigated further.

A solution of SeCA (1 mM) was subsequently oxidised by H2O2 (10 mM) and analysed under identical conditions to examine the oxidant-mediated modifications to the diselenide bond (305 nm) over time (Figure 4.2). Samples were left at room temperature (22 ˚C) in the dark between analyses, and the data presented are the average of three independent experiments, each performed in duplicate.

Figure 4.2: UV/vis spectra of SeCA (1 mM) oxidation by H2O2 (10 mM) over 7 days. Samples were prepared in buffer (0.1 M, pH 7.4) in duplicate and left at 22 ˚C in the dark between analyses. Data displayed are the average of three independent experiments.

The loss in absorbance of the diselenide peak at 305 nm is consistent with the H2O2-mediated oxidation of the diselenide bond and formation of products which absorb at shorter wavelengths (< 240 nm). The apparent isosbestic point at 249 nm, where the absorbance remains constant as the reaction proceeds, suggests that the oxidation of SeCA by H2O2 results in the formation of a single oxidation product. Subsequent experiments aimed to examine this reaction in greater detail and to determine a rate constant for reaction of H2O2 with SeCA and other diselenides.

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4.3.2. Calculation of Rate Constants for the Reaction of Diselenides with Hydrogen Peroxide

SeCA and DSePA (1 mM) were each exposed to increasing concentrations of H2O2 (10 to 50 mM, in 10 mM steps) and analysed over time as detailed in Section 2.8.1., whilst being maintained at 22 ˚C. Samples were analysed every 600 s for 100 repetitions (total 16.5 h; except for DSePA where shorter time-periods were used due to faster reaction) and all experiments were performed in triplicate. GSeSeG (1 mM) was also exposed to H2O2 (50 mM) but this experiment was performed only once due to the limited amounts of GSeSeG available, therefore, the rate constant obtained is an approximation.

The average of three independent UV/vis spectroscopy experiments for SeCA and DSePA at a single H2O2 concentration (20 mM) are shown in Figure 4.3, which show diselenide bond oxidation for both species, concomitant with the formation of products that absorb at shorter wavelengths (< 240 nm) and an isosbestic point at 249 nm. Increasing concentrations of H2O2 resulted in an increased rate of diselenide oxidation, which was then analysed to determine a rate constant, as detailed below.

-1 -1 57, 207 H2O2 is known to exhibit an absorbance maximum at ca. 240 nm (ɛ240 = 39.4 M cm ) and to decompose over time at neutral pH, particularly when in dilute solution.57 Therefore, the presence of H2O2 is not likely to interfere with the absorbance of the diselenide bond at 305 nm, but it is important to note that the concentration of H2O2 may decrease slightly over the experimental time course.

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Figure 4.3: Oxidation of diselenides (1 mM) by H2O2 (20 mM) over time; A) SeCA, B) DSePA. Each line represents spectra obtained every 100 min for SeCA and every 10 min for DSePA. The loss in absorbance at 305 nm indicates diselenide bond oxidation and the formation of products which absorb at shorter wavelengths.

For all spectroscopic data, the loss in absorbance was plotted against time for a range of wavelengths corresponding to the diselenide peak (295 to 315 nm, in 5 nm steps). These data were fitted with exponential decay curves and the observed rate constants (kobs) were obtained from these curves. Figure 4.4 shows examples of these plots for SeCA and DSePA in the presence of 20 mM H2O2. Analyses were performed in triplicate for each diselenide at each

H2O2 concentration.

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Figure 4.4: Diselenide (1 mM) absorbance loss over time (16.5 h) with H2O2 (20 mM); A) SeCA, B) DSePA. Wavelengths encompassing the diselenide region (305 nm) were taken in 5 nm steps and fitted with exponential curves to yield observed rate constants (kobs). The graphs display the exponential fit obtained from the average of three independent experiments, which were repeated over a range of H2O2 concentrations (10 – 50 mM, in 10 mM steps).

The kobs values obtained from these plots were then plotted against the concentration of H2O2, and the gradient of the resultant linear plot corresponds to the absolute rate constant (k) for the reaction of each diselenide with the oxidant (Figure 4.5). From these plots, k values for the -4 -1 - reaction of SeCA and DSePA with H2O2 were calculated to be kSeCA = (9.2 ± 0.4) x 10 M s 1 -2 -1 -1 and kDSePA = (1.5 ± 0.2) x 10 M s , respectively, with the errors expressed as 95% CI.

These values show the reaction of DSePA with H2O2 to be ca. 18-fold faster than that of SeCA, an effect which is attributed to the difference in the terminal functional group (-COO- for + DSePA vs -NH3 for SeCA).

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Figure 4.5: Pseudo first-order kinetic plots for the loss of diselenides (1 mM) with increasing concentrations of H2O2 (10 – 50 mM); A) SeCA, B) DSePA. Error bars are ± 1 SD. Data are the average kobs values obtained from three independent experiments plotted against the H2O2 concentration. The gradients of these plots yield the second-order rate -1 -1 constants for reaction of H2O2 with SeCA and DSePA respectively, in M s .

Pilot studies were also undertaken with GSeSeG. GSeSeG (1 mM) was added to H2O2 (50 mM) and analysed by UV/vis spectroscopy (Figure 4.6, A), as described above. Absorbance loss over the diselenide peak (295 – 315 nm, in 5 nm steps) was plotted against time and fitted with exponential decay curves (Figure 4.6, B).

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Figure 4.6: Oxidation of GSeSeG (1 mM) by H2O2 (50 mM) over time (16.5 hrs); A) UV/vis spectroscopy trace with each line representing spectra obtained at 100 min intervals. Loss in absorbance at 305 nm indicates diselenide bond oxidation. B) Exponential curves of absorbance loss over time across the diselenide peak (295 – 315 m). Data from a single pilot experiment.

-5 -1 Analysis of the absorbance loss data gives a kobs value of ca. 5 x 10 s . As the kobs was -3 -1 -1 determined with a H2O2 concentration of 50 mM, a value k1 value of ca. 1 x 10 M s can be estimated, which is similar to that for SeCA and ca. 20-fold slower than for DSePA. These rate constants are summarised in Table 4.2.

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Table 4.2: Absolute rate constants (k) for reaction of H2O2 with diselenides

-1 -1 Diselenide k /M s SeCA (9.2 ± 0.4) x 10-4 DSePA (1.5 ± 0.2) x 10-2 GSeSeG ~ 1 x 10-3

These kinetic data demonstrate the significance of diselenide structure on the reactivity with

H2O2. This effect is expounded upon in the literature, where the non-binding interactions of the selenium atom with other functional groups within the same molecule are shown to modulate 205, 241 reactivity. Subsequent experiments aimed to characterise the products of H2O2-induced SeCA oxidation by MS, before quantifying parent loss and product generation utilising ultrahigh-performance liquid chromatography (UPLC).

4.3.3. Characterisation of the Products of Selenocystamine Oxidation by Hydrogen Peroxide using MS

Samples were prepared in H2O containing control SeCA (1 mM) or SeCA (1 mM) pre-treated with H2O2 (1 or 10 mM) and were incubated in the dark at 22 ˚C prior to analysis, as detailed in Section 2.9.2. Spectra were generated from an average of 100 scans over m/z 100 to 400. The mass of the detected species was analysed in conjunction with the selenium isotope pattern to characterise the products generated.

Following injection of a control sample containing SeCA (1 mM), a diselenide isotope pattern was observed with the maximum peak intensity at m/z 249.0, consistent with singly-charged SeCA (i.e. [SeCA + H]+, Figure 4.7, A). After 3 days, the relative abundance of SeCA (m/z 249.0) had decreased concomitant with the formation of two major stable products which give rise to single selenium isotope patterns in the MS analysis with maximum intensity ions at m/z 158.1 and 172.1 (Figure 4.7, B). These two products were not detected at t = 0 min and increased in relative abundance over time when compared to untreated SeCA (Figure 4.7, C).

This demonstrates that these two products are generated via the oxidation of SeCA by H2O2 in a time-dependent manner.

The lower masses of the two resultant products (m/z 158.1 and 172.1) and the clear single selenium isotope pattern suggest that SeCA has undergone diselenide bond oxidation with the

128 addition of two or three oxygen atoms to form the seleninic acid (RSeO2H) and selenonic acid

(RSeO3H), respectively. The assignment of these species was supported by computational simulations using the chemical formulae of the compounds [Xcalibur and QualBrowser software, v1.4.; Figure 4.8], which shows a similar pattern to the experimentally-generated MS spectra. The proposed structures of these two products are given in Figure 4.9. In the presence of 1 mM H2O2, no SeCA product formation was detected, likely due to a slow rate of reaction.

Following the observation that SeCA oxidation by H2O2 results in the generation of the seleninic and selenonic acids, subsequent experiments were performed using other diselenides (Fmoc-SeCystine and GSeSeG). LC/MS experiments were not performed with SeCA as separation of the compounds could not be achieved by LC, as detailed in Chapter 3, Section 3.3.2.

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B) RSeO2H

RSeO3H SeCA

Figure 4.7: MS Spectra of SeCA (1 mM) on oxidation by H2O2 (10 mM) over time: A) SeCA peak at m/z 249.0, t = 0 min, B) SeCA loss and product formation, t = 3 days, C) SeCA loss and product formation, t = 6 days. Spectra obtained from a single experiment by direct injection are each the average of 100 scans from m/z 100 to 1000. Prior to injection, samples were diluted 1 in 5 into H2O, then added into an equal volume of 1% formic acid in MeOH to assist ionisation.

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Figure 4.8: Computational simulation of MS Spectra of SeCA, and the respective di- and tri-oxygenated species, RSeO2H and RSeO3H. Spectra obtained using the chemical formulae of the compounds [Xcalibur and QualBrowser software (v 1.4.)].

Figure 4.9: Proposed structures of the products of SeCA oxidation by H2O2. The m/z of the products of SeCA (1 mM) oxidation by H2O2 (10 mM) over time (3 days) are consistent with formation of the di- and tri-oxygenated mono-selenium species; the seleninic (RSeO2H) and selenonic acid (RSeO3H), respectively.

131

4.3.4. Quantification of the Oxidation of Fmoc-Selenocystine by Hydrogen Peroxide using UPLC

These experiments aimed to investigate the mechanisms by which Fmoc-SeCystine is oxidised 174 by H2O2 using a modified version of a UPLC protocol previously described elsewhere. This method was optimised as detailed in Chapter 3, Section 3.3.2., with the final protocol summarised in Section 2.7.

Initial UPLC experiments aimed to determine the retention time of Fmoc-SeCystine and any product/s generated by addition of H2O2, using the optimised method detailed in Section 2.7.3.

Samples containing Fmoc-SeCystine (100 µM) and increasing concentrations of H2O2 (0, 100, 200, 500, 1000 µM) were prepared alongside a blank [50% MeOH in buffer (0.1 M, pH 7.4)] and Fmoc-SeCystine (100 µM) and H2O2 (1000 µM) standards. Samples were incubated in the autosampler unit at 10 ˚C for 5 h before being analysed in the order of the blank, then the Fmoc-

SeCystine standard, oxidised samples from highest to lowest H2O2 concentration, and finally the H2O2 standard.

The blank sample generated baseline fluorescence which yielded significant peaks at the higher organic buffer percentages, presumably via interaction with buffer components. The buffer gradient caused the Fmoc-SeCystine to elute at 25.8 min, just prior to the wash phase, resulting in the diselenide co-eluting with buffer components (Figure 4.10, A). It also caused a primary oxidation product to elute at 11.2 min, which was generated in a H2O2 dose-dependent manner (Figure 4.10, B). These experiments demonstrated that Fmoc-SeCystine and the two primary stable product peaks observed eluted at the same times as the species generated with HOCl (Product 1, 11.2 min; Product 2, 6.2 min; see Chapter 3, Figure 3.15), indicating that both HOCl and H2O2 form the same products.

Despite the formation of significant amounts of detectable product, there was minimal apparent loss in Fmoc-SeCystine observed in oxidised samples relative to the standard (Figure 4.10, C).

No peak corresponding to unreacted H2O2 was detected.

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Figure 4.10: Representative UPLC chromatogram of Fmoc-SeCystine (100 µM) with H2O2 (0, 100, 200, 500, 1000 µM); A) Full chromatogram, B) Inset of primary product peak (11.2 min), C) Inset of Fmoc-SeCystine peak (25.8 min). Data were obtained using the newer Shim-Pack XR-ODS column (see Chapter 3, Section 3.3.2, cf. Figure 3.14), a binary buffer gradient (B = 10% at t = 0 min) with organic component increasing over time, and a -1 total flow rate 1.2 mL min for 30 min. The H2O2 (1000 µM) standard is indistinguishable from the baseline fluorescence of the blank (1:1 MeOH/buffer).

Samples containing Fmoc-SeCystine (100 µM) and increasing concentrations of H2O2 (0, 10 and 100 mM) were prepared and analysed over a period of ~ 24 h to gain insight into the rate of reaction. To prevent saturation of the fluorescence detector by the signal from Product 1 (cf. data from experiments with HOCl reported in Chapter 3, Section 3.3.3.), an additional automated analysis step was programmed into the UPLC protocol as detailed in Section 2.7.4.; immediately prior to injection onto the column, 5 or 10 µL of sample was aspirated and mixed into pre-prepared vials of diluent [50% MeOH in buffer (0.1 M, pH 7.4)] to achieve a final dilution factor of 1 in 40 or 1 in 20, respectively. Analysis was then carried out as described in

Section 2.7.3. The effect of both time and H2O2 dose on the oxidation of Fmoc-SeCystine was

133 analysed in terms of diselenide loss and product formation. Quantification of Product 1 was carried out on the samples subjected to the 1 in 40 dilution, whilst the less intense signal of Product 2 was quantified using the lower (1 in 20) dilution. The full elution profile of Fmoc- SeCystine and its oxidation products at a dilution factor of 1 in 20 is shown in the chromatogram in Figure 4.11. A large peak is seen at ca. 22 min, which was present in all samples including blanks and controls and was therefore deemed to be a component of buffer interactions and not analysed further. However, as the Fmoc-SeCystine co-eluted atop this buffer peak (labelled A, 25.8 min), the diselenide peak was integrated to the baseline fluorescence and not to a peak intensity of zero.

The representative chromatograms inset in Figure 4.11 show that the oxidation of Fmoc-

SeCystine and formation of Products 1 and 2 are H2O2-dose dependent, following a 24 h incubation period. No Fmoc-SeCystine fluorescence remained after 24 h incubation with 100 mM H2O2, but unreacted Fmoc-SeCystine was still detected with 10 mM H2O2 (Figure 4.11, A). The relative fluorescence intensity of Product 1 compared to that of Product 2 is evident in Figure 4.11 (B and C, respectively).

Data from these experiments were collected over 24 h, and the peak areas of Fmoc-SeCystine and its two major stable oxidation products were integrated. The results of four independent experimental replicates are shown in Figure 4.12. Fmoc-SeCystine loss is expressed as a percentage of the control (i.e. percentage peak area of the first sample in the analysis sequence for each H2O2 concentration), whereas the formation of Products 1 and 2 lack internal standards and are therefore expressed as integrated peak area.

Under these conditions, no significant loss in signal intensity for untreated control Fmoc- SeCystine was observed (P > 0.05), confirming that the diselenide remains stable over the experimental time frame (Figure 4.12, A). In the presence of 10 mM H2O2, Fmoc-SeCystine loss becomes significant (P < 0.05) at 8.5 h after oxidant addition, and at 1.5 h with 100 mM

H2O2, as determined by one-way ANOVA with Dunnett’s post-hoc test for multiple comparisons. This indicates that the oxidative loss of Fmoc-SeCystine by H2O2 is both time- and oxidant dose-dependent, consistent with the kinetic data generated for SeCA and DSePA, presented in Section 4.3.2.

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Figure 4.11: Full UPLC chromatogram of Fmoc-SeCystine (100 µM) with H2O2 (0 to 100 mM) at 24 h, with automated dilution (1 in 20) prior to injection, with insets showing; A) Fmoc-SeCystine, B) Product 1, C) Product 2. The loss in Fmoc-SeCystine and formation of products is H2O2-dose dependent at 24 h. The peak intensity of Product 1 is significantly greater than that of Product 2 (see vertical axes).

135

In the absence of oxidant, neither Product 1 nor Product 2 were detected. In the presence of

100 mM H2O2, over time (≥ 3 h), the fluorescence signal emitted by Product 1 saturated the fluorescence detector, making peak integration inaccurate (Figure 4.12, B). The observed plateau effect is likely to be the result of Fmoc-SeCystine depletion, which is complete after

14.5 h of exposure to 100 mM H2O2 (Figure 4.12, A). However, with 10 mM H2O2, ca. 50% of unreacted Fmoc-SeCystine remains at 24 h post-oxidant addition. Figure 4.12, B, shows that the formation of Product 1 displays a plateau effect even when the parent is not completely consumed and suggests that Product 1 is oxidised further to Product 2 in the presence of excess oxidant over time.

The extent of formation of Product 2 was low at 10 mM H2O2, with the peak area not reaching significance relative to t = 0 min within the experimental time frame (P > 0.05; Figure 4.12,

C). At a H2O2 concentration of 100 mM, Product 2 formation became significant at 18 h following oxidant addition and increased in a time-dependent manner without displaying the same plateau effect as Product 1 over the experimental time frame. This supports the suggestion that Product 2 is a secondary product.

To further investigate the plateau effect observed for Product 1, experiments were performed as above, with an adjusted pre-treatment protocol to give a final dilution of 1 in 40 (see Section 2.7.4.). As shown in Figure 4.13, the results from these experiments were consistent with those at the lower (1 in 20) dilution. Cumulatively, these data indicate that Fmoc-SeCystine is oxidised by H2O2 in a time- and oxidant dose-dependent manner, with the formation of Product 1 reaching a plateau at higher doses and over longer time periods. This implies that Product 1 is an intermediate species in the formation of Product 2. Subsequent experiments aimed to characterise these products of Fmoc-SeCystine oxidation by H2O2 using LC/MS.

136

Figure 4.12: Oxidation of Fmoc-SeCystine (100 µM) by H2O2 (0, 10, 100 mM) over time (24 h), with automated dilution (1 in 20) prior to analysis; A) Fmoc-SeCystine loss, B) Product 1 formation, C) Product 2 formation. Data are the average of 4 independent experimental replicates, error bars show ± SEM. Fmoc-SeCystine ‘Control’ is integrated peak area at t = 0 min.

137

138

4.3.5. Characterisation of the Products of Fmoc-Selenocystine Oxidation by Hydrogen Peroxide using LC/MS

The reaction of Fmoc-SeCystine (100 µM) with a five-fold excess of H2O2 (500 µM) was examined using the LC/MS protocol detailed in Section 2.9.3. Samples were prepared in 50%

MeOH in H2O and analysed immediately after preparation (t = 0 min) and again after 24 h of incubation in the dark at 22 ˚C. Immediately prior to analysis, samples were diluted 5-fold into

H2O and LC/MS analysis performed using a binary elution gradient with increasing organic buffer concentration over 85 min runs. As described in Chapter 3, Section 3.3.4., full-scan MS analysis was performed (m/z 150 – 2000) in conjunction with UV/vis spectroscopy (λ = 265 nm) to detect species containing Fmoc groups,69 as well as MS/MS analysis of the major ion peaks.

Representative data for a sample containing Fmoc-SeCystine (100 µM) and H2O2 (500 µM) are given in Figure 4.14. A large peak was detected by chromatographic separation at 33.3 min (A), for which the resulting MS spectrum displayed no notable ion peaks nor selenium isotope patterns, indicating that this species is likely an artefact, consistent with detection of this same peak in the presence of HOCl (see Chapter 3, Section 3.3.4.). The peak eluting at 46.1 min yielded a MS spectrum with a diselenide isotope pattern and a peak maximum at m/z 780.4 (Figure 4.14, B), consistent with the species being Fmoc-SeCystine, as confirmed by comparison of the experimental MS spectrum to a computational simulation for Fmoc- SeCystine using the known mass and chemical formula of the compound [778.6 g/mol;

(C18H16O4NSe)2; Figure 3.19]. No evidence for any oxidation product was observed at this

H2O2 concentration.

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Subsequent LC/MS experiments were performed in the same manner using higher concentrations of H2O2 (100 mM) in the presence of Fmoc-SeCystine (100 µM), and the concurrent UV/vis spectroscopy traces were examined to investigate the presence of Fmoc- containing species present. UV/vis data (λ = 265 nm) revealed the presence of fluorescent peaks eluting at 27.9, 37.6 and 39.7 min, in addition to the parent Fmoc-SeCystine at 46.1 min (Figure

4.15). The peaks at 27.9 min and 39.7 min increased in a H2O2 dose-dependent manner following 24 h of incubation, suggesting that these are Fmoc-containing oxidation products. Conversely, the small peak at 37.6 min was present at similar intensity in all samples, implying that it does not arise from the addition of H2O2. The MS spectra of these fluorescent peaks were examined to identify each species (Figure 4.16).

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Figure 4.15: Representative UV/vis traces (λ = 265 nm) showing Fmoc-SeCystine (100 µM) oxidation by increasing concentrations of H2O2; A) 0 mM, B) 10 mM, C) 100 mM. Data obtained concurrently with LC/MS data from a single independent experiment.

The MS spectrum of the peak eluting at 27.9 min displayed an isotope pattern typical of a species containing a single selenium atom with a peak maximum at m/z 424.0 (Figure 4.16, A), and the peak eluting at 46.1 min gave a MS spectrum with a typical diselenide isotope pattern and maximum m/z 780.4 (Figure 4.16, B). These data are consistent with these species being

RSeO2H and parent Fmoc-SeCystine, respectively, which is supported by computational simulation spectra generated using the masses and chemical formulae of the compounds (Figures 3.23 and 3.19, respectively). The elution times and MS spectra of the Fmoc-SeCystine and RSeO2H product are consistent with the HOCl data (Chapter 3, Section 3.3.4.), implying that both HOCl and H2O2 generate the same product.

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Figure 4.16: Representative MS spectra generated from the UV/vis (λ = 265 nm) chromatogram peaks eluting at; A) 27.9 min, B) 46.1 min, C) 37.6 min, D) 39.7 min. Spectra obtained from a single independent experiment where Fmoc-SeCystine (100 µM) was oxidised by H2O2 (10 mM).

The peak eluting at 37.6 min yielded a MS spectrum without a mono-selenium or diselenide isotope pattern (Figure 4.16, C). Whilst this species appears to contain at least one Fmoc group, as evidenced by its fluorescence at 265 nm, the detection of this species, even in the absence of oxidant, as well as the resulting MS spectrum indicates that this species is likely to be a Fmoc-containing contaminant, rather than an oxidation product. The peak eluting at 39.7 min was absent in the untreated Fmoc-SeCystine sample and appeared to increase with H2O2 dosage, yet the resulting MS spectrum displayed no mono-selenium nor diselenide isotope pattern (Figure 4.16, D). This implies that this is also a Fmoc-containing species formed by increasing concentrations of H2O2. As such, both species were discounted from further analysis. A similar effect was observed in the presence of increasing concentrations of HOCl, as discussed in Chapter 3, Section 3.3.4.

Extracted ion chromatograms were generated by selective ion monitoring (SIM) of the full MS scans (m/z 150 – 2000) of samples containing Fmoc-SeCystine (100 µM) and increasing concentrations of H2O2 (0 – 100 mM), with the mass ranges selected to correspond to Fmoc-

SeCystine (m/z 780.4 ± 7.5) and the RSeO2H product (m/z 424.0 ± 7.5). This wide isolation

142 width was employed to ensure detection of the mono- or di-selenium isotope pattern. The resulting extracted ion chromatograms (Figure 4.17, A and B, respectively) reveal a dose- dependent loss of Fmoc-SeCystine at 46.1 min and concomitant formation of RSeO2H eluting at 27.9 min, and the identity of these peaks was confirmed by analysis of the resulting MS spectra (Figure 4.17, C and D, respectively).

Figure 4.17: Representative LC/MS extracted ion chromatograms showing the oxidation of Fmoc-SeCystine (100 µM) by H2O2 (0 – 100 mM); A) Fmoc-SeCystine loss at t = 0 min (m/z 780.4 ± 7.5), B) RSeO2H formation at t = 24 h (m/z 424.0 ± 7.5), and MS spectra of these peaks; C) Fmoc-SeCystine (46.1 min), D) RSeO2H (27.9 min). Data obtained from full scan MS chromatograms (m/z 150 – 2000) with mass ranges selected for both Fmoc- SeCystine and the RSeO2H derivative.

MS/MS scans were performed on the major ion peaks corresponding to Fmoc-SeCystine (m/z

780.4 ± 7.5) and the RSeO2H derivative (m/z 424.0 ± 7.5), which gave data consistent with HOCl studies in Chapter 3, Section 3.3.4. Analysis of Fmoc-SeCystine yielded a single chromatogram peak at 46.1 min (Figure 4.18, A) with an MS/MS spectrum displaying a major ion fragment with a diselenide isotope pattern and maximum at m/z 556.9 (Figure 4.18, B), consistent with the loss of a single Fmoc group (223.3 g/mol) as a result of MS/MS analysis.

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Figure 4.18: MS/MS analysis of Fmoc-SeCystine (m/z 780.4 ± 7.5); A) MS/MS chromatogram, B) MS/MS spectrum of peak at 46.1 min. MS/MS analysis performed in conjunction with LC/MS, with NCE 28%., representative data taken from a single independent experiment.

MS/MS analysis of the RSeO2H derivative generated a chromatogram with two major peaks at 27.9 and 37.6 min (Figure 4.19, A). The MS/MS spectrum of the peak at 27.9 min yielded two peak maxima at m/z 405.7 and m/z 273.0 with Se isotope patterns (Figure 4.19, B), representing the loss of 18.3 and 151.0 mass units, respectively, from the RSeO2H molecule, which correspond to the loss of water and to Fmoc group fragmentation as a result of analysis and do not represent products generated as a result of diselenide oxidation. The chromatogram peak at 37.6 min corresponds to the contaminant detected by UV/vis analysis (Figure 4.16, C), for which the MS/MS spectrum gave fragments with maxima at m/z 422.1 and 376.2, neither of which display a Se isotope pattern (Figure 4.19, C); these species have not been characterised further.

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Figure 4.19: MS/MS analysis of RSeO2H (m/z 424.0 ± 7.5); A) Extracted ion chromatogram, B) MS/MS spectrum of peak at 27.9 min (RSeO2H), C) MS/MS spectrum of peak at 37.6 min (contaminant). MS/MS analysis performed in conjunction with LC/MS, with NCE 28%, representative data taken from a single experiment.

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No evidence was obtained from the full scan MS data to indicate the formation of the singly- oxygenated selenenic acid species (RSeOH), nor the tri-oxygenated selenonic acid derivative

(RSeO3H) with H2O2, even at the highest oxidant concentrations employed. This contrasted with the direct injection MS data obtained for SeCA, for which there was clear evidence of the formation of the selenonic acid derivative (RSeO3H; Figure 4.7). It is postulated that the

RSeOH form was not detected due to rapid oxidation to RSeO2H, whilst further reaction to 105 yield RSeO3H is suggested to occur very slowly.

These data indicate that in the presence of H2O2, Fmoc-SeCystine undergoes slow oxidant dose-dependent diselenide bond oxidation via the addition of two oxygen atoms (and one hydrogen atom) to form the major stable product, the seleninic acid (RSeO2H; Figure 3.24). It is proposed that oxidation of the diselenide precedes bond cleavage through the formation of unstable intermediates [e.g. RSe(=O)SeR, RSe(=O)2SeR)], as per the typical oxidative pathway of a disulfide.176 However, these species were not detected, likely due to their transient nature.

Subsequent experiments investigated the reaction of H2O2 with seleno-diglutathione (GSeSeG) in order to characterise the product/s formed.

4.3.6. Characterisation of the Products of Seleno-Diglutathione Oxidation by Hydrogen Peroxide using LC/MS

These experiments aimed to characterise the products of GSeSeG oxidation by H2O2, however, a lack of compound prevented a comprehensive quantitative analysis. Therefore, pilot studies were performed to provide an indication of which product(s) were generated by oxidation of

GSeSeG by H2O2, and to determine if these products differed from those generated by HOCl, which was characterised as the seleninic acid (RSeO2H), as detailed in Chapter 3 (Section 3.3.5.).

Initial experiments examined the oxidation of GSeSeG (100 µM) by H2O2 (1 mM) following 3 days of incubation in the dark at 22 ˚C and compared this to GSeSeG (100 µM) oxidised by HOCl (100 µM), analysed concurrently. Blank samples were analysed in parallel. This concentration of H2O2 was chosen because it was found to produce detectable amounts of product from SeCA. Samples were prepared and analysed as detailed in Section 2.9.4.

A full scan MS chromatogram (m/z 100 – 1500) yielded a peak eluting at 31.9 min, which decreased in intensity in the presence of H2O2 (Figure 4.20, A). The corresponding MS spectrum of this peak displayed a diselenide isotope pattern with a peak maximum at m/z 708.4,

146 consistent with this species being GSeSeG (707.5 g/mol; Figure 4.20, B). This assignment was supported by computational simulation of the MS spectrum of GSeSeG using the known molecular formula of the compound [(C10H16O6N3Se)2], which returned an identical MS spectrum [Xcalibur and QualBrowser software (v 1.4)]. A second peak was observed with half the m/z of GSeSeG at a maximum of m/z 355.1 with a compressed diselenide isotope pattern, indicating the presence of a doubly-charged GSeSeG ion (i.e. [GSeSeG + 2H+]), probably as a result of acidic buffers promoting sample ionisation. No oxidation product/s were detected in the full scan MS chromatogram in the presence of H2O2.

Figure 4.20: Representative LC/MS data of GSeSeG (100 µM) oxidation by H2O2 (0 or 1 mM); A) Full scan MS chromatogram (m/z 100 - 1500), B) MS spectrum of peak at 31.9 min (GSeSeG). Data for H2O2 acquired after 3 days of incubation in the dark at 22 ˚C.

147

Selective ion monitoring (SIM) was performed on these data at the m/z values corresponding to GSeSeG (m/z 708.4 ± 7.5) and to the major ion peak of the previously characterised product

(GSeO2H, m/z 387.8 ± 7.5), to generate extracted ion chromatograms for each compound

(Figure 4.21, A and B, respectively). GSeSeG was found to be lost in the presence of H2O2, concomitant with the formation of the GSeO2H derivative. MS spectra were generated for each extracted ion peak to confirm the presence of both GSeSeG and GSeO2H, by comparison of the resulting MS spectra (Figure 4.21, C and D, respectively) to simulated spectra (Figure 4.21, E and F). These MS spectra are consistent with those generated for the oxidation of GSeSeG by HOCl (Chapter 3, Section 3.3.5.), suggesting that the oxidation of GSeSeG by HOCl and

H2O2 generate the same major product.

The elution time of compounds in these experiments differed from those in the previous Chapter during the investigation of GSeSeG oxidation by HOCl (24.0 vs 32.0 min for GSeSeG,

5.5 vs 20.1 min for GSeO2H, respectively; see Chapter 3, Section 3.3.5.). This is postulated to be a result of using an older Hypercarb column in the analysis of H2O2 samples, causing an altered retention time.

No evidence was found to support the formation of mono- [selenenic acid (GSeOH)] or tri- oxygenated [selenonic acid (GSeO3H)] species. This is consistent with the generation of only the RSeO2H form from Fmoc-SeCystine (Section 4.3.5.). By analogy with the Fmoc-SeCystine oxidative pathway, it is proposed that the rapid oxidation of an unstable GSeSe(=O)G or

GSeSe(=O)2G intermediate precedes the GSeO2H derivative, but no GSeO3H is generated due to a slow rate of the second reaction.105 The steric bulk of the GSeSeG molecule may provide some protection to the selenium moiety, hindering oxidation to the tri-oxygenated species. However, this supposition is tentative, as the data require replication. No MS/MS scans were performed during these analyses.

These LC/MS experiments have indicated that the oxidation of GSeSeG by H2O2 generates the same oxidation product as that previously characterised for HOCl, although the experiments with H2O2 need to be confirmed. The major stable oxidation product with both oxidants was found to be the di-oxygenated GSeO2H derivative.

148

Figure 4.21: Representative LC/MS data of the oxidation of GSeSeG (100 µM) by H2O2 (0 or 1 mM). Data from a single experiment showing: extracted ion chromatograms of A) GSeSeG (m/z 708.4 ± 7.5) and B) GSeO2H (m/z 387.8 ± 7.5); MS spectra of C) peak at 31.9 min (GSeSeG) and D) peak at 20.1 min (GSeO2H); and simulated spectra (Xcalbur and QualBrowser software, v 1.4.) of E) GSeSeG and F) GSeO2H.

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4.4. Discussion

The experiments reported in this Chapter aimed to investigate the reactions of several low- molecular-mass diselenides with H2O2 by determining rate constants for, and characterising the products of, these reactions. The diselenide bond of SeCA (1 mM) was monitored by 220 UV/vis spectroscopy (λmax = 305 nm) and was found to be stable in the absence of oxidant 130 over 7 days, which is consistent with previous data. Addition of H2O2 (10 mM) caused the time-dependent loss of the diselenide bond absorbance, concomitant with the formation of products which absorb at shorter wavelengths (< 240 nm) with the presence of an isosbestic point consistent with the formation of a single primary product.

Absolute rate constants were then determined for the reactions of SeCA, DSePA and GSeSeG -4 -2 -3 -1 -1 with H2O2, with k = (9.2 ± 0.4) x 10 , (1.5 ± 0.2) x 10 and ca. 1 x 10 M s , respectively.

The reactivity of GSeSeG with H2O2 was similar to that of SeCA, but this requires experimental - + replication to validate this value. DSePA [(SeCH2CH2COO )2] and SeCA [(SeCH2CH2NH3 )2] are ionised and protonated, respectively, at pH 7.4. The reaction rate of DSePA represents an approximate 20-fold increase over that of SeCA. This is postulated to be due to substitution of the terminal amines for carboxyl groups, which suggests the importance of secondary structure and ionisation/protonation state in determining the reactivity of the diselenide moiety.

It has been reported previously that interaction of the Se atom with a nearby heteroatom (e.g. N, O, S, etc) can increase the reactivity of diselenides due to favourable non-covalent interactions during the course of reaction,203 and that diselenides exhibiting this property demonstrate enhanced antioxidant activity.204 It is proposed that the reaction intermediate - + interacts favourably with the terminal -COO groups of DSePA but not with the -NH3 groups of SeCA, accounting for the significant increase in observed reactivity with H2O2. It is suggested that the lone pairs of electrons on the ionised carboxyl groups of DSePA act to stabilise the diselenide bond through weak intramolecular non-bonding interactions.203 Furthermore, the relatively strong non-bonding interactions between the Se and O atoms of DSePA are proposed to increase the nucleophilic attack at the diselenide bond,203 thus, increasing the rate of reaction with H2O2.

The oxidation of SeCA by H2O2 was found to result in generation of the RSeO2H and RSeO3H derivatives as determined by MS. This contrasts to the H2O2-induced oxidation of Fmoc-

SeCystine and GSeSeG, which yielded only the seleninic acid forms (RSeO2H/GSeO2H) without concomitant detectable formation of RSeO3H/GSeO3H. It is suggested that Fmoc-

150

SeCystine and GSeSeG form unstable diselenide intermediates [RSeSe(=O)R or

RSeSe(=O)2R] which rapidly oxidise to the seleninic acid, which are relatively stable species that only react slowly with H2O2, thereby resulting in little or no formation of the selenonic acid over the experimental time-course.105 The steric bulk of the GSeSeG molecule and the large aromatic Fmoc groups may protect the Se atom in these species, whereas SeCA is a simple aliphatic diselenide that lacks groups which may physically inhibit further oxidation. The longer incubation times employed in the SeCA experiments is also likely to contribute to the detection of the tri-oxygenated product. Further experiments should examine the further oxidation of GSeSeG and Fmoc-SeCystine to assess the ratio of products formed and determine if the seleninic acid is indeed an intermediate in the formation of the selenonic acid derivative.

UPLC analysis was performed to quantify the loss of Fmoc-SeCystine and formation of stable oxidation products generated over time in the presence of increasing concentrations of H2O2.

The oxidation of Fmoc-SeCystine by H2O2 yielded a major (‘Product 1’) and a minor (‘Product

2’) stable oxidation product, believed to be the RSeO2H and RSeO3H derivatives, respectively, in a time- and oxidant dose-dependent manner. The RSeO2H product is suggested to be stable, slowly undergoing further oxidation to yield the RSeO3H derivative only in the presence of high excesses of H2O2, contributing to the observed plateau effect. A very low yield of RSeO3H fluorescence indicates limited formation, consistent with this species not being detected by

LC/MS analysis. The products detected with H2O2 are consistent with those formed in the presence of HOCl (Chapter 3, Section 3.3.3.), indicating that both HOCl and H2O2 generate the same products. Much higher doses of H2O2 were required to generate these species, consistent with the much slower reaction rates of this comparatively weak oxidant.80

Further experiments should aim to detect if the RSeH/GSeH species is formed as an intermediate in these reactions by addition of N-ethylmaleimide (NEM) to the reaction mixture to block any free selenol, thereby preventing its rapid oxidation and forming a stable adduct for LC/MS detection.87 Further work should also aim to detect if oxidised diselenide intermediate species such as RSe(=O)SeR and RSe(=O)2SeR are formed, which would be predicted by analogy with the corresponding disulfide chemistry.176

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4.5. Conclusions

The major stable oxidation product detected for SeCA, Fmoc-SeCystine and GSeSeG on reaction with H2O2 was the di-oxygenated seleninic acid (RSeO2H), which is proposed to form via the same mechanistic pathway as the sulfur species, but much more rapidly due to the increased reactivity of the selenium species and the resistance of the seleninic acid to further oxidation.

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Chapter 5

Reactions of Diselenides with Peroxides

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5.1. Introduction

One of the major products of protein oxidation are protein hydroperoxides (ROOH), formed • 1 215 1 by both radical (e.g. HO ) and non-radical (e.g. O2) reactions. O2 can be experimentally- generated by exposure of the photosensitive dye Rose Bengal (RB) to visible light and molecular oxygen, forming protein peroxides in vitro in high yield due to the rapidity of 242 55, 56 1 reaction, primarily on Trp, Tyr and His residues. Peroxides can also be formed by O2 on cholesterol, lipids and DNA.242 Direct evidence for the in vivo formation of protein hydroperoxides is scarce as these compounds decay rapidly under physiological conditions, i.e. at body temperature and in the presence of biological one- and two-electron reductants.74 Further biological reactions of hydroperoxides can perpetuate damage to other biomolecules and amplify the initial oxidative insult, resulting in DNA oxidation and fragmentation, and inactivation of crucial thiol-dependent enzymes.56 Furthermore, there is evidence of secondary oxidation events after initial photo-oxidation has ceased (‘dark reactions’), where relatively long-lived hydroperoxides diffuse from the initial site of generation to induce further biological damage, indicating that protein hydroperoxides can play a major role in both immediate and long-term cellular damage.74, 215

In the studies reported in this Chapter, the free amino acids Trp, Tyr and His were exposed to 1 visible light in the presence of a photo-sensitising agent (RB), causing the generation of O2 and subsequent formation of hydroperoxides on the amino acid residues. In order to compare the effect of free- versus protein-bound amino acids, hydroperoxides of TrpLys, LysTrp and LysTrpLys were also generated in this manner. The structures of the amino acids (Trp, Tyr, His) and peptides (LysTrp, TrpLys, LysTrpLys) investigated in this Chapter are shown in Figure 5.1. Data from these hydroperoxides were compared with commercial hydroperoxides

[tert-butyl hydroperoxide (tBuOOH) and cumene hydroperoxide (CHP)] as well as H2O2. tBuOOH and CHP are aliphatic and aromatic organic peroxides, respectively, that have been used as model inducers of oxidative stress.130, 243

There is substantial evidence to suggest that ROS-induced oxidation of proteins can induce fragmentation, aggregation and functional changes, leading to cellular and tissue dysfunction in the progression of inflammatory pathologies.37 Administering the appropriate exogenous antioxidant compounds to inflammatory loci at the appropriate intervention stage could potentially lessen the progression of inflammatory disease, by modulating oxidative stress, potentially leading to improved outcomes.

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5.2. Aims

In this Chapter, several diselenides (SeCA, DSePA, DPDSe and DMPSe; see Chapter 3, Figure 3.1) have been analysed in terms of their ability to scavenge a variety of stable reagent peroxides and labile photo-chemically generated hydroperoxides by determining the corresponding rate constants and examining the mechanisms of these reactions.

Figure 5.1: Structures of amino acids (Trp, Tyr, His) and peptides (LysTrp, TrpLys, LysTrpLys) investigated in this Chapter.

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5.3. Results

5.3.1. SeCA Oxidation by Reagent Peroxides

The FOX assay was employed to detect and quantify relative peroxide concentrations in the absence and presence of increasing amounts of various diselenides. This method relies upon the peroxides within a sample oxidising the Fe2+ ions in the FOX reagent to Fe3+, which then reacts with the XO dye to form a purple-blue complex which is detectable at 560 nm, providing an indirect measure of total peroxide concentration within the sample.209, 210 Reagent peroxides

(H2O2, tBuOOH and CHP; 25 mM) were analysed by FOX assay, detailed in Section 2.6, to determine their rates of intrinsic decay over the experimental time course (up to 7 days), measured as absorbance at 560 nm (Abs560). Analogous experiments were then carried out in the presence of increasing concentrations of SeCA (0 to 10 mM) in order to determine the efficacy of this diselenide in scavenging each peroxide. Pseudo first-order rate constants were calculated for these reactions.

Stock solutions of tBuOOH and CHP decay over time and the FOX assay is not specific to the species of peroxide present, therefore, concentrations of tBuOOH and CHP were expressed as

‘equivalents of H2O2’ as determined by a concomitant FOX assay of each peroxide in direct comparison to a standard curve of a known range of H2O2 concentrations, in order to provide a more accurate and consistent measure of peroxide concentration. The concentration of H2O2, tBuOOH and CHP (0 to 25 mM in H2O, in 5 mM steps) was then assayed to determine the optimal dilution factors for detection by FOX assay. H2O2 and CHP samples were diluted 1 in

1000 in H2O (final concentration 0 to 25 µM), and tBuOOH samples diluted 1 in 2000 in H2O (final concentration 0 to 12.5 µM) in two steps immediately prior to FOX analysis. It was observed that H2O2 and CHP exhibited similar absorbance measurements at these concentrations, whereas tBuOOH displayed a significantly higher absorbance reading (ca. two- fold) despite being half the final concentration of the other peroxides (Figure 5.2).

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3+ Figure 5.2: Absorbance of the Fe -XO complex generated by reagent peroxides (H2O2 and CHP, 0 to 25 µM; tBuOOH, 0 to 12.5 µM) measured using the FOX assay, as equivalents of H2O2. Abs560 data acquired by FOX assay, error bars are ± 1 SD of 4 independent experiments. Concentrations of stock peroxides determined via colorimetric assay or as given by the supplier. H2O2 and CHP diluted 1 in 1000 and tBuOOH diluted 1 in 2000 in H2O immediately prior to analysis.

The apparent molar extinction coefficients (ɛ) of the xylenol-orange complexes formed by 4 4 4 -1 H2O2, tBuOOH and CHP at 560 nm are reported to be 4.4 x 10 , 9.8 x 10 and 9.9 x 10 M -1 244 cm , respectively, using H2SO4 (25 mM) as solvent. In Figure 5.2, the two-fold higher absorbance of tBuOOH relative to H2O2 is consistent with its two-fold higher ɛ560 value.

However, the absorbance of CHP is very similar to that of H2O2 at almost half its predicted absorbance value, which may be due to decomposition of the stock peroxide over time or the relative immiscibility of CHP in water, making it less likely to react with the Fe2+ ions in the reagent, thereby generating less xylenol-orange complex for detection at 560 nm.

To examine the reaction of reagent peroxides (H2O2, tBuOOH, CHP; 25 mM) with diselenides, the peroxides were assayed in the absence or presence of SeCA (0 to 10 mM), by monitoring changes in peroxide absorbance (Abs560) over time. Samples were kept at 22 ˚C in the dark between sampling and were diluted into H2O (1000-fold for H2O2 and CHP, 2000-fold for tBuOOH) immediately prior to assay. The FOX assay was then performed at selected time points over a minimum period of 48 h, as follows: for H2O2 t = 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 24 and 48 h; for tBuOOH and CHP t = 0, 1, 2, 4 and 7 days.

Figure 5.3 shows that the changes in the quantity of untreated peroxides (0 mM SeCA, as % Control) were insignificant relative to t = 0 min (P > 0.05) over the time periods employed, which is consistent with the reported stabilities of these peroxides.130 This result implies that

157 any significant loss of peroxide in ensuing experiments was not due to intrinsic decay of the peroxides under the experimental conditions used.

Figure 5.3: Intrinsic decay of reagent peroxides (H2O2, tBuOOH and CHP; 25 mM) up to 7 days. Samples were diluted in H2O immediately prior to analysis to give final concentrations of 12.5 µM for tBuOOH and 25 µM for CHP and H2O2. The extent of decay of each peroxide at the experimental end-point (2 days for H2O2, 7 days for tBuOOH and CHP) is non-significant relative to t = 0 min (P > 0.05) under experimental conditions, as determined by one-way ANOVA and Tukey’s post-hoc test for multiple comparisons. Error bars show ± 1 SD from 3 independent experimental replicates.

Figure 5.4 shows the extent of loss of each peroxide (H2O2, tBuOOH, CHP; 25 mM) in the presence of added SeCA (0 to 10 mM) over time (48 h for H2O2, 7 days for tBuOOH and CHP), as determined by FOX assay. SeCA is shown to react with each peroxide at different rates, with the extent of peroxide loss being dependent upon both SeCA concentration and upon exposure time.

The reaction of SeCA with H2O2 proceeds rapidly, with the reaction appearing essentially complete at 7 h post-oxidant addition (Figure 5.4, A). At this time point, H2O2 loss is significant at all SeCA concentrations ≥ 2 mM relative to the untreated peroxide. Lower concentrations of

SeCA did not result in significant loss of H2O2, which is likely due to the comparatively low amounts of the diselenide (≤ 10 mM). The addition of 5 mM SeCA appears to decrease the absorbance of 25 mM H2O2 by ca. 40% (i.e. a decrease of ca. 10 mM H2O2), indicating that for each mole of diselenide, 2 moles of the peroxide are removed. Further changes in peroxide concentrations were not observed after 7 h and time periods longer than 48 h were not examined.

158

Figure 5.4: Extent of peroxide loss (H2O2, tBuOOH, CHP; initially 25 mM) over time in the absence or presence of SeCA (0 to 10 mM): A) H2O2 (48 h), B) tBuOOH (7 days), C) CHP (7 days). Error bars show ± 1 SD of 3 independent experiments, performed in duplicate. Legend shows SeCA concentration. ‘Control’ is 10 mM SeCA in the absence of peroxide. Lines represent exponential decay curves, from which kobs values were calculated.

The reaction of SeCA with tBuOOH is slow, with the reaction remaining incomplete 7 days

159 following oxidant addition (Figure 5.4, B), at which time concentrations of SeCA ≥ 2 mM demonstrate significance relative to the absence of diselenide. At 7 days, the addition of 10 mM SeCA decreased the Abs560 of 25 mM tBuOOH by ca. 50%. (i.e. a decrease of ca. 12.5 mM tBuOOH). However, this does not provide an accurate indication of the stoichiometry of the reaction, as the reaction remains incomplete over the experimental time frame (i.e. not all the peroxide in solution has been reacted with the SeCA within 7 days). The exponential decay curve of tBuOOH (25 mM) in the presence of SeCA (10 mM) was used to calculate a plateau value (0.3865) from which an approximation of the extent of total peroxide loss was calculated, yielding an estimation of ca. 60% tBuOOH loss (ca. 15 mM) upon completion of the reaction. This implies that the stoichiometry of the reaction is 2 moles of SeCA reacting with 3 moles of tBuOOH.

The reaction of SeCA with CHP proceeds more rapidly than that of tBuOOH, yet also remains incomplete at 7 days, as a plateau is not reached (Figure 5.4, C). At 7 days, the SeCA dose- dependent loss of CHP relative to the untreated sample is significant at SeCA concentrations ≥ 5 mM. As above, the non-linear one-phase decay curve of the reaction of CHP (25 mM) with SeCA (10 mM) was used to calculate a plateau value (0.1516), from which an approximation of ca. 80% total peroxide loss was obtained upon completion of the reaction. This is equivalent to a total loss of 20 mM CHP upon reaction with 10 mM SeCA, yielding a stoichiometry of 1 mole of SeCA reacting with 2 moles of CHP.

The absorbances from the FOX assay for each peroxide over time were fitted with exponential decay curves, from which kobs values were obtained. For tBuOOH and CHP, the decay curve was fitted having constrained the plateau value to the average Abs of the Control sample over 7 days (10 mM SeCA, untreated), to take the incomplete reaction into account. This is because the Control sample represents the background absorbance in the absence of peroxide; thus, it represents the predicted absorbance value of the sample once the peroxide had completely decayed. This step was not performed for H2O2 as the reaction reached completion within the experimental time frame. For the H2O2 system, points where [SeCA] < 0.5 mM were excluded from analysis due to the insignificant loss of peroxide detected at these concentrations.

The kobs values obtained in this way for the reaction of SeCA with H2O2, tBuOOH and CHP were plotted against SeCA concentration, and the gradient of these plots yielded absolute rate constants for these reactions (Figure 5.5), with these being in the order H2O2 >> CHP > tBuOOH (Table 5.1).

160

Figure 5.5: Observed loss (kobs) of peroxides (25 mM) with increasing concentrations of SeCA (0 to 10 mM); A) H2O2, B) tBuOOH, C) CHP. kobs values were obtained from exponential decay curves of absorbance loss data (560 nm). For H2O2, data where [SeCA] < 0.5 mM were excluded from analysis due to inaccuracy of fit. Data shown are from three independent experimental replicates. The gradient yields the absolute rate constants.

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Table 5.1: Absolute rate constants (k) for the reaction of SeCA with reagent peroxides

Peroxide k /M-1 s-1

-2 H2O2 (1.1 ± 0.5) x 10 CHP (5.4 ± 1.0) x 10-4 tBuOOH (1.4 ± 0.1) x 10-4

Subsequent experiments aimed to determine rate constants for the reaction of SeCA with photochemically-generated peroxides formed on amino acids (Trp, Tyr, His) and peptides (LysTrp, TrpLys, LysTrpLys), and to compare these values to those obtained for the reagent peroxides.

5.3.2. SeCA Oxidation by Photochemically-Generated Peroxides

Peroxides were generated on amino acids and peptides by the photolysis protocol detailed in Section 2.5. and the FOX assay was employed to determine the initial concentration of each peroxide immediately following their formation, as well as in the presence of increasing concentrations of diselenides, as detailed in Section 2.6. Briefly, each amino acid (Trp, Tyr,

His) or peptide (LysTrp, TrpLys, LysTrpLys) was prepared to 2.5 mM in 10 mL H2O, corrected to pH 7.2 – 7.4, with Rose Bengal (10 µM final concentration) added immediately before being placed on ice whilst being exposed to visible light and bubbled with compressed air for 30 55, 56, 61 -1 min. Catalase (10 µL of 1 mg mL solution) in H2O was added and samples incubated in the dark at room temperature (22 ˚C) to remove any H2O2 incidentally generated during photolysis. The hydroperoxide of each photolysed amino acid or peptide is denoted as such using the suffix -OOH.

The resultant hydroperoxide stocks were diluted into H2O (5-fold for Tyr, His and LysTrp; 20- fold for Trp, TrpLys and LysTrpLys), and the concentration of each stock was determined by -1 preparation of a series of standards of increasing concentrations (0 – 500 µL mL in H2O, in 100 µL steps) and subsequent FOX assay, as detailed in Section 2.6. Approximate hydroperoxide concentrations were calculated (assuming equivalent stoichiometry for the reactions) for each experiment by comparing the absorbance values of the assayed stocks to a

H2O2 standard curve (Figure 5.6). Final approximate concentrations of each stock hydroperoxide were calculated as; [TrpOOH] = 1.06 mM, [TyrOOH] = 219 µM, [HisOOH] =

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204 µM, [LysTrpOOH] = 245 µM, [TrpLysOOH] = 802 µM and [LysTrpLysOOH] = 685 µM.

Figure 5.6: Absorbance values of hydroperoxides expressed as equivalents of H2O2. See legend to Figure 5.2. Data are averages of at least 3 independent experiments, error bars are ± 1 SD. Absorbance data (560 nm) obtained by FOX assay of stock solutions of H2O2 and photochemically-generated peroxides immediately after preparation. H2O2 concentration is -1 -1 207 final concentration (assayed prior using ɛ240 = 39.4 M cm ). Dilution factors are; 5-fold (Tyr, His, LysTrp) or 20-fold (Trp, TrpLys, LysTrpLys).

In the absence of diselenide, untreated peroxides underwent rapid exponential decay in the order; His > TrpLys > LysTrp > Tyr > Trp > LysTrpLys (Figure 5.7), Data are expressed as %

Control (vs Abs560 at t = 0 min) as the initial absorbance varied between peroxide species and different dilution factors were used. The decay of His, TrpLys and LysTrp peroxides became significant relative to the Control (Abs560 at t = 0 min) at 15 min, whereas the loss of Tyr, Trp and LysTrpLys peroxides each reached significance after 4 h. These data were fitted with exponential decay curves, from which the observed rate constants (kobs, Table 5.2) were obtained.

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Figure 5.7: Intrinsic decay of photochemically-generated peroxides over time (48 h). Error bars are ± 1 SD from the average of 3 independent experiments. Lines are exponential decay curves from which kobs values were obtained. Decay is significant (P < 0.05) at 15 min for His, LysTrp and TrpLys, and at 4 hrs for Trp, Tyr and LysTrpLys, as determined by one-way ANOVA with Dunnett’s post-hoc test.

Table 5.2: Observed rate constants (kobs) for the intrinsic decay of photochemically- generated peroxides

-1 Peroxide kobs /s HisOOH 7.2 x 10-5 TrpLysOOH 4.0 x 10-5 LysTrpOOH 3.3 x 10-5 TyrOOH 1.8 x 10-5 TrpOOH 1.0 x 10-5 LysTrpLysOOH 6.6 x 10-6

The stock hydroperoxide solutions were added to increasing concentrations of SeCA (0 to 10 mM) in a 1:1 ratio, and samples were diluted in H2O to optimise the absorbance range for the FOX assay, as above. Peroxide loss was monitored by FOX assay at t = 0, 0.25, 0.5, 1, 2, 4, 6, 24 and 48 h and these data were used to calculate pseudo-first order rate constants for the reaction of SeCA with each hydroperoxide. The scavenging of all peroxides was found to depend on the parent amino acid or peptide, and to occur in both a time- and SeCA concentration-dependent manner (Figure 5.8).

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Figure 5.8: Loss in absorbance (560 nm) of photochemically-generated peroxides over time due to scavenging by SeCA (0 to 10 mM): A) TrpOOH, B) TyrOOH, C) HisOOH, D) LysTrpOOH, E) TrpLysOOH, and F) LysTrpLysOOH. Peroxide concentration determined by FOX assay. Error bars are ± 1 SD of at least 3 independent experiments. Lines are exponential one-phase decay curves, except for HisOOH, which is two-phase to account for the two competing reactions (i.e. intrinsic decay and reaction with SeCA).

165

To these data, non-linear one-phase decay curves were fitted for each peroxide, with the exception of HisOOH. In the case of HisOOH, the data did not fit well to a single-phase decay, probably because of the rapid intrinsic decay of this species being competitive with the SeCA- mediated reaction, as well as the probable formation of numerous initial His peroxide species which are likely to have different rates of decay56. Therefore, the HisOOH data was fitted with a two-phase decay curve, where the underlying slow reaction (kslow) was set to the value -5 -1 obtained for HisOOH in the absence of SeCA (kHis = 7.2 x 10 s ; Table 5.2). The kobs values obtained from the non-linear fits were then plotted for each peroxide against SeCA concentration (Figure 5.9). The probable formation of more than one species of His peroxide and the likelihood that they have different rates of decay may explain why the plot of HisOOH kobs vs SeCA concentration (Figure 5.9, C) does not intercept at (or near) zero, whilst the plots of the other peroxides do. The gradients of these plots yielded rate constants for the reaction of each peroxide with SeCA, with these decreasing in the order; HisOOH > TrpOOH > TyrOOH > TrpLysOOH ≈ LysTrpOOH > LysTrpLysOOH (Table 5.3).

Table 5.3: Absolute rate constants (k) for the reaction of SeCA with photochemically- generated peroxides (error is expressed as 95% CI)

-1 -1 Peroxide k /M s HisOOH (6.3 ± 1.3) x 10-2 TrpOOH (4.5 ± 0.2) x 10-2 TyrOOH (4.0 ± 0.3) x 10-2 TrpLysOOH (2.2 ± 0.1) x 10-2 LysTrpOOH (2.1 ± 0.1) x 10-2 LysTrpLysOOH (1.8 ± 0.2) x 10-2

These data show that whilst the rate of intrinsic decay of the peroxides varies with the structure of the amino acid or peptide upon which they are generated, the rate constants of reaction for each peroxide with SeCA vary only by ca. 3-fold. Subsequent experiments examined the structural dependence of the reaction of TrpOOH with various diselenides.

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Figure 5.9: Plots of observed rate constants (kobs) against concentration of SeCA for the reaction of SeCA (0 to 10 mM) with photochemically-generated hydroperoxides: A) TrpOOH, B) TyrOOH, C) HisOOH, D) LysTrpOOH, E) TrpLysOOH, F) LysTrpLysOOH. Data are the average of at least 3 independent experiments and error bars show ± 1 SD. Absolute rate constants are calculated from the gradient of these plots. kobs values were obtained from exponential decay curves of absorbance over time plots, all of which were fitted to a single exponential process, except for HisOOH, which was fitted to a double exponential curve to account for the two competing reactions (the rapid intrinsic decay of His peroxides and the reaction with SeCA).

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5.3.3. Diselenide Oxidation by Photochemically-Generated TrpOOH

In order to evaluate and compare the differing scavenging abilities of a range of structurally diverse diselenides, TrpOOH was selected as a model peroxide to react with these compounds because it is relatively stable and generated in higher yields than the other peroxides investigated.

TrpOOH was generated photochemically as detailed in Section 2.5. The initial TrpOOH stock concentration across all experiments (n = 9) was determined by FOX assay to be ca. 1 mM immediately following photolysis, by comparison to a standard curve of a known concentration of H2O2 assayed in the same way, expressed as H2O2 equivalents. The TrpOOH stock was added to increasing concentrations of DSePA (0 to 1 mM), DPDSe (0 to 3 mM in 6% DMSO) or DPMSe (0 to 5 mM in 20% DMSO) in H2O in a 1:1 ratio (ca. 500 µM peroxide). Samples were diluted 20-fold into H2O to optimise the absorbance range for the FOX assay, then peroxide absorbance (560 nm) was monitored to evaluate the diselenide-induced peroxide loss (initial concentration = 25 µM) over 48 h (Figure 5.10). Experiments were performed in duplicate, each with three replicates.

The reaction of TrpOOH with DSePA and DPDSe (Figure 5.10, A and C) was both time- and diselenide concentration-dependent. Whilst the addition of 0.5 mM DPMSe (Figure 5.10, B) substantially increased the extent of TrpOOH loss relative to the control (0 mM diselenide), higher concentrations (> 0.5 mM) showed very limited dose-dependence, with further increases in diselenide concentration inducing only minor increases in peroxide loss. Exponential decay curves were fitted to all data, yielding kobs values for the reaction of TrpOOH with each diselenide over the concentration ranges employed.

168

Figure 5.10: Loss of photochemically-generated TrpOOH (ca. 500 µM) with increasing concentrations of diselenides; A) DSePA (0 to 1 mM), B) DPMSe (0 to 10 mM), C) DPDSe (0 to 3 mM). Data shown are the average of 3 independent experiments, error bars are ± 1 SD. Peroxide concentration monitored over time by FOX assay. Lines are exponential decay curves.

169

The kobs values obtained from these data were then plotted against the concentration of each diselenide (Figure 5.11), producing linear trends for DSePA and DPDSe, the gradient of which yields the absolute rate constants for the reaction with TrpOOH (Figure 5.11, A and C). The -1 -1 rate constants obtained for these diselenides are kDSePA = (1.9 ± 0.1) M s and kDPDSe = (1.1 ± 0.3) x 10-2 M-1 s-1, where error is 95% CI.

In the case of DPMSe (Figure 5.11, B), the limited dose-dependence of the diselenide in scavenging TrpOOH is apparent, as the kobs values rapidly produce a plateau effect at concentrations > 1 mM DPMSe. It is probable that the limited dissolution of the compound in both DMSO and H2O is responsible for this effect and thus, this data does not provide an accurate estimate of the rate constant. Therefore, to calculate an approximate absolute rate constant for this reaction, only the lowest DPMSe concentrations (≤ 1 mM) were fitted with a linear regression, where solubility was not a limiting factor. The gradient of this plot provided an approximate absolute rate constant for the reaction of TrpOOH with low concentrations of -2 -1 -1 DPMSe, kDPMSe = (1.3 ± 0.5) x 10 M s . In future experiments, increasing the percentage of the DMSO solvent and adding additional DPMSe concentrations between 0 and 0.5 mM could be implemented to clarify if this reaction is truly independent of diselenide dose over this range, and to determine the order of reaction. The effect of higher DMSO concentrations on the accuracy of the FOX assay would also need to be assessed. A summary of the absolute rate constants for reaction of TrpOOH with the diselenides assessed is provided in Table 5.4.

Table 5.4: Absolute rate constants (k) for the reaction of various diselenides with photochemically-generated TrpOOH

-1 -1 Diselenide k /M s SeCA (4.5 ± 0.2) x 10-2 DSePA (1.9 ± 0.1) DPMSe (1.3 ± 0.5) x 10-2 DPDSe (1.1 ± 0.3) x 10-2

These data show that diselenides react with TrpOOH at different rates with this depending upon the structure of the diselenide. DSePA was shown to react the most rapidly with the peroxide, at a rate ca. 40-fold faster than that of SeCA, and ca. 150-fold faster than both DPMSe and DPDSe. This emphasises the critical role of secondary structure in the reactivity of the diselenide compound.

170

Figure 5.11: Plots of kobs against concentration of diselenide compounds upon reaction with TrpOOH: A) DSePA (0 to 1 mM), B) DPMSe (0 to 10 mM), C) DPDSe (0 to 3 mM). Data are average of three independent experiments, error bars show ± 1 SD. Each plot is fitted with a linear regression, for which the equation is given. DPMSe fits an exponential curve, consistent with its limited dissolution; its linear regression is plotted in red and includes concentrations from 0 to 1 mM DPMSe only. The gradients of each plot yield the absolute rate constants for these reactions.

171

5.3.4. The Products of Diselenide Oxidation are Consistent between Various Oxidant Species

These experiments aimed to determine the products of Fmoc-SeCystine oxidation by photochemically-generated amino acid hydroperoxides, using TrpOOH as a model peroxide owing to its relative stability. UPLC was used to detect the products of Fmoc-SeCystine oxidation by TrpOOH with these then compared to the products previously characterised for

HOCl and H2O2.

Control samples were prepared in order to confirm that any fluorescent peaks observed in the UPLC chromatograms were true oxidation products of diselenides, and not the result of TrpOOH degradation or the catalase or RB present in photolysed Trp solutions. Firstly, a sample was prepared containing 50% MeOH and 50% undiluted photochemically-generated TrpOOH (generated as described in Section 2.5., termed ‘TrpOOH Control’). A second sample was prepared in an identical way, but with the Trp omitted [‘Control’; H2O (pH 7.2 – 7.4) photolysed with RB, then incubated with catalase, in 50% MeOH], resulting in no hydroperoxide formation. These two samples were analysed concurrently with a ‘Blank’ sample (50% MeOH in H2O) by the UPLC methodology detailed in Section 2.7.3. (Figure 5.12,

A). To examine any time-dependent changes in the fluorescence profile (λex = 265 nm, λem = 275 – 500) of the photolysed Trp, the TrpOOH Control was analysed via UPLC at t = 0 and t = 72 h following oxidant generation (Figure 5.12, B).

The Control sample produced no significant additional fluorescence in comparison to the Blank sample (Figure 5.12, A). There was no apparent change in the fluorescence of the TrpOOH Control over time (Figure 5.12, B), which indicates that the peak at 1.6 min is residual Trp in the sample which had remained un-oxidised during photolysis treatment. This is consistent with the fluorescence maximum of Trp at 350 nm which was within with the fluorescence 245 detection conditions employed during analysis (λem = 275 – 500 nm). Conversely, no fluorescence was detected from the photochemically-generated peroxide of Trp, probably as a result of oxidative modification of the heterocyclic ring in this product.61

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Figure 5.12: UPLC Chromatograms of; A) Blank and Control samples, B) TrpOOH Control over time (t = 0 and 72 h). A) ‘Blank’ is 50% MeOH in H2O, ‘Control’ is H2O (pH 7.25) photolysed with RB plus catalase, in 50% MeOH. B) TrpOOH (ca. 500 µM) in 50% MeOH at t = 0 and 72 h. The peak at 1.6 min corresponds to residual Trp which did not undergo oxidation to TrpOOH when photolysed. No TrpOOH is detected, and no change in Trp 245 fluorescence (λem = 275 – 500 nm) was observed over the experimental time-course.

The oxidation of Fmoc-SeCystine by TrpOOH was then investigated. TrpOOH was prepared by photolysis as per Section 2.5. and its concentration quantified by the FOX assay (as detailed in Section 2.6.), before being reacted with increasing concentrations of Fmoc-SeCystine (0, 100, 200 or 500 µM) in a 1:1 ratio (final concentration ca. 500 µM TrpOOH) with a final solvent concentration of 50% MeOH and 50% buffer (0.1 M, pH 7.4) and analysed by UPLC as described in Section 2.7.3. The fluorescent peaks detected were compared to previous experiments with other oxidants in which the Fmoc-SeCystine oxidation products were characterised by LC/MS.

Control samples of Fmoc-SeCystine (500 µM in 50% MeOH) were injected onto the column at decreasing volumes (10 to 2 µL, in 2 µL steps) for UPLC analysis. The fluorescence intensity was proportional to the injected volume (and hence the concentration) of Fmoc-SeCystine, confirming that the peak at 25.1 min was Fmoc-SeCystine (Figure 5.13). Minor peaks from

173 contaminants were also observed in the samples containing Fmoc-SeCystine (e.g. peak at 22.5 min; Figure 5.13, inset). The background peaks at 24.2 min and > 27 min were present in all samples and disregarded.

Figure 5.13: UPLC chromatogram of Fmoc-SeCystine (500 µM in 50% MeOH) at decreasing injection volumes (10, 8, 6, 4, 2 µL). Volumes are represented by each respective line. Fmoc-SeCystine elutes strongly at 25.1 min, with minor baseline peaks each decreasing in proportion with the injection volume (e.g. peak at 22.5 min, inset).

To compare the products of Fmoc-SeCystine oxidation by TrpOOH and H2O2, TrpOOH (ca.

500 µM) or H2O2 (1 mM) were added to Fmoc-SeCystine (500 and 100 µM, respectively) in 50% MeOH and analysed by UPLC, as detailed in Section 2.7.3. (Figure 5.14). The concentration of Fmoc-SeCystine was limited to 100 µM for reaction with H2O2 to keep this consistent with previous experiments. However, the concentration of Fmoc-SeCystine was increased to 500 µM in the case of TrpOOH to ensure that sufficient concentrations of the diselenide were present to react with the peroxide, which decays rapidly.

Comparison of the UPLC traces in Figure 5.14 revealed that oxidation of Fmoc-SeCystine by both H2O2 and TrpOOH (and also HOCl, see Section 4.3.5.) yielded a fluorescent peak eluting at 11.2 min under identical analysis conditions, indicating that each oxidant generates the same primary product, the seleninic acid (RSeO2H).

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Figure 5.14: UPLC chromatogram showing oxidation of Fmoc-SeCystine (500 or 100 µM) by TrpOOH (ca. 500 µM) and H2O2 (1 mM). Black = 500 µM Fmoc-SeCystine (Standard), Red = Fmoc-SeCystine and H2O2 (at 10.5 h post oxidant-addition), Grey = Fmoc-SeCystine and TrpOOH (4 h post oxidant-addition). In the presence of each TrpOOH and H2O2, Fmoc- SeCystine yields a fluorescent peak at 11 min, indicating that both oxidants result in the same fluorescent Fmoc-SeCystine product.

5.4. Discussion

The studies reported in this Chapter have examined the oxidation of various diselenides by the photochemically-generated hydroperoxides of Trp, Tyr, His, LysTrp, TrpLys and LysTrpLys, as well as H2O2 and the model reagent peroxides tBuOOH and CHP. Rate constants were determined for reaction of TrpOOH with each diselenide and for all the hydroperoxides with SeCA (summarised in Table 5.5).

-2 -4 SeCA reacted with H2O2, CHP and tBuOOH with k1 = (1.1 ± 0.5) x 10 , (5.4 ± 1.0) x 10 and (1.4 ± 0.1) x 10-4 M-1 s-1, respectively. tBuOOH and CHP contain an aliphatic or aromatic group, respectively, which cause decreases in their rate constant of reaction with SeCA by ca.

80- fold and 20-fold, respectively, relative to H2O2, suggesting that rate constants for these reactions alter significantly with the moiety to which the hydroperoxide is attached.

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Table 5.5: Absolute rate constants (k) for the reaction of various diselenides with biological and model oxidants (error is expressed as 95% CI)

Peroxide SeCA k ± CI DSePA k ± CI DPMSe k ± CI DPDSe k ± CI (M-1 s-1) (M-1 s-1) (M-1 s-1) (M-1 s-1) -2 H2O2 (1.1 ± 0.5) x 10 tBuOOH (1.4 ± 0.1) x 10-4 CHP (5.4 ± 1.0) x 10-4 TrpOOH (4.5 ± 0.2) x 10-2 (1.9 ± 0.1) (1.3 ± 0.5) x 10-2 (1.1 ± 0.3) x 10-2 TyrOOH (4.0 ± 0.3) x 10-2 HisOOH (6.3 ± 1.3) x 10-2 LysTrpOOH (2.1 ± 0.1) x 10-2 TrpLysOOH (2.2 ± 0.1) x 10-2 LysTrpLysOOH (1.8 ± 0.2) x 10-2

1 Oxidation of protein residues by O2 can generate amino acid hydroperoxides (ROOH), which are unstable intermediates with the potential to propagate radical-initiated damage and contribute to oxidative stress in vivo. Protein peroxides are generated in high yields by many physiologically significant radicals (e.g. •OH), by formation of peroxyl radicals (ROO•) at the initial site of oxidation which subsequently react to generate the ROOH, which can persist for •- relatively long lifetimes, as well as acting as a source of radicals (e.g. O2 , C-centred) and other reactive species.246 It has been reported that GPx in the presence of reducing factors (e.g. GSH) appears to rapidly remove peptide, but not protein, peroxides.230 Conversely, protein thiols, GSH, low-molecular-mass thiols and ebselen react with both peptide and protein peroxides in a non-stoichiometric manner.230

1 Reaction of free and protein-bound Trp with O2 yields two hydroperoxides (a cis- and trans- diasteroisomer) by peroxidation of the five-membered ring structure, which undergo slow 247 1 decomposition to the corresponding alcohols. O2-mediated oxidation of Tyr, both free and protein-bound, yields one or more short-lived endoperoxides which can subsequently undergo ring-opening reactions.248 His residues are oxidised via initial formation of endoperoxide intermediates and subsequent ring-opening reactions involving the N lone pair, which ultimately result in the probable generation of multiple hydroperoxides.29, 51, 56 His-derived peroxides are unstable, with decay of these species complete after 24 h of incubation at 25 ˚C,56

176 which is consistent with the intrinsic peroxide decay observed in this study. The rapidity of HisOOH loss in both the presence and absence of SeCA is consistent with the endo- or hydroperoxides being unstable intermediates. This is supported by the fitting of a two-phase decay curve, indicating a more complex route of peroxide decay in which the diselenide-catalysed and spontaneous decay rates are close in value. This is consistent with previous data on the instability of His-derived peroxides.56

SeCA was oxidised by photochemically-generated hydroperoxides of Trp, Tyr, His, LysTrp, 1 TrpLys and LysTrpLys. Attack on Trp, Tyr and His residues by O2 results in the corresponding hydroperoxides as the major product.55, 61 The photo-oxidised peptides LysTrp, TrpLys and LysTrpLys were also examined to establish the importance of molecular structure and steric bulk on the reactivity of the peptide, in terms of both hydroperoxide formation and reaction with diselenides. SeCA reacted with hydroperoxides in the order of His > Trp > Tyr > TrpLys > LysTrp > LysTrpLys, with rate constants of k = (6.3 ± 1.3) x 10-2, (4.5 ± 0.2) x 10-2, (4.0 ± 0.3) x 10-2, (2.2 ± 0.1) x 10-2, (2.1 ± 0.1) x 10-2 and (1.8 ± 0.2) x 10-2 M-1 s-1, respectively (Table 5.5). The reaction of the comparatively stable and long-lived Trp hydroperoxide was also examined with DSePA, DPDSe and DPMSe to investigate the role of diselenide structure in their respective ability to scavenge the same peroxide. Rate constants were determined for the reaction of TrpOOH with DSePA, DPMSe and DPDSe, yielding k = (1.9 ± 0.1) M-1 s-1, (1.3 ± 0.5) x 10-2 and (1.1 ± 0.3) x 10-2 M-1 s-1, respectively (Table 5.5).

SeCA reacted with each of the six protein/peptide hydroperoxides (and H2O2) with rate constants in the order of 10-2 M-1 s-1, which implies that the diselenide is reacting with the peroxide group with a rate that is affected up to ca. 3-fold by the residues surrounding the peroxide. Whilst the amino acid peroxides reacted slightly faster than the Lys-bound peptides (ca. 2-fold), the steric bulk of the Lys residues did not drastically affect the reactivity with SeCA, with only a total ca. 4-fold difference in rate constant across all the peroxides investigated. This indicates that protein-bound and free amino acid hydroperoxides may share similar rate constants of reaction with a range of low-molecular-mass diselenides, but this proposal requires further investigation.

The secondary structure of each diselenide appears to play a key role in modulating the reactivity of the Se-Se group with TrpOOH. This is likely due to the accessibility and/or conformation of the diselenide group, which is strongly affected by the residues adjacent to the diselenide bond.203 Whilst SeCA, DPMSe and DPDSe reacted with TrpOOH at similar rates

177

(each having k = 10-2 M-1 s-1), DSePA was shown to react ~ 100-fold faster. SeCA and DSePA are both low-molecular-mass linear diselenides which differ only in their terminal groups (i.e. + - NH3 vs COO , respectively), the substitution of the carboxylic acid groups imbuing DSePA with a vastly enhanced capacity to scavenge TrpOOH compared to SeCA. These data provide valuable insight into the importance of secondary structure in the application of diselenide compounds in an anti-inflammatory context, with intramolecular interaction with nearby heteroatoms (e.g. N, O, S) proving to be crucial in determining diselenide reactivity.203

Ebselen is a low-molecular-mass selenium-containing heterocyclic compound with anti- inflammatory and anti-atherosclerotic properties that arise from its ability to mimic GPx in the 203 reduction of H2O2. It has been proposed that non-bonding interactions of Se with nearby N or O atoms are responsible for activating the active site selenium to a selenolate (RSe-) by H+ abstraction and for stabilising the selenenic acid (RSeOH) intermediate that is formed during the catalytic cycle of ebselen, thus, increasing the nucleophilic reactivity of the selenium atom to enhance the GPx-like activity of ebselen, as well as other Se species.203 Similarly, intramolecular interactions between sulfur atoms and nearby heteroatoms is proposed to increase the reactivity of disulfide compounds with hypohalous acids due to the favourable positioning of electron-donor groups which act to stabilise the positively-charged sulfur reaction centre.176 The similarities that exist between sulfur and selenium suggest that, like the corresponding disulfide compounds, the adjacent structure of acyclic (e.g. SeCA, DSePA) and aromatic-substituted (e.g. DPDSe, DPMSe) diselenides plays a key role in determining the reactivity of the diselenide moiety and that factors such as protonation and bond stabilisation by nearby heteroatoms are important considerations in the study of these compounds, as the microenvironment of these species markedly affects their reactivity.176 These data are consistent with the observation that the secondary structure of the diselenide is a key factor in determining its reactivity with a range of peroxide species. The secondary structure of the hydroperoxide species appears to play a less significant role than the structure of the diselenide in determining its reactivity with diselenides, a trend which is consistent with the reagent peroxides investigated.

The products of Fmoc-SeCystine oxidation by TrpOOH were compared to those characterised in the presence of HOCl and H2O2, detailed in the previous Chapters (Sections 3.3.4. and 4.3.5., respectively). UPLC analysis revealed a major fluorescent product peak with an elution time that was consistent across the different oxidants, indicating that Fmoc-SeCystine gives rise to the same primary stable oxidation product with each species. In the previous Chapters, LC/MS

178 analysis had revealed that the primary stable product of Fmoc-SeCystine oxidation by HOCl and H2O2 was the seleninic acid derivative (RSeO2H), strongly suggesting that TrpOOH also generates RSeO2H. Based on these data, it is suggested that the diselenide moiety of Fmoc- SeCystine is the primary site of oxidation across a range of biological oxidants, resulting in generation of the RSeO2H derivative. This finding provides insight into the mechanisms of diselenide oxidation and may be applied in the characterisation of the peroxide-mediated oxidation products of other diselenides (e.g. GSeSeG). Other, more unstable or intermediate products have not been identified here.

5.5. Conclusions

Low-molecular-mass diselenides, both aliphatic and aromatic, react with an array of peroxides with rates that depend upon the structure of both the diselenide and the peroxide. The rate of reaction appears to be dependent upon the structure of the diselenide compound, and favourable interaction with nearby heteroatoms may be a key determinant. It is postulated on the basis of the data obtained that substitution of the carboxylic acid groups demonstrates a more marked effect than the amine groups in enhancing the reaction rate. The reaction appears to occur at the hydroperoxide moiety of each species. The major stable product of diselenide oxidation is suggested to be the di-oxygenated seleninic acid (RSeO2H) derivative. Exogenous diselenide compounds, therefore, appear to react fairly rapidly with an array of biologically relevant peroxide species, both free and protein-bound, with an efficacy that depends upon their structure, predicating their potential therapeutic application in the treatment or prevention of oxidative stress-induced pathologies.

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Chapter 6

Discussion and Future Research

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6.1. General Overview of Oxidant Generation and Oxidative Stress

Oxidative stress is a deleterious state in which redox homeostasis is perturbed, favouring the balance of reactive oxygen species (ROS) over the antioxidant capabilities of an organism.246 ROS are chemically-reactive species containing oxygen and include both radical (one-electron) and non-radical (two-electron) oxidants, which are formed as part of normal cellular metabolism.24 ROS play a key role in the destruction of invading pathogens as part of the immune response, but can also have harmful effects on host tissue, inducing oxidative stress and contributing to the progression of inflammatory diseases including cancer, neurodegeneration and atherosclerosis.79

The oxidants examined in this thesis are hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and various protein peroxides (ROOR’), which were generated via the photo-oxidation of 1 amino acids and peptides by singlet oxygen ( O2). In biological systems, H2O2 is formed within •- the phagosome of the neutrophil via dismutation of superoxide (O2 ), either spontaneously or through the enzyme-catalysed reactions of myeloperoxidase (MPO) or superoxide dismutase 6 (SOD). The labile O-O bond of H2O2 can undergo homolysis in the presence of redox-active metal ions (e.g. Fe2+, Cu+) to yield the hydroxyl radical (HO•), which is one of the strongest reported oxidants with reactions that occur at near diffusion-controlled rates.75, 80 Reaction of

H2O2 with MPO forms the hypohalous acids (HOCl, HOBr, HOSCN), with approximately 45% of the H2O2 consumed by MPO being converted to HOCl, which reacts rapidly with a vast array of biological substrates including thiols (RSH) and amines (RNH2) to form disulfides, 27, 28 sulfinic (RSO2H) and sulfonic (RSO3H) acids, and chloramines (RNHCl), respectively. Oxidative stress arising from MPO-derived oxidants, particularly HOCl, has been associated with the onset and progression of inflammatory conditions such as cardiovascular disease (CVD) and atherosclerosis.12, 81

1 Further reaction of HOCl with H2O2 can yield O2. Singlet oxygen is the first excited state of •- molecular oxygen and a more potent oxidant than O2 , with a relatively long lifetime within 6, 29, 35 1 the range of microseconds. In addition to MPO-catalysed generation, O2 can be produced in biological systems via energy transfer from a sensitiser (such as various dyes and porphyrins)43 to molecular oxygen in the presence of light.29 Several methods of producing and 1 1 detecting O2 have been developed with the intention of applying O2 in a therapeutic approach, 50 49 1 e.g. photodynamic therapy of skin cancers. O2 reacts with a vast array of biomolecules, with reaction with Trp, Tyr, His, Met and Cys residues of proteins occurring more rapidly than

181 with most other cellular targets at physiological pH 7.4.51, 52 These protein-bound amino acid 1 residues can react with O2 either via direct chemical reaction or physical quenching, the latter of which results in energy transfer and de-excitation of the singlet state, with no chemical change in the amino acid.61 In proteins, only Trp gives rise to significant physical quenching, 1 whereas His, Tyr, Met and Cys (as well as Trp), react rapidly with O2 via chemical reaction at physiological pH 7.4.61

The role of MPO-derived oxidants in the onset and progression of inflammatory pathologies is compelling, with clinical studies detecting MPO in atherosclerotic lesions and revealing a strong correlation between blood levels of MPO and the incidence of disease, to such an extent that MPO is considered an independent risk factor for coronary artery disease.81 HOCl- mediated tissue damage in diseases such as atherosclerosis is evidenced by detection of the protein-derived biomarker, 3-chloro-tyrosine (3-ClTyr), as well as modified lipids and DNA.27 Cys (thiol, RSH) and Met (thioether, RSR’) residues also react with photochemically-generated 1 O2 at significant rates at physiological pH 7.4, becoming oxidised to the disulfide (RSSR) and sulfoxide [RS(=O)R’], respectively, resulting in inactivation of Cys-dependent enzymes and contributing to the pathogenesis of sunburn, skin cancer and cataract formation.29, 30, 52

6.2. Oxidation of Key Sulfur-Containing Amino Acid Residues

Methionine is an essential amino acid with a thioether moiety (RSR’) which is necessary for 84 1 protein synthesis. The initial oxidation of Met by HOCl, H2O2 and O2 generates methionine sulfoxide (MetO), which can be reduced by the methionine sulfoxide reductase (Msr) family of enzymes.117 This primary oxidation and reduction cycle of Met is proposed to regulate the biological activity of proteins, and as such, Met plays an important role in the redox regulation and antioxidant defence of cells, as well as contributing to protein initiation and structure.108, 114 However, in the presence of excess ROS, the further oxidation of MetO generates methionine sulfone (MetO2) which is biologically stable and cannot be enzymatically reduced, and as such, the accumulation of MetO2 has been associated with the development of neurodegenerative conditions.6, 84, 111, 119, 120

Cysteine is a sulfur-containing amino acid which has key roles in the structural integrity, function and regulation of proteins.249 The nucleophilic thiol moiety of Cys is necessary for the reversible formation of disulfide bonds, which are often required for the catalysis of key Cys- 250 dependent enzymes. Oxidation of thiols by H2O2 initially yields the sulfenic acid (RSOH)

182 and subsequent reaction with a second thiol forms the disulfide. Slow oxidation of the RSOH to yield the sulfinic acid (RSO2H) can also occur, though with rate constants that are typically ca. 1000-fold slower than oxidation of the parent thiol.80 Thiol oxidation by HOCl results in initial generation of sulfenyl chlorides (RSCl) which then form the disulfide (RSSR) and higher oxyacids [RSO2H and the sulfonic acid (RSO3H)], possibly cross-linking with Lys residues to 6 form sulfinamides [RS(O)NR’] and sulfonamides [RS(O2)NR’]. Reversible disulfide bond formation is a key post-translational mechanism by which thiol-containing proteins can modulate their functional activity in the presence of ROS, to act as regulators in cellular redox homeostasis.249 The oxidation of disulfides is typically slower than oxidation of the corresponding thiols due to disulfides being in a partially oxidised state.79

6.3. Endogenous Antioxidant Defence Mechanisms

Within biological systems, there exist a number of endogenous antioxidant defence mechanisms to delay or prevent the formation of excess ROS, or repair ROS-induced damage. These include non-enzymatic mechanisms, such as the scavenging of oxidants by low- molecular-mass species [e.g. glutathione (GSH), ascorbate and vitamin E], and enzymatic systems which: a) target oxidants or their precursors [e.g. catalase, SOD and glutathione peroxidase (GPx)], b) repair oxidised substrates (e.g. Msr and sulfiredoxins), or c) eliminate substrates which are irreparably damaged (e.g. proteases and phospholipases).40 Many of these mechanisms rely upon a reactive sulfur or selenium centre at the active site of a protein to carry out their antioxidant activity.

Glutathione is a ubiquitous low-molecular-mass thiol antioxidant which acts as a co-factor for 1 GPx, becoming oxidised (e.g. by HOCl and O2) to glutathione disulfide (GSSG) in the process, which is then reducible by NADPH-dependent enzymes such as glutathione reductase (GR).40, 84 • • •- One-electron oxidation of GSH by radicals such as OH, NO2 and CO3 generates the thiyl • • radical (GS ) which can then react with O2 to reversibly form a peroxyl radical (ROO ), or with - •- 79 •- •- GS to form a GSSG radical ion (GSSG ). GSSG can react rapidly with O2 to form O2 , which is rapidly detoxified by SOD, representing a viable in vivo mechanism by which GSH may scavenge protein-bound radicals.79 Two-electron oxidation of GSH (e.g. by ONOOH and HOCl) results in the initial formation of the sulfenic acid (RSOH), which is converted to the disulfide and higher oxyacids (RSO2H and RSO3H), presumably via formation of transient intermediate species which are difficult to detect (e.g. RSCl).79

183

Selenium-containing compounds often demonstrate significant analogy to the corresponding sulfur species in terms of reactivity due to the inherent similarity between the two elements (electron configuration, electronegativity, ionic radii, etc.).101, 102 However, selenium- containing species are typically more reactive, and thus, more susceptible to oxidation than their sulfur analogues due to having more favourable redox properties, i.e. the increased nucleophilicity and electronegativity of Se relative to S and the lower pKa of selenols (RSeH, 5.3) relative to thiols (8.3).102, 107, 177 This results in the major form present at physiological pH 7.4 being the selenolate ion (RSe-), which confers strong nucleophilicity to the molecule and enables the selenol to be oxidised to the selenenic acid, which is then readily reduced in the presence of reductants (e.g. GSH) in a stoichiometric manner to regenerate the active selenol form and water.157, 251 The faster rate constants of reaction suggest that selenocompounds could potentially act as competitive targets for biological oxidants in vivo, thus, selenocompounds have garnered considerable interest as potential catalytic oxidant scavengers in a therapeutic setting for mediating inflammation-associated pathologies.85, 89, 102

Whilst being more susceptible to oxidation than their sulfur-analogues (thiols, RSH), the oxidation of selenols (and selenoethers, RSeR’) is reversible upon reaction with GSH, unlike the more biologically stable, and hence irreversible, thiol (and thioether, RSR’) groups.85, 89, 96 Selenols are present as Sec at the active site of many selenium-dependent enzymes including GPx, TrxR and MsrB1, which typically function by initial oxidation of the selenol to the selenenic acid (RSeOH) and further oxidation to the seleninic acid (RSeO2H), which may be 105, 143 non-enzymatically reduced to the selenol by GSH (Figure 6.1). The RSeO2H is readily reduced and is strongly resistant to over-oxidation to the inactive selenonic acid (RSeO3H) species.252

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Figure 6.1: Mechanism of action and kinetics of Cys- vs Sec-dependent enzymes. The reduced Cys-SH or Sec-SeH is required for enzyme activity, which can be oxidised by H2O2 to generate the inactive Cys-SOH or Sec-SeOH, respectively, with addition of a second - - equivalent of H2O2 generating Cys-SO2 and Sec-SeO2 , respectively. Sec-SeH/SeOH oxidation is more rapid than that of Cys-SH/SOH. Reduction of these species (e.g. by GSH) regenerates - the active thiol/selenol with kred2 >> kred1, with the Cys-SO2 being highly resistant to reduction. - - - The Sec-SeO2 is resistant to further oxidation to Sec-SeO3 , whereas Cys-SO2 is rapidly - oxidised to Cys-SO3 , with kox2 << kox1. Image adapted from Ruggles, Snider and Hondal (2012)105

Cys-enzymes function in a similar manner to selenoenzymes, whereby the active thiol is oxidised to the RSOH and subsequent RSO2H; however, the RSO2H species is very resistant to GSH reduction to regenerate the thiol, but rapidly undergoes further oxidation to the RSO3H form, resulting in irreversible enzyme inactivation.105 These data demonstrate that selenoenzymes are more resistant to oxidative stress than their Cys-containing analogues, provided that adequate levels of GSH or other reductants are present.141 This is supported by 1 the observation of reversible O2-induced oxidation of the Sec residue of GPx and TrxR by addition of GSH, effectively restoring enzyme activity.30 Furthermore, selenoenyzmes have 1 been shown to resist oxidation of the Sec residue by H2O2, HOCl and O2 compared to a Cys- containing analogue, thereby potentially inhibiting irreversible enzyme inactivation.105 This represents a means by which Sec incorporation into proteins acts to protect against oxidative stress. However, the study and application of selenols is impeded by their inherent instability and rapid auto-oxidation to the corresponding diselenides (RSeSeR), making investigation difficult.79 Therefore, extensive research has focused on the reactions of selenoethers (e.g. SeMet) as opposed to the more labile selenols.

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6.4. Selenium Compounds as Exogenous Antioxidants

There is strong evidence to suggest that selenol (RSeH), selenoether (RSeR’) and diselenide (RSeSeR’) compounds have therapeutic potential as antioxidants by acting as mimetics of the Sec-enzyme GPx.178, 179, 182, 191, 194 Ebselen is a synthetic organoselenium compound which interacts with a vast range of molecular targets, exhibiting GPx-like activity in vivo to detoxify

H2O2, hydroperoxides and radicals, thereby protecting against ROS-mediated damage such as 104, 156 1 6 -1 - ONOOH-induced DNA strand breaks. Reaction of ebselen with O2 (k = 2.5 x 10 M s 1) is ca. 10-fold faster than reaction with its sulfur analogue.253 Its mode of action relies upon oxidation of the selenium moiety to RSeOH, and subsequent reduction by GSH (or other thiols, e.g. DTT) via generation of a selenenylsulfide (RSeSR) intermediate.154, 157, 158 The RSeSR intermediate may undergo unfavourable thiol-exchange reactions at the selenium centre which decreases GPx-like activity due to equilibrium favouring formation of the RSeSR/RSH pair rather than the RSSR/RSeH,152 an effect which may be modulated by the presence of strong S- N or S-O non-bonding interactions at the sulfur centre to encourage formation of the active 200 ebselen-selenol species. Ebselen and its diselenide [(EbSe)2] also act as substrates for mammalian TrxR to decompose H2O2 more effectively via this pathway than through its GPx- 159, 254 like activity. Ebselen reacts with ebselen-selenol to generate (EbSe)2, which is reduced by TrxR to regenerate the ebselen-selenol, stimulating the H2O2 reductase pathway of TrxR (in the presence of Trx).159

Ebselen has demonstrated antioxidant capabilities via its GPx-like activity and its ability to act as a substrate for TrxR and as such, has been shown to act as a neuroprotective agent and a modulator of inflammation with promising potential as a therapeutic agent in multiple inflammatory pathologies including diabetes and the progression of atherosclerosis.160, 161, 163 However, the efficacy of ebselen is often limited by its low solubility in water and potential for causing cellular toxicity, associated with thiol oxidation and depletion.147 Therefore, alternative selenocompounds have been investigated which may demonstrate greater biocompatibility in a therapeutic setting.

6.4.1. Diselenide Species

Diselenides have been investigated as shelf-stable compounds which may be reduced to yield the more reactive selenol derivative which is responsible for the GPx-like activity of many selenium-containing antioxidants.79 Oxidation of diselenide compounds is more kinetically

186 favourable than oxidation of the analogous disulfides, which initially yield the thiosulfinate

[RSS(=O)R’] and thiosulfonate [RSS(=O)2R’] species, with subsequent reaction and bond 79, 86, 102, 175, 176 cleavage generating the RSO3H derivative. Kinetic data for the direct oxidation of diselenides is scarce, but these compounds have been shown to exhibit antioxidant effects in an inflammatory context through their GPx-like activity. For example, the oral administration of diphenyl diselenide [(PhSe)2] to rats prevented the formation and reversed the symptoms of ethanol-induced gastric ulcers, by modulation of catalase and SOD activity, suggesting that diselenides have the potential to alter inflammatory processes.179 Injection of

(PhSe)2 into mice demonstrated anti-viral action against herpes simplex virus 2 (HSV-2) in vivo by modulating the GPx, SOD and catalase activity and reducing the inflammatory 255 response to viral infection and subsequent oxidative injury. (PhSe)2 also reduced hepatic damage in mice caused by chemically-induced oxidative stress, both when administered orally prior to oxidative insult183 and following intraperitoneal injection after oxidative insult.256 Other disubstituted diaryl diselenides have shown greater antioxidant capabilities than ebselen which are not attributed to their GPx-like activity, instead acting to detoxify peroxides by acting as a substrate for TrxR.182

The favourable reaction kinetics of selenium species results in compounds which consistently display elevated rate constants of reaction with biological oxidants (including HOCl and HOSCN) compared to the corresponding sulfur compounds, and are generally believed to form the analogous products.69, 79, 174, 222 However, evidence indicates that alternative products may be generated by oxidation of the selenium species. For example, the oxidation of GPx1 by H2O2 resulted in the Sec residue at the active site being irreversibly converted to dehydroalanine 143 (DHA) via the loss of H2SeO2 from the seleninic acid (RSeO2H) by β-elimination, rather than yielding the selenonic acid product predicted by thiol chemistry,27 indicating that selenol oxidation may not necessarily mirror thiol chemistry, with the reaction proceeding via different pathways to yield alternative products. This suggests that further research is needed to characterise the products of selenocompound oxidation, to assess the ability of these 1 compounds to act as competitive scavengers of HOCl, H2O2 and O2-induced amino acid/peptide hydroperoxides, to mitigate oxidative stress-associated pathologies.

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6.5. Kinetics and Products of the Oxidation of Selenium Compounds

The efficacy of antioxidant species is predicated on their ability to kinetically compete with the biological targets of ROS, thereby reducing the potential for oxidative damage. Some of the fastest reported reactions are between HOCl and sulfur-containing species, with rate constants for Met (thioether) and Cys (thiol) being k = 3.4 x 107 M-1 s-1 and 3.6 x 108 M-1 s-1, respectively.69 Reaction of HOCl with the other amino acids His, Trp and Tyr, occur at rates that are significantly slower, with k = 1.0 x 105, 1.1 x 104 and 44 M-1 s-1, respectively.69 Alternative targets of HOCl include amine groups (k = 103 – 105 M-1 s-1) and disulfides.6 The rate constants of reaction of H2O2 with biological substrates are consistently slower than for -1 -1 many other ROS, with k = 0.89, 2.9 and 1.05 M s reported for the reaction of H2O2 with the thiol compounds GSH, Cys and Trx, respectively.80, 237

The experiments undertaken in this thesis have investigated the reactions of various diselenides [selenocystamine (SeCA), 3,3’-diselenodipropionic acid (DSePA), 1-(9-fluorenyl)methyl chloroformate-tagged selenocystine (Fmoc-SeCystine), seleno-diglutathione (GSeSeG), 2,2’- dipyridyl diselenide (DPDSe) and dipyrimidin-2-yl diselenide (DPMSe), see Figure 3.1.] with

HOCl, H2O2 and photochemically-generated hydroperoxides (ROOH) of Trp, Tyr, His, LysTrp, TrpLys and LysTrpLys in order to evaluate the antioxidant capacity of the diselenides. The rate constants determined for the reaction of various diselenides with biological oxidants

(HOCl, H2O2 and TrpOOH) are given in Table 6.1, and those of SeCA with a range of peroxides in Table 6.2.

Table 6.1: Absolute rate constants (k) for the reaction of various diselenides with biological oxidants

Diselenide HOCl k ± CI H2O2 k ± CI TrpOOH k ± CI (M-1 s-1) a (M-1 s-1) b (M-1 s-1) c SeCA (3.6 ± 0.6) x 105 (9.2 ± 0.4) x 10-4 (4.5 ± 0.2) x 10-2 DSePA (1.8 ± 0.2) x 105 (1.5 ± 0.2) x 10-2 (1.9 ± 0.1) DPMSe (3.9 ± 0.8) x 103 ND (1.3 ± 0.5) x 10-2 DPDSe (6.9 ± 1.2) x 104 ND (1.1 ± 0.3) x 10-2 GSeSeG ND ~ 1 x 10-3 ND

Data obtained by; a competition kinetics (Section 2.10.2), b UV/vis spectroscopy (Section 2.8.1), c FOX assay (Section 2.6). ND = not determined. Error is expressed as 95% CI.

188

Table 6.2: Absolute rate constants (k) for the reaction of SeCA with various peroxides

Peroxide SeCA k ± CI (M-1 s-1)

-2 H2O2 (1.1 ± 0.5) x 10

-4 tBuOOH (1.4 ± 0.1) x 10 CHP (5.4 ± 1.0) x 10-4 TrpOOH (4.5 ± 0.2) x 10-2 TyrOOH (4.0 ± 0.3) x 10-2 HisOOH (6.3 ± 1.3) x 10-2 LysTrpOOH (2.1 ± 0.1) x 10-2 TrpLysOOH (2.2 ± 0.1) x 10-2 LysTrpLysOOH (1.8 ± 0.2) x 10-2

Rate constants determined by FOX assay (Section 2.6). Error is expressed as 95% CI.

6.5.1. Oxidation of Diselenides by Hydrogen Peroxide

The oxidation of SeCA by H2O2 returns two different k values, depending on the methodology used (FOX assay or UV/vis spectroscopy; cf. Table 6.1 and 6.2). For the FOX assay, hydroperoxide samples were prepared in unbuffered H2O (pH 7.2 – 7.4) prior to addition of the acidic working solution and the assay indirectly measured the loss in total peroxide absorbance. Conversely, the UV/vis spectroscopy samples were prepared in buffered solution (pH 7.4) and this method directly measured the loss in diselenide bond absorbance. The difference in the rate constants obtained is attributed to the pH variation and these two methods measuring different processes. As such, the UV/vis spectroscopy method is believed to yield a more accurate rate constant of reaction.

Oxidation of SeCA by H2O2 resulted in loss of the diselenide bond with a rate constant of reaction, k = (9.2 ± 0.4) x 10-4 M-1 s-1, with the products characterised by LC/MS to be the seleninic (RSeO2H) and selenonic acids (RSeO3H), postulated to form via oxidation of the diselenide bond and its subsequent cleavage. It is suggested that the RSeO2H species slowly undergoes further oxidation to the RSeO3H derivative, which was detectable following 3 days of incubation post-oxidant addition. The slow generation of the RSeO3H species is consistent with UV/vis spectroscopy data which displayed an isosbestic point, indicating formation of only a single oxidation product (RSeO2H) over the shorter experimental time-course (16.5 h).

189

Oxidation of DSePA and GSeSeG by H2O2 yielded rate constants of reaction, k = (1.5 ± 0.2) x 10-2 and ~ 1 x 10-3 M-1 s-1, respectively, with the products of the latter reaction characterised by LC/MS to be the analogous di-oxygenated seleninic acid (GSeO2H), with no evidence to suggest formation of the mono- or tri-oxygenated species (GSeOH or GSeO3H). Similarly, the oxidation of Fmoc-SeCystine by H2O2 generated RSeO2H without detection of other species

(RSeOH/RSeO3H). It is suggested that diselenide bond oxidation occurs via the generation of a transient mono-oxygenated diselenide intermediate [RSeSe(=O)R’] with subsequent reaction giving a di-oxygenated diselenide intermediate [RSeSe(=O)2R’] preceding diselenide bond cleavage to yield the stable RSeO2H derivative. The data are consistent with diselenide oxidation occurring via an analogous pathway to that of disulfide oxidation, whereby oxygen addition precedes -S-S- bond cleavage.176 However, unlike the corresponding sulfinic acids

(RSO2H) which rapidly undergo further oxidation to the sulfonic acids (RSO3H), the analogous seleninic acids (GSeO2H/RSeO2H) are suggested to be resistant to further oxidation by H2O2, 79, 105, 176 reacting only very slowly to yield the selenonic acids (GSeO3H/RSeO3H). UPLC analysis of the H2O2-induced oxidation of Fmoc-SeCystine revealed a major stable oxidation product believed to be the RSeO2H species, as well as a minor product with low fluorescence yield, consistent with the very limited formation of RSeO3H, which was not detectable by

LC/MS. These species were both formed in a time- and H2O2-dose dependent manner.

The steric bulk of GSeSeG and large aromatic Fmoc groups of Fmoc-SeCystine were postulated to provide protection to the Se moiety of these compounds, whereas SeCA is a simple aliphatic species which lacks secondary groups which may sterically inhibit further oxidation; this may account for the detection of RSeO3H upon oxidation of SeCA, whilst the analogous selenonic acids were not detected with GSeSeG or Fmoc-SeCystine.

6.5.2. Oxidation of Diselenides by Reagent and Photochemically- Generated Peroxides

Singlet oxygen reacts preferentially with the aromatic and sulfur-containing side chains of Trp, Tyr, His, Met and Cys residues, which react at physiological pH values with k = 106 – 107 M-1 s-1, resulting in formation of endoperoxides and zwitterionic species as the major products, which can undergo further reaction via radical and ring-opening mechanisms to ultimately induce protein aggregation.37, 257 Photo-oxidation of Trp, Tyr and His, as well as the peptides 1 LysTrp, TrpLys and LysTrpLys, resulted in generation of O2 and subsequent formation of

190 amino acid/peptide hydroperoxides. The k values reported in Table 6.2 demonstrate that the variety of peroxides investigated react relatively slowly with SeCA, with an approximate 3- fold difference in rate constant across the range of photochemically-generated amino acid and -2 -1 -1 peptide peroxides (k = 1.8 – 6.3 x 10 M s ), with a comparable rate of reaction with H2O2 determined via this method (1.1 x 10-2 M-1 s-1) and much slower rates with tBuOOH and CHP (ca. 100-fold).

These data suggest that the accessibility of the peroxide bond plays a role in determining the reactivity of each species with the diselenide, a factor which is heavily dependent upon the secondary structure of the amino acid or peptide. The relatively bulky secondary structure of CHP (aromatic) and tBuOOH (aliphatic) may limit the accessibility of the peroxide bond, thus, decreasing the rate of reaction compared to the amino acid/peptide hydroperoxides, which typically form the hydroperoxide on the exposed aromatic ring moiety.37 Consequently, the favourable secondary structure of the amino acid hydroperoxides results in rate constants with

SeCA that are similar to H2O2 yet much higher than those of tBuOOH and CHP.

The hydroperoxides of LysTrp, TrpLys and LysTrpLys each displayed a ~ 2-fold decrease in reactivity with SeCA relative to that of free Trp. This is presumably due to the introduction of steric hindrance from the Lys groups, lessening the accessibility of the peroxide moiety to attack by the diselenide. Another factor which may be affecting reactivity is the electrostatic repulsion between the Lys side chains and SeCA, both of which hold a positive charge. This effect was observed during the dimerisation of Trp radicals in the presence of nearby Lys side- chains.258

Addition of a Lys residue to Trp at either the amine group (LysTrp) or the carboxyl group (TrpLys), or both (LysTrpLys; see Chapter 5, Figure 5.1), did not vastly affect the rate of reaction between the peptide species (k = 1.8 – 2.2 x 10-2 M-1 s-1), indicating that neither the amine nor carboxylic acid group of the Trp residue play a significant role in determining the kinetics of reaction, as evidenced by Lys binding at either site yielding similar rate constants. The addition of a Lys group is predicted to change the electronic structure of the amino acid, affecting the site of initial reaction due to hydrogen abstraction (and other factors),215 but this had little effect on the rate constants of reaction with SeCA between the Lys-bound peptides.

The rate constants reported in Table 6.1 show that DSePA reacts with H2O2 ca. 20-fold faster than both SeCA and GSeSeG. Similarly, DSePA reacted with photochemically-generated TrpOOH much more rapidly than did the other diselenides investigated, with k = (1.9 ± 0.1)

191

M-1 s-1, compared to k = (4.5 ± 0.2) x 10-2, (1.3 ± 0.5) x 10-2 and (1.1 ± 0.3) x 10-2 M-1 s-1 determined for SeCA, DPMSe and DPDSe, respectively. These data indicate that the nature of the secondary structure of the diselenide plays a key role in determining its reactivity with each oxidant. This is consistent with studies investigating the antioxidant capacity of water-soluble aliphatic selenoethers, which showed that substitution of a -COO- group increased the GPx- like activity and free radical scavenging ability of the compound as a result of the electron- donating nature of this substituent, which elevates the energy level of the highest occupied molecular orbital, making the selenium reaction centre more nucleophilic, and thus, more 259 + reactive. Substitution of an alcohol (-OH) or amine (-NH3 ) group to the selenoether decreased this energy level, respectively, and it was suggested that electron-donating groups destabilise the reactive selenium centre, whereas electron-withdrawing groups stabilise the selenium, supporting the hypothesis that oxidation of the selenium moiety is the most crucial aspect in determining the antioxidant activity of these compounds.259

6.5.3. Oxidation of Diselenides by Hypochlorous Acid

The studies reported in this thesis have determined a rate constant of reaction for the oxidation of coumarin boronic acid (CBA) with HOCl, with stopped-flow experiments yielding k = (4.2 ± 0.1) x 104 M-1 s-1. This value is intermediate between the reported rate constants of reaction - 6 -1 -1 of CBA with ONOO and H2O2, which are k = (1.1 ± 0.2) x 10 and 1.5 ± 0.2 M s , respectively.230 CBA is a pro-fluorescent probe that has recently been applied in an assay for the real-time detection of amino acid and protein hydroperoxides, using photochemically generated TyrOOH as a model peroxide for investigation, giving k = 15.4 ± 0.3 M-1 s-1, which is ca. 10-fold faster than the CBA/H2O2 reaction and 4 orders of magnitude slower than the CBA/HOCl reaction reported here.230

Using this data, rate constants were obtained for the reactions of DSePA and SeCA with HOCl, via a competition kinetics approach using CBA, giving k = (1.8 ± 0.2) x 105 and (3.6 ± 0.6) x 105 M-1 s-1, respectively. Competition kinetics approaches typically use at least ca. 5-fold excesses of reactant and scavenger over oxidant to give true pseudo first-order kinetics, so that the change in concentrations of the competing reactants are negligible as the reaction proceeds.69 However, it must be noted that the conditions of this study used equal concentrations of reactant (CBA) and oxidant (HOCl), and the scavenger (diselenide) was present in excess.

192

The k values reported in Table 6.1 for the reaction of diselenides with HOCl show significant variation (ca. 100-fold) depending upon the species of diselenide, and thus, upon its secondary structure. The linear diselenides (SeCA and DSePA) gave comparable rate constants (k ~ 105 M-1 s-1) whilst the diselenides containing aromatic functional groups (DPMSe and DPDSe) showed less reactivity, as evidenced by slower reactions (k = 3.9 x 103 and 6.9 x 104 M-1 s-1, respectively; Table 6.1) and a lesser overall extent of oxidant scavenging. These rates of reaction are too slow to compete effectively with the rapid oxidation of Cys and Met by HOCl (k ~ 108 M-1 s-1), but may provide protection to other amino acids (His, Trp, Tyr) and amine groups which demonstrate comparable (or significantly slower) rate constants (k ≤ 105 M-1 s- 1).6, 69 The presence of the aromatic groups will decrease the electron density of the diselenide bond, thus decreasing its reactivity. Also, the structural conformation of the aromatic 6- membered rings may induce a non-favourable electronic interaction with the diselenide bond, decreasing its reactivity.176

It is believed that factors such as the size of the aliphatic chain, steric hindrance and potential Se-heteroatom interactions are key in determining the reactivity of diselenides.205 This is supported by studies investigating the oxidation of disulfides by hypohalous acids (HOCl, HOBr, HOSCN), which showed significant variation in rate constant between cyclic and acyclic species, with increased reactivity attributed to appropriately-positioned heteroatoms providing stabilisation to the disulfide moiety.176 Favourable intramolecular interactions increased the reactivity of five-membered ring structures up to 20,000-fold that of the acyclic disulfides, and up to 3,000-fold that of the six-membered rings, with favourable structural conformations facilitating electron donation from the sulfur lone pair due to better alignment of the orbitals, decreasing the overall reaction energy.176 For acyclic disulfides, increases in rate constant were attributed to the interaction of suitably positioned electron-donor groups with the positively-charged sulfur reaction centre, acting to stabilise the molecule.176 The rate constant of reaction of 3,3’-dithiodipropionic acid (DTDPA; the S analogue of DSePA) increased with deprotonation of the neutral carboxylic acid groups, an effect which is much less marked for the amine groups of cystamine (the S analogue of SeCA).176

130, 208 The pKa of the oxidising species (11.75 vs 7.6 for H2O2 and HOCl, respectively) is also suggested to play a key role in reaction rate as this affects the ionisation state of the species at neutral pH; e.g. at pH 7.4 HOCl is present as a mixture of ca. 1:1 HOCl to OCl-, the former of which is more reactive.208 Therefore, these reactions are significantly pH-dependent due to the protonation/deprotonation equilibria of both the oxidant and the disulfide substrate.176 The

193 comparatively slow reactivity of H2O2 relative to HOCl with disulfide/diselenide compounds may also be partially attributed to HO- being a poor leaving group relative to Cl-. The similarities between sulfur and selenium suggest that, like the corresponding disulfides, the adjacent structure of acyclic (e.g. SeCA, DSePA) and aromatic-substituted (e.g. DPDSe, DPMSe) diselenides also plays a major role in determining the reactivity of the diselenide moiety and factors such as protonation and bond stabilisation by nearby heteroatoms are important considerations in the study and development of these compounds, as the microenvironment of these species markedly affects their reactivity.176

The oxidation of Fmoc-SeCystine by HOCl resulted in the oxidant dose-dependent generation of a major and a minor oxidation product observable by UPLC, believed to be the RSeO2H and

RSeO3H derivatives, respectively, with only the former being detected and characterised by

LC/MS. Similarly, oxidation of GSeSeG by HOCl resulted in formation of GSeO2H. LC/MS analysis gave no evidence for the formation of the mono- or tri-oxygenated selenenic or selenonic acids for either diselenide, suggestive of the very limited formation of the RSeO3H species. These results are consistent with H2O2 data, above, indicating that both HOCl and

H2O2 generate the seleninic acid derivatives upon oxidation of both diselenides. Furthermore, the oxidation of Fmoc-SeCystine by TrpOOH also yielded this product, indicating that all three oxidants (HOCl, H2O2, TrpOOH) generate the RSeO2H derivative.

The major detectable oxidation product of each of the diselenides investigated was characterised to be the seleninic acid derivative (RSeO2H). This is in contrast to the major HOCl-induced oxidation products of the analogous disulfide species, suggested to be the thiosulfinate [RSS(=O)R’, disulfide S-oxide] and thiosulfonate [RSS(=O)2R], with further reaction generating the sulfonic acid (RSO3H) concomitant with cleavage of the disulfide 175, 176, 228 bond. The analogous selenium species [RSeSe(=O)R and RSeSe(=O)2R] were not detected, which suggests that the diselenide bond readily fragments following oxidation, or that these species, if formed, are transient intermediates which rapidly undergo further reaction to yield the RSeO2H derivative.

Based on these data and analogy with disulfide chemistry, it is suggested that the diselenides investigated are oxidised to form a transient mono-oxygenated intermediate [RSeSe(=O)R], which undergoes rapid secondary oxidation of the diselenide bond to form a di-oxygenated intermediate [RSeSe(=O)2R] with subsequent diselenide bond cleavage yielding detectable

RSeO2H. The RSeO2H derivative is postulated to be resistant to further oxidation to the tri-

194 oxygenated species (RSeO3H), which accounts for the lack of RSeO3H detected by LC/MS.

This is in contrast to the reported formation of the analogous sulfur species (RSO3H) which is 105 readily formed upon oxidation of the corresponding sulfinic acid (RSO2H). It is also suggested that the selenonic acid of GSeSeG (GSeO3H) was not detected because, if formed, - this species may eliminate the selenate (SeO3 ) moiety to generate dehydroalanine (DHA), which is not detectable by the LC/MS method used here, which relied primarily upon detection of a selenium isotope pattern.139

6.6. Comparison of the Reactions of Sulfur- and Selenium-Containing Compounds with Various Biological Oxidants

Rate constants have been reported for the reaction of HOCl, HOSCN and ONOOH with various selenium species, which have consistently demonstrated greater rate constants of reaction than those reported for the analogous sulfur compounds, some of which are summarised in Table 6.3.39, 69, 77, 104, 174, 222, 260 The rate constants of reaction determined for many of the selenium species are sufficiently high as to be kinetically competitive substrates of ROS in a biological setting, having the potential to mitigate oxidative stress and prevent the onset or progression of inflammatory pathologies.

195

Table 6.3: Comparison of rate constants (k) for the reactions of sulfur compounds (and some of their selenium analogues) with biological oxidants.

Substrate HOCl k HOSCN k ONOOH k (M-1 s-1) (M-1 s-1) (M-1 s-1) Cys 3.1 x 108 69 7.8 x 104 77 2.6 x 103 39 Sec ND 1.2 x 106 222 6.6 x 105 39 Cystamine 2.6 x 105 176 7.8 x 104 77 2.3 x 103 39 SeCA 3.6 x 105 ^ ND 7.2 x 102 39 DTDPA 1.7 x 105 176 1.9 x 103 176 ND DSePA 1.8 x 105 ^ ND 1.3 x 103 39 Met 3.4 x 107 69 < 103 77 1.6 x 102 39 SeMet 3.2 x 108 174 2.8 x 103 222 2.5 x 103 39 Ebselen ND 3 x 101 222 2 x 106 260 GPx ND 5 x 105 222 8 x 106 104 SeTal 1.0 x 108 174 ND 2.5 x 103 39 SeGul 9.4 x 107 174 ND 2.5 x 103 39 GSH 1.1 x 108 69 2.5 x 104 77 7.3 x 102 39 GSSG 1.4 x 105 176 6.7 x 102 176 ND

ND = not determined; ^ Determined in Chapter 3.

Exchange reactions are common in systems which contain both sulfur and selenium, and the formation of a selenenylsulfide intermediate (RSeSR) is common during the catalytic cycle of selenoenzymes (e.g. GPx, see Figure 1.2).103 Sulfur and selenium can undergo similar chemical exchange reactions and it has thus been proposed that the selenium atom can accelerate thiol/disulfide exchange reactions such as those catalysed by selenoenzymes by; a) increasing the nucleophilicity of an attacking thiolate (RS-), b) increasing the electrophilicity of reactive sulfur centre, and c) increasing the stability of the leaving group sulfur atom.177 Studies comparing thiol/disulfide and selenol/diselenide exchange reactions using SeCA and cystamine (and their selenol/thiol derivatives; Figure 6.2) concluded that the substitution of selenium enhanced the nucleophilic and electrophilic exchange reactions by 2-3 and 4 orders of magnitude, respectively, relative to sulfur, and that selenium and sulfur are comparable as leaving groups.103 Rate constants were determined for the exchange reactions of 5 7 SeCA/cystamine and the corresponding selenol/thiol to be k1 = 1.3 x 10 , k-1 = 2.6 x 10 , k2 =

196

-3 -1 -1 103 11 and k-2 = 1.4 x 10 M s (Figure 6.2), as measured by stopped-flow spectrophotometry. Additionally, this study determined that the selenenylsulfide species had a lower electrode potential than the corresponding disulfide, indicating that the former are more difficult to reduce.103

Figure 6.2: Thiol/disulfide and selenol/diselenide exchange reactions. Image adapted from Steinmann, Nauser and Koppenol (2010)103

Other physical properties of selenium can increase these rate constants of reaction, including the longer length of the Se-S bond relative to the S-S bond, rendering the former more liable 177 to diatomic cleavage. The pKa of the selenol (5.3) relative to the thiol (8.3) also highlights a strong pH-dependence of the reaction and suggests that the microenvironment of a protein plays a key role in influencing the kinetics of the reaction.157, 177, 251

6.6.1. Disulfides and Diselenides

The oxidation of disulfides and diselenides is characteristically slower than oxidation of the corresponding reduced thiol/selenol and thioether/selenoether species.79 Disulfides typically react slowly with many two-electron oxidants (e.g. ONOOH, HOSCN); however, the reactions of HOCl with cystine (the disulfide of Cys) and with 3,3’-dithiodipropionic acid (DTDPA) and cystamine (the sulfur analogues of DSePA and SeCA, respectively) occur relatively rapidly, each having k ~ 2 x 105 M-1 s-1 (Table 6.3).71, 176 These values are comparable to the rate constants obtained in the present study for the reactions of DSePA and SeCA with HOCl (k ~ 105 M-1 s-1, Table 6.1). It has not yet been established if the thiosulfinates [RSS(=O)R’] that result from disulfide oxidation can be directly repaired, although these species can undergo further reaction to give the sulfenic acid (RSOH), which can be repaired.175, 176

Seleno-diglutathione (GSeSeG; the Se-analogue of GSSG) has been investigated as a GPx mimetic, which can be reduced to selenoglutathione (GSeH) by NADPH in the presence of

197

90, 197 glutathione reductase (GR). In the presence of excess GSH, reaction of a 3:1 ratio of H2O2 to GSeSeG yielded 2 molecules of the seleninic acid (GSeO2H) via formation of transient selenenic acid (GSeOH) intermediate, concomitant with the generation of the selenenylsulfide species (GSeSG).199 However, GSeSG was unable to be reduced by GSH to regenerate GSeH, due to the GSH lacking sufficient reducing potential to cleave the Se-S bond.199 Despite the presence of the intrinsically more reactive Se centre, the S atom in GSeSG is kinetically favoured by attack with a thiol substrate (e.g. DTT) to cleave the Se-S bond and generate a disulfide and RSe-, suggesting that the secondary structure of the diselenide and presence of other species in the reaction mixture has the potential to modify the rate of reaction and potentially, the products formed, thereby altering the antioxidant ability of certain selenocompounds.200 These data demonstrate that thiol/disulfide and selenol/diselenide exchange reactions have the potential to modulate the reactivity of certain species, and these reactions are important to consider when designing potential antioxidant compounds for application in a system that contains both sulfur- and selenium-containing residues, such as those present in biological systems.

Furthermore, GSeH displayed greater potential than GSH as an oxidant scavenger, with rate constants of reaction with HOSCN determined to be k = 1.7 x 106 M-1 s-1 and k = 2.5 x 104 M- 1 s-1 for the selenium and sulfur derivates respectively, representing a ca. 70-fold increase in rate constant due to substitution of the selenium moiety.77, 79 Oxidation of GSeH generates GSeSeG which can be reduced to by GR (in the presence of NADPH) in the same manner as GSH, indicating that GSeH may act as a more efficient antioxidant than GSH in vivo.197

To date, the cellular concentrations of diselenides required to afford protection to cells against oxidative damage remains unclear. Thiol concentrations within cells are in the millimolar range (e.g. GSH is typically 2 – 10 mM and protein thiols can reach 40 mM),84, 85 so given the rate constants for the scavenging of HOCl and peroxides by diselenides obtained within this thesis, it may be that millimolar concentrations of the diselenides are necessary for competitive oxidant scavenging. However, within cells diselenides can be reduced via multiple pathways to yield the corresponding selenols, which are much more reactive than the parent diselenide and have rate constants that are consistently 10- to 100-fold higher than those of the analogous thiol species39, 136, 141, 174, 253. This suggests that lower concentrations of diselenides may be required for effective oxidant scavenging. Furthermore, the selenol-diselenide pair are able to redox cycle and may therefore act in a catalytic manner to remove oxidants, indicating that millimolar concentrations of diselenide may not be necessary for cellular protection. It is

198 unclear how readily diselenides enter cells and what concentrations can be achieved; although the monoselenides (e.g. SeTal) can enter cells very rapidly and be detected at steady-state levels in the micromolar range, at which concentration they do provide protection against HOCl- induced damage (T. Zacharias, unpublished data).

6.6.2. Thiols and Selenols

The reduced selenol forms of SeCA (SeHCH2CH2NH2) and DSePA (SeHCH2CH2COOH) were prepared by reduction of each diselenide by addition of a 10-fold excess of NaBH4 under an N2 or argon atmosphere, to prevent the selenols from rapidly auto-oxidising under atmospheric oxygen concentrations.261 The rate constants of reaction with ONOOH were significantly elevated for the selenol form of each compound relative to the diselenide, increasing from k = 7.2 x 102 and 1.3 x 103 M-1 s-1 for SeCA and DSePA, respectively, to 1.9 x 106 and 5.1 x 105 M-1 s-1 for the respective selenols.39 ONOOH reacted relatively slowly with 4 -1 -1 diphenyl diselenide [(PhSe)2] (k2 < 10 M s ), but more rapidly with the reduced selenol form 5 -1 -1 (selenophenol; k2 = 2.7 x 10 M s ), which exhibited GPx-like activity to protect against ONOOH-mediated endothelial cell death.39, 262 The selenol forms of SeCA and DSePA also reacted rapidly with HOSCN, with k = 5.8 x 106 and 2.0 x 106 M-1 s-1, respectively, which are at least 10-fold faster than the rate constants of the corresponding thiols (7.1 x 104 – 1.6 x 105 M-1 s-1).77, 222 These data highlight the favourable reaction kinetics of selenol compounds as competitive scavengers of biological oxidants. However, the rapid auto-oxidation of selenols in atmospheric conditions highlights the prudency of initially implementing the diselenide form, which must then undergo in situ reduction to yield low steady-state levels of the more reactive selenol for enhanced effectiveness in a therapeutic application.79

6.6.3. Synthetic Antioxidants

Competition kinetics studies have shown that selenium-containing sugar derivatives [e.g. 1,4- dideoxy-4-seleno-L-talitol (SeTal) and 1,5-dideoxy-5-seleno-L-gulitol (SeGul)] react with HOCl (k ~ 108 M-1 s-1; Table 6.3) considerably faster than the analogous sulfur-sugars (k ~ 106 M-1 s-1), thereby decreasing the extent of oxidation of Met, His, Trp, Lys and Tyr residues by acting as competitive substrates.174 These water-soluble novel seleno-sugars therefore have the potential to act as scavengers of HOCl to modulate acute and chronic inflammation-induced damage in multiple human pathologies.174

199

A model of synthetic selenoenzyme generation includes a ‘dendrimer’ model containing a catalytic diselenide core, which facilitates the GPx-like reduction of H2O2 (2 mM) in the presence of PhSH (1 mM), at a rate constant reaching a ca. 1400-fold increase over that of ebselen under similar experimental conditions (3:7 chloroform/methanol).263 This is attributed to the favourable microenvironment of the synthetic diselenide, which demonstrates good binding affinity to the substrate.263 These works provide a basis for the use of synthetic (or chemically-modified) selenocompounds as effective GPx-mimics, yet further refinement is required to apply these techniques to more physiologically-relevant conditions by increasing water solubility whilst simultaneously retaining a high reactivity.

Dihydroxy-1-selenolane (DHS) is an organoselenium compound consisting of a five- membered ring structure with a monoselenide moiety which has shown radioprotective and anti-inflammatory effects in vivo, whilst proving to be non-toxic and water-soluble.165-167 DHS exhibits biological activities including free-radical scavenging, catalysing the folding of denatured proteins and acting as a mimetic of GPx,264 suggesting that this compound is a good candidate for further research into its potential use as an anti-inflammatory supplement.

It becomes apparent that the pharmacochemical properties of potential anti-inflammatory selenocompounds require thorough investigation in the development of selenium-containing species, to maximise GPx-like (or TrxR-like) activity whilst maintaining therapeutic viability by minimising metabolic toxicity and maximising water-solubility, for example.

6.7. Future Directions of Research

The selenocompounds investigated in this study should be examined following oxidation by other ROS (ONOOH, radicals, HOSCN, etc) to compare the rate constants of reaction and products formed. Other selenium species (e.g. cyclic diselenides, 5- and 6-membered ring structures) should also be investigated with a range of oxidants to examine the effect of side chain length and composition on diselenide reactivity. The pH-dependence of the reactions between various diselenides and oxidants should also be investigated.

Further LC/MS analysis of both Fmoc-SeCystine and GSeSeG products should be performed to identify other selenium analogues of well-characterised sulfur species [e.g. dehydroalanine (DHA) analogues, in which selenium is absent] and the selenium analogue of glutathione sulfonamide (GSA).87 Other methods of detecting transient mono- and di-oxygenated - diselenides [RSeSe(=O)R’ and RSeSe(=O)2R’] and RSeO2 intermediates should also be

200 attempted to identify if these species are formed during oxidation, such as time-resolved studies and Raman spectroscopy. Further experiments should also aim to determine if these species are reducible and to calculate rate constants of reduction in the presence of various reductants.

The selenenic acid species (RSeOH), like their sulfur counterparts, are presumed to be unstable in the presence of acids and the lack of a characteristic UV-visible absorbance profile and fluorescence properties makes them difficult to detect.57 Identification of any unstable intermediates should be performed at physiological pH 7.4 and facilitated by using chemical traps that can potentially stabilise these species, such as N-ethylmaleimide (NEM) and dimedone, allowing detection by MS. Dimedone and NEM react selectively with the sulfur atom in RSOH and RSH groups, respectively, to form stable thioether (RSR’) products, and thus, could potentially trap unstable selenenic acids (e.g. GSeOH) and selenols (e.g. GSeH) by the same mechanism.265-267 This chemical trapping method is necessary due to the transitory nature of the intermediates making them difficult to detect.

Furthermore, recent advances have been made in stabilising sulfenic acids (RSOH) for detection, by sterically or electronically impeding the formation of inter- or intra-molecular hydrogen bonding, which initially causes formation of the thiosulfinate [RSS(=O)R’].250 For example, the addition of electron-withdrawing groups adjacent to the sulfenic acid moiety decreased the nucleophilicity of the sulfur centre, stabilising the aromatic molecule in its mono- oxygenated (RSOH) form.250 These methods may potentially be applied to the stabilisation and subsequent detection of the corresponding selenenic acids (RSeOH). Further experiments should also be performed examining the potential regeneration of the selenol or diselenide species from the seleninic acid products using thiol reductants (e.g. GSH) to fully elucidate the stoichiometry of these reactions under various experimental conditions that reflect physiological environments.

The diselenides investigated in this thesis could potentially be applied to an ex vivo model of protein oxidation, in an attempt to modulate the extent of HOCl-induced Tyr oxidation in human blood, by acting as competitive scavengers of HOCl, as the rate of reaction of HOCl with Tyr is very slow (k = 44 M-1 s-1) relative to all the diselenides investigated (k ≥ 103 M-1 s- 1).69, 268 The ability of diselenides to modulate the extent of ONOOH-mediated oxidation of Tyr residues in human tropoelastin also warrants investigation.269

Studies examining the in vitro antioxidant properties of diselenides should be performed by investigating the effect of diselenide species and concentrations on oxidative stress markers

201

•- (e.g. O2 , protein carbonylation and lipid peroxidation) in human blood samples under conditions of oxidative stress (e.g. in the presence of ONOO- or Fe2+).270 These results may be compared to the antioxidant properties of synthetic disubstituted diselenides [bis(2- hydroxyphenyl) diselenide, bis(2-aminophenyl) diselenide, etc] and ebselen, characterised previously.270

It is suggested that DSePA may be an appropriate candidate for in vivo studies owing to the more rapid rates of reaction with both HOCl and H2O2 elucidated herein, as well as this diselenide showing antioxidant properties without inducing toxicity in an animal model.193 However, the physicochemical properties of the diselenides should be investigated thoroughly (i.e. water solubility, toxicity to cells and tissues, bioavailability and metabolism) prior to performing any in vivo studies.

6.8. Conclusions

It is apparent that many sulfur- and selenium-containing species are critical for the maintenance of redox homeostasis within biological systems, and the presence of the selenium atom often results in favourable reaction kinetics to drive enzymatic activity and generate products which are biologically reversible. As such, selenocompounds represent a potential mechanism for protection against oxidative stress- and inflammation-associated pathologies induced by one- and two-electron oxidation events. The efficacy of these compounds relies upon numerous influences including substrate specificity, pKa value (and pH dependency), the presence of non- bonding interactions between the reactive selenium centre and intramolecular heteroatoms, and physicochemical factors such as metabolism, bioavailability and toxicity.

The studies presented in this thesis have outlined a potential role of diselenide compounds as potent shelf-stable alternatives to selenols as scavengers of some biological oxidants; HOCl, 1 H2O2 and O2-derived amino acid/peptide hydroperoxides. SeCA and DSePA (and to a lesser extent, DPMSe and DPDSe) reacted relatively rapidly with HOCl, yielding rate constants that are fast enough to be kinetically competitive with certain amino acid residues (e.g. His, Trp, Tyr), amine groups and disulfides, but are not fast enough to be competitive with the rapid HOCl-induced oxidation of Cys or Met residues.

Oxidation of Fmoc-SeCystine and GSeSeG by both HOCl and H2O2 were shown to result in generation of the seleninic acid as the major stable detectable product, which is postulated to arise via formation of a mono-oxygenated intermediate preceding diselenide bond cleavage.

202

-4 Rate constants were determined for the oxidation of SeCA and DSePA with H2O2 (k ~ 10 and 10-2 M-1 s-1, respectively) and approximated for GSeSeG (k ~ 10-3 M-1 s-1), which revealed DSePA to react at a rate approaching ca. 20-fold faster, likely due to the presence of the terminal carboxyl groups.

The reaction of SeCA with a range of photochemically-generated peroxides was investigated, each yielding rate constants which were comparable to those obtained for the reaction of DPDSe and DPMSe with a model hydroperoxide, TrpOOH (k ~ 10-2 M-1 s-1). However, reaction of TrpOOH with DSePA gave a rate constant ca. 100-fold faster than the other diselenides investigated, further highlighting the importance of secondary structure on the reactivity of the diselenide moiety. Further research is required to elucidate the efficacy of these compounds as scavengers of biological oxidants within more complex in vitro systems, and to characterise any additional oxidation products not detected in the current study.

The data presented in this thesis have contributed to a wider understanding of the chemistry of low-molecular-mass diselenide compounds as potential scavengers of biologically relevant oxidants, by determining rate constants of reaction and characterising the products of oxidation. These findings may assist the future development of selenium-containing compounds as antioxidants in an inflammatory context, where these species have the potential to modulate oxidative damage and decrease inflammatory pathologies.

203

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