Open GYK Dissertation Final.Pdf

The Pennsylvania State University

The Graduate School

College of Agricultural Sciences

REACTION MECHANISMS OF TRANSITION METALS WITH

HYDROGEN SULFIDE AND THIOLS IN WINE

A Dissertation in

Food Science

Gal Y. Kreitman

 2016 Gal Y. Kreitman

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

August 2016

The dissertation of Gal Y. Kreitman was reviewed and approved* by the following:

Ryan J. Elias Associate Professor of Food Science Dissertation Advisor Chair of Committee

Joshua D. Lambert Associate Professor of Food Science

John N. Coupland Professor of Food Science

Michela Centinari Assistant Professor of Horticulture

David W. Jeffery Senior Lecturer in Wine Science Special Member

John C. Danilewicz Special Signatory

Robert F. Roberts Professor of Food Science Head of the Department of Food Science

*Signatures are on file in the Graduate School

ABSTRACT

Sulfidic off-odors due to hydrogen sulfide (H2S) and low molecular weight thiols are commonly encountered in wine production. These odors are a serious quality issue in wine and may result in consumer rejection. Therefore, sulfidic off-odors are generally controlled prior to bottling, and are frequently removed by the process of Cu(II) fining – a process that remains poorly understood. Cu(II) is effective at binding with sulfhydryl functionalities and forming nonvolatile complexes thereby removing aroma associated with the compound. However, this technique leaves residual copper in the wine which catalyzes non-enzymatic wine oxidations. Furthermore, elevated copper concentrations are usually associated with increased sulfidic off-odors under anaerobic aging conditions.

In this work, I elucidated the underlying mechanisms by which Cu(II) interacts with H2S and thiol compounds under wine-like conditions. Adding Cu(II) sulfate to air saturated model wine containing H2S, cysteine (Cys), 6-sulfanylhexan-1-ol (6SH), or 3-sulfanylhexan-1-ol (3SH) led to a rapid formation of ~1.4:1 H2S:Cu and ~2:1 thiol:Cu complexes. This resulted in the oxidation of

H2S and thiols, and reduction of Cu(II) to Cu(I) without oxygen uptake. Both H2S and thiols resulted in the formation of Cu(I)-SR complexes, and subsequent reactions with oxygen led to the oxidation of H2S rather than the formation of insoluble copper sulfide, which has been previously assumed. The proposed reaction mechanisms provide an insight into the extent to which H2S can be selectively removed in the presence of thiols in wine.

The interaction of iron and copper is also known to play an important synergistic role in mediating non-enzymatic wine oxidation. Therefore, I assessed the interaction of these two metals in the oxidation of H2S and thiols (Cys, 6SH, and 3SH) under wine-like conditions. H2S and thiols were shown to be slowly oxidized in the presence of Fe(III) alone, and were not bound to Fe(III) under model wine conditions. However, Cu(II) added to model wine containing Fe(III) was quickly

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reduced by H2S and thiols to form Cu(I)-complexes, which then rapidly reduced Fe(III) to Fe(II).

Oxidation of Fe(II) in the presence of oxygen regenerated Fe(III) and completed the iron redox cycle. This work clearly demonstrated a synergistic effect between Fe and Cu during the oxidation of H2S and thiols. In addition, sulfur-derived oxidation products were observed, and the formation of organic polysulfanes was demonstrated for the first time under wine-like conditions.

Manganese has a modest activity in catalyzing polyphenol and sulfite oxidation in wine.

Furthermore, manganese is known to have a catalytic activity at mediating thiol and H2S oxidation in aquatic systems. Thus, the interaction of manganese with iron and copper was investigated in relation to thiol and H2S oxidation in model wine. The reaction of thiols with Mn alone or in combination with Fe resulted in radical chain reaction paired with large oxygen uptake and generation of sulfur oxyanions. H2S did not generate free thiyl radicals, and had minimal interaction with Mn(II). When Cu(II) was introduced, Cu-mediated oxidation dominated in all treatments and

Mn-mediated radical reaction was limited. Mn demonstrated a different reaction mechanism with thiols compared to Cu and Fe, and may generate transient thiyl radicals during wine oxidation.

Demonstrating that Cu(II) addition to model systems containing H2S and thiols resulted in the generation of polysulfanes led to an investigation of the formation of mixed disulfides and polysulfanes in model and white wine samples. I found that at relatively low concentrations of H2S and methanethiol (MeSH, 100 µg/L each), Cu(II)-fining resulted in the generation of MeSH- glutathione disulfide and trisulfane in white wine. The reduction of the resulting nonvolatile disulfides may then play a role in the generation of undesirable sulfidic off-odors. Therefore,the ability of Fe and Cu in combination of bisulfite (SO2), ascorbic acid, and Cys to promote the catalytic scission of diethyl disulfide (DEDS). I found that the combination of SO2 along with Fe and Cu depleted more DEDS than the other treatments. Furthermore, a method for releasing volatile sulfur compounds from their precursors was investigated using tris(2-carboxyethyl)phosphine (a

reducing agent) and bathocuproine disulfonic acid (a chelator). The addition of the reagents successfully released H2S and MeSH from red and white wines that were free of reductive faults at the time of addition.

I have demonstrated the underlying reaction mechanisms of H2S and thiols with Cu, Fe, and Mn under wine-like conditions. I showed that Cu(II) was readily reduced by H2S and thiols, and that this complex remained redox active and reduced oxygen. The reaction of Cu with H2S and thiols is further accelerated by the presence of Fe and Mn. While the initial Cu(II) fining process removed volatile sulfhydryl compounds, it generated disulfides, polysulfanes, and Cu(I)-

SR complexes that remain in the wine. I showed that disulfide scission is accelerated by the presence of metals and reducing agents under wine conditions. Furthermore, I provided a strategy to quickly reduce or dissociate disulfides, polysulfanes, and metal complexes for the release of volatile sulfur compounds in both red and white wines. This can be used by winemakers to predict a wine’s potential to exhibit sulfidic odors and take further action. Overall, a better understanding of the underlying reaction mechanisms with H2S and thiols provided a foundation for future strategies to better control sulfidic off-odors in wine.

TABLE OF CONTENTS

LIST OF FIGURES ...... x

LIST OF TABLES ...... xv

ACKNOWLEDGEMENTS ...... xvii

Chapter 1 Literature Review...... 1

1.1 Introduction ...... 1 1.2 Metal-catalyzed redox reactions ...... 6 1.2.1 Copper ...... 10 1.2.1.1 Copper fining ...... 10 1.2.1.2 Redox cycling of copper ...... 11 1.2.2 Iron ...... 12 1.2.3 Manganese ...... 14 1.2.4 Other transition metals ...... 15 1.2.5 Release of metal sulfide and metal thiol complexes ...... 16 1.3 Thiol/disulfide couple ...... 18 1.3.1 Occurrence and oxidation of disulfides ...... 18 1.3.2 Thiol-disulfide interchange ...... 21 1.3.3 Sulfitolysis ...... 22 1.3.4 Metal catalyzed disulfide scission ...... 24 1.3.5 Ascorbic acid ...... 26 1.4 Reactions of sulfhydryls with organic wine constituents ...... 28 1.5 Thioester hydrolysis...... 29 1.6 Strecker degradation of amino acids ...... 30 1.7 Further reactions of sulfur containing compounds ...... 30 1.8 Research overview, significance, and hypotheses ...... 31

Chapter 2 Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model Wine. Part 1: Copper Catalyzed Oxidation...... 33

2.1 ABSTRACT ...... 33 2.2 INTRODUCTION ...... 34

2.3 MATERIALS AND METHODS ...... 36 2.3.1 Chemicals ...... 36 2.3.2 Model wine experiments ...... 37 2.3.3 Determination of oxygen consumption ...... 38 2.3.4 Cu-complex formation and dissolution ...... 39

2.3.5 Spectrophotometric measurements of thiols and H2S ...... 39 2.3.6 Spectrophotometric measurement of Cu(I)-BCDA ...... 39

2.3.7 HPLC analyses of thiols and H2S...... 40 2.3.8 HPLC analysis of catechols ...... 42 2.3.9 HPLC analysis of acetaldehyde ...... 42 2.3.10 Copper determination ...... 42 2.3.11 EPR analysis ...... 43 2.4 RESULTS ...... 43 2.5 DISCUSSION ...... 50 2.5.1 Cu reduction and complex formation ...... 50 2.5.2 Disulfide formation ...... 54 2.5.3 Oxidation of the Cu(I)-complex ...... 56 2.6 Acknowledgments ...... 61

Chapter 3 Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model Wine. Part 2: Iron and Copper Catalyzed Oxidation...... 62

3.1 ABSTRACT ...... 62 3.2 INTRODUCTION ...... 63 3.3 MATERIALS AND METHODS ...... 66 3.3.1 Chemicals ...... 66 3.3.2 Model Wine Experiments ...... 67 3.3.3 Determination of oxygen consumption ...... 68 3.3.4 Spectrophotometric measurements ...... 68 3.3.5 HPLC Analyses...... 69 3.4 RESULTS AND DISCUSSION ...... 71

3.4.1 Reaction of Fe(III) with H2S and thiols in model wine ...... 71

3.4.2 Fe(III) reduction by thiols and H2S ...... 73 3.4.3 Fe(II) oxidation and oxygen consumption ...... 74 vii

3.4.4 Fe(III) and Cu(II) reduction by thiols and H2S ...... 76 3.4.5 Fe(II)/Cu(I) oxidation, oxygen consumption, and acetaldehyde formation..... 79

3.4.6 Reaction of Fe(III)/Cu(II) with H2S in combination with thiols in model wine ...... 80 3.4.7 Formation of mixed organic polysulfanes ...... 83

Chapter 4 Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model Wine. Part 3: Manganese Catalyzed Oxidation and Interaction with Iron and Copper...... 85

4.1 ABSTRACT ...... 85 4.2 INTRODUCTION ...... 85 4.3 MATERIALS AND METHODS ...... 87 4.3.1 Chemicals ...... 87 4.3.2 Model Wine Experiments ...... 88 4.3.3 Determination of oxygen consumption ...... 89 4.3.4 Spectrophotometric measurements ...... 89 4.3.5 HPLC Analyses...... 90 4.4 RESULTS AND DISCUSSION ...... 90 4.4.1 Reaction of Cys with Mn ...... 90 4.4.2 Reaction of Cys with Mn+Fe ...... 94 4.4.3 Reaction of Cys with Mn+Fe+Cu ...... 95 4.4.4 Reaction of 6SH ...... 96

4.4.5 Reaction of H2S...... 99 4.5 CONCLUSIONS ...... 101

Chapter 5 Investigating Volatile Sulfur Compound Precursors and Practical Applications ...... 103

5.1 ABSTRACT ...... 103 5.2 INTRODUCTION ...... 104 5.3 MATERIALS AND METHODS ...... 106 5.3.1 Materials ...... 106 5.3.2 Preparation of model wine and real wine samples ...... 106 5.3.2.1 Disulfide and polysulfane generation ...... 106 5.3.2.2 Disulfide scission by Cu(II) and bathocuproine disulfonic acid ...... 107

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5.3.2.3 Diethyl disulfide scission in the presence of metals and reducing agents ...... 108 5.3.2.4 Release and reduction of bound VSCs ...... 109 5.3.3 Methods of analysis...... 110 5.3.3.1 HPLC ...... 110 5.3.3.2 GC ...... 110 5.3.3.3 UV-Vis ...... 111 5.4 RESULTS AND DISCUSSION ...... 111 5.4.1 Disulfide and polysulfane generation ...... 111 5.4.2 Disulfide scission ...... 116 5.4.3 Reactivity of diethyl disulfide...... 119 5.4.4 Predicting a wine’s ability to exhibit reductive off-odors ...... 123

Chapter 6 Conclusions and Recommendations for Future Work...... 129

6.1 Summary ...... 129 6.2 Future Work ...... 130

6.2.1 Interaction of H2S and Thiols with Zinc ...... 130 6.2.2 Interaction of reducing agents and disulfides ...... 131 6.2.3 Using alternative treatments to Cu(II) fining ...... 131 6.3 Concluding Remarks ...... 131

REFERENCES ...... 133

Appendix A. Supplementary information for Chapter 2 ...... 157

Appendix B: Supplementary information for Chapter 3...... 160

Appendix C. Supplementary information for Chapter 4 ...... 163

Appendix D. Supplementary information for Chapter 5 ...... 166

Appendix E. Preliminary studies using Cu(II) sulfate alternatives for the control sulfidic odors in wine ...... 170

LIST OF FIGURES

Figure 1.1. Proposed reaction mechanism of Fe(II) with oxygen to produce hydrogen peroxide, followed by Fenton reaction and oxidation of ethanol to acetaldehyde in wine...... 6

Figure 1.2. Oxidation of o-catechol to o-quinone in the presence of Fe(III) and subsequent Michael type addition reaction of sulfhydryl to give a catechol-thiol adduct...... 7

Figure 1.3. Proposed reaction mechanism of hydrogen peroxide thiols to generate sulfenic acid (A) which subsequently reacts with thiol to generate disulfide (B). Bisulfite will react with hydrogen peroxide to generate sulfuric acid, which will exist as sulfate in wine...... 19

Figure 1.4. (A) Generation of thiyl radical under wine conditions by a one electron oxidant and subsequent (B) dimerization to a disulfide, or (C) reaction with oxygen to generate disulfide anion radical followed by (D) disproportionation to disulfide and peroxyl radical. Alternatively, (E) the thiyl radical can be scavenged by a catechol moiety...... 20

Figure 1.5. Reaction of thiols with Cu(II) to produce disulfides without free radical generation...... 21

Figure 1.6. Reaction mechanism of thiol-disulfide interchange via trisulfide like transition state to generate a new disulfide and corresponding thiol...... 21

Figure 1.7. Example of transition metal assisted thiol-disulfide interchange resulting in the generation of a new Cu(I)-SR complex...... 22

Figure 1.8. Sulfitolysis followed by acid-catalyzed cleavage of an organic thiosulfate...... 23

Figure 1.9. Concurrent electrophilic and nucleophilic assisted disulfide bond scission...... 24

Figure 1.10. Reversible reactions of aldehydes with bisulfite in wine to generate hydroxyalkylsulfonates or with thiols to generate hemithioacetals and thioacetals...... 29

Figure 2.1. Removal of H2S by addition of Cu(II) and formation of insoluble CuS...... 35

Figure 2.2. H2S and thiols used throughout this study...... 37

Figure 2.3. Loss of thiol/H2S by Ellman’s assay in air saturated model wine upon addition of Cu(II) (50 µM) to 6SH, H2S, Cys (300 µM) and Cu(II) (100 µM) to 3SH (300 µM). Error bars indicate standard deviation of triplicate treatments...... 44

thiol/H2S treatments (1.5 mM), and (B) UV-spectra of the thiols/H2S (300 μM) in model wine after mixing with Cu(II) (50 μM)...... 45

Figure 2.5. (A) UV-Vis spectra over time of air saturated model wine after addition of 6SH (300 uM) and Cu(II) (50 uM) in model wine. Removal of the Cu(I) complex by filtration. (B) Cu concentration after filtration after having added 6SH, H2S, Cys (300 µM) to Cu(II) (50 µM) and 3SH (300 µM) to Cu(II) (100 µM) at each respective time point. Error bars indicate standard deviation of triplicate treatments. .... 46

Figure 2.6. Loss of H2S and Cys in air saturated model wine upon adding Cu(II) (100 µM) to H2S (~100 µM) in combination with Cys (~400 µM). Error bars indicate standard deviation of triplicate treatments...... 47

Figure 2.7. O2 and 6SH consumption, and 6SH-disulfide formation in air saturated model wine containing 240 μM 6SH and 50 μM Cu(II). Error bars indicate standard deviation of triplicate treatments...... 48

Figure 2.8. O2 consumption in air saturated model wine upon addition of Cu(II) (50 µM) to 6SH, H2S, and Cys (300 µM), and addition of Cu(II) (100 µM) to 3SH (300 µM). Error bars indicate standard deviation of triplicate treatments...... 49

Figure 2.9. Acetaldehyde produced in air saturated model wine upon addition of Cu(II) (50 µM) to 6SH, H2S, and Cys (300 µM), and addition of Cu(II) (100 µM) to 3SH (300 µM). Error bars indicate standard deviation of triplicate treatments...... 50

Figure 2.10. Proposed mechanism for initial reaction of thiols with Cu(II) and Cu(I)-thiol complex formation. Only the thiol ligands are shown...... 51

Figure 2.11. Proposed thiyl radical formation and subsequent scavenging with 4-MeC and DMPO...... 55

Figure 2.12. Four electron steps in the reduction of O2 to H2O via the hydroperoxyl radical, hydrogen peroxide and the hydroxyl radical...... 57

Figure 2.13. Proposed Cu(I)-SH complex catalyzed two-electron reduction of O2 to H2O2...... 57

Figure 2.14. Proposed Cu(I)-SH complex catalyzed two-electron reduction of H2O2 to H2O...... 58

Figure 2.15. One-electron reduction of H2O2 to produce hydroxyl radicals, and the oxidation of ethanol by the Fenton reaction to form 1-hydroxyethyl radicals. 1- hydroxyethyl radicals are oxidized by oxygen and subsequently reduced by metals to yield acetaldehyde...... 59

Figure 3.1. Reduction of oxygen by Fe(II) to yield hydrogen peroxide without the release of hydroperoxyl radicals...... 64

Figure 3.2. Reduction of hydrogen peroxide to produce hydroxyl radicals by the Fenton reaction and subsequent formation of the 1-hydroxyethyl radical. 1-hydroxyethyl radical is further oxidized by oxygen or Fe(III) to eventually yield acetaldehyde...... 64

Figure 3.3. Proposed mechanism for initial Fe(III) reduction by thiols showing that the resulting Fe(II) is not coordinated to sulfur after the disulfide is formed...... 65

Figure 3.4. Reaction of H2S or thiols on addition of Fe(III) (200 µM) to 6SH, H2S, Cys, or 3SH (300 µM) in air saturated model wine. (A) Consumption of H2S or thiols; (B) %Fe(III)-tartrate based on absorbance at 336 nm; (C) O2 consumption. Error bars indicate standard deviation of triplicate treatments...... 72

Figure 3.5. Reaction of H2S or thiols on addition of Fe(III) (200 µM) and Cu(II) (50 µM) to H2S, 6SH, 3SH (300 µM), and Fe(III) (100 µM) and Cu(II) (25 µM) to Cys (300 µM) to air saturated model wine. (A) %Fe(III)-tartrate based on absorbance at 336 nm; (B) Consumption of H2S or thiols; (C) O2 consumption; (D) AC generation. Error bars indicate standard deviation of triplicate treatments...... 78

Figure 3.6. Proposed mechanism demonstrating initial Cu(II) reduction by thiols and H2S to yield Cu(I)-SR complex and subsequent oxidation of the complex by Fe(III). Fe(II) then reduces oxygen to hydrogen peroxide. Subsequent reaction of H2O2 is depicted in Figure 2...... 78

Figure 3.7. Total thiol and H2S loss on addition of Fe(III) (200 µM) and Cu(II) (50 µM) to (A) 6SH (300 µM) + H2S (100 µM); (B) 3SH (300 µM) + H2S (100 µM); (C) Cys (300 µM) + H2S (100 µM); (D) Fe(III) (100 µM) and Cu(II) (25 µM) to Cys (300 µM) + H2S (50 µM) to air saturated model wine. Error bars indicate standard deviation of triplicate treatments...... 81

Figure 3.8. Total concentrations of Fe(III), Fe(II), O2 (consumed), thiol, and AC in Cys+H2S treatment containing low and high metal concentration. (A) Low Fe (100 µM) and Cu (25 µM), (B) High Fe (200 µM) and Cu (50 µM). Error bars indicate standard deviation of triplicate treatments...... 82

Figure 4.1. Fe(III) initiated sulfite oxidation and subsequent Mn-catalyzed radical chain reaction resulting in sulfite oxidation and sulfate generation...... 86

Figure 4.2. Reaction of Mn(II) with Fe(III)-superoxo complex to generate Mn(III) and H2O2...... 87

Figure 4.3. Reaction of Cys (150 or 200 μM) with Mn(II) (100 μM), Fe(III) (100 μM), and Cu(II) (25 μM) in air saturated model wine. (A) Cysteine consumption, (B) O2 consumption, (C) acetaldehyde generation, and (D) %Fe(III)-tartrate based on absorbance at 336 nm. Error bars indicate standard deviation of triplicate treatments. .. 91

Figure 4.4. Proposed mechanism of Mn(III)-catalyzed radical chain reactions of thiols in air saturated model wine resulting in thiyl radical intermediates which subsequently oxygen and ethanol...... 93

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Figure 4.5: Reaction of 6SH (150 or 200 μM) with Mn(II) (100 μM), Fe(III) (100 μM), and Cu(II) (25 μM) in air saturated model wine. (A) 6SH consumption, (B) O2 consumption, (C) acetaldehyde generation, and (D) %Fe(III)-tartrate based on absorbance at 336 nm. Error bars indicate standard deviation of triplicate treatments. .. 97

Figure 4.6. Reaction of H2S (150 or 200 μM) with Mn(II) (100 μM), Fe(III) (100 μM), and Cu(II) (25 μM) in air saturated model wine. (A) H2S consumption, (B) O2 consumption, (C) acetaldehyde generation, and (D) %Fe(III)-tartrate based on absorbance at 336 nm. Error bars indicate standard deviation of triplicate treatments. .. 100

Figure 5.1. Cu(I)-BCDA generation over time in the presence of cystine (400 µM), Cu(II) (100 µM), and BCDA (1 mM) in air saturated model wine over different pH values...... 118

Figure 5.2. Reduction of disulfides in the presence of TCEP...... 124

Figure A.1. Fragmentation pattern of Cys-bimane...... 157

Figure A.2. Fragmentation pattern of sulfide-dibimane...... 158

Figure A.3. Chromatographic profile of combined MRM spectra. Rt 7.97 min – Cys- bimane (m/z 310→223); 12.59 min – sulfide-dibimane (m/z 413→191); 13.63 min – 6SH-bimane (m/z 323→222)...... 159

Figure B.1. HPLC chromatogram with detection at 210 nm showing organic polysulfanes (identified by MS) obtained from reaction of 6SH (300 µM and H2S 100 µM) with Fe(III) (200 µM) and Cu(II) (50 µM)...... 160

Figure B.2. Fragmentation pattern of organic polysulfanes shown in Figure S1...... 161

Figure B.3. ESI- mass spectrum of S5-bimane obtained from reaction of H2S (300 µM) with Fe(III) (200 µM) and Cu(II) (50 µM) followed by MBB derivatization...... 162

Figure C.1. LC-MS/MS monitoring fragmentation of 6SH-sulfonic acid (181>81 m/z) during the oxidation of 6SH in the presence of (top) Fe(III), Cu(II), and Mn(II) or (bottom) Fe(III) and Mn(II)...... 163

Figure C.2. Peak corresponding to 6SH-disulfide, thiol-sulfinate, thiol-sulfonate, sulfinyl-sulfone, and α-disulfone in 6SH oxidation by Fe(III) and Mn(II) after ~190 hr...... 164

Figure C.2. Lack of peaks for the Mn+Fe+Cu system after 144 hr ...... 165

Figure D.1. Identified Cys-polysulfanes by LC-QTOF after reacting Cys (500 µM) and H2S (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model wine. The insert shows the maximum abundance based on percent of each given mass...... 166

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Figure D.2. Identified GSH-polysulfanes by LC-QTOF after reacting GSH (500 µM) and H2S (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model wine. The insert shows the maximum abundance based on percent of each given mass...... 167

Figure D.3. Identified mixed Cys-MeSH disulfide and polysulfanes by LC-QTOF after reacting Cys (500 µM), H2S (250 µM), and MeSH (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model wine. The insert shows the maximum abundance based on percent of each given mass...... 168

Figure D.4. Identified mixed GSH-MeSH disulfide and polysulfanes by LC-QTOF after reacting GSH (500 µM), H2S (250 µM), and MeSH (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model wine. The insert shows the maximum abundance based on percent of each given mass...... 169

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LIST OF TABLES

Table 1.1. Odor descriptors and thresholds for volatile sulfur compounds in wine...... 2

Table 1.2. Occurrence and oxidation states of various sulfur species which may be present in wine...... 4

Table 1.4. Calculated solubilities of metal sulfides at 25 °C, 1.013 atm total pressure, and pH 7 in pure water. Values adapted from Ricard and Luther75 ...... 9

Table 1.5. Diagnostic test and sensory screening of sulfidic odors in wine utilizing copper, cadmium, and ascorbic acid...... 27

Table 5.1. Treatment addition to anaerobic model wine containing 50 µg/L diethyl disulfide...... 108

Table 5.2. Cys-polysulfanes identified by LC-QTOF after reacting Cys (500 µM) and H2S (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model wine...... 113

Table 5.3. GSH-polysulfanes identified by LC-QTOF after reacting GSH (500 µM) and H2S (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model wine...... 113

Table 5.6. Identified mixed GSH-MeSH disulfide and polysulfanes in white wine spiked at various concentrations of H2S and MeSH by LC-QTOF...... 115

Table 5.7. Decrease in DEDS concentration over time with respective treatments.* ...... 119

Table 5.8. Peak area for each corresponding compound after addition of treatments in air saturated model wine...... 124

Table 5.9. Peak area for H2S after addition of treatments in anaerobic model wine...... 125

Table E.1. Observations for H2S. *relative to control ...... 171

Table E.2. Observations for EtSH. *relative to control ...... 172

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ACKNOWLEDGEMENTS

I am very grateful to my advisor, Dr. Ryan Elias, for providing me the opportunity to undertake this research project under his guidance. Ryan was supportive of my ideas and provided me with the freedom to fully explore my research interests. I thank my committee members, Dr.

Josh Lambert, Dr. John Coupland, and Dr. Michela Centinari for their guidance. Their knowledge on aspects outside of wine chemistry helped me realize a larger context to my work.

I am deeply indebted to Dr. John Danilewicz for continually guiding me throughout my research project. John has been giving me stimulating suggestions and encouraged me throughout my PhD. I greatly appreciate John’s feedback and I believe he helped tremendously in my growth as a scientist.

I also want to thank Dr. David Jeffery for serving on my committee. Dave provided me with the opportunity to work with him in Adelaide, which ultimately led to the conception of this project. Dave’s expertise in wine chemistry and his critiques had greatly improved my communication skills as a scientist.

I would like to thank the Department of Food Science for providing salary and tuition support. I would also like to thank the Pennsylvania Wine Research and Marketing Board for providing some funding support for this project.

I thank all my lab mates and classmates for being supportive of me. They have taught me many valuable skills and helped me develop as a scientist. They have made my experiences here much more enjoyable by being great friends socially and academically. My family and friends at home have always provided love and support, and for that, I am eternally grateful.

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Chapter 1

Literature Review

1.1 Introduction

Volatile sulfur containing compounds (VSCs) are a group of aroma compounds that have a tremendous impact on the sensory quality of wine.1–4 Typically, VSCs have low odor detection thresholds and, depending on their chemical structures, can have beneficial or detrimental effects on the sensory quality of wine. In general, VSCs containing the sulfhydryl (-SH) functionality have lower detection thresholds than other forms and are commonly responsible for sulfurous aromas in wine. However, disulfides, thioethers, and thioesters have important contributions to overall wine aroma as well.

Sulfur-containing compounds such as 3-sulfanylhexan-1-ol (3SH) and 4-methyl-4- sulfanylpentan-2-one (4MSP) contribute to pleasant aromas in wine, such as grapefruit, passionfruit, and blackcurrant.5–7 The yeast generates these compounds by cleaving 3SH and 4MSP from odorless precursors in the must.8,9 These compounds are often referred to as varietal thiols as they typify certain grape varieties (e.g. Sauvignon Blanc) and have aroma detection thresholds at nanogram-per-liter concentrations (Table 1.1).7,10,11 On the other hand, fermentative VSCs such as hydrogen sulfide (H2S), methanethiol (MeSH), and ethanethiol (EtSH) are considered defects as they contribute to “reductive” sulfidic off-odors that are associated with rotten egg, sewage, and burnt rubber (Table 1.1). The alcoholic fermentation process of juice or must to wine by the yeast

Saccharomyces cerevisiae is the main factor in the accumulation of H2S and other organic sulfur

12–16 compounds in the final wine. H2S is produced as a byproduct during normal yeast metabolism

via the sulfate reduction pathway, in which H2S acts as an intermediate in sulfur-containing amino

17 acid biosynthesis. The production of excess H2S depends on the fermentation and nutrition conditions, as well as yeast strain, and can lead to the formation of other VSCs such as MeSH and

EtSH17–21 as well as dimethylsulfide (DMS) and dimethyl disulfide (DMDS), which are reminiscent of rotten cabbage or canned vegetables.1,22,21 Wine yeast can also form thioacetates by enzymatic action.17,23 These VSCs have relatively low detection thresholds (i.e. low microgram-per-liter)

(Table 1.1), and have a negative effect on wine quality.1,24–28 DMS may positively impact the bouquet of the wine at subthreshold concentrations, although this is generally not the case.1,29 In depth examination of the flavor impact of VSCs in wines, associated aromas, and detection thresholds are outside of the scope of this review, and have been thoroughly reviewed elsewhere.1,6,22

Table 1.1. Odor descriptors and thresholds for volatile sulfur compounds in wine. Compound Odor descriptor Odor detection threshold Hydrogen sulfide Rotten egg 1.1 – 1.6 µg/L30 Methanethiol Cabbage, sewage 1.8 – 3.1 µg/L31 Ethanethiol Onion, rubber, fecal 1.1 µg/L27 Dimethylsulfide Cabbage, asparagus, corn, 25 µg/L27 blackcurrant Dimethyldisulfide Cooked cabbage, sulfurous, onion 29 µg/L27 Diethyldisulfide Onion, garlic, rubber 4.3 µg/L27 Methylthioacetate Sulfurous, cheesy 50 µg/L32 Ethylthioacetate Cabbage, cauliflower 10 µg/L32 4-Methyl-4-sulfanylpentan-2- Box tree, guava, cat urine 3.3 ng/L33 one 3-Sulfanylhexan-1-ol Passionfruit, grapefruit 60 ng/L5

Many of the sulfur compounds occurring in wine due to viticultural practices and subsequent yeast fermentation remain redox-active in wine during aging, where they are able to participate in one- and two-electron transfer, radical processes, and exchange reactions. Many of these compounds, particularly species containing sulfhydryl moieties, can also bind to metals and result in a range of metal complexes that are commonly found in biological and geochemical

systems.34,35 Indeed, sulfur plays an important in vivo role in redox systems that is critical for all organisms (e.g. plants, bacteria, fungi, yeast).35,36 As such, the presence of these various sulfur compounds in wine is a combination of overall grape and yeast metabolism. The major changes occurring during grape maturation and grape juice/must fermentation are due to enzymatic processes that have been (and remain) the focus of much research with the ultimate goal of predicting and improving wine quality.4,37 However, once a finished wine is bottled, enzymatic action ceases yet subsequent non-enzymatic chemical reactions may result in nuanced aroma changes over time.

Many non-enzymatic wine oxidation reactions in wine occur due to oxygen, and can result in loss of pleasant fruity aromas containing sulfhydryl functionality (e.g. 3SH and 4MSP)38 and the generation of various undesirable aldehydes that derive from ethanol, organic acids, and sugars in wine.39 To avoid excessive wine oxidation, modern winemakers take great care to minimize oxygen exposure throughout the winemaking process.40 Unfortunately, the increasing use of reductive winemaking (i.e. minimizing O2 exposure) and use of low oxygen transmission rate (OTR) closures in recent years has made post-bottling generation of sulfidic off-odors more common. The generation of H2S and MeSH above their odor detection threshold in wine may occur when O2 is limited and can result in consumer rejection of the wine.37,41 It appears that an intricate balance of

O2 ingress through the wine’s packaging system (e.g., its closure) is needed to prevent wine spoilage due to either oxidation or reduction; however, no model currently exists that can accurately predict what such an O2 balance should be based on a given wine’s chemical composition, its closure type, the environmental conditions to which it is exposed, and its time in-bottle.

Sulfur-containing compounds can possess various oxidation states and can remain redox active in wine. These species can have either reducing or oxidizing capacity which is influenced by factors such as the overall redox state of the wine, dissolved O2 concentration, and the presence of

transition metals and polyphenols. Various sulfur species and their oxidation states in wine are

listed in Table 1.2. Numerous sulfur oxyanions could originate from grapes or yeast metabolism,

but can result from non-enzymatic oxidation. Comprehensive reviews of biogenesis and sensory

properties are covered elsewhere.4,42

Table 1.2. Occurrence and oxidation states of various sulfur species which may be present in wine. Sulfur Species Structure Sulfur Occurrence Reactivity Oxidation State Sulfhydryl H2S, RSH -2 Grapes and yeast Reducing agent metabolism Thiyl radical -1 Transient Reducing or oxidizing, can dimerize to RSSR Perthiol RSSH -1 Reduction of Strongly reducing polysulfanes Disulfide RSSR -1 Naturally present, Mild oxidant, can be further oxidation of RSH oxidized Organic RSSnSR -1,0,-1 Oxidation of RSH and Mildly oxidizing polysulfanes H2S Elemental sulfur S8 0 Pesticide residue, Very weak oxidant, can be oxidation of H2S reduced by RSH Sulfenic acid RSOH 0 Transient Condenses to disulfide Sulfinic acid RSO2H +2 Oxidation product of Adds to quinones RSH Sulfonic acid RSO3H +4 Oxidation product of Unreactive RSH - Sulfite HSO3 +4 Yeast byproduct, Reducing agent, antioxidant winemaking additions 2- Sulfate SO4 +6 Sulfite oxidation, Unreactive yeast and grapes - Thiosulfate RSSO3 -1,+4 Sulfitolysis of Hydrolyze to sulfate and disulfides43,44 free thiol

Thiosulfinate +1,-1 Unknown Oxidizing

Thiosulfonate +3, -1 Unknown Oxidizing

Sulfinylsulfone +3, +1 Unknown Oxidizing

Disulfone +3, +3 Unknown Oxidizing

Thioethers, RSR -2 Dimethylsulfide, dialkylsulfides thioesters, etc. Sulfoxide +2 dimethylsulfoxide45

Sulfone +4 dimethylsulfone46

Metal sulfides MnSn varies Various complexes Reducing, oxidizing, or with first row inert transition metals

The generation of H2S and MeSH have been implicated as the compounds responsible for

47–50 post-bottling reduction which occurs when O2 ingress is low. In recent years, numerous studies

attempted to identify precursors and conditions needed for the generation undesirable sulfidic off-

odors. However, the precursors of these undesirable sulfidic odors and the storage conditions

involved in their release remain ambiguous. Some reactions may be equilibrium-driven, such as

those involving acid hydrolysis or disproportionation. However, the interaction of sulfur

compounds with transition metals and generation of subsequent metal complexes appears to play a

critical role in mediating redox reactions and generating sulfidic off-odors in the post-bottle period.

This review focusses on non-enzymatic reactions occurring post-fermentation that are

associated with the loss and formation of sulfhydryl containing compounds. An overview on the

redox chemistry underlying the reactions between these sulfhydrdryl compounds and transition

metals will be covered in significant detail. In addition, the reaction of sulfhydryls, disulfides, and

other sulfur compounds that result in the generation of volatile sulfhydryls will be discussed. The

proposed relevance of previous research on sulfur chemistry within physiological and

biogeochemical contexts will be presented in relation to reactions under wine conditions.

1.2. Metal-catalyzed redox reactions

Transition metals are well known to catalyze redox reactions in wine.51,52 Under wine

53 conditions, O2 is reduced to H2O in a 4-electron step manner in the presence of transition metals, and the process is coupled with the oxidation of wine constituents, notably polyphenols, ethanol, and sulfhydryl compounds.51,54–56 The overall rate of non-enzymatic wine oxidation is generally

57 3 dictated by the rate of O2 ingress. O2 is stable in its triplet ground state (i.e., O2) and its direct reaction with organic compounds (singlet state) is spin forbidden; however, O2 can be reduced by transition metals prior to its reaction with wine constituents. It has recently been argued that Fe(II) and Cu(I) can mediate the concerted reduction of O2 to H2O2 without the release of hydroperoxyl

55,58 radicals or oxidation of catechols (Figure 1.1). Once H2O2 is generated it may undergo reduction via Fenton reaction involving Fe(II) (or other reduced metals) to generate hydroxyl radicals (HO·).51,59 The highly reactive hydroxyl radical reacts at diffusion limiting rates with organic compounds in proportion to their concentrion. As ethanol is the most abudant organic species in wine (ca. 2 M), it has been shown to be the most likely target of hydroxyl radicals in wine. This reaction results in ethanol oxidation and the formation of the intermediate 1- hydroxyethyl radical (1-HER) which can subsequently be oxidized to acetaldehyde.59–61

Figure 1.1. Proposed reaction mechanism of Fe(II) with oxygen to produce hydrogen peroxide, followed by Fenton reaction and oxidation of ethanol to acetaldehyde in wine.

During the O2 reduction process, transition metals are oxidized and can subsequently oxidize polyphenols or sulfhydryls. The quinones that result from polyphenol oxidation can

undergo Michael-type addition reaction with sulfhydryls, resulting in another mechanism for the loss of aroma through binding of the sulfhydryl functionality (Figure 1.2).62–64 The presence of transitions metals is needed to drive this reaction forward,65 and it has been shown that the presence of nucleophiles, such as sulfhydryls, can drastically increases the rate of reaction as it drives the

54,66 reaction forwards. It appears that the relationship between sulfhydryls and O2 is facilitated by redox cycling of transition metals (especially Fe and Cu), but some studies indicate that radical intermediates, such as 1-HER, may react directly with thiols.67,68

Figure 1.2. Oxidation of o-catechol to o-quinone in the presence of Fe(III) and subsequent Michael type addition reaction of sulfhydryl to give a catechol-thiol adduct.

Clearly transition metals play a critical role in mediating wine oxidation, and many oxidation intermediates may result in loss of sulfhydryl compounds. Ribéreau-Gayon showed that the rate of oxidation could be slowed and eventually stopped in wine by the removal of iron and copper with potassium ferrocyanide.69 This was more recently confirmed in another study by

Danilewicz and Wallbridge.65

On the other hand, in the absence of O2, VSCs that contribute to reductive sulfidic odors can accumulate, particularly in the presence of transition metals.48,50,70,71 The formation of sulfidic odors is attributed to H2S and MeSH, but the mechanism for their formation and involvement of transition metals remains poorly understood.

In addition to their redox cycling capability, transition metals and sulfhydryls are also capable of forming ionic bonds. This is especially important in the case of H2S, which can react with transition metals, and upon further rearrangment, may result in crystal structure formation and

subsequent mineral precipitation.72,73 The ability of sulfhydryls to both dissociate bulk minerals and generate metal-sulfide structures has been heavily studied in geochemical processes.34,74–78 Some of these metal sulfide structures are relatively inert, wheras others remain redox active and can effectively behave as aqueous species.73 It is relatively well known that the majority of sulfide (over

90%) in bodies of water is complexed to copper, iron, and zinc.79 The importance of these complexes in the context of wine chemistry remains poorly understood, but has piqued interest in recent years.80,81

The stability constants for metal sulfide complexes of wine relevant transition metals are reported in Table 1.3. Generally speaking, the larger the stability constant, the more likely it is for the transition metal to bind with H2S, and potentially with thiol compounds too. These values are reported for sea water conditions but this information may still be applicable to wine. For example, log K values for Cu(I), Cu(II), and Zn(II) are higher than Fe(II) and Mn(II), and this is consistent with recent studies in wine showing Cu and Zn species correlate with H2S concentrations moreso than Fe and Mn.70,80

Table 1.3. Experimental stability constants (log K) for metal sulfides at 25 °C in water with ionic strength of 0.7 at pH 7. Values adapted from Ricard and Luther75 and sources within.82–85 Metal Complex Log K Mn(II) [MnHS]+ 4.5 Fe(II) [FeHS]+ 5.4 Co(II) [CoHS]+ 5.5 Ni(II) [NiHS]+ 5.0 Cu(II)* [CuHS]+ 6.5 [CuS]0 11.2 Cu(I) [CuHS]0 12.1 Zn(II) [ZnHS]+ 6.1 [ZnS]0 11.7 Ag(I) [AgHS]0 11.2 [AgS]- 22.8 Au(I) [AuHS]0 24.5 *Cu(II) likely reduced to Cu(I) to some extent during analysis.

Furthermore, the solubilities of the metal sulfides are reported in Table 1.4. These values are calculated for pure water and may give an indication of the general solubilities of some metal sulfides under wine conditions. For example, as can be seen from this table, CuS and ZnS are predicted to be considerably less soluble than FeS and MnS. However, there are limitations to this table as it does not consider other wine constituents (e.g. organic acids, polyphenols, thiols) which may limit the formation of metal sulfide solids. Futhermore, metastable metal sulfide clusters may be kinetically significant in wine and have higher solubilities compared to their more stable solid forms.75 The misconception that the comlexes are virtually insoluble is especially important in copper fining, where CuS is reported to have an exceedingly low solubility, yet is not readily formed in wine. This is discussed further in Section 1.2.1.1.

Table 1.4. Calculated solubilities of metal sulfides at 25 °C, 1.013 atm total pressure, and pH 7 in pure water. Values adapted from Ricard and Luther75 Metal sulfide Solubility (mg/L) MnS 6×100 FeS 6×10-2 CoS 5×10-3 NiS 2×10-5 CuS 3×10-14 ZnS 8×10-9 AgS 2×10-14 AuS 2×10-27

The importance of transition metals in wine with respect to the loss and formation of sulfhydryl compounds is two-fold. One is the ability of the metals to redox cycle sulfur, and the other is forming ionic bonds and corresponding metal sulfides and metal thiol complexes. Catalytic oxidation of organic thiols by O2 in the presence of metals was investigated in borate-phosphate buffer at a wide range (pH 2 – 13) where it was found to follow the trend of Cu > Mn > Fe > Ni >>

Co.86 However, a sharp decrease in reactivity occurs when the pH is close to that of wine pH (pH

3 – 4). On the other hand, the formation of metal sulfhydryl complexes may follow the order of Cu

> Zn > Fe > Mn (Tables 1.3 and 1.4). Again, the formation and constants may change when at wine-relevant pH.

The nature of redox reactions and ionic bonding under wine conditions remains poorly understood; however, it is critical to understand these reactions in order to better control and predict sulfhydryl compound loss and regeneration in wine. The importance of some first row transition metals and their relevance to wine is elaborated in the following sections.

1.2.1 Copper

Cu is naturally present in grapes, and Cu based fungicide treatments in the vineyard may cause carryover into the juice;87 however, the concentration of Cu is known to decrease during fermentation due to Cu adsorption and removal by yeast cells.88,89 The major source of Cu in finished, packaged wine is the intentional addition of Cu salts during the process known as Cu fining. The legal limits globally for Cu in finished wine generally vary between 0.5 – 1 mg/L, but may be as high as 10 mg/L.90

1.2.1.1 Copper fining

The accumulation of sulfidic off-odors is a common problem in wine production, and the addition of Cu(II) salts for their removal has been used as a standard procedure in winemaking for

2,41,90 many decades. Sulfidic off-odors are typically attributed to H2S (and thiols such as MeSH) and it is generally assumed that reacting Cu with H2S would result in formation and complete precipitation (and removal) of CuS, due to its low solubility product (3×10-14 mg/L, Table 1.4).

However, it has been noted that this precipitate is not always formed and that tartaric acid might inhibit the aggregation of CuS.71,90,91 A recent study Clark et al. demonstrated the practical difficulty 10

of removing CuS from wine, even with filtration.91 In fresh and saltwater it has been shown that the reaction of H2S with Cu results in CuS nanoclusters that effectively behave as soluble species.

Their condensation results in Cu(I)S covellite that precipitates out of solution and becomes chemically inert.72

It has been suggested that other agents, such as nonvolatile thiols, could interfere with precipitation during the fining process by competing for Cu(II).55,56,91 For example, the average combined concentration of cysteine (Cys), N-acetylcysteine and homocysteine was reported to be ca. 20 µM in a survey of white wines, while the average concentration of glutathione (GSH) was reported to be ca. 40 µM in wines made from Sauvignon blanc grapes.92–95 These nonvolatile thiols would be in large molar excess to the exogenous Cu (3–6 µM) used in a fining operation, and would

30 far exceed the concentration of H2S (ca. 300 nM) when copper fining is considered.

In addition to the ambiguity of Cu fining for the removal of sulfhydryl compounds, there are known disadvantages to the process. In the case of disulfides, thioacetates, and cyclic sulfur compounds, which can also contribute unpleasant sulfidic off-odors, Cu fining is ineffective due to the absence of a free sulfhydryl functionality.2,41 Cu fining can also cause significant losses of beneficial thiol compounds (e.g. 3SH, 4MSP) that are important to the varietal character of a wine.48

Although the precipitation of chemically inert CuS would be ideal under wine conditions, it has become clear that this is not the case and that residual CuS nanoparticles remain redox active in wine which may result in deleterious reactions.

1.2.1.2 Redox cycling of copper

Trace concentrations of Cu are now known to act synergistically with Fe in mediating non- enzymatic wine oxidation reactions, particularly by accelerating oxygen consumption and polyphenol oxidation.52 As described above, polyphenol oxidation generates quinones which may 11

undergo subsequent Michael-type addition reaction and trap sulfhydryl compounds (Figure

1.2).38,64,96–98 Furthermore, the importance of Cu(II) in bridging reactions involving catechin with glyoxylic acid with a quinone intermediate has been demonstrated.99

Surprisingly, limited research has been conducted under wine conditions that focuses on the direct interaction of Cu with sulfhydryl compounds. When H2S, MeSH, and EtSH were oxidized in model brandy by Cu(II), the formation of mixed disulfides and trisulfanes was observed.100

Recent work by Franco-Luesma and Ferreira found that virtually all H2S is bound when Cu(II) is added, forming an inert Cu(II)S complex that remains in solution and is resistant to aerial oxidation.80,81,101 However, biologically relevant thiols have been shown to readily reduce Cu(II) to

Cu(I) with their concomitant oxidation to disulfides at pH 7.4.102,103 Similarly, under biogeochemical conditions, H2S reduces Cu(II) to Cu(I) during Cu3S3 ring formation, and these species remain in solution as polynuclear nanoclusters72. The relevance of these reactions and their redox activity is thoroughly investigated in Chapter 2.

1.2.2 Iron

Fe has been focused on heavily by wine chemists because it mediates many wine oxidation reactions involving oxygen, polyphenols, and sulfite (Figures 1.1 and 1.2). The overall rate of non- enzymatic wine oxidation is highly dependent on the reduction potential of the Fe(III)/Fe(II) couple, which is lowered by tartaric acid.51,58,59,104 The lower the reduction potential, the greater the reducing power; therefore, if the reduction potential of the Fe(III)/Fe(II) couple is low, O2 will be reduced to H2O2 more readily. A relatively low Fe(III)/Fe(II) reduction potential will also facilitate the reduction of H2O2 to hydroxyl radicals via the Fenton reaction (Figure 1.1). When Fe(II) is oxidized, the Fe(III) formed is quickly reduced back to Fe(II) in the presence of sulfite and phenolics, both which are abundant in wine.59 Fe speciation in wine has been examined and it has 12

been suggested that the majority of free Fe is present as Fe(II),59,105 although Fe remains bound to the organic fraction of wine such as tartrate106 and tannins.107 Fe(II) is the major species of Fe in wine due to wine’s low pH and abundance of phenolics, which has been recently confirmed in a variety of wines.108

Although Fe has been shown to play an important role in the generation of reactive intermediates that are subsequently capable of reacting with sulfhydryl compounds in wine, the amount of research that focuses on the direct reaction of Fe with sulfhydryl compounds is sparse.

It has been proposed that the oxidation of thiols by Fe(III) may be radical-mediated with the generation of disulfides.54 Studies performed with GSH in a range of pH conditions (3-7) have shown that Fe(II) is spontaneously produced when GSH is added to Fe(III).109,110 The same has been shown with Cys at low pH, as the Fe(III)-Cys complex is unstable and quickly reacts to yield

Fe(II) and cystine.111 After the reduction of Fe(III) to Fe(II), GSH and Cys appear to be coordinated with the carboxylate group under wine’s acidic conditions (pH<4), and not the sulfhydryl group.

109,110 Therefore, under wine conditions it is unlikely that the sulfhydryl compounds remain bound to Fe(II) due to competition by excess tartaric acid as the dominant ligand, which is addressed directly in Chapter 3. H2S may behave differently than thiols and remain bound to Fe(II) to some degree. It has been shown that Fe(II) can form a complex with H2S, and FeS does not exhibit odors

80,101 associated with H2S. The binding of H2S is likely to form subunits of Fe2S2 similar to mackinawite structure, however, under acidic conditions it does not appear to be sufficiently stable

112,113 73 to aggregate as a solid. Furthermore, FeS clusters are reactive in the presence of O2.

Generally, elevated Fe levels are associated with a decrease in volatile sulfhydryl concentrations.57 This is likely due to formation of quinones and their subsequent reactions, as their

97 reaction rates with some sulfhydryls, particularly H2S, is very high. Although Fe(III)-catalyzed

oxidation of suldhydryls is possible,54 it is unlikely this reaction will occur to a considerable degree relative to other chemical reactions (e.g. Figure 1.2) that may occur under real wine conditions.

1.2.3 Manganese

Mn is typically present in wines at concentrations that are comparable to Fe,114 and has been suggested to play an important role in non-enzymatic wine oxidation. Cacho et al. showed that Mn, along with Fe, affected the rate of non-enzymatic oxidation in white wine.115 The presence of Mn resulted in elevated acetaldehyde concentrations, suggesting the ability of Mn to catalyze

Fenton-like reactions in wine (Figure 1.1).115 The exact mechanism for reaction of Mn in wine conditions remains poorly understood, but it may behave in a similar manner to Fe.

Recent work has investigated the Mn(II)-mediated oxidation of polyphenols and sulfite in wine. The Mn(III)/Mn(II) couple has a high reduction potential and is difficult to redox cycle under wine conditions. However, once Mn(III) is formed, presumably due to interaction with Fe-superoxo complex, it is capable of oxidizing wine constituents.116 In a system without polyphenols, Mn(III) has been shown to initiate radical chain reaction with sulfites.116

Based on work in non-wine model systems, it would appear that sulfhydryls are more susceptible to oxidation by Mn than Fe.86 It was recently reported that Mn was responsible for the oxidative degradation of MeSH.117 Mn(III) may be more selective towards sulfhydryl compared to other wine constituents, and promote their oxidation. This mechanism is investigated further in

Chapter 4.

1.2.4 Other transition metals

Zinc concentrations average between 0.3 – 0.7 mg/L and can exceed 1 mg/L, as such it

114 may be present at comparable concentrations to Cu in wine. Zn has been shown to effect H2S and MeSH concentrations in beer and wine.70,80,118 However, unlike the other metals described above, Zn(II) does not redox cycle and is unlikely to have an effect on rate of oxidation reactions in wine, but needs to be investigated further. Nonetheless, Zn(II) binding with H2S is comparable to Cu, as it has a high stability constant (1×10-13) and low solubility (8×10-9 mg/L).34 Similarly to

119 Cu, it forms a Zn3S3 ring structure that further condenses to Zn4S6 under aquatic conditions.

However, unlike the reaction with Cu(II), which involves an electron transfer, the reaction displayed by Zn(II) is a simple substitution reaction.72,119 This can result in fast binding of sulfhydryls, particularly H2S, and formation of a relatively stable complex that effectively renders the sulfhydryl group unavailable for reaction (or volatilization).

The binding of H2S to Zn(II) has been demonstrated in synthetic wine solutions and

80,118 70 beer. Furthermore, the generation of H2S was positively correlated with Zn(II), suggesting that ZnS complex could be responsible for subsequent release in wine under reductive conditions.

However, in accelerated aging studies in wine, Zn was negatively correlated with H2S production, which may not necessarily be due to post-bottling chemical reactions,81 but rather that low Zn concentrations resulted in sluggish fermentations which generated more H2S in the wine prior to

120 bottling. Therefore higher Zn concentrations may result in lower H2S production during fermentation, but this needs to be investigated further.

Other first row transition metals including chromium, cobalt, and nickel are less understood under wine conditions. While they have catalytic abilities and binding affinities with sulfhydryls, these metals are generally present at concentrations far below 0.1 mg/L. Due to their low natural abundance they may be of lesser importance compared to the transition metals discussed above.

1.2.5 Release of metal sulfide and metal thiol complexes

Transition metal catalyzed wine oxidation has been fairly well studied in recent years. As described above, elevated concentrations of any transition metals cause a decrease in sulfhydryl concentration in the presence of O2. Although the mechanisms by which these metals promote wine oxidation have been elucidated to varying degrees, the most abundant oxidation products arising from metal-catalyzed reactions are disulfides, catechol-thiol adducts, and metal complexes. The reduction and dissociation of these compounds has been hypothesized to generate sulfidic off-odors

48,57,70 due to H2S and MeSH, especially when O2 ingress is low. However, up until recently, the driving mechanism for the generation of these compounds was unknown.

Recent work by Ferreira’s group has demonstrated that the major factor for the release of

81,101 H2S and MeSH is the dissociation of bound metal species. In that study, diluting wine in a strong brine solution has been demonstrated to release the metal-bound forms of sulfhydryl compounds.80 Indeed, it has been previously shown that chloride anions can ligate, stabilize, and

2- 3- 121,122 solubilize Cu to generate the corresponding CuCl3 and CuCl4 complexes, effectively displacing organic thiols.122 Similarly, chloride can cause dissociation of bulk metal sulfide minerals by displacing sulfur.123 The results from brine addition demonstrated that on average 94% and 47% of H2S and MeSH, respectively, are effectively bound to the metals under wine conditions.80,101

Of the first row transition metals present in wine, Cu is the one that binds most strongly to sulfhydryls (Table 1.3). Perhaps counterintuitively though, elevated Cu concentrations in a finished wine are associated with higher generation of H2S and MeSH. The formation of soluble CuS nanoclusters is likely a major contributing factor for the subsequent release of H2S and MeSH.

Zn(II) reacts in a similar fashion to Cu and is also important for binding of H2S. Fe(II) has been shown to have some ability at binding to H2S, although as described above (section 1.2.2), it forms

a different metal sulfide complex likely consisting of Fe2S2. The binding of H2S and MeSH correlate with the stability constants of the corresponding metal sulfides (Table 1.3).

Given that metal sulfides are non-volatile and therefore odorless, a wine may appear free of faults until the complexes dissociate. Further research is needed to understand what drives these dissociation reactions, but it is clear that anaerobic conditions are the key driving force for the dissociation and release of H2S and MeSH. Studies in which H2S release was monitored in wine have indicated that during an anoxic 18 month aging period of a wine, free H2S increased with time

101 while total H2S concentration remained unchanged. One hypothesis is that polyphenolic

101 compounds may reduce the CuS complex to release free H2S and Cu(0), however, there are other strongly reducing agents in wine which may play a role, including sulfite, thiols (e.g. Cys and

GSH), and ascorbic acid in the case of some wines.

While a large proportion of H2S and MeSH release could be attributed to the dissociation of metal sulfide complexes, it has been shown that up to 42% and 76% of H2S and MeSH, respectively, are generated due to de novo formation.81 There are several hypotheses for the generation mechanisms of these sulfidic compounds, and these are discussed in depth in the following sections.

1.3 Thiol/disulfide couple

In general, reduced sulfur species (with S2-, Table 1.2) have considerably lower detection thresholds than their corresponding oxidized species, and thus have a greater impact on overall wine aroma. Several of these oxidized species including disulfides (S1-), elemental sulfur (S0), sulfoxides (S2+) and sulfite (S4+), are naturally occurring and are present post-fermentation in wine, and their chemical reduction post-bottling can result in the appearance of undesirable sulfidic off- odors in wine previously deemed to be free of apparent faults.

Winemakers are advised to avoid aerating their wines or utilizing Cu fining in the presence of O2 as it may result in the generation of disulfides that can be subsequently reduced, thus adversely affecting wine quality.43,124,125 The implication of disulfides on wine reduction has been commonly referred to and accepted in enology text books. However, the generation of symmetrical disulfides from MeSH and EtSH (that is, DMDS and DEDS, respectively) are rarely observed, if ever, post-fermentation.49,126–128 In general, the majority of disulfides are formed during yeast metabolism21,129 although there is some evidence for the generation of disulfides and polysulfanes under wine and model wine conditions during Cu(II) addition and subsequent aging.55,100,130

1.3.1 Occurrence and oxidation of disulfides

Sulfhydryls cannot be directly oxidized by O2 due to Pauli’s exclusion principle and require transition metals to facilitate oxidation reactions. They can however, be oxidized by two-electron

131 oxidants such as H2O2 to yield a sulfenic acid (RSOH) and water (Figure 1.3A). Sulfenic acids are transient species that can condense with thiols to form disulfides (Figure 1.3B).131,132 However, the initial reaction with H2O2 is relatively slow under wine conditions and will likely be

133 outcompeted by sulfite to form sulfate (Figure 1.3C). As such, the oxidation of thiols by H2O2 is most likely of little relevance in wine.

Radical-mediated reactions present another pathway by which sulfhydryl compounds can be oxidized to disulfides. Thiyl radicals can be generated by electron transfer after sulfhydryl compounds form unstable complexes with oxidized transition metals (Figure 1.4A). Alternatively, studies in wine and beer suggest that thiols may reduce 1-HER, resulting in the formation of thiyl radical and ethanol. Once the thiyl radical is formed, it may result in either dimerization of thiyl radicals67,68 (Figure 1.4B) or reaction of thiyl radical with a thiol to form the disulfide anion radical, which further reacts with oxygen to yield a disulfide and peroxyl radical (Figures 1.4C and

1.4D).54,131,134 However, wine contains an excess of polyphenolics containing the catechol and galloyl moieties that will quickly scavenge the thiyl radical (Figure 1.4E).67 Alternatively, the thiyl radical may further react with α,β-unsaturated side chains.135

As described in the reactions involving Fe and Cu above, metal catalyzed oxidation of sulfhydryls may result in a concerted oxidation to the disulfide without the release of free thiyl radicals, resulting in the generation of the corresponding reduced metals along with disulfides

(Figure 1.5). This has been shown to occur under physiological conditions with Cu(II),103 and more recently described under wine conditions as well (Chapter 2).55 Furthermore, Cu(II) fining does not strictly result in symmetrical disulfide generation. It would be expected that H2S, MeSH, and EtSH would be present at concentrations below 100 nM, whereas Cys and its analogues may be present at concentrations up to 0.1 mM. Therefore, it is likely that mixed disulfides and polysulfanes with

S-containing amino acids would be generated rather than DMDS and DEDS. These effectively nonvolatile disulfides may result in release of H2S, MeSH, and EtSH upon their reduction during anoxic storage. In the presence of H2S, oxidation of H2S and thiols may result in the insertion of sulfur into disulfides and subsequent formation of polysulfanes. In model solutions containing 20% ethanol, H2S was shown to react with MeSH and EtSH in the presence of Cu(II) to form mixed di- and trisulfanes.100 It has been suggested that this is formed with the generation of a perthiol (RSSH) intermediate followed by oxidation in the presence of a thiols to generate the trisulfane (RSSSR).100

Alternatively, H2S is oxidized to elemental sulfur followed by its insertion into the disulfide to generate the trisulfane.136

Figure 1.5. Reaction of thiols with Cu(II) to produce disulfides without free radical generation.

1.3.2 Thiol-disulfide interchange

Thiol-disulfide interchange reactions are biologically important, and have been studied extensively as they are responsible for intracellular redox homeostasis, and play a critical roles in antioxidant defense and redox regulation of cell signaling in vivo.137 These interchange reactions involve a nucleophilic substitution of a free thiol with a thiol from the disulfide. The reaction follows a one-step SN2 mechanism with a trisulfide-like transition state complex and delocalized negative charge (Figure 1.6).131,138–141

Figure 1.6. Reaction mechanism of thiol-disulfide interchange via trisulfide like transition state to generate a new disulfide and corresponding thiol.

In the above describe reaction, the thiolate anion serves as a nucleophile because it is a stronger nucleophile than its corresponding thiol. The nucelophilicty of a thiol is inversely dependent upon its pKa, and these reactions typically proceed at or above physiological pH. The pKa of cysteine’s and glutathione’s respective thiol groups are ca. ~8-9, whereas simpler thiols are

142 closer to 10. However, due to the linear-free energy relationship, increasing pKa is directly correlated with thiol nucleophlicity.131

If the interchange reaction were to proceed in wine, DMDS or DEDS would potentially undergo thiol-disulfide interchange with the abundant concentrations of Cys (or its analogs) and

GSH, which would generate a mixed disulfide and release of EtSH and MeSH. While the pKa is higher for EtSH and MeSH, they make a better leaving group due to their higher linear free energy.

Furthermore, concentrations may play a role in driving the reaction,139 and Cys and GSH are present in molar excess compared to DMDS and DEDS. However, given the pH of wine is well below the pKa of thiols, the unassisted reaction is prohibitively slow.

Thiol-disulfide interchange may be assisted at wine pH by transition metals (Figure 1.7).

Recent work has shown that phosphine Au(I) thiolate complexes accelerated thiol-disulfide interchange reactions.143 Although phosphine is a strongly electron withdrawing group, a similar pathway may occur by Cu(I) or Zn(II) thiolate complex. Because of the abundance of transition metals in wine, these reactions, and their potential relevance to wine thiol phenomena, should be the topic of future research.

Figure 1.7. Example of transition metal assisted thiol-disulfide interchange resulting in the generation of a new Cu(I)-SR complex.

1.3.3 Sulfitolysis

Sulfitolysis works in a similar manner to thiol-disulfide interchange wherein sulfite substitutes one of the thiols of a disulfide and forms an organic thiosulfate, also known as Bunte salt (Figure 1.8).144 The organic thiosulfate may then undergo acid-catalyzed scission over time to yield the other thiol that was present in the original disulfide. This reaction was initially proposed by Bobet et al. to be feasible under wine conditions.43 However, results from their study indicate that the release of EtSH to reach above threshold concentrations would require over 2 years with

30 mg/L free SO2 and 50 µg/L DEDS. 22

Figure 1.8. Sulfitolysis followed by acid-catalyzed cleavage of an organic thiosulfate.

The mechanisms by Bobet et al. are predicted on the assumption that the formation of the organic thiosulfate is rate limiting, and not its acid-catalyzed hydrolysis (Figure 1.8). This is a reasonable assumption, as the bisulfite ion is a considerably stronger nucleophile at higher pH when

2- its fully deprotonated SO3 form would dominate, and like thiol-disulfide interchange this reaction appears to be driven by higher pH. The reaction comes to completion in a matter of hours at pH

7.2, but would take years to detect any differences at pH 3.5.43 In contrast, the acid-catalyzed cleavage of the thiosulfate would be expected to be much faster at wine pH compared to the initial bisulfite substitution (Figure 1.8).

Recent work has shown the formation of organic thiosulfates in wine due to sulfitolysis of

GSH disulfide and cystine (i.e., the disulfide of cysteine).44 However, unlike the slow sulfitolysis of DEDS, GSH disulfide was shown to react with sulfite to generate detectable concentrations of free GSH and GSH S-sulfonate in a matter of hours. Furthermore, GSH disulfide was not detectable in wines, but GSH S-sulfonate was detectable, which would suggest that the acid-catalyzed hydrolysis of GSH S-sulfonate is not as fast as the initial sulfite substitution.

131 Due to its higher pKa, EtSH is a better leaving group than GSH. However, the concentrations of GSH disulfide in wine should far exceed that of DEDS, and as described above for thiol-disulfide interchange (Section 1.3.2), may serve to drive the reaction forward. Sulfitolysis may therefore prove to be important in terms of the presence in wine of both symmetrical and asymmetrical disulfides as well as polysulfanes, which may result in release of H2S, MeSH, and

EtSH due to hydrolysis of the corresponding organic thiosulfates. It may be that sulfitolysis is

accelerated at wine pH by the presence of transition metals, similar to disulfide-interchange (Figure

1.7). However, this proposition needs to be investigated further to understand the conditions that could drive such reactions.

1.3.4 Metal catalyzed disulfide scission

Transition metals may play a role in assisting thiol-disulfide interchange and sulfitolysis

(Sections 1.3.2 and 1.3.3). This reaction may proceed because of the metal’s ability to catalyze electrophilic and nucleophilic reactions of the disulfide bond (Figure 1.9).144 The binding of an electrophilic species (e.g. oxidized metals) makes one sulfur on the disulfide a better leaving group, facilitating its subsequent displacement by nucleophilic attack of the other sulfur moiety.144 This may be sufficient in cleaving the disulfide in the presence of wine nucleophiles including bisulfite, ascorbic acid, and perhaps polyphenolic compounds. A reduced metal can also bind to a thiol, as is the case with Cu(I)-SR, effectively making the thiol more nucleophilic (Figure 1.7). This will be more prevalent if the metal is simultaneously bound to an electron withdrawing group.143 Cobalt has been implicated in metal-assisted nucleophilic cleavage of disulfides.145

Figure 1.9. Concurrent electrophilic and nucleophilic assisted disulfide bond scission.

It appears that metals may play a role in both oxidative and reductive cleavage of disulfides, consistent with studies investigating DMDS and DEDS in wine that have demonstrated that concentrations of the disulfides decrease over time regardless of anaerobic or aerobic

conditions.49,117 It is likely that both reductive and oxidative cleavage mechanisms could occur, but would depend on the redox status of the wine.

In a study investigating disulfide bonds in wheat proteins, the combination of Mn and Cu- containing proteins (Cu(I) in particular) was found to be responsible for the reduction of the disulfide bond.146 In hydro(solvo)thermal conditions, the addition of transition metals including

Cu(II), Cu(I), Ni(II), Co(II), and Mn(II) to a disulfide resulted in the generation of multiple reaction products including the corresponding free thiols, trisulfides, and even new thiols, and generally with the corresponding metal-sulfur cluster coordination.147–150 Although these reactions are generally carried out under extreme conditions, they have been shown to also occur at room temperature.145,151 In some experiments, the cleavage of cystamine in the presence of Cu(II) was nearly instantaneous with water as the nucleophile.152,153

In general, the reactions described above are base-catalyzed, as the anionic form of water, thiols, and sulfite are much stronger nucleophiles that drive the reaction forward. However, the combination of both metal-assisted electrophilic and metal-assisted nucleophilic reactions may drastically accelerate the rates, which would be faster than the predicted year-long disulfide scission under simple model wine conditions.43

The interaction of polysulfanes may further drive metal-catalyzed scission reactions forward. The binding energy generally increases as the S-chain gets longer, and the maximum coordination number also increases corresponding with the number of S-atoms.154 Therefore, the interaction of polysulfanes with transition metals and possible release of H2S may be significant.

155 The release of H2S from elemental sulfur has been previously shown in wine, and it is likely that this reaction will be accelerated with assistance of transition metals, yeast-derived thiols, and reducing agents such as ascorbic acid.

1.3.5 Ascorbic acid

Ascorbic acid has been extensively studied in food systems and under physiological conditions as an antioxidant. Ascorbic acid has both antioxidant and pro-oxidant activities under wine conditions, and its chemistry as it relates to wine has been recently reviewed.156,157

Dehydroascorbic acid, the oxidized form of ascorbic acid, is well known to be reduced by GSH under physiological conditions to generate the corresponding GSH disulfide.158 However, there is also evidence for the reverse, where ascorbic acid reduces disulfide bridges.159 It has been speculated that the disulfide-reducing ability of ascorbic acid could occur under wine conditions with generation of undesirable sulfhydryl compounds.156

Winemakers wanting to screen their wine for VSCs often utilize ascorbic acid to test for the presence of disulfides. Screening for VSCs involves the addition of solutions of cadmium sulfate, copper sulfate, and ascorbic acid to the wine, with informal sensory analysis after each treatment addition.124 The expected sensory results of such testing are presented in Table 1.5. The role of ascorbic acid in this assay is to reduce disulfides in order to give the analyst an indication as to whether or not their wines contain DMDS and DEDS.124 Surprisingly, while this screening test and its potential use for treatment of disulfides has been practiced for several decades, the mechanism of disulfide reduction is unknown. Literature searches revealed there had been no published work that investigated the mechanism of disulfide reduction under wine conditions and the extent to which it proceeds. Winemakers are advised that the addition of Cu(II) sulfate and ascorbic acid may eliminate disulfides, but it may take several weeks for equilibrium to be established. However, this work remains mostly anecdotal with no or limited research available.

Table 1.5. Diagnostic test and sensory screening of sulfidic odors in wine utilizing copper, cadmium, and ascorbic acid. Control Cu(II) (0.2 g/L) Cd(II) (0.2 g/L) Ascorbic acid (1 g/L) + Sulfidic Cu(II) (0.2 g/L) compound Presence of Odor gone Odor gone Odor gone H2S sulfidic off-odors Odor gone No change Odor gone Thiols Odor gone Slight Odor gone H2S and thiols improvement No change No change Odor gone Disulfides No change No change No change Dimethyl sulfide

Ascorbic acid may reduce disulfide bonds, but like sulfitolysis and thiol-disulfide

interchange, it appears to proceed faster at higher pH. The reaction likely occurs via the mono- and

di-anion of ascorbic acid, whereas the undissociated acid has negligible reactivity in cleaving RSSR

as well as RSNO, with the latter possibly having a similar reaction pathway to the disulfide.159–161

Ascorbic acid’s first ionizable proton has a pKa of 4.25, which would mean that at pH 3.5 about

85% of ascorbic will remain non-ionized, whereas the other 15% would exist as the mono-anion

form.156

Rates of reduction of biological disulfides have been found to lie between ~3–5

× 10−5 M−1 s−1 at physiological pH (7.4).159 However, studies investigating the role of pH on RSNO,

which likely cleaves in the same way RSSR, found that the rate at pH 3.0 – 3.5 is 1000-fold lower

than at physiological pH,161 so the unassisted reaction will likely proceed extremely slowly in wine.

It has been suggested that the presence of transition metal ions, such as Cu and Fe, facilitate

disulfide cleavage.159 Given the concentrations of Cu and Fe in wine, as well as intentional addition

of ascorbic acid, this may play a crucial role in disulfide reduction at wine pH. While the

mechanism of disulfide reduction by ascorbic acid remains unknown, it is well known that ascorbic

acid can reduce Cu(II) to Cu(I), and this has been utilized in organic synthesis.162–165 It has been

suggested that in the ascorbic acid/copper system, Cu(I) drives the reduction of disulfides.161,164

Ascorbic acid also efficiently scavenges O2 by accelerating its reduction, and it promotes the anoxic conditions in bottled wine which are generally associated with release of VSCs. It is also possible that ascorbic acid plays a role in reducing metal sulfide complexes. Further studies should be conducted to decipher the mechanism of VSC generation as it relates to ascorbic acid.

1.4. Reactions of sulfhydryls with organic wine constituents

The reaction of sulfhydryls with organic compounds in wine results in C-S bond formation, and depending on the compound, may create a new aroma-active compounds or become nonvolatile and therefore eliminate the odor. Sulfhydryl compounds are nucleophilic species, especially H2S, and may react with electrophilic compounds in either reversible or non-reversible reactions. Wine contains a host of electrophilic compounds for such reactions, including quinones and aldehydes.

There is abundant research in wine showing the formation of catechol-thiol adducts during the wine oxidation process.62–64 These are formed by the reaction of thiol and quinone via a

Michael-type addition reaction, as shown in Figure 1.2. Given that the catechol-thiol adduct is nonvolatile, it effectively causes loss of aroma associated with the compound. The reaction is reversible, but whether this can be driven backward remains poorly understood. Preliminary results involving the H2S adduct of 4-methylcatechol (4-methyl-5-sulfanylcatechol) demonstrated that the

155 release of H2S occurs at pH 6 in the presence of reducing agents. Given that catechol-H2S adducts can exist in equilibrium with the catechol and H2S, it is possible that reducing conditions would result in H2S when O2 is limited.

It is well known that sulfite can react reversibly with aldehydes, forming a strong covalent bond (Figure 1.10).166,167 Reaction of sulfhydryls with aldehydes may also occur, resulting in 28

hemithioacetals and thioacetals under acidic conditions (Figure 1.10). Due to the abundance of carbonyl compounds in wine (e.g. acetaldehyde, glyceraldehyde, etc.),168,169 these may play a role in reversibly binding to sulfhydryls. It has been demonstrated that Cys may reversibly bind to aldehydes, and that the dissociation of these compounds is responsible for the generation of odor defects associated with aldehyde that are observed during beer aging.170 The bisubstitutional ability

171 of H2S may result in its reaction with multiple aldehydes.

Figure 1.10. Reversible reactions of aldehydes with bisulfite in wine to generate hydroxyalkylsulfonates or with thiols to generate hemithioacetals and thioacetals.

Wines contain abundant amounts of hydroxycinnamic acids bearing the electrophilic α,β- unsaturated carboxylic side chain, and their reversible reactions with sulfhydryls may be relevant in wine. Bouzanquet et al. have demonstrated an irreversible GSH-hydroxycinnamic acid product under wine conditions which involve free radicals.135 Another group investigated the reaction of

Cys with ferulic acid in wheat flour doughs and found that a cysteine-ferulic acid adduct is formed

172 which may later decompose in the dough. The equilibrium of H2S and thiols with the hydroxycinnamic acids may exist under wine conditions, but would need to be investigated further.

1.5 Thioester hydrolysis

Thioacetates are present in wine and are primarily generated by yeast during primary alcoholic fermentation. The formation of thioacetates is thermodynamically unfavorable and therefore unlikely to form without enzymatic action. However, thioesters can be hydrolyzed to their corresponding thiols at low pH, and given the lower detection threshold of thiols released, this may

have a significant impact on a wine’s aroma.173 The thioacetates of MeSH and EtSH have been observed in wines, and their hydrolysis could be an explanation for their release, however, there have been no studies showing conclusive evidence for their cleavage. On the other hand, thiol- thioester exchange may also have implications with respect to the generation of VSCs;174 for example, sulfite may react with methyl thioacetate to generate the corresponding sulfonate, with the release of MeSH.

1.6 Strecker degradation of amino acids

Strecker degradation of amino acids is known to occur in the presence of a dicarbonyl compound. It was first suggested that an o-quinone can play this role in tea leaves,175 and has since been shown to occur in synthetic solution and model wine.176,177 It has been demonstrated that Cys can generate H2S, and formation of MeSH from methional and methionine was also reported under wine-like conditions.178 Recent work supports the idea that methionine is one of the most important precursors for the formation of MeSH post-fermentation.117 These reactions are non-reversible, and transition metals play an important role in generating the o-quinone as the starting reactant for

Strecker degradation compounds.

1.7 Further reactions of sulfur containing compounds

There are likely numerous yet-to-be identified sulfur-containing compounds in wine that may further contribute to wine aroma. Oxidation of MeSH in the presence of H2S may yield potent polysulfanes, dimethyl trisulfane and tetrasulfane, which have detection thresholds of 100 ng/L and

1,179 60 ng/L, respectively. Reaction of H2S with benzaldehyde generates benzyl mercaptan, which has a smoky odor,180 whereas reaction with furfural generates furfurylthiol that is reminiscent of 30

roasted coffee.181 In food systems other than wine, sulfur compounds with extremely low threshold have been identified; for example, (S)-1-p-menthene-8-thiol (grapefruit mercaptan) has an odor threshold of 6.6×10-6 ng/L in air. Furthermore, modification of grapefruit mercaptan structure by changing the location of the sulfur atom resulted in unique odors described as sulfury, rubber-like, burned, soapy, and mushroom-like.182 Some of these compounds would generally be considered as defects in food and beverages. The occurrence of sulfur compounds may be specific for certain wine styles, and the contribution of unidentified compounds may be important in explaining the phenomenon of ‘reduction’ of certain wines.

1.8 Research overview, significance, and hypotheses

Wine is a globally consumed alcoholic beverage with tremendous economic value. In the

US alone, the estimated retail value of all wine produced in 2014 amounted to US$37.6billion.183

Because wine is an important agricultural commodity, wine quality and long shelf life are crucial for consumers. The generation of reductive sulfidic off-odors is not an uncommon fault in wines, reportedly accounting for 25% of faults in wine shows.184 The presence of sulfidic off-odors in wine can adversely affect sales and brand image with consumers.

The overall aim of this thesis is to elucidate some key mechanisms that govern the redox cycling of sulfhydryl compounds in the presence of transition metals in wine. VSCs are amongst the most important aroma compounds in wine, as they can either contribute pleasant varietal aromas or deleterious sulfidic off-odors, depending on their structures. I hypothesize that the decline of these compounds in wine is linked to oxidation reactions mediated by transition metals.

Furthermore, I hypothesize that the reappearance of unwanted sulfidic off-odors is linked to the reduction of disulfides, polysulfanes, and metal sulfide complexes, which is also mediated by transition metals. 31

The objectives needed to achieve the aims of this research are to:

1. Elucidate the oxidation mechanism of H2S and thiols during Cu(II) fining

2. Investigate the oxidation of sulfhydryl compounds in the presence of a combination of

copper, iron, and manganese

3. Uncover the reactions and conditions responsible for release of sulfhydryl-bearing

compounds

4. Provide winemakers with tools to predict and control a wine’s quality from a VSC

perspective

Chapter 2

Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model Wine. Part 1: Copper Catalyzed Oxidation.

Published as:

Kreitman, G.Y.; Danilewicz, J.C.; Jeffery, D.W.; Elias, R.J. Reaction Mechanisms of Metals with

Hydrogen Sulfide and Thiols in Model Wine. Part 1: Copper Catalyzed Oxidation. J. Agric. Food

Chem. 2016, 64, 4095-4104.

2.1 ABSTRACT

Sulfidic off-odors due to hydrogen sulfide (H2S) and low molecular weight thiols are commonly encountered in wine production. These odors are usually removed by the process of Cu(II) fining

– a process that remains poorly understood. The present study aims to elucidate the underlying mechanisms by which Cu(II) interacts with H2S and thiol compounds (RSH) under wine-like conditions. Copper complex formation was monitored along with H2S, thiol, oxygen, and acetaldehyde concentrations after addition of Cu(II) (50 or 100 μM) to air saturated model wine solutions containing H2S, cysteine, 6-sulfanylhexan-1-ol, or 3-sulfanylhexan-1-ol (300 μM each).

The presence of H2S and thiols in excess to Cu(II) led to the rapid formation of ~1.4:1 H2S:Cu and

~2:1 thiol:Cu complexes, resulting in the oxidation of H2S and thiols, and reduction of Cu(II) to

Cu(I) which reacted with oxygen. H2S was observed to initially oxidize rather than form insoluble copper sulfide. The proposed reaction mechanisms provide an insight into the extent to which H2S can be selectively removed in the presence of thiols in wine.

2.2 INTRODUCTION

Volatile sulfur containing compounds (VSCs) have a major impact on the sensory quality of wine.1–3 Typically, VSCs have exceedingly low aroma detection thresholds (i.e., μg/L to ng/L) and, depending on their structure, can have beneficial or deleterious effects with respect to consumer acceptance. Grape-derived varietal thiols, such as 3-sulfanylhexan-1-ol (3SH), 3- sulfanylhexyl acetate (3SHA), and 4-methyl-4-sulfanypentan-2-one (4MSP), contribute pleasant aromas (e.g., grapefruit, passionfruit, and blackcurrant).5–7 On the other hand, the production of fermentation-related VSCs, such as H2S, methanethiol (MeSH), and ethanethiol (EtSH), can result in the development of undesirable odors, often described as rotten egg, putrefaction, sewage and burnt rubber, that are obviously detrimental to wine quality.1,41,185 These odors are generally most evident at low oxygen concentrations and are described to be sulfidic off-odors. Wines that display such odors are described as having reductive character.

The accumulation of sulfidic off-odors is a common problem for winemakers and is usually remedied by splash racking in order to volatilize and/or oxidize VSCs or, classically, by the use of copper fining.2,41,90 In this latter practice, Cu(II) is added as its sulfate or citrate salt whereby it is

90,167 assumed to remove H2S by forming a highly insoluble colloidal CuS precipitate (Figure 2.1), which can be subsequently removed from the wine by racking and/or filtration. The mechanism for copper fining remains poorly understood and there are known disadvantages to the process. In the case of disulfides, thioacetates, and cyclic sulfur compounds, which can also contribute unpleasant sulfidic off-odors, copper fining is ineffective due to the absence of a free thiol group.2,41 Copper fining can also cause significant losses of beneficial thiol compounds (e.g. 3SH, 3SHA, 4MSP) that are important to the varietal character of a wine.48 Furthermore, other thiols could interfere with the fining process by competing for Cu(II) given that the average combined concentration of cysteine

(Cys), N-acetylcysteine and homocysteine is reported to be ca. 20 µM in a number of white wines,

while the average concentration of glutathione (GSH) is reported to be ca. 40 µM in wines made from Sauvignon blanc.92–95 These nonvolatile thiols would be in large molar excess to the exogenous copper (3–6 µM) used in a fining operation, and would far exceed the concentration of

30 91 H2S (ca. 300 nM) when copper fining is considered. Furthermore, a recent study by Clark et al. demonstrated the practical difficulty of removing CuS from wine, even with filtration, as the precipitate may not be observed.167 This lack of precipitate formation would leave residual copper in wine that can contribute to a series of redox-mediated reactions in the post-bottling period, as elaborated below.

Figure 2.1. Removal of H2S by addition of Cu(II) and formation of insoluble CuS.

After bottling, the concentration of sulfidic off-odors can increase, especially under reductive conditions when oxygen exposure is limited such as when screw cap closures are used.47,48,186 Although the causative mechanism remains unclear, wine appears to contain precursors

50,57 that are able to produce H2S and MeSH. The formation of H2S from the Strecker degradation

178 of Cys has been previously reported, while some have suggested that H2S may be formed by the direct reduction of sulfate or sulfite.47 It has also been shown that thiols can be reversibly bound by iron and copper,80,81 and that wines containing higher copper concentrations can accumulate sulfidic off-odors during bottle aging.48,70 While transition metals are known to be essential for catalyzing oxidation reactions in wine,51 Cu, Fe, Mn, Zn, and Al have more recently been shown to synergistically affect the evolution of VSCs under anaerobic storage conditions.70

In order to understand how wines develop sulfidic off-odors during storage, it is essential to understand how H2S and thiols react in the presence of oxygen and transition metals prior to bottling. The identification of reaction products may then allow potentially troublesome precursors 35

to be targeted. Recent studies in this area have advanced our general mechanistic understanding of iron-catalyzed wine oxidation; however, the role of copper remains poorly understood. The goal of this present study is to determine the underlying mechanism of Cu-catalyzed H2S and thiol oxidation under wine conditions.

2.3 MATERIALS AND METHODS

2.3.1 Chemicals

4-Methylcatechol (4-MeC), L-cysteine (Cys), monobromobimane (MBB), 5,5- dimethyl-1-pyrroline N-oxide (DMPO), bathocuproinedisulfonic acid (BCDA) disodium salt, 6-sulfanylhexan-1-ol (6SH), and diethylenetriaminepentaacetic acid (DTPA) were obtained from Sigma-Aldrich (St. Louis, MO). 2,4-Dinitrophenylhydrazine (DNPH) was purchased from MCB laboratory chemicals (Norwood, OH) and L-tartaric acid, 3SH, and

5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) were obtained from Alfa Aesar (Ward Hill,

MA). Cupric sulfate pentahydrate was purchased from EMD Chemicals (Gibbstown, NJ),

TRIS hydrochloride from J.T. Baker (Center Valley, PA), and sodium hydrosulfide hydrate

(as a source of H2S) was purchased from Acros Organics (Geel, Belgium). Water was purified through a Millipore Q-Plus system (Milipore Corp., Bedford, MA). All other chemicals and solvents were of analytical or HPLC grade, and solutions were prepared volumetrically, with the balance made up with Milli-Q water unless specified otherwise.

2.3.2 Model wine experiments

Model wine was prepared by dissolving tartaric acid (5 g/L) in water, followed by the addition of ethanol to yield a final concentration of 12% v/v. The solution was adjusted to pH 3.6 with sodium hydroxide (10 M) and brought to volume with water. For H2S and Cys, an aqueous stock solution of each (0.5 M) was freshly prepared, whereas 6SH and 3SH were added directly by syringe during experimentation (Figure 2.2). An aqueous stock solution of Cu(II) sulfate (0.1 M) was prepared freshly. In certain experiments, 4-MeC (1 mM) was added prior to the addition of

H2S and thiol compounds, and Cu(II). H2S, Cys, 6SH, or 3SH were added to air saturated model wine (1 L, 300 μM) followed by thorough mixing. Cu(II) was added to H2S, Cys, and 6SH (50 μM) or 3SH (100 μM) and thoroughly mixed. For mixed H2S and Cys system, H2S (100 µM) and Cys

(400 µM) were added to air saturated model wine (1 L), followed by the addition of Cu(II) (100

µM) and thorough mixing. The solution was immediately transferred to 60 mL glass Biological

Oxygen Demand (B.O.D.) bottles (Wheaton, Millville, NJ), allowing the solution to overflow, and bottles were capped immediately with ground glass stoppers, thereby eliminating headspace. The glass reservoir of the B.O.D. bottles was topped off with water daily. The bottles were stored in the dark at ambient temperature. One B.O.D. bottle was sacrificed per time point per replicate and used for further analyses. All experiments were conducted in triplicate and had their own series of sacrificial bottles.

Figure 2.2. H2S and thiols used throughout this study.

For experiments focusing on 6SH-disulfide formation, one experiment was prepared as described above and followed over time. For additional experiments for deciphering immediate disulfide generation, model wine (3 mL) containing 6SH (600 μM) in a glass test tube was deoxygenated for 2 min under argon with stirring. After sparging,

Cu(II) was added at varying concentrations (50, 100, or 200 µM) under argon and reacted with stirring for 5 minutes. The solution was then immediately analyzed to determine 6SH and 6SH-disulfide concentrations (described below). In experiments involving 4-MeC or

DMPO, these compounds were dissolved directly into model wine to achieve a final concentration of 1 mM prior to addition of Cu(II) (100 µM).

2.3.3 Determination of oxygen consumption

Prior to the experiment, 60 mL glass B.O.D. bottles containing PSt3 oxidots (Nomacorc

LLC, Zublon, NC) were filled with air saturated model wine for a minimum of 2 hours to allow the oxidots to equilibrate. One B.O.D. bottle was used as a model wine control (i.e., did not contain a treatment) and two other bottles were used as technical duplicates to determine oxygen concentration for each treatment replicate (3 treatment replicates total). Thus, immediately after the addition of Cu(II) solution, the model wine used for equilibration was discarded and the respective treatment solution was instantly transferred into the bottles. Oxygen readings were taken per time point using NomaSense O2 P6000 meter (Nomacorc LLC, Zublon, NC), and data were normalized to the model wine reference sample. Starting oxygen concentrations were approximately 7 mg/L

(~220 µM) in all solutions.

2.3.4 Cu-complex formation and dissolution

6SH-Cu(I) complex was prepared by adding Cu(II) (100 µM) to model wine (1 L) containing 6SH (400 µM). The immediately formed precipitate was vacuum filtered with a 0.45

µm nylon membrane (Wheaton, Millville, NJ), washed with water followed by ethyl acetate in order to remove residual disulfide, and dried under vacuum. In an anaerobic chamber (95% Ar, 5%

H2), ~1 mg of the solid was added to water containing approximately 5× molar excess of BCDA.

This mixture was stirred for approximately 30 min until all of the solid dissolved. 6SH, 6SH- disulfide, and Cu(I) concentrations were measured as described below.

2.3.5 Spectrophotometric measurements of thiols and H2S

UV-vis spectra were recorded on an Agilent 8453 UV-Vis spectrophotometer (Agilent, Santa Clara,

CA). Determination of Cu binding to H2S and thiols was determined by measurement over 200-

700 nm. The concentration of H2S, Cys, 6SH, and 3SH was determined using Ellman’s reagent

(DTNB).187 An aliquot of sample (100 μL) diluted with model wine (900 μL) was treated with a solution of DTNB (400 μL, 2 mM) in phosphate buffer (10 mM, pH 7.0) followed by addition of

TRIS-phosphate buffer (100 μL, 1 M, pH 8.1). The mixture was left at ambient temperature for 30 min before the absorbance was measured at 412 nm against a blank consisting of model wine,

DTNB solution, and TRIS-phosphate buffer in the proportions specified above.

2.3.6 Spectrophotometric measurement of Cu(I)-BCDA

Cu(I) concentration was analyzed using the BCDA assay.188 Treatment and standard solutions consisted of excess Cys (5 mM) to ensure Cu(I) remained in its reduced state. An external

standard curve of the Cu(I)-BCDA complex was prepared in model wine, and absorbance values were recorded at 484 nm against a model wine blank.

2.3.7 HPLC analyses of thiols and H2S

MBB derivatization was used to determine each H2S and Cys concentrations in the mixed system based on a modification of a previous method.189 MBB reagent (40 mM) was prepared anaerobically by dissolving the solid in acetonitrile. Aliquots of the reagent were stored at -80 °C.

Briefly, a sample aliquot (70 μL) was mixed with an equal volume of TRIS-HCl buffer (100 mM) containing DTPA (0.1 mM) at pH 9.5, followed by the immediate addition of MBB (10 μL; 40 mM). The reaction was allowed to proceed aerobically at room temperature in the dark for 30 min before the addition of sulfuric acid (50 μL, 200 mM) and 6SH-bimane internal standard (50 μL).

6SH-bimane was prepared following a sulfide-dibimane synthesis described previously.189 Samples were filtered through PTFE syringe tip filters (0.45 μm, 13 mm filter diameter; AcrodiscTM, Ann

Arbor, MI) prior to analysis by HPLC-MS/MS.

Quantitative analysis was performed with a Shimadzu LC-VP series HPLC

(Columbia, MD) interfaced to a Waters Quattro micro triple quadrupole mass spectrometer

(Milford, MA) that was operated with MassLynx software. Bimane adducts were separated on a ZORBAX Eclipse Plus C18 column (2.1 x 150 mm, 5 μm) with a guard column of the same material at a flow rate of 0.2 mL/min with mobile phases consisting of 0.1% v/v formic acid (A) and 0.1% v/v formic acid in acetonitrile (B) and a linear gradient according to the following program: 0 min, 2% B; 9 min, 50% B; 14 min, 100% B; 18 min, 100% B;

19 min, 2% B; 26 min, 2% B.

Detection of bimane adducts was performed using negative ion electrospray ionization

(ESI-) with multiple reaction monitoring (MRM) (Figures A.1-A.3). The ESI capillary spray voltage was set to 4 kV, the sample cone voltage was set to 25 V, and the source temperature was

120 °C. The desolvation gas flow was 450 L/h and collision energy was set to 20 eV. The mass transition of sulfide-dibimane was monitored at m/z 413→191, cysteine-bimane was monitored at m/z 310→223, and the internal standard 6SH-bimane was monitored at m/z 323.2→222.2. An external standard curve was prepared for sulfide-dibimane and Cys-bimane and data were normalized to the 6SH-bimane internal standard.

For experiments involving 6SH and its disulfide, quantitative analysis was performed using the HPLC system described above and UV detection at 210 nm with external standard calibration curves. Separation was achieved at a flow rate of 0.2 mL/min with mobile phases consisting of 0.1% v/v formic acid (A) and 0.1% v/v formic acid in acetonitrile (B) and a linear gradient according to the following program: 0 min, 5% B; 20 min, 95% B; 28 min, 95% B; 28.1 min, 5% B; 38 min, 5% B.

For experiments involving dissolution of 6SH-Cu complex with BCDA, the same chromatographic conditions described for 6SH and its disulfide were followed. However, the BCDA peak could not be resolved from that of 6SH at 210 nm, therefore detection of

6SH was performed using ESI+ with selective ion monitoring (SIM) at m/z 135 with an external calibration curve. The ESI capillary spray voltage was set at 4 kV, the sample cone voltage was set to 25 V and the source temperature was 120 °C. The desolvation gas flow was 650 L/h.

2.3.8 HPLC analysis of catechols

For experiments containing 4-MeC, quantitative analysis was performed with the HPLC system described above and UV detection at 280 nm with an external standard calibration curve.

4-MeC was separated on an Ultra Aromax column (2.1 x 150 mm, 5 μm) with a guard column of the same material at a flow rate of 0.2 mL/min with mobile phases consisting of 0.1% v/v formic acid (A) and 0.1% v/v formic acid in acetonitrile (B) and a linear gradient according to the following program: 0 min, 30% B; 3 min, 30% B; 12 min, 100% B; 20 min, 100% B; 20.1 min, 30% B; 25 min, 30% B. The putative formation of oxidation products including catechol-thiol adducts and condensed units was monitored both at 280 nm and with negative ion ESI-MS (total ion chromatogram m/z 100-1000).

2.3.9 HPLC analysis of acetaldehyde

Acetaldehyde was measured in model wine treatment solutions as its 2,4- dinitrophenylhydrazone (DNPH) derivative by HPLC as described previously67 with the following modification: the sample was centrifuged at 15000 × g at 4 °C for 10 min. The supernatant was then transferred to an HPLC vial for further analysis.

2.3.10 Copper determination

For each given time point, samples were mixed in B.O.D. bottles and then filtered through a 0.45 um PTFE syringe filter. The resulting filtrate (5 mL) was digested by the addition of 30% hydrogen peroxide (3 mL) and sulfuric acid (100 μL) based on modification of previous reported methodology.190 The samples were heated in a convection oven at 110 °C overnight before being

reconstituted to 5 mL with 0.1 M nitric acid. Samples were analyzed by inductively coupled plasma optical emission spectroscopy (Agilent 700 Series, Santa Clara, CA) using a vertically aligned torch and with monitoring at 324.7 nm.

2.3.11 EPR analysis

Loss of the electron paramagnetic resonance (EPR) signal for active Cu(II) (0.5 mM) in model wine was monitored after the metal solution was mixed with the respective H2S and thiol treatments (1.5 mM). Samples were transferred to a cuvette and snap frozen in liquid nitrogen.

Continuous wave EPR spectra were acquired on a Bruker ESP300 X-band spectrometer (Billerica,

MA) equipped with a ER 041MR microwave bridge and a Bruker ER 4102ST resonator.

Temperature was controlled by a variable temperature helium flow cryostat (ER 4112-HV, Oxford

Instruments, Abingdon, UK). Data acquisition and control of experimental parameters were performed using the EWWIN 2012 software package. Instrument settings were as follows: temperature, 100 K; microwave power, 2 mW; modulation frequency, 9480 MHz; modulation amplitude, 20 dB; scan range, 2000 G.

2.4 RESULTS

The reactivity of Cu(II) with H2S, which is the primary target of Cu fining, and the following three thiols was investigated under wine conditions (Figure 2.2): (1) Cys, which also represented homo-Cys and Cys derivatives, (2) 6SH to represent primary thiols, and (3) 3SH to represent secondary thiols. With H2S Cu(II) addition resulted in an immediate uptake of ~1.4 (72

µM) mole equivalents of H2S, the remainder was then fully consumed within 72 h. However, with the thiols, the immediate uptake increased to approximately two equivalents (Figure 2.3), with 43

initial consumption of 101 and 121 µM for Cys and 6SH, respectively, the remainder then being fully consumed within 48 h. The varietal thiol 3SH reacted in the same manner but more slowly, with 2 mole equivalent of 3SH (210 µM) consumed relative to Cu(II) added after 2 hours, and was not fully reacted after 168 h (Figure 2.3).

EPR analysis showed that Cu(II) was immediately reduced to Cu(I) due to loss of paramagnetic Cu(II) signal by Cys, 6SH and H2S; again, 3SH reacted more slowly (Figure 2.4A), with Cu(II) reduction being complete after 2 h (data not shown). The apparent formation of a Cu(I) complex was observed by UV spectroscopy (Figure 2.4B). Absorbance increased markedly from

200-400 nm by the addition of H2S and Cys to model wine containing Cu(II), but did not produce a distinct absorbance maximum above 220 nm. In contrast, 6SH showed a maximum at 353 nm, and 3SH had absorbance maxima at 282 and 311 nm (Figure 2.4B). 44

Figure 2.4. Reaction of Cu(II) in (a) model wine and treatments containing (b) 3SH, (c) 6SH, (d) Cys, and (e) H2S, showing (A) loss of electron paramagnetic resonance (EPR) active Cu(II) (0.5 mM) signal in model wine after mixing with the respective thiol/H2S treatments (1.5 mM), and (B) UV-spectra of the thiols/H2S (300 μM) in model wine after mixing with Cu(II) (50 μM).

The addition of Cu(II) to H2S in model wine resulted in a clear golden colored solution that yielded a green/black precipitate over time, whereas a haze that developed with the three thiol treatments (Cys, 6SH, 3SH) aggregated to form a fine white/yellow precipitate. This was particularly evident for 6SH, as essentially all the Cu(I) complex was removed by filtration (0.45

µm) from 5 to 45 min after mixing (Figure 2.5A). Filtration at earlier time points and measurement of residual copper remaining in solution confirmed that the 6SH aggregate formed rapidly and could be removed from solution by filtration after 5 min (Figure 2.5B). However, at the last time point, copper had been released from the insoluble Cu(I) complex in a copper form that could not be removed by a 0.45 µm filter. 3SH reacted in the same manner, but more slowly. For the H2S treatment, ca. 60% of the copper was removed by filtration within 5 min and up to 24 h. After 72 h, there was a green-black precipitate. Approximately 90% of copper was then removed from solution (Figure 2.5B).

The aggregate initially formed from the reaction between Cu(II) and 6SH on drying gave a fine powder, which was solubilized in water containing BCDA (a Cu(I) selective chelator188). The insoluble Cu(I)-complex dissolved as BCDA displaced the thiolate ligand, yielding 1.17 ± 0.02 mM Cu(I), as determined by UV spectrophotometry, and 1.17 ± 0.13 mM 6SH was released, as determined by HPLC-MS, giving a ~1:1 Cu(I):6SH molar ratio with minimal disulfide formation

(data not shown).

When H2S (75 µM) and Cys (468 µM) were added together to model wine in the presence of Cu(II), ca. 53 and 135 µM of H2S and Cys, respectively, were consumed within 5 min (Figure

2.6). Together this gives 189 µM of sulfhydryl compounds consumed with added 100 µM Cu(II) which translates to a ~2:1 binding ratio of H2S + Cys:Cu(II). Subsequent reaction resulted in complete loss of H2S within 40 min and Cys after 48 h. While a visible precipitate was observed at the end of the reaction (74 h), it was not observed to the same extent as was the case with H2S alone.

The 6SH/Cu(II) system was used to monitor disulfide formation under argon. Addition of

Cu(II) at 50, 100, and 200 µM resulted in disulfide generation of 19.7 ± 3.6, 43.4 ± 3.1, and 98.2 ±

3.6 µM, respectively (data not shown). In addition, the oxidation of 6SH (240 μM), in the presence of 50 µM Cu(II) was monitored over time in air saturated model wine (Figure 2.7). After 262 h,

231 ± 2.5 µM of the thiol reacted and 116 ± 2.7 µM disulfide was produced. Approximately 69 ±

8.0 µM O2 was consumed in this reaction (Figure 2.7), giving an O2:thiol molar reaction ratio of

~1:3.3.

Figure 2.7. O2 and 6SH consumption, and 6SH-disulfide formation in air saturated model wine containing 240 μM 6SH and 50 μM Cu(II). Error bars indicate standard deviation of triplicate treatments.

To further examine the mechanism of disulfide formation using 6SH as a model, an attempt was made to intercept potential intermediate thiyl radicals with the o-quinone-producing 4-MeC, and the radical trap DMPO. However, no change in disulfide formation was observed by HPLC upon addition of Cu(II) (100 µM) to model wine containing 6SH (600 µM) and 4-MeC or DMPO

(1.0 mM) under anaerobic conditions (data not shown).

Oxygen consumption was also measured in model wines containing the H2S and thiol treatments, as well as a combination treatment consisting of Cys+H2S (Figure 2.8). Minimal O2 uptake (<5 µM in all treatments) was observed within the first 30 min of the reaction. During the course of the experiments, H2S had the highest O2 consumption (175 ± 9 µM), followed by 6SH and Cys, which showed similar O2 consumption patterns (76 ± 6 and 66 ± 6 µM, respectively), and lastly 3SH, which consumed the least O2 (23 ± 1 µM). The treatment containing both Cys and H2S resulted in an O2 consumption of 117 ± 5.2 µM. Separately H2S or Cys were oxidized in the 48

presence of Cu(II) and excess 4-MeC and monitored over time. The rate of O2 consumption was not effeceted by the presence of the catechol, and its concentration did not decrease over time.

There was also no evidence of catechol-thiol adduct formation as assessed by HPLC-MS (data not shown).

Complementing the range of measurements described above, acetaldehyde (AC) generation was monitored over time (Figure 2.9). At the end of the experiment, the H2S containing system had accumulated the highest concentration of AC (79 ± 2 µM), followed by 6SH with 52 ±

4 µM, Cys at 26 ± 0.3 µM, and 3SH at 13 ± 0.8 µM. The combination of Cys + H2S yielded an AC concentration of 54 ± 3 µM.

2.5 DISCUSSION

2.5.1 Cu reduction and complex formation

From the above results, it is proposed that when a thiol is added to Cu(II), Cu(II) coordinates with two thiol moieties to give product (1, Figure 2.10). Electron transfer from sulfur gives the Cu(I) intermediate, two of which associate to (2) allowing bond formation between the two sulfur atoms to form the disulfide bound to Cu(I) (3), without release of free thiyl radicals. The released Cu(I)-complex then associates to give the sparingly soluble aggregate (4). H2S is proposed to react similarly with the formation of an initial complex, which could be Cu3S3, as discussed below.

Figure 2.10. Proposed mechanism for initial reaction of thiols with Cu(II) and Cu(I)-thiol complex formation. Only the thiol ligands are shown.

The initial binding of H2S and thiols to Cu(II) (Figure 2.3), therefore, appears to coincide with the reduction of Cu(II) to Cu(I) as seen by the rapid loss of the cupric species’ paramagnetic signal (Figure 2.4A). Of note is that with H2S a signal due solid Cu(II)S is not evident; furthermore, there was no appreciable oxygen consumption within this time frame (Figure 2.8). The immediate reduction of Cu(II) by Cys to form a Cu(I) complex has previously been demonstrated in phosphate buffer (pH 7.4) by EPR.102 No paramagnetic Cu(II) signal was observed immediately after thiol addition but returned as the Cu(I) was allowed to oxidize in air. In a previous study, EPR was used to show that GSH reduced Cu(II) in the pH range of 4-7, while the 1H-NMR spectrum of a 1:2 mixture of Cu(II):GSH in H2O-D2O (pH 7.5) indicated that one GSH was coordinated to Cu(I), while a second GSH had been oxidized to the corresponding disulfide.103 This also demonstrated that the stoichiometry required for complete loss of the Cu(II) signal was 1:2 Cu(II):RSH. Similar results were obtained with Cys, N-acetyl-cysteine and 2-mercaptoethanol, in which disulfide peaks were observed in the absence of Cu(II).103 Our results obtained in model wine were consistent with these studies, despite the large molar excess of tartaric acid, which did not appear to interfere with

H2S or thiol coordination by Cu(II).

Previous studies in phosphate buffer (pH 7.4) have shown that the Cu(I)-Cys complex has an absorbance maximum at 260 nm with a characteristic shoulder at 300 nm.102 In the present study, 51

the addition of H2S and Cys to model wine containing Cu(II) did not produce a distinct absorbance maximum above 220 nm, although the absorbance increased markedly (Figure 2.4B). The H2S- containing system’s UV spectrum had an elevated baseline, which could be due to the presence of

Cu(I) complex nanoparticles, some of which are sufficiently small to behave as dissolved species capable of absorbing energy in the UV region of the spectrum.34 In contrast, 6SH showed an absorbance maximum of 353 nm, and 3SH had absorbance maxima at 282 and 311 nm (Figure

2.4B). The formation of an insoluble Cu-complex (4) was evident upon the addition of Cu(II) to

6SH (Figure 2.10) and the complex was retained on a 0.45 µm filter, causing complete loss of absorbance in the UV region (Figure 2.5A), including that due to the Cu(II)-tartrate species (240 nm). As the Cu(I)-complex was allowed to slowly oxidize from the initial air saturation, a fraction of Cu(II) was shown to be released back into solution as particles smaller than 0.45 μm, as was evident by the increase in total Cu concentration at later time points (Figure 2.5B). Previous studies using X-ray absorption spectroscopy found that the aggregated GSH-Cu(I) complex was coordinated to three sulfur atoms with a stoichiometry of [CuS1.2], suggesting that the structure was polymeric with a thiolate sulfur serving as a bridge.191 This complex, however, did not have a single rigid cluster structure but was comprised of a mixture of various polymers.191 The triply-bridged

Cu(I) likely binds to water to satisfy its four-coordinate geometry. The dissolution of the Cu(I)-

6SH complex with BCDA revealed a ~1:1 Cu(I):6SH molar ratio, which is in agreement with previous work.191

The reaction between H2S and Cu(II) has been shown to be different from that of thiols, and has been studied in some detail. Initial coordination and reduction of Cu(II) to Cu(I), which is proposed to occur by inner-sphere electron transfer, is relatively fast.72 The resulting Cu(I) complex forms clusters composed of neutral 6-membered Cu3S3 ring systems that adopt a chair-like conformation.72 As discussed above, these polynuclear nanoclusters are sufficiently small to behave

like dissolved species.34 This process is consistent with our observation of a clear golden-brown solution in model wine, the UV-spectrum of which showed a broad increase in absorbance with an elevated baseline (Figure 2.4B), and thus indicative of light scattering by nanoparticles. Over time,

- these rings are known to condense, yielding Cu-S-S or Cu-S-Cu linkages and formation of [Cu4S5]

4 -4 72 and [Cu4S6] polynuclear nanoclusters that can further condense and precipitate as dark green or bluish covellite containing only Cu(I).34,36,192 The reduction of Cu(I) occurs prior to aggregation, and the rate of aggregation of these nanoparticles is relatively slow at ambient temperature,

192 although the presence of O2 at various concentrations has been shown to alter the rate of reaction.

The presence of excess H2S may favor formation of higher order clusters and further binding of S by Cu,72 which results in aggregation and may explain why approximately 40% of Cu was able to be filtered from solution after mixing (Figure 2.5B). A similar effect has been previously observed in model wine solutions when the ratio of H2S to Cu(II) exceeded 2.5:1, in which Cu was shown to aggregate and was able to be partially filtered from solution.91 An important consideration is that Cu(II) is typically added in excess to H2S in winemaking, which would limit ring formation and further aggregation of the Cu(I)-complex. In addition, other thiols also present in wine may compete with H2S for Cu coordination.

When H2S and Cys were added in combination in the presence of Cu(II), a 2:1 binding ratio of H2S + Cys:Cu(II) was still observed (Figure 2.6). Cu(II) binds rapidly to H2S and relatively more strongly than Cys, which is a benefit for winemakers wanting to remove H2S. While there was a visible precipitate towards the end of the reaction, it was not observed to the same extent as was the case with H2S alone. This could be due to the presence of Cys, which may prevent further aggregation of the Cu(I)-complex, as organic thiols are capable of terminating the highly ordered polymerization and condensation of the bulk metal sulfide complex.75 This process may account

91 for the apparent lack of a precipitate when Cu(II) is added to wine in order to remove H2S.

2.5.2 Disulfide formation

The formation of 6SH-disulfide as a model for disulfide formation by other volatile thiols was monitored to confirm the proposed mechanism. No appreciable uptake of O2 was observed during the first phase of the reaction of 6SH (or any of the treatments) in which Cu(II) was reduced

(Figure 2.8), suggesting that the thiol was initially oxidized directly by Cu(II) to its disulfide

(Figure 2.10). When Cu(II) was added to model wine containing excess 6SH at increasing concentrations (50, 100, and 200 µM) under argon, 0.5 moles of disulfide was produced (19.7 ±

3.6, 43.4 ± 3.1, and 98.2 ± 3.6 µM, respectively) for each mole of Cu(II) that was present. One thiol would be oxidized to yield half an equivalent of disulfide while the other would coordinate to Cu(I), which supports our proposed mechanism (Figure 2.10). Evidently, this Cu(I)-bound thiol can be removed from solution by filtration (0.45 μm) prior to HPLC analysis and does not react with

Ellman’s reagent, which was used to measure thiol concentration. 6SH was also oxidized in air saturated model wine in the presence of Cu(II) and monitored over time (Figure 2.7). The entirety of the thiol appeared to have reacted after 74 h, leaving an equimolar quantity bound to Cu(I) (50

µM). O2 uptake and disulfide formation then continued as this remaining thiol was oxidized. The aggregate had settled over time, and the heterogeneous nature of the system likely accounts for the slowness of the reaction. After 262 h, the reaction was complete and the 1:0.5 RSH:RSSR molar ratio showed that the disulfide was essentially the sole product. This was paired with 69 µM of O2 uptake, giving an O2:thiol molar reaction ratio of ~1:3.3.

We further examined disulfide formation by ascertaining whether free thiyl radicals were produced in the thiol/Cu(II) systems, as recently suggested,50 using 6SH/Cu(II) system. Wine contains various compounds such as polyphenols that could preferentially react with radicals, thereby preventing the formation of disulfides. Experiments were therefore conducted with 4-MeC and 6SH in anaerobic model wine prior to addition of Cu(II); if free thiyl radicals were formed

under such condition, the catechol would be expected to scavenge those radicals to yield semiquinone radicals (Figure 2.11) and ultimately o-quinones that could undergo 1,4-Michael addition with thiols to yield a catechol-thiol adducts.96 However, this was not observed as disulfide concentration remained unchanged and no catechol-thiol adducts were detected (data not shown).

In a separate experiment, DMPO was added to anaerobic model wine prior to Cu(II)-catalyzed 6SH oxidation, which should have yielded DMPO-thiyl radical adducts at the expense of disulfide formation (Figure 2.11), yet no depression in disulfide formation was observed (data not shown).

Based on the lack of evidence of thiyl radical formation in this, as well as from previous studies conducted at physiological pH,122,193 it appears that such radicals are not produced during the initial

Cu(II) reduction. Instead, it is proposed that disulfides arise through bond formation between two sulfur atoms in the Cu(I)(SR)2 dimer (2) without release of free thiyl radicals (Figure 2.10).

Figure 2.11. Proposed thiyl radical formation and subsequent scavenging with 4-MeC and DMPO.

2.5.3 Oxidation of the Cu(I)-complex

Oxygen consumption was determined as Cu-mediated H2S and thiol oxidation proceeded

(Figure 2.8). 3SH (307 µM) reacted slowly and incompletely up to 168 h. When 100 µM Cu(II) was added, an equimolar concentration (i.e. 100 µM) of the thiol would have initially been oxidized to the disulfide in the production of the Cu(I) complex, leaving 100 µM of thiol coordinated to the

Cu(I) according to our proposed mechanism (Figure 2.10). It can be estimated from the 3SH that remained, and accounting for the 100 µM of the thiol bound to Cu(I), that ~74 µM of thiol would have reacted to correspond to a consumption of 28 µM of O2, resulting in a 1:2.6 O2:thiol molar reaction ratio. The presence of free 3SH indicated that all the Cu remained as Cu(I) at the end of the reaction. In comparison, Cys (299 µM) reacted completely but consumed relatively less O2 (66

µM), giving a ~1:4.5 O2:Cys molar reaction ratio. H2S (284 µM) also reacted completely but resulted in much greater O2 consumption, affording an O2:H2S molar reaction ratio of ~1:1.6. This

0 can be explained on the basis that H2S is capable of being oxidized to ground state S , effectively reducing two equivalents of Cu(II). It is also possible for H2S to be fully oxidized to sulfate, or to form partially oxidized polysulfides.194

Oxygen may be reduced in four discrete one-electron steps in metal-catalyzed wine oxidation (Figure 2.12). The possibility that hydroperoxyl radicals were generated under this scenario was tested by oxidizing H2S or Cys in the presence of excess 4-MeC, wherein the catechol would quench hydroperoxyl radicals to generate the o-quinone.51 However, the concentration of 4-

MeC did not change as oxidation proceeded, and formation of catechol-thiol adducts was not observed (data not shown). Thus, it appears that hydroperoxyl radicals are not produced and so O2 was reduced directly to hydrogen peroxide (H2O2) in a two electron process. It is proposed that the close proximity of two Cu(I) ions in aggregate (4) allows for such a process to occur (Figure 2.13).

Similarly, it has previously been concluded that the Fe(II) reduction of O2 to H2O2 in model wine also proceeds without the release of hydroperoxyl radicals or oxidation of catechols.58

Figure 2.12. Four electron steps in the reduction of O2 to H2O via the hydroperoxyl radical, hydrogen peroxide and the hydroxyl radical.

Figure 2.13. Proposed Cu(I)-SH complex catalyzed two-electron reduction of O2 to H2O2.

Previous studies of the copper-catalyzed H2O2 oxidation of Cys similarly failed to detect hydroxyl radicals, and it was suggested that H2O2 was also reduced in a two-electron step (Figure

2.14). However, it was proposed that at higher dilution rates, when the Cu(I) complex is less aggregated, the usual Fenton pathway would be favored (Figure 2.15).103 Without the hydroxyl radical, the Fenton reaction-mediated oxidation of ethanol in model wine would not occur and no

AC should be produced. Overall a 1:4 molar reaction ratio of O2:thiol would result, with all four electrons being derived from the thiol to reduce O2 to two equivalents of H2O (Figures 2.13 and

2.14). If H2O2 was reduced in a one-electron step, hydroxyl radicals would result (Figure 2.15). As these radicals are powerful, non-selective oxidants that react at diffusion-controlled rates, they would be expected to react with solution components in proportion to their concentration. As the most abundant oxidizable constituent in model wine, ethanol would serve as the likely target of hydroxyl radical oxidation, from which 1-hydroxyethyl radicals (1-HER) would be generated.59 In the Fe-catalyzed Fenton reaction, 1-HER would be oxidized to AC by Fe(III) at very low O2 concentrations, resulting in a 1:1 molar ratio of O2:AC. However, the presence of O2 in the system 57

would favor the formation of the 1-hydroxyethylperoxyl radical (1-HEPR).60,61 It has been previously proposed that 1-HEPR can release the hydroperoxyl radical and form acetaldehyde; however, the lack of 4-MeC oxidation suggests that again the hydroperoxyl radical is not formed.

Instead, it is proposed that 1-HEPR is quickly reduced in the presence of Cu(I)-complex, yielding the corresponding peroxide (Figure 2.15).195 This peroxide may then be reduced to the alkoxyl radical, and quickly reduced to 1,1-dihydroxyethane by the Cu(I)-complex due to its close proximity rather than reacting with 4-MeC. 1,1-Dihydroxyethane (i.e. acetaldehyde hydrate) is then expected to dehydrate under wine conditions to yield acetaldehyde (Figure 2.15). This route would result in a 2:1 O2:AC molar ratio and a 1:3 O2:thiol molar reaction ratio, with three electrons being provided by RSH, one electron being provided by ethanol, and O2 accepting four electrons.

Figure 2.14. Proposed Cu(I)-SH complex catalyzed two-electron reduction of H2O2 to H2O.

Figure 2.15. One-electron reduction of H2O2 to produce hydroxyl radicals, and the oxidation of ethanol by the Fenton reaction to form 1-hydroxyethyl radicals. 1-hydroxyethyl radicals are oxidized by oxygen and subsequently reduced by metals to yield acetaldehyde.

H2S oxidation produced the most AC (Figure 2.9), and with an O2:AC molar ratio of 2.2:1, oxidation could have proceeded mainly as shown in Figure 2.15. This uptake of O2 and production of AC clearly showed that Cu(II) did not simply form Cu(II)S. The oxidation of Cys resulted in lower AC formation, with an O2:AC molar ratio of 2.5:1, while that of Cys+H2S resulted in a ratio of 2.1:1. The O2:AC molar ratios of 6SH and 3SH were 1.5:1 and 1.8:1, respectively. Cys produced relatively less AC, and it may be inferred that the mechanisms shown in Figures 2.13 and 2.14 might operate to a greater extent, although there is some uncertainty as to the fate of AC and a closer examination of AC production in these systems is warranted. Nonetheless, it can be concluded that the Fenton reaction does occur during H2S and thiol oxidation in model wine, albeit to varying degrees.

In conclusion, we show that Cu(II) is reduced by H2S and thiols in air saturated model wine, while thiols, which are present in relative excess to added Cu(II), as well as H2S, are oxidized.

These studies were conducted at initial aerial O2 saturation in order to follow the oxidative

processes. These conditions are unlikely to occur during the fining process. However, it should be noted that the reactions were followed down to ~50% and 25% air saturation. Furthermore, the

EPR study showed that Cu(II) is very rapidly reduced to Cu(I) and when Cu(II) was reacted with

6SH, the Cu(I)-SR complex precipitated immediately, before any oxygen reacted. Similarly, when the Cu(I)-6SH complex was formed under argon, quantitative yields of disulfide were obtained in

5 min.

It can therefore be concluded that if fining were conducted under anaerobic conditions, all the Cu(II) would be quickly reduced to Cu(I) by H2S and thiols, which would be oxidized. The present work, therefore, provides a mechanistic foundation for future studies in both model and real wine systems, which would contain sulfite, as well as in other alcoholic beverages in which thiols and H2S play an important role with respect to quality (e.g. beer and cider). In part 2 of this investigation, it is shown that Cu(I) complexes react rapidly with Fe(III); as such, any Fe(III) that remained in these conditions would be reduced to Fe(II) and Cu(I) would recycle until no Fe(III) remained. The reaction would then stop until O2 is introduced as a result of racking or filtration.

2.6 Acknowledgments

The authors thank Alexey Silakov from the Department of Chemistry at The Pennsylvania

State University for his assistance with EPR analysis.

Chapter 3

Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model Wine. Part 2: Iron and Copper Catalyzed Oxidation.

Published as:

Kreitman, G.Y.; Danilewicz, J.C.; Jeffery, D.W.; Elias, R.J. Reaction Mechanisms of Metals with

Hydrogen Sulfide and Thiols in Model Wine. Part 2: Iron- and Copper- Catalyzed Oxidation. J.

Agric. Food Chem. 2016, 64, 4105-4113.

3.1 ABSTRACT

Sulfidic off-odors arising during wine production are frequently removed by Cu(II) fining.

In Part 1 of this study, the reaction of H2S and thiols with Cu(II) was examined; however, the interaction of iron and copper is also known to play an important synergistic role in mediating non- enzymatic wine oxidation. The interaction of these two metals in the oxidation of H2S and thiols

(cysteine, 3-sulfanylhexan-1-ol, and 6-sulfanylhexan-1-ol) was therefore examined under wine-like conditions. H2S and thiols (300 μM) were reacted with Fe(III) (100 or 200 μM) alone and in combination with Cu(II) (25 or 50 μM), and concentrations of H2S and thiols, oxygen, and acetaldehyde were monitored over time. H2S and thiols were shown to be slowly oxidized in the presence of Fe(III) alone, and were not bound to Fe(III) under model wine conditions. However,

Cu(II) added to model wine containing Fe(III) was quickly reduced by H2S and thiols to form Cu(I)- complexes, which then rapidly reduced Fe(III) to Fe(II). Oxidation of Fe(II) in the presence of oxygen regenerated Fe(III) and completed the iron redox cycle. In addition, sulfur-derived oxidation products were observed, and the formation of organic polysulfanes was demonstrated.

3.2 INTRODUCTION

Non-enzymatic wine oxidation, in which polyphenols interact with oxygen, is now known to be catalyzed by trace concentrations of transition metals in wine, particularly iron (Fe) and

51,52 copper (Cu). During this oxidation process, O2 can be reduced to water in four discrete one- electron steps,51 resulting in the formation of reactive intermediate oxygen species53 that can oxidize wine constituents.39,59,196 However, recently, it was proposed that under wine-like conditions, Fe(II) reduces an intermediate Fe(III)-oxygen complex in a concerted 2-electron reduction to produce H2O2 from O2 without the formation of an intermediate hydroperoxyl radical

(Figure 3.1).58 Similar results were obtained for the Cu(I)-mediated reduction of oxygen, where no evidence of an intermediate hydroperoxyl radical was observed.55 In combination, these metals act synergistically, with copper playing an important role in the overall wine oxidation process by accelerating the reaction of Fe(II) with oxygen to regenerate Fe(III),52 presumably, copper facilitates Fe(III)/Fe(II) redox cycling. Once H2O2 is formed, it is reduced by Fe(II) through the

Fenton reaction to yield the highly reactive hydroxyl radical, which results in ethanol oxidation by

60 forming the intermediate 1-hydroxyethyl radical (1-HER). In low O2 concentrations, 1-HER will be oxidized by Fe(III) to yield acetaldehyde (AC); however, at higher O2 concentrations, O2 is known to add to 1-HER to yield the 1-hydroxyethylperoxyl radical (1-HEPR) (Figure 3.2). Recent work suggests that rather than 1-HEPR releasing AC and hydroperoxyl radicals, 1-HEPR is reduced to the peroxide by the presence of reduced metal complexes.55 The peroxide can then undergo a

Fenton-like reaction to form the alkoxyl radical that will subsequently be reduced to 1,1- dihydroxyethane that dehydrates to AC.

Figure 3.1. Reduction of oxygen by Fe(II) to yield hydrogen peroxide without the release of hydroperoxyl radicals.

Fe(III) catalyzes the oxidation of wine polyphenols containing catechol or pyrogallol moieties to form intermediate semiquinone radicals, which are further oxidized to o-quinones. The reaction is accelerated by nucleophiles such as bisulfite and thiols.54,65 In this latter process, quinones are reduced back to catechols by reaction with sulfite54 or undergo Michael-type addition reactions with sulfite or thiols96,97, effectively driving the reaction forward by consuming the product of phenolic oxidation. Fe(III) may also interact with thiols directly, which could either have deleterious effects by causing the oxidative loss of important aroma compounds such as 3-

54,112 sulfanylhexan-1-ol (3SH), or a beneficial effect by reacting with hydrogen sulfide (H2S). The 64

presence of thiols in wine may, therefore, play an important role in mediating wine oxidation, although the mechanism by which sulfhydryl compounds (i.e., species containing an –SH moiety) directly interact with iron and copper in wine remains poorly understood. Such information is important to winemakers in order for them to make informed decisions about managing oxidation to improve wine quality.

Studies performed with glutathione (GSH) in a wine pH range (3-7) have shown that Fe(II) is spontaneously produced when GSH is added to Fe(III) (Figure 3.3).109,110 The same has been shown with Cys at low pH, as the Fe(III)-Cys complex is unstable and quickly reacts to yield Fe(II) and cystine.111 Previous work has failed to provide evidence of free thiyl radical generation under those conditions,109 and the disulfide is seemingly formed in situ before being released from the metal complex. The resulting Fe(II) remains bound to GSSG and is only released when excess GSH is present; however, unlike Cu(I), which coordinates strongly with thiols, Mössbauer spectroscopy showed that Fe(II) is not bound to sulfur. It was concluded that coordination to GSSG, GSH and also Cys occurred by interaction with carboxylate groups under acidic conditions (pH<4).109,110 As discussed above, the Fe(II) produced can be reoxidized to Fe(III) by reacting with O2, with the reaction markedly accelerated by copper.

Figure 3.3. Proposed mechanism for initial Fe(III) reduction by thiols showing that the resulting Fe(II) is not coordinated to sulfur after the disulfide is formed.

Recent work in model systems has demonstrated that tartaric acid determines the reduction potential of the Fe(III)/Fe(II) couple in wine,197 but it may be possible that thiols also affect that potential. This is of particular interest to copper-containing systems, as H2S and thiols keep copper in its reduced Cu(I) state under wine-like conditions.55 In view of the known interaction of iron and copper in relation to wine oxidation, it is of interest to examine the effect of the metal combination in the removal of undesirable sulfidic off-odors in comparison to copper alone. Recent work has

91 examined the reaction of H2S with Cu(II), but did not take into account the presence of iron, which could be present in ~10 fold excess in wine compared to copper.114

The aim of this present study was to elucidate the mechanism underlying Fe-mediated thiol oxidation under wine-like conditions, which builds on the findings of the first part of this larger study involving copper alone. Since the interaction of iron and copper plays an important role in polyphenol oxidation, it was of interest to understand whether these metals also interacted

8 synergistically in the oxidation of H2S and thiols. As noted previously , the concentration of thiols, such as glutathione and cysteine analogues, far exceeds that of H2S that at likely to occur in wine.

The oxidation of H2S in the presence of greater concentrations of Cys, as a representative thiol, was therefore investigated due to its relevance to the copper fining operation in winemaking.

3.3 MATERIALS AND METHODS

3.3.1 Chemicals

L-Cysteine (Cys), monobromobimane (MBB), 6-sulfanylhexan-1-ol (6SH), and diethylenetriaminepentaacetic acid (DTPA) were obtained from Sigma-Aldrich (St. Louis,

MO). 2,4-Dinitrophenylhydrazine (DNPH) was purchased from MCB laboratory chemicals (Norwood, OH) and L-tartaric acid, 3SH, and 5,5’-dithiobis(2-nitrobenzoic acid)

(DTNB) were obtained from Alfa Aesar (Ward Hill, MA). Copper(II) sulfate pentahydrate was purchased from EMD Chemicals (Gibbstown, NJ), TRIS hydrochloride from J.T.

Baker (Center Valley, PA), and sodium hydrosulfide hydrate (as a source of H2S) was purchased from Acros Organics (Geel, Belgium). Iron(III) chloride hexahydrate was purchased from Mallinckrodt Chemicals (St. Louis, MO). Water was purified through a

Millipore Q-Plus system (Milipore Corp., Bedford, MA). All other chemicals and solvents were of analytical or HPLC grade and solutions were prepared volumetrically, with the balance made up with Milli-Q water unless specified otherwise.

3.3.2 Model Wine Experiments

For H2S and Cys, an aqueous stock solution of each (approximately 0.5 M) were freshly prepared, whereas 6SH and 3SH were added directly by syringe during experimentation. Aqueous stock solutions of Cu(II) sulfate and Fe(III) chloride (0.1 M and 0.4 M, respectively) were freshly prepared. H2S, Cys, 6SH, or 3SH were added to air saturated model wine (1 L, 300 μM) followed by thorough mixing.

For Fe experiments, Fe(III) (200 μM) was added to all H2S and thiol treatments and thoroughly mixed. For Fe and Cu combination experiments, Fe(III) (200 μM) and Cu(II) (50 μM) were consecutively added to H2S, 6SH, or 3SH solutions. For Cys experiments, Fe(III) (100 μM) 67

and Cu(II) (25 μM) were consecutively added and mixed thoroughly. For thiol experiments in combination with H2S and Fe/Cu, H2S was added to the thiol treatment and mixed prior to the addition of metal stock solutions. H2S (100 μM), Fe(III) (200 μM), and Cu(II) (50 μM) were added to Cys, 6SH, and 3SH. For Cys experiments with low metal concentrations, H2S (50 μM), Fe(III)

(100 μM), and Cu(II) (25 μM) were added and thoroughly mixed.

The resulting treatment solutions were immediately transferred to 60 mL glass Biological

Oxygen Demand (B.O.D.) bottles (Wheaton, Millville, NJ), allowing the solution to overflow, and bottles were capped immediately with ground glass stoppers, eliminating headspace. The glass reservoir of the B.O.D. bottles was topped off with water daily. The bottles were stored in the dark at ambient temperature. One B.O.D. bottle was sacrificed per time point per replicate and used for further analyses. All experiments were conducted in triplicate and contained their own series of sacrificial bottles.

3.3.3 Determination of oxygen consumption

Glass B.O.D. bottles were fitted with PSt3 oxidots and oxygen readings were taken per time point using a NomaSense O2 P6000 meter (Nomacorc LLC, Zublon, NC). Further details were reported in Part 1.55

3.3.4 Spectrophotometric measurements

UV-vis spectra of the treatments were recorded at each time point using 10 mm quartz cuvettes (model wine blank) and measured using Agilent 8453 UV-Vis spectrophotometer

(Agilent, Santa Clara, CA). Determination of Fe(III) concentration was achieved by measurement of absorbance at 336 nm associated with the Fe(III)-tartrate complex.58 68

For H2S, Cys, 6SH, and 3SH, total concentration was analyzed using Ellman’s assay.

Further details were reported in Part 1.55

3.3.5 HPLC Analyses

For the mixed H2S and thiol treatments, MBB derivatization and analysis of thiol concentration was performed using negative electrospray ionization (ESI-) HPLC-MS/MS as described in Part 1.55 The mass transition of sulfide-dibimane was monitored at m/z 413→191, Cys- bimane was monitored at m/z 310→223, 3SH-bimane at m/z 323→222 and the internal standard

6SH-bimane was monitored at m/z 323→222. External standard curves prepared for sulfide- dibimane, Cys-bimane, and 3SH-bimane were normalized to the 6SH-bimane internal standard. In the case of 6SH/H2S combination experiment, external calibration curves were made the same day prior to analysis and used without addition of 6SH-bimane internal standard.

Acetaldehyde was measured in model wine treatment solutions as its 2,4- dinitrophenylhydrazone (DNPH) derivative with an external standard curve (10 – 220 μM) by

HPLC as described in Part 1.55

Polysulfides were formed by the reaction of H2S (300 μM) with Cu(II) (50 μM) and

Fe(III) (200 μM). A sample was derivatized using MBB as described above with the same

HPLC separation parameters. Mass spectra were obtained using ESI- and full scan between m/z 100-1000. 6SH and 3SH polysulfanes were obtained by adding H2S (100 μM), Fe(III)

(200 μM), and Cu(II) (50 μM) to 6SH or 3SH (300 μM). The organic polysulfanes were detected by UV absorbance at 210 nm and verified using MS detection with ESI+ and full scan between m/z 100-1000. Mobile phases consisted of 0.1% v/v formic acid (A) and 0.1% v/v formic acid in acetonitrile (B) with a linear gradient according to the following

program: 0 min, 5% B; 20 min, 95% B; 28 min, 95% B; 28.1 min, 5% B; 38 min, 5% B.

The ESI capillary spray voltage was set to 4 kV, the sample cone voltage was 25 V, the source temperature was 120 °C, and the desolvation gas flow was 650 L/h.

3.4 RESULTS AND DISCUSSION

3.4.1 Reaction of Fe(III) with H2S and thiols in model wine

The reactivity of Fe(III) with the following treatments was investigated in model wine: (1)

Cys, which also represents homo-Cys and Cys derivatives; (2) 6SH, to represent primary thiols; (3)

3SH, to represent secondary thiols; (4) H2S, as it is one of the primary targets associated with sulfidic off-odors. Unlike the Cu(II) experiments described in Part 1, in which 2 mole equivalents

55 of thiols and 1.4 equivalents of H2S were immediately consumed (i.e. within 5 min), there was no initial uptake of these substances when Fe(III) was added (Figure 3.4A). In the case of H2S, although there was no appreciable consumption observed within the first few hours of the experiment, it reacted faster than the other thiol compounds, its concentration declining as Fe(III) was reduced and O2 was consumed (Figures 3.4B and 3.4C). A total of 262 µM of H2S was consumed after 144 h elapsed, and 192 µM of Cys was consumed after 193 h. Both 6SH and 3SH reacted extremely slowly, with negligible losses (<15 µM) throughout the time course of the experiments.

3.4.2 Fe(III) reduction by thiols and H2S

The Fe(III)-tartrate complex shows an absorbance maximum at 336 nm due to a d→d electronic transition, which can be used to obtain Fe(III):Fe(II) ratios in model wine systems.58

Fe(II)-tartrate complex does not absorb light in the UV spectral range. The absorbance of the

Fe(III)-complex was followed by UV spectroscopy over time upon adding Fe(III) to thiol or H2S treatments in model wine (Figure 3.4B). For the H2S treatment, Fe(III) was gradually reduced up to a maximum of approximately 66% of Fe(II) within 96 h. For the Cys treatment, a maximum of approximately 17% of Fe(III) was reduced to Fe(II) within 24 h, before apparently reaching an equilibrium state wherein the rates of Fe(II) oxidation and Fe(III) reduction equalized. This difference was consistent with a slower rate of Fe(III) reduction compared to that produced by H2S.

Minimal Fe(III) reduction was observed in experiments involving 6SH and 3SH, which was matched by minimal thiol and O2 uptake (Figures 3.4A and 3.4C) None of the treatments showed changes in absorbance maxima compared to Fe(III)-tartrate in model wine or resulted in the appearance of additional peaks, which indicated that these treatments did not displace tartaric acid from its Fe(III) complex.

Based on these results obtained in model wine (Figure 3.4B), and compared to previous studies where GSH and Cys were shown to reduce Fe(III) in simple aqueous systems,109,110 it is apparent that tartaric acid inhibits both the coordination of thiols with Fe(III) and its subsequent reduction to Fe(II). Furthermore, as Fe(III) coordinates preferentially with carboxylate moieties rather than with the thiolate function at wine pH,110 it would appear that Fe(III) remains bound to tartaric acid. However, due to its carboxylate function, Cys can presumably compete for Fe to displace tartrate ligands. In contrast, 6SH and 3SH, which lack a carboxylate function, are unable to displace tartaric acid in the Fe-containing systems, which would account for their low reactivity.

This behavior is quite different from that of Cu(II), which was very rapidly reduced to Cu(I) by

55 thiols and H2S in model wine.

Notably, H2S behaves differently than thiols, as it is capable of reducing Fe(III) in the

2+ presence of tartaric acid (Figure 3.4B). Fe(II) can bind H2S to yield [Fe-H2S] which would

34 deprotonate to yield FeS in the form of a [Fe2S2]n mackinawite to drive the reaction forward.

Under acidic conditions, FeS aggregates to form metastable nanoparticles (<150 Fe2S2 subunits) that behave like dissolved species but will quickly dissociate under low pH conditions,75 such as those encountered in wine. This will prevent further FeS aggregation and precipitation, and would explain why bulk FeS formation is not observed in wine, furthermore, FeS solubility is

12 75 approximately 10 -fold higher than CuS. Tartaric acid should also prevent H2S coordination, but the ligated acid does not limit the ability of H2S to reduce Fe(III), in contrast to what occurs with

6SH and 3SH. Recent work suggests that H2S can remain bound to Fe(II), causing loss of its free

80,81 sulfhydryl functionality and aroma associated with H2S.

3.4.3 Fe(II) oxidation and oxygen consumption

The ratio at which Fe(III)/Fe(II) reaches equilibrium is determined by the relative rate of

Fe(III) reduction by thiols or H2S, and that of Fe(II) reoxidation by O2. As tartaric acid determines the reduction potential of the Fe(III)/Fe(II) redox couple in the model system described here, it is likely that the reoxidation of Fe(II) will proceed as described previously (Figure 3.1).58 Fe(II) is expected to reduce O2 by a concerted 2-electron mechanism, yielding a Fe(III)-dioxygen complex that directly hydrolyzes to H2O2 without release of hydroperoxyl radicals. H2O2 should then undergo reduction via the Fenton reaction in the presence of Fe(II) to yield hydroxyl radicals that will subsequently oxidize ethanol (Figures 3.1 and 3.2). Fe behaves as a redox catalyst, cycling electrons from thiols and H2S to O2. Based on the overall sequence of reactions, it would be 74

expected that 3 electrons would come from thiols or H2S and 1 electron from ethanol to reduce O2 to water. Consequently, it would be expected that the O2:thiol molar reaction ratio would be 1:3, and the O2:H2S ratio would be 1:1.5 as H2S is capable of reducing 2 equivalents of Fe(III) as it is oxidized to ground state sulfur.73

The treatment containing H2S resulted in the greatest uptake of O2 in the presence of Fe(III).

Of the 262 µM H2S that reacted (Figure 3.4A), 135 µM of O2 was consumed (Figure 3.4C), giving a 1:1.9 O2:H2S molar reaction ratio. However, roughly 66% of Fe(III) had also been reduced to

Fe(II) (~132 µM) (Figure 3.4B), which would have required ~66 µM of H2S. Subtracting that amount from total reacted H2S would give ~196 µM uptake corresponding to the 135 µM O2 uptake, thus lowering the O2:H2S molar reaction ratio to ~1:1.5, as anticipated from the proposed mechanism (Figures 3.1 and 3.2). Fe(III) is reduced to some extent by Cys, likely in the same manner proposed in Figure 3.3, and 192 µM Cys (Figure 3.4A) reacted to reduce Fe(III) with subsequent consumption of 49 µM of O2 (Figure 3.4C). However, roughly 17.5% (35 µM) of Fe(II) remained at the end of the reaction, which corresponded to 35 µM Cys uptake. Subtracting this amount results in 157 µM Cys oxidized with the corresponding 49 µM O2 uptake, giving a O2:thiol molar ratio of ~1:3.2, which is in agreement with the proposed mechanism. (Figures 3.1 and 3.2).

Due to the inability of 6SH and 3SH to outcompete tartaric acid to form an Fe(III) complex, the oxidation of 6SH and 3SH was extremely slow and the O2:thiol molar reaction ratios could not be calculated (Figures 3.4A and 3.4C).

Low concentrations of acetaldehyde (AC) (15 – 30 μM) were formed in the Cys and H2S systems (data not shown), demonstrating that the Fenton reaction does proceed in the system described. The formation of AC is thought to proceed as described in Figure 3.2. It was expected that a higher concentration of acetaldehyde would be formed in the H2S system. In a previous study in which the Fenton reaction was investigated in model wine with iron only, up to 90% of 1-HER

radical was intercepted by thiol-containing compounds, the resulting thiyl radical likely then quickly dimerizing to yield a disulfide.67

3.4.4 Fe(III) and Cu(II) reduction by thiols and H2S

The interaction of iron and copper plays an important synergistic role in wine oxidation, and it was important to investigate whether these metals impact H2S and thiol oxidation. The treatments described above were employed again, this time using a combination Cu(II) (50 µM) and Fe(III) (200 µM). Cu(II) concentration was chosen to remain consistent with Part 1 of this investigation, and these concentration ratios were chosen as wines typically have 5–10-fold higher relative concentrations of iron to copper.114 In this experiment, Cys reacted rapidly and was completely consumed within 5 min (data not shown); therefore, the concentrations of Fe(III) and

Cu(II) were halved to 100 µM and 25 µM, respectively, to allow Cys oxidation to be more conveniently monitored.

In the presence of Fe(III) alone, Cys was slowly oxidized, with the reaction remaining incomplete after 200 h (Figure 3.4A). It was also determined that Cys did not coordinate to any significant extent to Fe(III) under the experimental conditions, with the metal center remaining largely bound to tartaric acid (Figure 3.4B). The addition of Cu(II) markedly increased the rate of the reaction, and Fe(III) was almost fully reduced within 5 min in the Cys system (Figure 3.5A), as less than 5% of the absorbance at 336 nm due to Fe(III)-tartrate complex was observed. Despite the fact that the concentration of Cu(II) and Fe(III) had to be decreased in this experiment, oxidation of Cys (296 µM) was complete within 7 h (Figure 3.5B). It was concluded that Fe(III) was not reduced by Cys directly but by the Cu(I)-Cys complex (Figure 3.6), which was rapidly formed.55

Given that 25 µM of Cu(II) was added initially, 25 µM of the Cu(I) complex would have been immediately produced and then oxidized by Fe(III). Recycling of copper three further times (with 76

the consumption of Cys) would rapidly reduce nearly all 100 µM of Fe(III) within 5 min (Figure

3.5A). At this point, the resulting Cu(II) would oxidize 25 µM of Cys to cystine, and 25 µM of Cys would be bound in the Cu(I) complex. In total, 150 µM of Cys would be consumed when all Fe(III) and Cu(II) were reduced, in accordance with the amount actually consumed during the initial rapid

Cys uptake phase (Figure 3.5B). It is noted that at this point no O2 had yet reacted (Figure 3.5C).

3SH and 6SH were less reactive than Cys, and led to an initial ~40% reduction of Fe(III) to Fe(II), with iron speciation reaching equilibrium at ~25% Fe(II) (Figure 3.5A). 6SH (273 µM) was fully oxidized within 7 h whereas 3SH, as a secondary thiol, oxidized more slowly and the reaction was incomplete at the 150 h time point (Figure 3.5B). The limiting factor for 3SH oxidation could potentially be the rate of formation of the Cu(I)-complex due to steric hindrance of the thiol.55

However, the reaction for 3SH proceeded more quickly in the iron/copper combination treatment compared to the systems with Fe(III) (or Cu(II)55) alone, resulting in the consumption of 267 µM of 3SH. H2S caused a rapid and near complete reduction of Fe(III) to Fe(II) within 30 min, corresponding to the loss of the absorbance peak at 336 nm (Figure 3.5A) along with a sharp initial drop (~135 µM) in H2S concentration (Figure 3.5B). However, the formation of Cu(I)-complex nanoparticles resulted in an elevated baseline, therefore the data were normalized to the baseline.55

It appears that iron remained reduced until no free H2S remained (308 µM consumed) at ~48 h, after which Fe(II) re-oxidized to Fe(III) in the presence of O2 (Figures 3.5A and 3.5B).

3.4.5 Fe(II)/Cu(I) oxidation, oxygen consumption, and acetaldehyde formation

It is proposed that with copper alone, overall thiol oxidation is dependent on the rate of reaction of

O2 with the Cu(I)-complex; however, when iron is present, the reaction rate is dependent on the oxidation rate of the Fe(II)-tartrate complex, which is known to be fast.197 When the two metals are present in combination, Fe(III) rapidly oxidizes Cu(I) first (Figure 3.6) and the Fe(II) produced is oxidized by O2 (Figure 3.3), markedly increasing the rate of Cu(I) oxidation. The degree of

55 consumption of H2S with copper determined previously was similar to that when Fe(III) was added in combination with Cu(II) (Figure 3.5B). It would appear that, in this case, the rate of oxidation of the Cu(I)-H2S complex was similar to that of the Fe(II)-tartrate complex.

O2 consumption was monitored as thiol and H2S oxidation proceeded (Figure 3.5C). In the

H2S system, around 46% (92 µM) of iron remained reduced after 120 hr (Figure 3.5A), which would require 46 µM of H2S. As a result, 262 µM of H2S would be left to react with 160 µM of O2 consumed, giving a ~1:1.6 O2:H2S molar reaction ratio, approximately the same as the Fe(III) or

Cu(II) treatment alone. As for the Cys treatment, roughly 12% (12 µM) of Fe remained reduced, which would require 12 µM Cys. Therefore, 284 µM Cys reacted with 110 µM O2, giving a 1:2.6

O2:Cys ratio. Applying the same reasoning, 223 µM of 6SH and 217 µM of 3SH reacted with an

O2 consumption of 106 µM and 82 µM, respectively. This afforded a ~1:2.1 O2:RSH molar ratio in the 6SH system and ~1:2.6 in the 3SH system. As with H2S, reaction ratios were comparable to those involving Cu(II) alone. Given that treatments involving the combination of Fe(III) and Cu(II) resulted in quicker thiol consumption than Fe(III) alone, it would suggest that the Cu(I)-SR aggregate reacts more slowly with O2 than with Fe(III), with the overall reaction rate being dictated by Fe(II)-tartrate oxidation, as alluded to above. However, the similarity in the molar ratio of O2 and thiol or H2S consumed may indicate that both iron and copper behave in the same mechanistic manner with respect to O2.

The ~1:3 O2:RSH molar reaction ratio observed in the Cys and 3SH systems is indicative of a combination of 2-electron reduction of O2 to H2O2, as well as the 1-electron reduction of H2O2 to hydroxyl radicals and subsequent one electron ethanol oxidation (i.e., Figures 3.1 and 3.2). The

H2S treatment resulted in the generation of 100 µM AC, whereas the Cys treatment resulted in 60

µM AC, giving O2 to AC molar reaction ratios of approximately 1.6:1 and 2:1, respectively (Figure

3.5D). This was in accord with the Fenton-catalyzed wine oxidation described from Part 1,55 in which 1-HEPR is formed and subsequently reduced by metals. However, in the case of 6SH, in which 146 µM of AC was formed, the ratio was closer to 1:1 O2:AC, which would suggest direct

Fe(III) oxidation of 1-HER, as Fe(III) is present at higher concentrations than that of the Cys and

H2S system (Figure 3.2). Furthermore, reduction of Fe(III) by 1-HER generates Fe(II) that subsequently react with O2, explaining why the molar ratios for the 6SH system, as well as 3SH and Cys, were lower than 1:3 O2:RSH.

3.4.6 Reaction of Fe(III)/Cu(II) with H2S in combination with thiols in model wine

Under normal conditions, the concentration of H2S in wine (0.3 – 1 µM) would generally be lower than that of other thiols, such as the combined pool of GSH (up to 40 µM) and Cys, homo-

Cys and Cys analogues (20 µM).92–94,185,198 Therefore, to better model a real wine situation, the oxidation of H2S in the presence of an excess of thiols (Cys, 6SH, and 3SH) was examined in model wine with the combination of Fe/Cu described above (Figures 3.7A-D). The final concentration of added H2S was targeted to be double that of the Cu(II) concentration that was established in the model wine, based on the initial 2:1 H2S:Cu(II) molar ratio. In these experiments, a haze was formed initially, presumably due to insoluble Cu(I)-thiol complexes.55 However, no black-green

CuS precipitate was observed at the end of the reaction, indicating that the Cu(I)-complex did not aggregate to the point of precipitation under conditions that were designed to closely mimic real 80

wine conditions. This observation may explain why precipitates are not observed when Cu(II) is added to wine containing H2S. The reduction of Cu(II) also explains the absence of the highly

91 insoluble Cu(II)S, which may have been expected to form. Compared to H2S, the three thiols were present in large molar excess, but H2S was still quickly oxidized, with at least 60% of free H2S removed within 5 min in all treatments (Figures 3.7A-D). By 24 h, there was virtually no H2S remaining in the four experiments, and even after all free H2S was depleted, the remaining free thiol continued to oxidize without precipitation of a copper-complex.

The Cys+H2S system was conducted at high (200 µM Fe(III) and 50 µM Cu(II)) and low

(100 µM Fe(III) and 25 µM Cu(II)) metal concentrations (Figured 3.7C and 3.7D); iron speciation,

O2 consumption, thiol consumption, and AC generation were measured to further examine the 81

reaction ratios (Figured 3.8A and 3.8B). Under both conditions (i.e., high and low metal concentrations), virtually all Fe(III) was reduced to Fe(II) within the first few minutes of the experiment; however, in the high metal treatment, Fe(II) quickly reoxidized to Fe(III). The high metal concentration treatment caused all H2S and Cys to be oxidized within 2 h whereas the low metal treatment required 24 h. The total combined Cys+H2S consumption was 302 and 326 µM for the high and low treatments, respectively, with corresponding total O2 consumption of 132 and 138

µM for high and low treatments. This resulted in approximately the same molar reaction ratios, at

~1:2.3 O2:Cys+H2S, irrespective of metal concentration, and was intermediate between the expected 1:3 ratio for Cys and 1:1.5 ratio for H2S. However, the total concentration of AC generated was quite different between the two systems. The high metal concentration treatment resulted in

150 µM of generated AC, whereas the low metal treatment resulted in 81 µM of AC. Figures 3.8A and 3.8B correspond to approximately 1:1 AC:O2 ratio in the high metal system and a 1:2 AC:O2 ratio in the low metal system. This could be explained by the fact that a higher concentration of

Fe(III) would favor the oxidation of 1-HER to AC, rather than the formation of 1-HEPR by O2

(Figure 3.3).

3.4.7 Formation of mixed organic polysulfanes

When H2S and 6SH were oxidized together in the presence of Cu(II) and Fe(III), the formation of 6SH-polysulfane was evident; these were present with up to five linking S atoms

(n=5), as determined by HPLC-MS (Figures B.1 and B.2). These were not detected when 6SH was oxidized in the absence of H2S. Similar results were obtained with H2S and 3SH (data not shown), revealing that in a mixed thiol system, as is typical of wines, the formation of mixed disulfides and polysulfanes would be expected in the initial Cu(II) fining process. This is consistent with the

Cu(II)-catalyzed formation of trisulfides that was previously reported in model brandy containing

100 H2S, methanethiol, and ethanethiol. When H2S was oxidized alone, MBB derivatization followed by HPLC-MS analysis indicated the presence of up to S5-bimane, with sequential fragmentation losses of m/z 32 (Figure B.3). These species would likely remain bound to Cu(I)72 or potentially to

Fe(II),112 but importantly, mixed-thiol disulfides and organic polysulfanes could contribute to the recurrence of H2S post-bottling. The release of thiols from disulfides via sulfitolysis is a likely scenario invoked by the presence of sulfite, which was recently found to react with disulfides resulting in the release of a free thiol and the formation S-sulfonated products in wine.44 Further research is underway to investigate the importance of these compounds on the evolution of sulfidic off-odors in wine.

Overall, it was observed that copper and iron act synergistically to catalyze the oxidation of

H2S and thiols. Accordingly, the presence of H2S and thiols was shown to rapidly reduce Cu(II), with the resulting Cu(I) then able to rapidly reduce Fe(III). This process occurs more quickly than when H2S and thiols react directly with Fe(III). The iron redox cycle is then completed as Fe(II) is re-oxidized to Fe(III) by oxygen. Oxygen reacts in the Fenton reaction to produce acetaldehyde so it is unlikely that it adds to sulfur to form sulfur oxyanions to any significant extent.

Though these studies were conducted at initial air saturation in order better to follow the oxidative processes, it was argued in Part 1 of this investigation that aspects of the proposed mechanisms would apply to Cu fining conducted under anaerobic conditions. Under such conditions, all the Cu(II) would be quickly reduced to Cu(I) by H2S and thiols, and the Cu(I) would be oxidized by any Fe(III) that might remain. The reaction would then be expected to stop until O2 was introduced as a result of racking, filtration, or bottling.

Copper fining quickly oxidizes H2S, but the subsequent interaction with other transition metals and wine constituents needs to be better understood. The interaction of other metals in wine including Zn, Al, and Mn, which are present at an average of 0.54, 0.41, and 0.97 mg/L, respectively, should also be considered in future studies, as they are present in significant quantities and have been shown to influence the evolution of volatile sulfur compounds in wine over time.70

Chapter 4

Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model Wine. Part 3: Manganese Catalyzed Oxidation and Interaction with Iron and Copper.

4.1 ABSTRACT

Recent work suggests that manganese has a modest activity in catalyzing polyphenol and sulfite oxidation in wine. Furthermore, manganese is known to mediate thiol and H2S oxidation in aquatic systems. It was therefore of interest to investigate the interaction of manganese with iron and copper toward catalyzing thiol and H2S oxidation under wine-like conditions. Sulfhydryl compounds (cysteine, 6-sulfanylhexan-1-ol, and H2S) were reacted with Mn(II) alone or in combination of Fe(III) and Cu(II) in model wine, and the concentrations of sulfhydryl, oxygen, and acetaldehyde were monitored over time. The reaction of thiols with manganese resulted in radical chain reaction paired with large oxygen uptake and generation of sulfur oxyanions. H2S did not generate free thiyl radicals, and had minimal interaction with Mn(II). When Cu(II) was introduced,

Cu-mediated oxidation dominated in all treatments and Mn-mediated radical reaction was limited.

4.2 INTRODUCTION

Iron and copper catalyze non-enzymatic wine oxidation by reducing oxygen, which is paired with oxidation of ethanol, polyphenolics, and sulfhydryls.52,54–56,59 However, few studies have examined the mechanistic involvement of other transition metals on the oxidation in wine.

Manganese has been proposed to have an effect at mediating wine oxidation, and is present at concentrations similar to Fe (~1 mg/L average around the world114,199). Mn has been reported to catalyze browning in sherry wine in combination with iron,200 increase acetaldehyde production in 85

red wines,115 and decrease volatile sulfur compounds concentrations during storage in both red and white wines.70,117 Furthermore, recent work demonstrated modest catalytic activity of Mn in model wine and Sauvignon Blanc in the presence of Fe and Cu.116

Mn(III) is a strongly oxidizing species which can be readily reduced to Mn(II) by wine constituents. Recent work demonstrated that when Mn(III) is added to model wine, it forms a

Mn(III)-tartrate complex with a UV-absorbance maximum at ~240 nm and a shoulder at ~300 nm.116 Under wine pH conditions the Mn(III)-tartrate complex is unstable, with Mn(III) being reduced, presumably by the tartaric acid ligand.116 It is therefore expected that essentially all Mn should exist as Mn(II) under wine conditions, and likely remains bound to organic acids (i.e. tartaric and malic acid).

The reduction potential of the Mn(III)/Mn(II) redox couple is considerably higher than that of the Fe(III)/Fe(II) system and Mn cannot readily redox cycle in wine conditions.116 The reaction of O2, H2O2, or Fe(III) with Mn(II) to generate Mn(III) is thermodynamically disfavored and is found to proceed very slowly if at all in model wine.116 However, Mn(II) is a very effective catalyst

201 of SO2 autoxidation. Its catalytic action is initiated by traces of Fe(III), which oxidizes SO2 to

•- the sulfite radical (SO3 ), which in turn reacts with O2 to produce the peroxomonosulfate radical

•- (SO5 ), It is proposed that this strongly oxidizing radical oxidizes Mn(II) to Mn(III), which allows

116 the Mn catalyzed process to proceed (Figure 4.1). The generated Fe(II) is able to react with O2 to regenerate Fe(III) to continue the process.58

Figure 4.1. Fe(III) initiated sulfite oxidation and subsequent Mn-catalyzed radical chain reaction resulting in sulfite oxidation and sulfate generation.

58 Fe(II) reacts with O2 forming an intermediate Fe(III)-superoxo complex. The reduction

58 of the complex is inhibited by the presence of Fe(III) as it competes with Fe(II) to generate H2O2.

It was found that Mn(II) may play a role in reacting with Fe(III)-superoxo intermediate and driving the reaction forward (Figure 4.2).116 The reduction of this complex regenerates Mn(III) which can further oxidize wine constituents. It was found that added Mn(II) does not affect the Fenton reaction under wine conditions, but it may play a role in directly oxidizing tartaric acid.116

Figure 4.2. Reaction of Mn(II) with Fe(III)-superoxo complex to generate Mn(III) and H2O2.

Under aquatic environments, the reaction of organic thiols and H2S with Mn(III) has been shown to be faster than that of organic acids.74,202 It is therefore possible that these substrates may be preferentially oxidized even in the presence of excess tartaric and malic acids. Based on recent work on the interaction of Fe, Cu, and Mn in wine oxidation, it would be of interest to investigate the possible catalytic action of Mn in mediating the oxidation of thiols and H2S and its interaction with Fe and Cu in wine conditions.

4.3 MATERIALS AND METHODS

4.3.1 Chemicals

4-methylcatechol (4-MeC), L-Cysteine (Cys), 6-sulfanylhexan-1-ol (6SH), and manganese(II) sulfate monohydrate, and iron(II) sulfate heptahydrate were obtained from Sigma-

Aldrich (St. Louis, MO). 2,4-Dinitrophenylhydrazine (DNPH) was purchased from MCB laboratory chemicals (Norwood, OH), and L-tartaric acid and 5,5’-dithiobis(2-nitrobenzoic acid)

(DTNB) were obtained from Alfa Aesar (Ward Hill, MA). Copper(II) sulfate pentahydrate was purchased from EMD Chemicals (Gibbstown, NJ), and sodium hydrosulfide hydrate (as a source of H2S) was purchased from Acros Organics (Geel, Belgium). Iron(III) chloride hexahydrate was purchased from Mallinckrodt Chemicals (St. Louis, MO). Water was purified through a Millipore

Q-Plus system (Milipore Corp., Bedford, MA). All other chemicals and solvents were of analytical or HPLC grade and solutions were prepared volumetrically, with the balance made up with Milli-

Q water unless specified otherwise.

4.3.2 Model Wine Experiments

For H2S and Cys, an aqueous stock solution of each (approximately 0.4 M) were freshly prepared, whereas 6SH was added directly by syringe during experimentation. Aqueous stock solutions of Cu(II) sulfate (~50 mM), Fe(II) sulfate (~50 mM), Fe(III) chloride(~200 mM), and

Mn(II) sulfate (~200 mM) were freshly prepared. For Mn experiments, Mn(II) (100 μM) was added to air saturated model wine containing H2S, 6SH, or Cys treatments (1 L, 150 μM each) and thoroughly mixed. An additional treatment was prepared with Cys containing 4-MeC (1 mM) prior to the addition of Mn(II). For Mn and Fe combination experiments, Mn(II) (100 μM) and Fe(III)

(100 μM) were consecutively added to model wine containing H2S, 6SH, or Cys solutions (1 L,

150 μM each). An additional treatment for Cys was prepared with Fe(II) (10 μM) instead of Fe(III)

(100 μM). The experiments containing the combination of Mn(II) (100 μM), Fe(III) (100 μM), and 88

Cu(II) (25 μM) had the metals added consecutively to a model wine solution containing the sulfhydryl treatments (1 L, 200 μM each).

The resulting treatment solutions were immediately transferred to 60 mL glass Biological

Oxygen Demand (B.O.D.) bottles (Wheaton, Millville, NJ), allowing the solution to overflow, and bottles were capped immediately with ground glass stoppers, eliminating headspace. The glass reservoir of the B.O.D. bottles was topped off with water daily. The bottles were stored in the dark at ambient temperature. One B.O.D. bottle was sacrificed per time point per replicate and sample aliquots were stored at -80 °C until further analyses. All experiments were conducted in triplicate and contained their own series of sacrificial bottles.

4.3.3 Determination of oxygen consumption

Glass B.O.D. bottles were fitted with PSt3 oxidots and oxygen readings were taken per time point using a NomaSense O2 P6000 meter (Nomacorc LLC, Zublon, NC). Initial O2 concentrations ranged from 6.6 – 7.0 mg/L. Further details were reported in Chapter 2.

4.3.4 Spectrophotometric measurements

UV-vis spectra of the treatments were recorded at each time point using 10 mm quartz cuvettes (model wine blank) and measured using Agilent 8453 UV-Vis spectrophotometer

(Agilent, Santa Clara, CA). Determination of Fe(III) concentration was achieved by measurement of absorbance at 336 nm associated with the Fe(III)-tartrate complex.197

For H2S, Cys, 6SH, and 3SH, total concentration was analyzed using Ellman’s assay.

Further details were reported in Chapter 2.

4.3.5 HPLC Analyses

Acetaldehyde was measured in model wine treatment solutions as its 2,4- dinitrophenylhydrazone (DNPH) derivative with an external standard curve (10 – 220 μM) by

HPLC as described in Chapter 2.

Oxidized species formed by the reaction of 6SH were monitored using LC-MS/MS. Mass spectra were obtained using ESI- and ESI+ and full scan between m/z 100-1000. The compounds were also monitored by UV absorbance at 210 nm. Mobile phases consisted of 0.1% v/v formic acid (A) and 0.1% v/v formic acid in acetonitrile (B) with a linear gradient according to the following program: 0 min, 5% B; 8 min, 95% B; 10 min, 95% B; 10.1 min, 5% B; 12 min, 5% B.

The ESI capillary spray voltage was set to 4 kV, the sample cone voltage was 25 V, the source temperature was 120 °C, and the desolvation gas flow was 650 L/h. ESI- with multiple reaction monitoring was utilized for detection of the 6SH-sulfonate using the same parameters described above and collision energy of 20 eV. The 6SH-sulfonate was monitored at m/z 181→81.

4.4 RESULTS AND DISCUSSION

4.4.1 Reaction of Cys with Mn

When Cys (150 μM) was oxidized in the presence of Mn(II) (100 μM) in air-saturated model wine, it was found that the consumption of Cys (118 µM) was accompanied with a large O2

(208 µM) uptake (Figure 4.3A and 4.3B). As with sulfite autoxidation (Figure 4.1), Mn(II) would have to be oxidized for the process to proceed. It seems likely the oxidation of Cys is also initiated by traces of iron contaminating the model wine used in this study. However, Fe(III)-mediated oxidation of Cys does not appear to generate free thiyl radicals in model wine (Chapter 3). It is

proposed, therefore, that the reaction is initiated by the oxidation of Mn(II) to Mn(III) by a stronger oxidant such as the Fe(III)-superoxo complex that is proposed to be generated when Fe(II) is oxidized (Figure 4.2).116

Mn(III) has a fast ligand exchange rate with sulfhydryls and is competitive with carboxylate and amino functional groups,74 so once Mn(III) is generated, Cys may displace the

Mn(III)-tartrate complex. Studies investigating MnO2 mediated thiol oxidation and dissolution of the polymeric complex suggest that oxygen in MnO2 is displaced by thiols, resulting in

Mn(IV)SR.74 Subsequent intra-molecular electron transfer generates Mn(III)OH and a thiyl radical.74 The resulting Mn(III)OH complex, which may be analogous to Mn(III)-tartrate in wine,

readily co-ordinates with thiols and the resulting Mn(III)SR quickly dissociates releasing Mn(II) and a thiyl radical.74 Mn(III) could directly oxidize tartaric acid, but the reaction rate between

Mn(III) and carboxylic acid ligands is slower than with sulfhydryl compounds.202

During the oxidation of Cys, there was an initial induction period (approximately 8 hr) in which minimal Cys and O2 were consumed (Figure 4.3A and 4.3B). Presumably, during this time build-up of reactive intermediates could have occurred, similar to sulfite autoxidation.201 Oxygen was quickly consumed until the system became anoxic, containing less than 50 μg/L (~1.5 µM) O2.

After approximately 120 h, 118 µM of Cys were consumed along with 208 µM O2, giving a O2:Cys molar ratio of ~1.8:1. This overall molar ratio suggests that a large amount of O2 adds to Cys, resulting ultimately in the formation of cysteine sulfonic acid, but other oxidation products could include disulfides and sulfinic acids.203

A mechanism approximating to the following description is suggested (Figure 4.4).

Mn(III) initiates one-electron oxidation of the thiol to produce free thiyl radicals. The presence of

• 203,204 O2 in the system would favor the formation of a thiol peroxyl radical (RSOO ). Studies have shown that this radical may undergo isomerization in the presence of visible light,204 however the samples were stored in the dark. It is also possible for the radical to undergo thermal isomerization at 300 K, which is near the temperature at which the experiments were conducted, resulting in

• 203,204 generation of the sulfonyl radical (RSO2 ). This radical can also rapidly react with O2 to

• 203,204 generate the sulfonyl peroxyl radical (RSO2OO ), which is a very strong oxidant, and could oxidize Mn(II). The sulfonyl peroxide (RSO2OOH) would be generated, which could undergo

• Fenton-like reaction to yield the sulfonic acid (RSO3H) and hydroxyl radicals (HO ). Previous work

116 • demonstrated that H2O2 is not a sufficiently strong oxidant to oxidize Mn(II), however, RSO2OO could be capable of oxidizing Mn(II) to Mn(III). HO• radicals would in turn abstract hydrogen from ethanol to yield a hydroxyethyl radical (1-HER), finally producing acetaldehyde (AC).

Figure 4.4. Proposed mechanism of Mn(III)-catalyzed radical chain reactions of thiols in air saturated model wine resulting in thiyl radical intermediates which subsequently oxygen and ethanol.

There are difficulties associated with measuring initial thiyl radical generation by Mn- mediated oxidation, but there has been some indirect evidence through sulfur addition products to double bonds.205 The thiyl radicals may dimerize to yield a disulfide, and this would be the case if the Mn(II)-thiol radical complex polymerized and disulfide formation occurred in situ as is the case in Cu(II)- and Fe(III)-mediated thiol oxidation (Chapters 2 and 3). However, this was not the outcome in the case of Mn(III). It would appear that the free thiyl radical is released, which quickly reacts with O2 as discussed above. The possibility that Cys coordinates with Mn(II) to catalyze the reduction of O2 was considered, but it is not expected that the thiolate group will bind to Mn(II) at wine pH,206 similar to Fe(II).109,110

The reaction observed for Mn is unlike that which was observed in the previous studies focusing on Fe and Cu mediated oxidation, which appeared to result in a concerted oxidation of sulfhydryls and the generation of the corresponding disulfide (Chapters 2 and 3). No evidence was found for the formation of thiyl radicals or subsequent formation of sulfonic acids in these latter systems described. However, Mn(III)-mediated oxidation shows convincing, yet indirect evidence for the formation of thiyl radical which may subsequently react with O2 and eventually yield a sulfonic acid (see results for 6SH).

Along with the O2 consumption, AC was generated (Figure 4.1C), and with 184 µM generated it gives an AC:O2 ratio of 1:1.1. The above mechanism (Figure 4.4) would indicate a 1:2

AC:O2 reaction ratio, but it is possible that AC could also be formed by the oxidation of ethanol by

• 203 the sulfonyl peroxyl radical (RSO2OO ), which is a strong hydrogen abstractor.

The reaction initiated by Mn is readily quenched by the introduction of polyphenolic compounds, which can react with thiyl and derived radicals to form the resonance-stabilized semiquinone (Figure 4.4), which in turn would disproportionate to yield a quinone. The addition of 1 mM 4-MeC (a model polyphenol) to the system resulted in minimal O2 consumption (Figure

4.3B). As a radical scavenger, the catechol intercepts intermediate radicals and, as in sulfite autoxidation, prevents radical chain propagation. Consequently, Mn alone should not catalyze thiol oxidation in wine, where polyphenols are present. A more detailed examination of the Mn- catalyzed reaction products was therefore not undertaken.

4.4.2 Reaction of Cys with Mn+Fe

When Mn(II) and Fe(III) (100 µM each) were combined there was a longer induction period compared to Mn(II) alone, which could be explained by the presence of a large excess of

Fe(III), which would delay Fe(II) oxidation.197 Despite the longer induction period, it appears that overall molar ratios in the presence of Fe(III) remained similar (Figures 4.3A-C): 201 µM of O2 was consumed along with 118 µM Cys, giving a total of 1.7:1 O2:Cys molar reaction ratio. This again would suggest that O2 is incorporated into the Cys molecule to form cysteine oxyanions, presumably with cysteine sulfonic acid being a major product.

The Fe(III)-tartrate absorbance was also measured (Figure 4.3D), and approximately 15% of Fe(III) had been reduced shortly after initiating the reaction. The concentration started to decrease at the last time point (168 h) and approximately 18% of Fe(III) was reduced to Fe(II), presumably due to the absence of O2 at that point with residual Cys reducing Fe(III). The measured

AC concentration (268 µM) was higher at the last time point compared to that of Mn(II) alone, 94

which gave a molar ratio of 1.33:1 AC:O2. This could be attributed to Fe(III) oxidizing 1-HER to

AC directly, especially when O2 concentrations were low.

Although the presence of traces of iron was thought necessary to initiate Mn(II) oxidation to Mn(III) (Figure 4.2), an addition of a small (10 µM) amount of Fe(II) to the solution along with

Mn(II) was investigated to see its effect on the induction period. However, the results were similar to that of Mn(II) alone (Figure 4.3B), which suggests that the reduction of trace amounts of Fe(III) to Fe(II) by Cys is not the rate limiting step for the initial reactive intermediate buildup.

4.4.3 Reaction of Cys with Mn+Fe+Cu

When Cu(II) (25 µM) was added along with Fe(III) and Mn(II) (100 µM each), there was a rapid consumption of Cys with small amount of O2 uptake (Figures 4.3A and 4.3B). Based on previous work, it would be expected that Cu(II) would be rapidly reduced by Cys to Cu(I), which would subsequently reduce Fe(III), cycling Cu(II) until all Fe and Cu are reduced (Chapter 3). The concentration of Cys was increased from 150 µM to 200 µM to account for the initial rapid uptake of 150 µM Cys, and to allow subsequent oxidation to be monitored.

After initiating the reaction, the majority of Fe(III) (80%) was reduced to Fe(II) within 5 min (Figure 4.3D), which was paired with the reaction of 133 µM Cys and minimal O2 consumption (Figures 4.3A and 4.3B). The initial and subsequent reaction appear to be dominated by the presence of Cu, preventing Mn-mediated thiol oxidation and subsequent radical formation.

After 48 h, 184 μM Cys was consumed along with the 74 μM O2 consumed to give ~1:2.5 O2:Cys molar ratio. This ratio was slightly lower but consistent with that of Fe+Cu system, which resulted in a ~1:2.7 O2:Cys molar ratio (Chapter 3). A total of 46 μM of AC was generated (Figure 4.3C), giving a AC:O2 molar ratio of ~1.6:1, which was lower than the ~2:1 ratio observed in the Fe+Cu system alone. 95

It appears that with Mn(II) alone or Fe(III)+Mn(II), Mn promotes the generation of cysteinyl radicals which quickly react with oxygen and result in large O2 uptake (Figure 4.4).

However, when Cu is present it appears to dominate and oxidation reverts to the Cu catalyzed mechanism that would yield disulfide. Nonetheless, it does appear that the presence of Mn(II) catalyzed the reoxidation of Fe(II) in the presence of O2, as observed by the fast reoxidation of

Fe(II) to Fe(III) (Figure 4.3D).

4.4.4 Reaction of 6SH

Previous work on Fe(III)-mediated oxidation of 6SH showed that the reaction proceeded extremely slowly, which would affect Fe(II) generation (Chapter 3). Consequently, the oxidation of 6SH was found to proceed relatively slowly with Mn(II) (Figure 4.5A-C). This may indicate the importance of Fe(III) reduction to Fe(II) and subsequent formation of the Fe(III)-superoxo complex to generate Mn(III) and drive the reaction forward (Figure 4.2). The Mn(II)-catalyzed oxidation of Cys is much faster than that of 6SH, and may be explained by the greater ability of

Cys to reduce Fe(III).

Nonetheless, the reaction proceeded over time with 6SH and resulted in O2 consumption

(Figures 4.5A and 4.5B). In the case of Mn(II)-mediated oxidation, which is expected to be initiated by trace iron contamination, approximately 14 μM O2 and 23 μM of 6SH were consumed over a 192 h period. This resulted in a ~1:1.6 O2:6SH ratio, which is lower than that observed with

Cys (Figure 4.3). This perhaps indicates that not as much O2 is incorporated into the thiol. A small amount of AC (7 μM) was generated, which would correspond AC:O2 molar ratio of 1:2.

When Fe(III) (100 μM) was added along with Mn(II) (100 μM), the reaction proceeded more quickly (Figures 4.5A and 4.5B), indicating a synergistic effect between the metals, unlike the case of Cys (Figure 4.3A). There was a total consumption of 46 μM O2 and 55 μM 6SH. This resulted in a ~1:1.2 O2:6SH ratio, which is higher than that with Mn(II) alone. AC (49 μM) was 97

generated to give a ~1.1:1 AC:O2 molar ratio, which was more in line with what was observed for

Cys. Monitoring the Fe(III)-tartrate concentration over time indicated that virtually all Fe remained as Fe(III) throughout the experiment (Figure 4.5D). Evidently any Fe(II) generated was rapidly re- oxidized.

6SH (200 µM) was oxidized much faster with a combination of Fe(III) (100 µM), Mn(II)

(100 µM) and Cu(II) (25 µM) compared to the other two metal combinations (Mn or Mn+Fe). With the Fe+Mn+Cu combination, 65 µM of O2 was consumed with 189 of 6SH within 72 h, giving a

1:2.9 O2:6SH molar reaction ratio (Figure 4.5A and 4.5B). AC (58 µM) was also produced giving a ~1.1:1 AC:O2 molar reaction ratio (Figure 4.5D). These ratios, which are similar to those obtained with the Fe+Cu system (Chapter 3) indicate that Cu catalysis dominated in the presence of Mn, which was similarly observed for the Cys system. The low O2 uptake relative to thiol oxidation points to the disulfide being the main product and that the O2 is reduced to H2O2 to produce an equivalent of AC.

Mn(II) (100 µM) alone produced a slow oxidation of 6SH (150 µM), with a 1:1.6 O2:6SH molar reaction ratio. The reaction is accelerated by Fe(III) (100 µM) with a 1:1.2 O2:6SH molar reaction ratio. The higher O2 uptake relative to that of the Cu containing system (1:2.9 O2:6SH molar reaction ratio) points to the formation of oxyanions as with Cys. Clearly, Fe and Mn interact as Fe(III) (200 µM) alone does not catalyze the oxidation of 6SH (Chapter 3).

The oxyanion products of 6SH were analyzed by LC-MS. Using MS/MS, the 6SH-sulfonic acid was observed near the column void volume in the Mn+Fe system (Figure C.1), whereas it was not present in the initial 6SH stock or Mn+Fe+Cu mediated oxidation. Furthermore, it was observed that several oxidized disulfide species were formed including thiol-sulfinate, thiol-sulfonate, sulfinyl-sulfone, and α-disulfone (Figure C.2) in the Fe+Mn system. The same species were observed in the Mn-only system except for the α-disulfone, presumably due to the relatively slow

reaction and insufficient concentration for detection. In the Mn+Fe+Cu system, the thiol-sulfinate was observed, but this was a smaller response than the other two systems, despite the higher consumption of 6SH. This may indicate that Mn(III)-mediated oxidation did occur to some extent, but the disulfide due to Cu- mediated oxidation was still deemed to be the major product in the system as discussed above.

Several mechanisms could be proposed to explain how these oxidation products arise; it is possible that the 6SH-sulfinate was one of the predominant intermediates that can then disproportionate to the various observed oxidation products.144 However, as with Cys, the conditions in which O2 is present in large excess to form sulfur oxyanion species is unlikely under real wine conditions. Nonetheless, the thiyl radical is the likely precursor for the formation of these products. Furthermore, the formation of a glutathione-hydroxycinnamic acid product has been observed and proposed to be initiated by the glutathione thiyl radical.135

4.4.5 Reaction of H2S

When H2S (150 µM) was oxidized in the presence of Mn(II) (100 µM) alone, there was no

O2 consumption or appreciable amount of H2S consumed (Figures 4.6A and 4.6B). It would be expected that trace contamination by Fe would be present in this system as well, resulting in generation of trace amounts of Mn(III). However, unlike Cys and 6SH, H2S can be considered a sulfhydryl compound capable of donating two electrons. Furthermore, the generation of a hydrosulfide radical would be thermodynamically unfavorable and it would quickly react with

207 metals to either reform H2S or lose an electron to form elemental sulfur. The reduction of Mn(III) by H2S likely proceeds through an inner-sphere mechanism. In this process, two equivalents of

Mn(III) would be reduced as H2S is oxidized to elemental sulfur, resulting in no radical

202,208 generation, and therefore negligible O2 consumption over time. Due to the presence of only 99

trace amounts of Fe, which would be capable of oxidizing Mn(II), and the fact that H2S would not result in buildup of reactive intermediates, Mn-mediated oxidation of H2S does not occur under the conditions described. Similarly, no AC was generated (Figure 4.6C).

When Fe(III) (100 µM) was added in combination to Mn(II), H2S was slowly consumed over time, along with O2 (Figures 4.6A and 4.6B). Presumably, the interaction occurs in the same manner as described previously with Fe(III) whereby H2S reduces two equivalents of Fe(III) to

0 Fe(II) with its oxidation to S (Chapter 3). Over time, Fe(II)-tartrate reduces O2, resulting in generation of H2O2, and subsequent Fenton reaction to generate hydroxyl radicals. Overall, 54 µM of O2 were consumed in conjunction with 84 µm of H2S, giving an O2:H2S molar ratio of ~1:1.6,

100

which is consistent with the results for Fe alone (Chapter 3). This result, along with Mn-only oxidation, suggests that the reaction is driven primarily by Fe in this system. However, Mn does play a role in reoxidizing Fe(II), and the relative amount of Fe(II) in the system was much lower than with Fe-alone (Figure 4.6D and Chapter 3).

When Cu(II) (25 µM) was added along with Mn(II) and Fe(III) to H2S (200 µM), the results were similar to those obtained with the Fe+Cu combination (Chapter 3). During the process, there was a fast initial uptake of H2S, with approximately 58 µM consumed within 5 min. At the end of the reaction at around 120 h, there was 115 µM O2 consumed along with 180 µM of H2S (Figures

4.6A and 4.6B), again resulting in a ~1:1.6 O2:H2S molar reaction ratio. Therefore, it would appear that the addition of Mn to the H2S system does not alter the course of the reaction. H2S is likely

0 207 oxidized to S and the reduced metals are re-oxidized by O2. However, if other thiols were also present, it would be expected that polysulfanes would be formed (Chapters 3 and 5). Mn(II) seems to play an important role in oxidizing Fe(II), as virtually all Fe was re-oxidized at the end of the reaction, whereas in the Fe+Cu system approximately 40% of Fe(II) remained reduced at the end of the reaction (Figure 4.6D, Chapter 3). Approximately 53 µM of acetaldehyde was generated

(Figure 4.6C), which gave a ~1:2.2 AC:O2 molar ratio that is consistent with previous findings.

4.5 CONCLUSIONS

Mn(II) was found to catalyze Cys and 6SH oxidation with high O2 consumption relative to that of the thiol. It is proposed, therefore, that thiyl radicals are released and subsequently add O2 to produce sulfur oxyanions. It may be concluded that Mn(II)-catalyzed oxidation is a radical chain reaction initiated by traces of Fe, in a similar manner to sulfite autoxidation. Consequently, 4-MeC was found to inhibit the Mn(II) catalyzed reaction, presumably by intercepting intermediate radicals so preventing radical chain propagation. 101

Previous studies have shown that the Cu-catalyzed thiol oxidation proceeds with disulfide formation, as the initially formed thiyl radicals condense before they can be released from an aggregated Cu(I) complex. Cu(I) reduces Fe(III) and the resulting Cu(II) is itself reduced by the thiol so that Cu redox cycles until all the available Fe(III) is reduced. The process appears to be

0 similar for H2S and occurs without O2 consumption and likely generates S . When present, O2 is reduced by Cu(I) or Fe(II) to produce H2O2, which undergoes the Fenton reaction to generate AC.

When Fe, Mn and Cu are combined, the catalytic activity of Cu dominates so that thiol oxidation by Cu(II) occurs with minimal radical formation. Therefore, Mn(II) alone should not catalyze thiol oxidation in wine. Nonetheless, Mn(II) appears to promote reoxidation of Fe(II); whether Mn is capable of specifically catalyzing thiol oxidation needs to be investigated further using a more complete model wine system and in real wines.

102

Chapter 5

Investigating Volatile Sulfur Compound Precursors and Practical Applications

5.1 ABSTRACT

The addition of Cu(II) to model systems containing H2S and thiols demonstrated the generation of polysulfanes, rather than simply forming insoluble Cu(II)S as previously assumed. It was therefore of interest to investigate the formation of mixed disulfides and polysulfanes in model and white wine samples. It was found that at relatively low concentrations of H2S and methanethiol

(MeSH) (100 µg/L of each), Cu(II)-fining resulted in the generation of MeSH-glutathione disulfide and trisulfane in white wine as determined by qTOF LC/MS. The reduction of the resulting nonvolatile disulfides may then play a role in the recurrence of undesirable sulfidic odors. Therefore, the ability of Cu(II) and bisulfite (SO2), ascorbic acid, and cysteine to promote the catalytic scission of diethyl disulfide (DEDS) was investigated. It was found that the combination of SO2 along with

Fe and Cu depleted more DEDS than the other treatments. Furthermore, a method for releasing volatile sulfur compounds from their precursors as a diagnostic test was investigated using tris(2- carboxyethyl)phosphine (a reducing agent) and bathocuproine disulfonic acid (a chelator). The addition of the reagents successfully released H2S and MeSH from red and white wines that were free of reductive faults at the time of addition.

103

5.2 INTRODUCTION

Sulfidic off-odors in wine have been a serious quality issue for decades and, when detected in the course of winemaking, are generally controlled by sparging, aerative pump overs, splash racking, and/or the use of copper fining.2,41,90 Chapters 2 – 4 of this dissertation focused heavily on elucidating the initial mechanisms of oxidation responsible for removal of these undesirable compounds using H2S and model thiols. It was found that the addition of Cu(II) oxidized thiols to disulfides and the presence of H2S together with thiols resulted in polysulfanes as a result of oxidation. Furthermore, the complete loss of aroma – but not necessarily redox activity – occurs when thiols and H2S are bound to a metal complex as Cu(I)-SR. It is therefore apparent that the volatile sulfur compounds (VSCs) are not readily removed from wine in an insoluble complex that can be filtered, but rather generate redox active compounds that remain in the wine as soluble components.

In the post-bottling period, in which a wine is assumed to be free of faults, it has been well established that wine may accumulate undesirable sulfidic odors during the aging period, especially

47,48,186 when O2 ingress is limited. There have been numerous studies suggesting that the most common VSCs responsible for post-bottling reductive aroma are H2S, MeSH, and dimethyl sulfide

(DMS).50,57,70 There have been several hypotheses proposed to explain the mechanism(s) that underlie the generation of these sulfidic off-odors; these include bisulfite reduction,209 thioacetate and thioether hydrolysis,41 and sulfidic off-aroma generation from strecker degradation of sulfur- containing amino acids.71,178 Another well accepted hypothesis is the reduction of symmetrical disulfides of MeSH and ethanethiol (EtSH), which typically have 10-50-fold higher sensory detection thresholds than their respective free thiols.1,185 However, the rates that influence these reactions, and their relevance under wine conditions remain unknown.

104

Recent work has suggested that upwards of 99% H2S and ~70% MeSH may be effectively bound with transition metals (e.g., Cu, Fe, Zn) in wine, and that accelerated anaerobic aging results in the release of these complexes.80,81 From the work described in chapters 2 – 4 of this thesis, it is apparent that Cu(I)-SR complex generation is fast. Although this mechanism has not been studied under anaerobic conditions, Cu(I)-SR is unlikely to easily react or oxidize in the absence of O2.

Nonetheless, disulfides and polysulfanes are generated in the presence of H2S and thiols in the initial Cu(II) fining process with no O2 uptake. Subsequent oxidation of Cu(I)-SR upon O2 ingress likely results in further generation of disulfides and polysulfanes.

It was therefore of interest to further investigate the generation of disulfides and polysulfanes under real wine conditions, and to examine how they may contribute to reduced off- odors in wine. Given that thiols typically have lower detection thresholds compared to their corresponding disulfides, and that mixed disulfides may have no perceptible odor, the release of free thiols via disulfide reduction or scission reactions could result in reductive odors becoming apparent in a wine that had previously been free of faults. This was examined for diethyl disulfide

(DEDS) in the presence of Fe and Cu as well as reducing agents.

Furthermore, working under the assumption that metal complexes and disulfides/polysulfanes play a crucial role as potential precursors for these sulfidic odors, a method for their quick release has been developed and validated with the ultimate goal of informing winemakers if their product is susceptible to reductive off-aromas in the post-bottling period. This would afford them the opportunity to take steps to control this – for example, through proper bottle closure selection.

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5.3 MATERIALS AND METHODS

5.3.1 Materials

L-Cysteine (Cys), L-cystine, ethanethiol (EtSH), diethyl disulfide (DEDS), sodium thiomethoxide (as a source of MeSH), ferrous sulfate hexhydrate, tris(2-carboxyethyl)phosphine

(TCEP) and bathocuproinedisulfonic acid (BCDA) disodium salt) were obtained from Sigma-

Aldrich (St. Louis, MO). L-tartaric acid and L-glutathione (GSH) were obtained from Alfa Aesar

(Ward Hill, MA). Cupric sulfate pentahydrate was purchased from EMD Chemicals (Gibbstown,

NJ), TRIS hydrochloride from J.T. Baker (Center Valley, PA), and sodium hydrosulfide hydrate

(as a source of H2S) was purchased from Acros Organics (Geel, Belgium). Iron(III) chloride hexahydrate was purchased from Mallinckrodt Chemicals (St. Louis, MO). Water was purified through a Millipore Q-Plus system (Milipore Corp., Bedford, MA). All other chemicals and solvents were of analytical or HPLC grade, and solutions were prepared volumetrically, with the balance made up with Milli-Q water unless specified otherwise.

5.3.2 Preparation of model wine and real wine samples

5.3.2.1 Disulfide and polysulfane generation

Either glutathione (GSH, 500 µM) or cysteine (Cys, 500 µM) were added to model wine and mixed thoroughly. H2S (250 µM) and/or MeSH (250 µM) were subsequently added to the

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solutions to give a total of four treatments: (1) Cys+H2S, (2) Cys+H2S+MeSH, (3) GSH+H2S, and

(4) GSH+H2S+MeSH. Once the respective sulfhydryls were added to their respective solutions,

Fe(III) (100 µM) and Cu(II) (50 µM) were subsequently added and thoroughly mixed. The solutions (25 mL) were stored in the dark in capped 50 mL capacity polypropylene tubes under air.

The samples were analyzed the following day by HPLC-QTOF, as described below.

Commercial white wine blend was purchased locally to which GSH was added to achieve a final concentration of 50 µM. H2S and MeSH were subsequently added to achieve the following three treatment concentrations: 100 µg/L, 500 µg/L, and 5000 µg/L. Following the addition of the sulfhydryl-containing compounds, Fe(III) (5 mg/L) and Cu(II) (1 mg/L) were added and the resulting solutions were mixed thoroughly. The samples (100 mL) were stored in the dark in stoppered 100 mL volumetric flasks and analyzed the following day by HPLC-QTOF.

5.3.2.2 Disulfide scission by Cu(II) and bathocuproine disulfonic acid

Model wine was prepared as described above; however, cystine (400 µM) was added prior to pH adjustment for this experiment and mixed until it dissolved. Afterwards, sample aliquots (~30 mL) were adjusted to pH 2, 3, 4, 5, or 11 using hydrochloric acid (5 M) or sodium hydroxide (10

M). Following pH adjustment, BCDA (1 mM) was added followed by Cu(II) (100 µM). A control sample was prepared which contained only BCDA (1 mM) and Cu(II) (100 µM) over the pH range

2, 3, 4, 5, and 11. A positive control was also prepared and contained cysteine (400 µM), BCDA

(1mM), and Cu(II) (100 µM) over the pH range described above. The samples were analyzed over time for BCDA-Cu(I) generation as described below. Experiments were conducted in triplicate.

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5.3.2.3 Diethyl disulfide scission in the presence of metals and reducing agents

Model wine (pH 3.6) was prepared as described above and deoxygenated with argon until the dissolved oxygen concentration fell below 50 µg/L as measured by a NomaSense O2 P6000 meter (Nomacorc LLC, Zublon, NC) equipped with a dipping probe. Following deoxygenation, model wine solutions were transferred to an anaerobic chamber to equilibrate overnight. The following day, diethyl disulfide (DEDS, 50 µg/L) was added from a stock solution by syringe to

250 mL samples of model wine. To the solution, Cys, potassium metabisulfite (SO2), ascorbic acid

(AA), Cu(II) sulfate, and Fe(II) sulfate were added from freshly made stock solution to yield final concentrations outlined in Table 5.1.

Table 5.1. Treatment addition to anaerobic model wine containing 50 µg/L diethyl disulfide. Treatment Cys SO2 AA Cu(II) Fe(II) T1 - - - - - T2 - - 50 mg/L - - T3 - 50 mg/L - - - T4 - - - 1 mg/L 5 mg/L T5 12 mg/L - - 1 mg/L 5 mg/L T6 - - 50 mg/L 1 mg/L 5 mg/L T7 - 50 mg/L - 1 mg/L 5 mg/L T8 12 mg/L 50 mg/L 50 mg/L 1 mg/L 5 mg/L

The resulting treatment solutions were immediately transferred to 60 mL capacity glass

Biological Oxygen Demand (B.O.D.) bottles (Wheaton, Millville, NJ), allowing the solution to overflow, at which point the bottles were immediately capped with ground glass stoppers in order to completely eliminate headspace. The glass reservoir of the B.O.D. bottles was topped off with water and covered with 2 layers of parafilm and aluminum foil to prevent evaporation. The bottles were covered in aluminum foil and stored at 40 °C. One B.O.D. bottle was sacrificed per time point and used for further GC analysis as described below. Samples were prepared by transferring 1 mL of sample aliquot into a 20 mL amber GC vial containing 9 mL of saturated brine (350 g/L NaCl)

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and capped immediately based on previously described methodology in order to release metal-thiol complexes.80 Experiments were conducted in duplicate.

5.3.2.4 Release and reduction of bound VSCs

Initial experiments were conducted in either air saturated model wine (dissolved [O2]: 7 –

8 mg/L) or in an anaerobic chamber (dissolved [O2]: <100 µg/L). The model wine was spiked with a combination of H2S (100 µg/L), MeSH (100 µg/L), and EtSH (100 µg/L). One sample aliquot was transferred to a 60 mL B.O.D. bottle and capped without headspace using the procedure described above. The remaining sample fraction was spiked with Cu(II) sulfate (1 mg/L) and the resulting solution was transferred to a B.O.D. bottle and stored overnight. The following day, 10 mL sample aliquot of the control was transferred to a 20 mL amber GC vial and capped immediately. Sample aliquots (10 mL) of the Cu(II) sulfate-containing sample were transferred to five 20 mL amber GC vials. One sample was used as a positive control (i.e. no reagents added) and capped. The other four treatments included: TCEP (tris(2-carboxyethyl)phosphine, 1 mM), BCDA

(1 mM), TCEP (1 mM) + BCDA (1 mM), and TCEP (1 mM) + BCDA (1 mM) + Cys (1 mM).

After the addition of the reagents the vials were capped and analyzed by GC as described below.

The experiments were conducted in triplicate.

Six commercial Pennsylvania wines were obtained locally. The bottles were opened, and ca. 50 mL of wine were carefully transferred to beakers using a serological pipette while taking care to avoid agitation, and these aliquots were immediately transferred to an anaerobic chamber. One 10 mL sample aliquot was used as a control for determination of free VSCs in the original wine samples. The other four treatments (TCEP, BCDA, TCEP+BCDA, TCEP+BCDA+Cys) were prepared as described above.

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5.3.3 Methods of analysis

5.3.3.1 HPLC

Samples (5 µL) were separated by reversed-phase HPLC using a Prominence 20 UFLCXR system (Shimadzu, Columbia MD) with a Waters (Milford, MA) BEH C18 column (100 mm × 2.1 mm, 1.7 µm particle size) maintained at 55 °C and a 20 minute aqueous acetonitrile gradient, at a flow rate of 250 µL/min. Solvent A was HPLC grade water with 0.1% formic acid and Solvent B was HPLC grade acetonitrile with 0.1% formic acid. The initial mobile phase conditions were 97%

A and 3 % B, increasing to 45% B at 10 min, then to 75% B at 12 min, and holding at 75% B until

17.5 min before returning to the initial conditions. The eluate was delivered into a 5600 TripleTOF

(QTOF) MS with Duospray™ ion source (AB Sciex, Framingham, MA) using electrospray ionization (ESI) conditions. The ESI capillary voltage was set at 5.5 kV in positive ion mode or 4.5 kV in negative ion mode, with a declustering potential of 80 V. The mass spectrometer was operated in Information-Dependent Acquisition (IDA ) mode with a 100 ms survey scan from 100 to 1200 m/z, and up to 20 MS/MS product ion scans (100 ms) per duty cycle using a collision energy of 50 eV with a 20 eV spread.

5.3.3.2 GC

Samples were analyzed using an Aglient 5890 gas chromatograph (Santa Clara, CA) equipped with a Gerstel MPS2 autosampler (Linthicum, MD) and coupled to a pulsed flame photometric detector (PFPD). Instrument control and data analysis were performed with Agilent

GC Chemstation. The column was an Rxi-1ms from Restek (Bellefonte, PA), 30 m × 0.32 mm with

4.0 µm film thickness. Carrier gas was He at a constant flow of 1.7 ml/min. The initial temperature

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was 35 °C, which was held for 3 min, and then ramped to 100 °C at a rate of 10 °C/min, and finally ramped to to 220 °C at a rate of 20 °C/min. The programmable temperature vaporizer (PTV) inlet

(Gerstel, Linthicum, MD) was held at 60 °C. The 5380 PFPD (O.I. Analytical, College Station,

TX, USA) detector was maintained at 250 °C using the default flow rates suggested by the manufacturer. Emission was monitored from 6 to 24.9 msec.

The samples were stored in a cooled sample tray at 4 °C. The vial was incubated at 60 °C for 10 min with agitation at 500 rpm. Using a Gerstel 1.0 mL headspace (HS) syringe kept at 60

°C, a 500 µL static HS sample was injected at 500 µL/s into the PTV injector using split mode at a

1:2 split ratio.

5.3.3.3 UV-Vis

Cu(I) concentration was analyzed using a BCDA assay, as described previously.188

Standard solution consisted of excess Cys (5 mM), which was added in order to ensure that Cu(I) remained in its reduced state. An external standard curve of the Cu(I)-BCDA complex was prepared in model wine, and absorbance values were recorded at 484 nm using a 10 mm quartz cuvette against a model wine blank. The baseline measurements of the control samples were subtracted from the treatment samples for each pH value.

5.4 RESULTS AND DISCUSSION

5.4.1 Disulfide and polysulfane generation

We showed that Cu(II) fining results in near immediate Cu(II) reduction along with oxidation of H2S and thiols in Chapter 2 of this thesis. We subsequently showed that the oxidation 111

of 6-sulfanylhexan-1-ol (6SH) and H2S resulted in the formation of 6SH polysulfane with up to 5 linking sulfur atoms between the 6SH molecules in Chapter 3. With this knowledge, we investigated whether mixed disulfides and polysulfanes could be formed with wine relevant thiols.

Two non-volatile thiols, Cys and GSH, were used in these experiments as they represent the major fraction of free sulfhydryl functionality in wine and are typically present at concentrations that far exceed those of VSCs. MeSH and H2S, which are two of the primary sulfhydryl-containing compounds associated with sulfidic off odors in wine were also added. Fe(III) and Cu(II) were then added to mimic copper fining and wine oxidation. Although these experiments were conducted under air, it is expected that the initial oxidation reaction of the sulfhydryls paired with Cu(II) reduction will occur in the same manner as would be expected in the absence of O2. The concentrations of sulfhydryls used in this model system far exceed those found in wine, but were used to readily assess and detect oxidation products.

Test solutions were allowed to oxidize overnight, after which point they were analyzed using LC-Q-TOF. Cys polysulfanes were observed up to n=6 (Table 5.2) for the treatment containing the combination of Cys+H2S. Similarly, the oxidation of the GSH+H2S combination treatment resulted in GSH polysulfanes up to n=7 (Table 5.3). When MeSH was added along with

H2S, the symmetrical polysulfanes for Cys and GSH (Tables 5.2 and 5.3) were formed, and the presence of the mixed disulfide and polysulfanes was also readily observed. In the case of Cys,

Cys-MeSH disulfide and polysulfanes were observed up to n=6 (Table 5.4), and GSH-MeSH was observed up to n=8 (Table 5.5). The corresponding spectrum can be found in the appendix (Figures

D.1 – D.4). The Cu(II)-mediated oxidation process results in disulfides, but it is clear that it does not result strictly in the generation of symmetrical disulfides. Furthermore, it appears that when

H2S is present, it results in the incorporation of sulfur to the disulfide, and results in generation of polysulfanes.

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Table 5.2. Cys-polysulfanes identified by LC-QTOF after reacting Cys (500 µM) and H2S (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model wine. S (n) Molecular M+H monoisotopic Retention time S/N Intensity (ion formula mass (min) ratio count) 1 C3H7NO2S 122.027 ± 0.005 0.99 1027.4 52270

2 C6H12N2O4S2 241.031 ± 0.005 0.99 6820.7 685100

3 C6H12N2O4S3 273.003 ± 0.005 0.99 3737.2 319400

4 C6H12N2O4S4 304.975 ± 0.005 1.22 39805.8 190900

5 C6H12N2O4S5 336.947 ± 0.005 2.38 203.6 9045

6 C6H12N2O4S6 368.919 ± 0.005 3.41 47.4 612.2

Table 5.3. GSH-polysulfanes identified by LC-QTOF after reacting GSH (500 µM) and H2S (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model wine. S (n) Molecular M+H monoisotopic Retention S/N ratio Intensity (ion formula mass time (min) count) 1 C10H17N3O6S 308.091 ± 0.005 1.28,1.42 4650, 1855 2308000, 1180000

2 C20H32N6O12S2 613.159 ± 0.005 1.29, 1.49, 6070.9, 3741.3, 2166000, 1019000, 1.66 6289.6 1143000 3 C20H32N6O12S3 645.131 ± 0.005 2.29, 2.51 6033.1, 1382000, 1413000 13107.5 4 C20H32N6O12S4 677.103 ± 0.005 3.46 8178.4 634300

5 C20H32N6O12S5 709.075 ± 0.005 4.25 1150.6 28550

6 C20H32N6O12S6 741.043 ± 0.005 5.1 161.4 1513

7 C20H32N6O12S7 773.020 ± 0.005 5.87 27.2 67.57

Table 5.4. Mixed Cys-MeSH disulfide and polysulfanes identified by LC-QTOF after reacting Cys (500 µM), H2S (250 µM), and MeSH (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model wine. S (n) Molecular M+H monoisotopic Retention S/N ratio Intensity formula mass time (min) (ion count) 2 C4H9NO2S2 168.015 ± 0.005 1.48 2683.8 201400

3 C4H9NO2S3 199.987 ± 0.005 3.1 3843.1 134200

4 C4H9NO2S4 231.959 ± 0.005 4.68 1154.5 31140

5 C4H9NO2S5 263.931 ± 0.005 6.27 805.5 6398

6 C4H9NO2S6 295.903 ± 0.005 7.75 146.7 915

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Table 5.5. Mixed GSH-MeSH disulfide and polysulfanes identified by LC-QTOF after reacting GSH (500 µM), H2S (250 µM), and MeSH (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model wine. S (n) Molecular formula M+H monoisotopic Retention time S/N ratio Intensity mass (min) (ion count) 2 C11H19N3O6S2 354.079 ± 0.005 3.32 68539.5 3601000

3 C11H19N3O6S3 386.051 ± 0.005 4.69 36202.7 2277000

4 C11H19N3O6S4 418.023 ± 0.005 6.03 19465.6 703200

5 C11H19N3O6S5 449.995 ± 0.005 7.33 5645.9 120200

6 C11H19N3O6S6 481.967 ± 0.005 8.48 13701.3 17660

7 C11H19N3O6S7 513.939 ± 0.005 9.5 1293.9 2361

8 C11H19N3O6S8 545.911 ± 0.005 10.47 40 337

The masses associated with the higher oxidation states of sulfur, including sulfenic, sulfinic, and sulfonic acids, as well as oxidized disulfides, could not be observed. This may indicate that during the process of Fe(III) and Cu(II) oxidation, free sulfur radicals are not generated to an appreciable degree that would result in a detectable amount of sulfur oxyanions. As discussed in

Chapter 2 and 3, the sulfhydryl likely remains anchored onto the metal center during the electron transfer oxidation process, giving disulfides and polysulfanes as the exclusive products. This also indicates that while O2 plays an important role in the re-oxidation of the metals and in accepting electrons via metal catalysis, O2 does not play a “direct” role in sulfhydryl-mediated oxidation in the case of Fe(III) and Cu(II), which is unlike that of Mn(III) which results in free radical generation and subsequent O2 uptake (Chapter 4).

The recognition that both symmetrical and asymmetrical disulfides and polysulfanes are generated under the conditions described above is important, as winemakers generally assume the that symmetrical disulfides are exclusively generated during wine oxidation.210 Research that has focused on reduction of symmetrical disulfides (DMDS and DEDS) to explain the generation of

MeSH and EtSH has not found good correlation between the two.49,126,127 It is possible that large amounts of MeSH and EtSH are, in fact, bound as non-volatile disulfides in combination with Cys

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and/or GSH, which would not be detectable by the standard analytical practices (e.g., GC analysis) that are typically used for VSCs.1

It was important to verify that the reactions described above are also relevant and possible under real wine conditions. In order to test this, GSH (50 µM) was added to a commercial white wine blend (i.e, the average GSH concentration in young Sauvignon blanc wines).211 Along with

GSH, MeSH and H2S were also added in order to establish the following three final concentrations:

100, 500, and 5000 µg/L. The wines were subsequently oxidized by the addition of Cu(II) (1 mg/L) and Fe(III) (5 mg/L). At the highest treatment level (5000 µg/L each of H2S and MeSH), the mixed

GSH-MeSH disulfide was readily observed, along with the corresponding polysulfanes up to n=8

(Table 5.6). At 500 µg/L, the formation of polysulfanes up to n=5 was detected. At the lowest concentration, the peak corresponding to the mixed MeSH-GSH was apparent and the trisulfane was detected (Table 5.6).

Table 5.6. Identified mixed GSH-MeSH disulfide and polysulfanes in white wine spiked at various concentrations of H2S and MeSH by LC-QTOF. H2S and MeSH added S(n) Retention time 100 µg/L 500 µg/L 5000 µg/L (min) S/N ratio intensity S/N ratio intensity S/N ratio intensity 2 4.5 74.3 3732 259.6 9736 105.6 5623 3 5.9 33.2 885 280.1 8833 207.3 10800 4 7.3 - - 146 2903 245.4 7373 5 8.6 - - 36 251.5 222 2273 6 9.7 - - - - 25.5 588.8 7 10.67 - - - - 26.1 400.1 8 11.57 - - - - 11.9 126

Winemakers are advised to avoid and minimize O2 throughout the Cu(II) fining process to prevent disulfide generation. However, we have demonstrated that the initial Cu(II) reduction will result in inevitable formation of disulfides and mixed disulfides in a manner that is independent of 115

O2. The presence of O2 will reoxidize the metals and cause further oxidation of sulfhydryl compounds. More realistically, MeSH will typically be present in wine at ~1 – 5 µg/L; however, the concentration of GSH and the transition metals used here are in molar excess to MeSH, and so the reaction is expected to be similar under wine conditions. The generation of mixed disulfides at trace concentrations that are nonvolatile, as described here, could potentially act as precursors for reductive odor generation post-bottling, and needs to be further investigated.

5.4.2 Disulfide scission

The mechanisms for disulfide reduction in wine, as well as the conditions and parameters that favor this reduction, remain ambiguous. The involvement of transition metals, bisulfite, and ascorbic acid all appear to be capable of playing a role in the redox status of sulfur compounds

(Chapter 1). It has been hypothesized that disulfide reduction in wine generates volatile thiols with low detection thresholds; however, recent work has failed to show depletion of symmetrical disulfides and corresponding thiol generation.49,127 As described above, mixed disulfides are expected to form, and may play a role in thiol generation.

Recent studies have shown that elevated Cu concentrations are associated with elevated

VSC in wine during the post-bottling period. It is possible that Cu and other transition metals may be involved in disulfide bond scission via concomitant electrophilic and nucleophilic attack (Figure

1.9 – pg 39).144 In this mechanism, an electrophilic species (E+), such as Cu(II), may bind to the disulfide bond making the overall complex more electrophilic and causing the disulfide bond to become more susceptible towards nucleophilic attack. The nucleophilic species (Nu-) could be water, but under wine conditions, sulfite, other thiols, and ascorbic acid may play a more important role as nucleophiles. This reaction could potentially result in the release of potent VSCs. If Cu(II) and a thiol behave as the electrophilic and nucleophilic species, respectively, the reaction with the 116

disulfide would yield a new mixed disulfide and a Cu(I)SR complex. Although it is still unclear which conditions drive the release of Cu(I)SR complex, recent work has demonstrated that the complex dissociates with accelerated anaerobic aging conditions.81

BCDA was used in combination with cystine and Cu(II) to examine whether cystine can undergo oxidative scission. A positive control wherein cysteine was added in excess to Cu(II) resulted in a near immediate and complete reduction of Cu(II) to Cu(I); the generated Cu(I)SR complex was displaced by BCDA to give the BCDA-Cu(I) complex, which was evident due to corresponding absorbance increase at 484 nm (data not shown). The oxidative cleavage of cystine should similarly yield a Cu(I)SR complex that will be displaced by BCDA, and this results in an increase in BCDA-Cu(I) absorbance at 484 nm over time.

The oxidative scission mechanism was investigated over a pH range of 2 – 5 as well as at pH 11. At pH 11, approximately 30 µM of Cu(I) was generated within 30 min, and by 24 hours, almost all Cu(II) in solution had been reduced to Cu(I) (Figure 5.1). The results at varying pH levels showed a decrease in reactivity as the pH was lowered, with pH 2, 3, 4, and 5 resulting in the generation of 3, 6.9, 18.6, and 55.2 µM of Cu(I), respectively after 97 hours (Figure 5.1).

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1 5 0 p H 2

p H 3

) 1 0 0 p H 4

 (

p H 5

)

I (

u p H 1 1

C 5 0

0 0 5 0 1 0 0 1 5 0 T im e (h o u rs )

Figure 5.1. Cu(I)-BCDA generation over time in the presence of cystine (400 µM), Cu(II) (100 µM), and BCDA (1 mM) in air saturated model wine over different pH values.

These results clearly demonstrate that the reaction proceeds quickly at high pH, which is expected as the nucleophilic species would be HO-. Basicity is the main determining factor of the reaction rate in metal-assisted nucleophilic disulfide cleavage, although steric effects can account for rates of reaction.145 Nevertheless, there appears to be some activity at a pH range that is relevant to wine (i.e., pH range of 3-4). The effect of pH on the generation of VSCs had been recently

71 investigated, and it was found that low pH is associated with a lower generation of H2S and MeSH.

The possibility that disulfides are cleaved at higher pH to generate H2S and MeSH is, therefore, consistent with the results shown here.

One confounding factor that needs to be taken into account is that BCDA makes Cu(II) a much stronger oxidant, driving the reaction forward in a matter of days. It may be expected that this reaction could also occur under wine conditions in the absence of BCDA, but it would be a much slower process over several weeks to months. The ability of other nucleophilic species (e.g., thiols, sulfite, ascorbic acid) to accelerate the reaction could not be tested using this protocol as they are capable of directly reducing Cu(II) to Cu(I).

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5.4.3 Reactivity of diethyl disulfide

To determine the practical relevance of the above described mechanism (i.e., the proposed electrophile-assisted nucleophilic cleavage of disulfides), the reaction was further investigated under model wine conditions. In this experiment, 50 µg/L of DEDS was used as a model disulfide.

The treatments added were common nucleophilic species in wine that included cysteine, bisulfite, and ascorbic acid in the presence or absence Fe(II) and Cu(II) (refer to Table 5.1). The samples were stored anaerobically at 40 °C to mimic accelerated reductive aging and were monitored over time by GC-PFPD (Table 5.7). The samples were diluted with a strong brine prior to analysis to release thiols from their metal complex as described previously.80 It is expected that Cu(I)SR would be formed upon the cleavage of the disulfide.

Table 5.7. Decrease in DEDS concentration over time with respective treatments.* Diethyl disulfide (µg/L) Treatment Day 4 Day 8 Day 18 T1 47.9 ± 3.0 Aa 42.1 ± 2.7 Aa 45.4 ± 0.0 Aa T2 45.9 ± 4.1 Aa 39.6 ± 2.4 Bab 41.2 ± 4.2 ABab T3 44.4 ± 2.9 Aa 36.4 ± 3.7 Bab 36.4 ± 0.6 Bbc T4 44.6 ± 2.9 Aa 36.4 ± 0.6 Bab 37.4 ± 0.0 Bbc T5 40.7 ± 1.1 Aa 35.7 ± 1.0 ABab 34.7 ± 1.5 Bbc T6 44.6 ± 0.8 Aa 35.1 ± 0.2 Bab 32.9 ± 0.7 Bc T7 40.4 ± 1.6 Aa 32.6 ± 1.0 Bb 24.4 ± 5.0 Cd T8 42.4 ± 2.0 Aa 36.0 ± 1.5 Bab 34.5 ± 2.8 Bbc * Results are shown ± standard deviation of the means. Rows with different capital letters indicate significant differences over time (p < 0.05), whereas columns with different lower case letters case indicate significant differences between treatments (p < 0.05).

The concentration of DEDS was observed to fluctuate in the control treatment (T1) during this experiment; however, there was no significant difference in its concentration over the 18 day period. T2 was not significantly different than the control, but all other treatments had significantly

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(p<0.05) lower DEDS concentration compared to control at day 18, which was particularly evident for T7. There was no detectable concentration of EtSH generated in any of the samples.

The decrease of DEDS over time in the sample treatments could indicate disulfide scission, however, the fact that EtSH was not detected was surprising. A possible explanation is that the brine dilution could have brought the concentration of EtSH to below the detection limit of the instrument. It is also possible that the generated EtSH reacted further to form the corresponding nonvolatile mixed disulfides with Cys and organic thiosulfate with sulfite. In a previous study where aging trials were performed with EtSH and DEDS using stable isotope dilution, it was found that even without aeration both EtSH and DEDS concentrations were decreased.127

Sulfite was observed to play a role in decreasing DEDS concentration, with a significantly lower value for T3 measured compared to control at day 18 (Table 5.7). Furthermore, the combination of sulfite and transition metals (T7) were significantly lower than the control (T1) and sulfite without metals (T3), suggesting a synergistic effect in the reaction with DEDS.

The interconversion of DEDS in the presence of sulfite (sulfitolysis) to form free EtSH and the corresponding organic thiosulfate (Bunte salt) has been previously investigated in model wine, and it has been claimed that ca. 700 days would be necessary to generate EtSH to a level that exceeds the odor detection threshold.43 Simiar to thiol-disulfide interchange, sulfitolysis is a base- catalyzed reaction and is not expected to occur to a significant degree under wine conditions.

Sulfitolysis proceeds as shown in Figure 1.8 (pg 38), with sulfite cleaving the disulfide to generate a free thiol and corresponding Bunte salt. The Bunte salt may then undergo acid-catalyzed cleavage to generate the corresponding free thiol and sulfate. Recent reports have shown sulfitolysis occurs under wine conditions causing the cleavage of glutathione disulfide and cystine to generate the corresponding Bunte salt, which appeared to be relatively stable,44 although previous work with

DEDS assumes that the rate limiting step is the formation of the Bunte salt and not its hydrolysis.43

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Together with transition metals, the reaction could be accelerated based on the reaction depicted in

Figure 1.9 (pg 39).

Ascorbic acid alone (T2) did not result in a decrease in DEDS concentration over the 18 day period, although the combination of ascorbic acid and transition metals (T6) did cause a significant decrease compared to the control and T2 within that same period. However, while the value for T6 was lower than T4, this was not statistically significant and so the effect between transition metals with or without ascorbic acid could not be differentiated. Ascorbic acid is frequently used during bench trials to assess and compare aroma of wines in order to determine if disulfides are present in the wine. In the trial, ascorbic acid is added in excess to release disulfides with an incubation time of a few minutes, followed by the addition of Cu(II) sulfate to remove the generated thiols.124 If the resulting odor disappears after the addition of Cu, the type of reductive compound is attributed to disulfides in wine. Surprisingly, much like the copper fining practice, the aforementioned practice has been commonplace in the wine industry for several decades, yet the mechanism that causes the reduction under wine conditions, and the degree to which it proceeds, remains unknown. Recent work suggests that Cu(I)SR is an important nonvolatile precursor for releasing H2S and thiols, and that ascorbic acid may have an effect at reducing or displacing these complexes. Based on the results described here, ascorbic acid in a simple model system is not capable of reducing disulfides, and may require the involvement of transition metals.

Ascorbic acid may be capable of reducing disulfide bonds, and like sulfitolysis and thiol- disulfide interchange, it appears to proceed faster under high pH conditions. The reaction likely occurs via the involvement of the mono- and di-anion of ascorbic acid, whereas the undissociated acid has negligible reactivity in cleaving RSSR as well as RSNO, which may have a similar reaction pathway to the disulfide.159–161 The mechanism for ascorbate-mediated cleavage of the disulfide is

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unknown, but it has been suggested that the presence of transition metal ions, such as copper and iron, facilitate disulfide cleavage.159

The treatment containing Cys and transition metals (T5) was significantly lower than control at day 18, and while it was lower than T4, this was not statistically significant (Table 5.7).

Interestingly, similar results were obtained for T8, which contained sulfite, AA, and Cys. It was expected that the combination would play a role at further decreasing DEDS concentrations; however, this was not the case and the decrease was inhibited compared to T7.

These results demonstrate that transition metals and sulfite play an important role in loss of disulfides over time under wine conditions. However, the results relating to the generation of the corresponding thiols remain inconclusive and need to be further investigated. As a simple disulfide,

DEDS may not be as reactive as mixed disulfides containing GSH or Cys with VSCs (e.g., MeSH or EtSH), as their tridentate ability may bind to the metal more effectively and drive the reaction forward. We have shown that the generation of these mixed disulfides is possible, and their reaction should be investigated further. Furthermore, sulfitolysis of disulfides of either symmetrical or assymetrical disulfides containing MeSH and EtSH may generate the corresponding Bunte salt with

MeSH and EtSH, and these compounds may be susceptible to acid-catalyzed cleavage and subsequent release of VSCs.

Although it is not expected that polysulfanes would be generated at sufficiently high concentrations to contribute to the generation of sulfidic off odors in wine, these species are likely to be more reactive due to their ability to simultaneously coordinate with several sulfur atoms and, therefore, act as a multi-dentate ligands to metal ions. The ability of metals to bind directly to the sulfur chain may therefore promote subsequent reductive or oxidative cleavage.154

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5.4.4 Predicting a wine’s ability to exhibit reductive off-odors

At present, winemakers have limited options for controlling, or even predicting, the development of reductive off-odors in the post-bottling period. Cu(II) additions are common for the control of free thiols prior to bottling, but little can be done once the wine is bottled. There are methods for quantifying various reductive aroma precursors in wine (e.g., disulfides and thioesters), however, this practice is both time consuming and expensive, and is thus not practical for most winemakers. Providing winemakers with the tools for predicting the evolution of VSCs in a specific lot of wine would be extremely useful and would inform further remedial actions.

We have demonstrated that the Cu-fining process may generate non-volatile mixed disulfides and metal complexes. A wine may, therefore, lack a reduced aroma profile despite the presence of significant amounts of disulfides and metal complexes; however, once these molecules are cleaved, as described above, the resulting thiol compounds are capable of causing wine spoilage. The objective of this project was to develop a simple, fast, inexpensive, and reliable method kit for testing a wine’s ability to exhibit reductive odors during the post-bottling period by the dissociation of VSCs. Our goal was to demonstrate a practical application of the fundamental, mechanistic work described in previous chapters of this thesis.

Commonly encountered VSCs (H2S, MeSH, and EtSH) were added to model wine at a final concentration of 100 µg/L, at which point Cu(II) sulfate was added at 1 mg/L to simulate copper fining process. As described previously, this would result in the formation of the corresponding disulfides, polysulfanes, and Cu(I)SR complexes (Chapter 2). Afterwards, treatments for their reduction were added and then analyzed using GC-PFPD (Table 5.8). As expected, Cu(II) addition, which was in molar excess to the VSCs, resulted in a complete loss of all sulfhydryl compounds.

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Table 5.8. Peak area for each corresponding compound after addition of treatments in air saturated model wine. H2S MeSH EtSH Treatment Average Recovery Average Recovery Average peak Recovery peak area (%) peak area (%) area (%) control 6977.2 ± 151 100 485.2 ± 47.9 100 4857.3 ± 92.2 100 Cu(II) 0 ± 0 0 0 ± 0 0 0 ± 0 0 Cu+TCEP 155.6 ± 60.8 2.23 337.0 ± 22.2 69.47 3457.4 ± 182.1 71.18 Cu+BCDA 0 ± 0 0 0 ± 0 0 0 ± 0 0 Cu+TCEP+BCDA 254.8 ± 54.3 3.65 328.7 ± 30.0 67.74 3454.1 ± 235.2 71.11 Cu+TCEP+BCDA 242.1 ± 21.1 3.47 353.7 ± 34.1 72.90 3682.1 ± 276.5 75.81 +Cys

The addition of TCEP resulted in the release of ~70% of MeSH and EtSH, but was

relatively ineffective in releasing H2S (~2% release). TCEP is a reagent capable of reducing a

disulfide (S-S) into two free thiols (-SH), and the strength of the resulting phosphorus-oxygen bond

makes the reaction irreversible (Figure 5.2).212,213 The reagent is practically odorless and will not

interfere with the aroma associated with free thiol compounds, and can quickly react at acidic wine

conditions and reduce disulfides and polysulfanes.

Figure 5.2. Reduction of disulfides in the presence of TCEP.

Surprisingly, BCDA alone failed to result in the release of any of the tested sulfhydryl

compounds, which would have been expected to be bound as Cu(I)SR complexes to some extent.

We had previously shown that BCDA is capable of displacing the insoluble Cu(I)-6SH aggregate

(Chapter 2). Recent work has shown that the metal complex-bound forms of H2S and MeSH could

be responsible for VSC generation.80,81 It appears that anaerobic aging results in a decrease of bound

forms and the release of the volatile fraction. As the experimental conditions were conducted under

air, the lack of release of the corresponding sulfhydryls could therefore be attributed to their 124

oxidation. In a separate experiment, large amounts of H2S and Cu(II) sulfate were combined in model wine to form the non-volatile CuS nanoparticles. The addition of TCEP resulted in the release of H2S as noted by smell, however, BCDA addition did not result in H2S release. Addition of barium hydroxide to the solution after BCDA addition resulted in a fine white precipitate due to

BaSO4, suggesting that H2S had been oxidized to sulfate.

The use of BCDA and TCEP in combination yielded results similar to that of TCEP alone.

The addition of Cys in combination of BCDA and TCEP resulted in a slight increase in the recovery of the thiols (Table 5.8). Cys was added in excess to act as a reducing agent for Cu(II) and to serve as a sacrificial thiol. If excess Cu(II) remains, BCDA may oxidize the released volatile thiol fraction to subsequently reduce Cu(II) to Cu(I).

Results obtained under aerobic conditions showed reasonable recovery of MeSH and EtSH, but were insufficient in the case of H2S. Even in the presence of excess reducing agents, it appears that O2 interferes with the recovery of labile H2S, and so the experiment was repeated under anaerobic conditions for H2S (Table 5.9).

Table 5.9. Peak area for H2S after addition of treatments in anaerobic model wine. Treatment Average peak area Recovery (%) control 6355.8 ± 740.1 100 Cu(II) 0 ± 0 0 Cu+TCEP 4348 ± 121.7 68.41 Cu+BCDA 16.7 ± 14.6 0.26 Cu+TCEP+BCDA 4798.5 ± 392.8 75.50 Cu+TCEP+BCDA+Cys 5935.6 ± 23.5 93.39

A markedly higher recovery was observed with TCEP in the absence of O2, resulting in 68% recovery compared to ~2% in the presence of oxygen. For BCDA, virtually no H2S was recovered, presumably due to the presence of excess Cu(II) in solution. The combination of BCDA and TCEP resulted in a 75% recovery of H2S, giving a slight increase compared to TCEP alone. When Cys

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was present along with TCEP and BCDA, the recovery was further increased to 93%. This is

apparently due to the presence of excess Cys that was capable of reducing Cu(II) to Cu(I), thereby

preventing the oxidation of released H2S by Cu(II).

Having established the conditions for optimal sulfhydryl compound release and recovery, these

conditions and reagents were then used to analyze six commercial Pennsylvania red and white

wines in order to determine their ability to release VSCs (Table 5.10).

Table 5.10: Concentrations of H2S and MeSH in three PA white wines and three PA red wines before and after addition of treatment reagents. None of the wines released detectable amounts of EtSH before or after the kit was used. WW1 WW2 WW3 H2S (µg/L) MeSH (µg/L) H2S (µg/L) MeSH H2S (µg/L) MeSH (µg/L) (µg/L) control 2.50 ± 0.11 2.00 ± 0.07 2.25 ± 0.07 2.57 ± 0.01 ND ND Cu+TCEP+ 50.34 ± 2.16 2.93 ± 0.01 79.61 ± 5.72 4.25 ± 0.43 43.28 ± 5.70 2.43 ± 0.05 BCDA Cu+TCEP+ 51.94 ± 4.14 2.69 ± 0.09 79.61 ± 1.84 4.25 ± 0.46 46.02 ± 4.12 2.33 ± 0.03 BCDA+Cys

RW1 RW2 RW3 H2S (µg/L) MeSH (µg/L) H2S (µg/L) MeSH H2S (µg/L) MeSH (µg/L) (µg/L) control ND ND ND ND 2.22 ± 0.01 ND Cu+TCEP+ 26.94 ± 0.88 87.93 ± 0.54 45.74 ± 2.80 3.89 ± 0.08 4.47 ± 0.24 8.04 ± 0 BCDA Cu+TCEP+ 32.83 ± 1.76 88.94 ± 0.75 46.35 ± 2.86 3.56 ± 0.04 5.19 ± 0.49 8.37 ± 0.09 BCDA+Cys

EtSH was not detected in any of the samples before or after the addition of the reagents,

however free H2S and MeSH ranged from undetectable concentrations to 2.50 µg/L and 2.57 µg/L,

respectively. In all cases, H2S and MeSH were released in the wines above their reported threshold

upon the addition of the test reagents. H2S release ranged from 5.19 to 79.61 µg/L, and MeSH

ranged from 2.33 to 8.37 µg/L, with an outlier at 88.94 µg/L. These concentrations were consistent

with the study reported by Franco-Luesma and Ferreira, and were consistent with the fact that over

80 50% of MeSH and 90% of H2S are bound. It appears that the addition of Cys improved recovery 126

slightly in some of the wines, however, it was mostly ineffective. This could be explained by the fact that wines would likely already contain thiols such as Cys and GSH in excess, and that copper will likely be present in its reduced Cu(I) form under reductive conditions.

TCEP may have some activity with respect to reducing copper and dissociating its thiol complex, and can also reduce sulfoxides (e.g. DMSO to DMS), although these were not quantified.

The precise mechanism governing the release of VSCs cannot be elucidated from the results outlined here; however, recent work suggests that 60 – 90% of H2S release and 24 – 48% of MeSH release is attributed to metal complex dissociation.81 The remaining portion is due to de novo formation, which could be attributed to disulfide reduction, although there are also other pathways proposed for generation of VSCs.

While the anaerobic preparation of the samples is not practical from a winery perspective, these results can easily be adapted to work as a kit in a winery setting. The samples can be prepared by transferring ~20 – 30 mL of wine to a 50 mL polypropylene tube with a screw cap. The sample can be deoxygenated with nitrogen, argon, or sodium bicarbonate. Alternatively a sample of the wine can be taken from the bottom of the tank and carefully transferred to avoid oxygen ingress.

The reagents can be made into a kit with a packet containing 5 mg each of TCEP, BCDA, and Cys.

The reagents are added to the wine, followed by capping the tube and mixing. After 5 – 10 min, the wine sample is evaluated by informal sensory analysis; if VSCs are present above their odor detection thresholds, they will be readily apparent.

The use of the reagents described above is an effective way of quickly releasing VSCs, which are indicative of a wine’s ability and potential to exhibit reductive odors after bottling. Such a kit needs to be tested compared to natural reductive bottle aging processes to verify that any of the results obtained correlate with VSC generation. The dissociation of the metal complexes as well as reduction disulfide and polysulfanes is done at a very high efficiency by the reagents, and it is

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unlikely that the generation will proceed to such extent under typical wine aging. Nevertheless, such a semi-quantitative kit may be able to predict potential for a wine to exhibit reductive odors post-bottling. If the wine exhibits reductive off odors, the winemaker can take preventative measures including consideration for copper additions, sparging, bottle closures, and wine aging.

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Chapter 6

Conclusions and Recommendations for Future Work

6.1 Summary

In this dissertation, I examined the interaction of transition metals with H2S and thiols in model wine conditions. I found that copper plays a central role at mediating redox reactions of sulfhydryl compounds, and is capable of oxidizing thiols and H2S to disulfides and polysulfanes and form Cu(I)-SR metal complexes. The formation of disulfides, polysulfanes, and Cu(I)-SR complexes occurs without oxygen uptake, and will therefore similarly occur in wineries when

Cu(II) fining is employed. I observed that the presence of thiols also inhibits the precipitation of

CuS, presumably by interfering with bulk crystal formation. Furthermore, Cu(I)-SR is not inert, and can react in the presence of oxygen and catalyze Fenton-like reaction and subsequent ethanol oxidation.

I found that when Fe(III) is added in combination of H2S and thiols, the oxidation of H2S and thiols and reduction of Fe(III) to Fe(II) occurs with the generation of disulfides. However, the reaction is drastically slower compare to that of Cu(II), furthermore, Fe(II) did not appear to play a major role in binding to H2S and thiols. When Fe(III) and Cu(II) used in combination, the reaction was much faster than either of the metal alone, suggesting a synergistic reaction. It was found that

Cu(I)-SR is generated within seconds, and is subsequently oxidized by Fe(III). Cu(II) is reduced again to Cu(I)-SR in the presence of excess H2S and thiols, resulting in fast reduction of both Cu(II) and Fe(III). Fe(II) appeared to react faster with oxygen than Cu(I)-SR, driving the overall reaction faster in the presence of oxygen. When H2S and 6SH were oxidized in the presence of Fe and Cu,

I was able to detect polysulfanes up to 5 linking sulfur atoms.

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I had also investigated the effect of manganese at catalyzing thiol and H2S oxidation. I found that unlike the reaction with Fe and Cu, Mn resulted in the generation of free thiyl radicals and subsequent radical chain reaction. This resulted in the generation of sulfonic acids and various oxidized disulfide species. However, in the presence of polyphenolics, which are abundant in wine, the thiyl radicals are quickly scavenged. Furthermore, when Cu(II) is added, it appears that the Cu- driven reaction dominates and limits thiyl radical formation. Nonetheless, it appears that Mn may accelerate the reaction and also generate transient thiyl radicals during wine oxidation.

Lastly, I had demonstrated that applying Cu-fining in white wine which had added H2S and

MeSH resulted in the generation of mixed GSH-MeSH disulfide and trisulfane. This compound is nonvolatile and may release MeSH under post-bottling conditions. I have demonstrated that Fe and

Cu in combination of reducing agents (SO2, Cys, and ascorbic acid) play a key role in disulfide scission under anaerobic conditions. Given that disulfides, polysulfanes, and metal sulfide complexes may play a crucial role in the generation of sulfidic odors post-bottling, I developed a method kit to force their reduction and dissociation. I have successfully released H2S and MeSH from wines previously free of sulfidic faults. This protocol may aid winemakers in predicting their wine’s ability to exhibit sulfidic odors and therefore take action.

6.2 Future Work

6.2.1 Interaction of H2S and Thiols with Zinc

Zn(II) is known to have similar binding properties with sulfide as Cu(II), but it does not redox cycle. The reaction displayed by Zn(II) is a simple substitution reaction generating Zn(II)S.

There is evidence showing the binding of Zn with H2S in wine and beer, but whether it effects overall redox reactions in wine need to be further investigated. 130

6.2.2 Interaction of reducing agents and disulfides

Under physiological conditions, ascorbic acid and glutathione have an intricate relationship, with glutathione reducing dehydroascorbic acid to ascorbic acid. However, it has also been suggested that ascorbic acid could reduce disulfide bridges with release of free thiols. In my work investigating DEDS reduction, ascorbic acid alone was ineffective at reducing DEDS without the addition of Fe and Cu. Further work should investigate the interaction of transition metals and ascorbic acid at reducing and/or dissociating VSC precursors.

6.2.3 Using alternative treatments to Cu(II) fining

Cu(II) salts are extremely effective at removing free sulfhydryl functionalities, but they may result in accumulation of copper and oxidation products that release post-bottling. The use of physically bound copper could prove effective at providing the beneficial effects of copper while minimizing its downsides. Preliminary work reported in Appendix E showed promising results but this needs to be investigated further. The work has shown that the use of a bound Cu-iminodiacetic acid complex encapsulated in a PDMS material was effective at removing free H2S and EtSH while limiting the accumulation of metal sulfides and disulfides. There are numerous types of support materials and methods for synthesizing copper particles, and it is worthwhile to explore further to avoid the use of the free Cu(II) salt.

6.3 Concluding Remarks

Cu(II) fining is a commonly utilized process for the control of sulfidic odors in wine in both small and largescale wineries. This work demonstrates how Cu(II) interacts with both H2S and

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thiols and which major products are formed. It was found that disulfides, polysulfanes, and Cu(I)-

SR complexes are readily formed regardless of oxygen concentration. Fe and Mn play a role at catalyzing the redox reactions, but do not change the resulting oxidation products. Because the oxidation products remain redox active, they may reduce and/or dissociate under reductive wine conditions, resulting in the release of H2S and MeSH. Fe and Cu in combination of reducing agents in wine play a key role at mediating the reduction of these compounds. This work provides a foundation and basis for future work in effectively controlling sulfidic odors in wine post-bottling.

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Appendix A. Supplementary information for Chapter 2 mBBr + cys + h2s

310 100

309

223

225 312 113 149 191 238 367 378 117 153 211 247 288 292 313 345 394 397 425 0 m/z 100 125 150 175 200 225 250 275 300 325 350 375 400 425

Figure A.1. Fragmentation pattern of Cys-bimane.

157 mBBr + cys + h2s

413 100

412

% 412

191 415 113 190 450 531 694 149 192 221 267 300 310 357379 481 511 549 605627 673 0 m/z 100 150 200 250 300 350 400 450 500 550 600 650 700

Figure A.2. Fragmentation pattern of sulfide-dibimane.

158 t=0m R2 D

12.59 100 7.97

13.63

0 Time 5.00 10.00 15.00 20.00 25.00

Figure A.3. Chromatographic profile of combined MRM spectra. Rt 7.97 min – Cys-bimane (m/z 310→223); 12.59 min – sulfide-dibimane (m/z 413→191); 13.63 min – 6SH-bimane (m/z 323→222).

159

Appendix B: Supplementary information for Chapter 3. 6MH+H2S Ox

n = 2 n = 3 100

n = 4

% n = 5

0 Time 20.00 21.00 22.00 23.00 24.00 25.00

160

6MH+H2S Ox

214 100 n = 5 214 215

353

% 124 158 345 216 363 301 329 141 171197199 236249 282 309 365 0

n = 4 313 100 331 331 214

% 315 116 158 333 215 352 124 159 179 199 249 317 237 279 334 0

n = 3 281 100

% 299 116 147 282 214 301 124 158 0

n = 2 249 100

% 267 116 0 m/z 125 150 175 200 225 250 275 300 325 350 375 400

Figure B.2. Fragmentation pattern of organic polysulfanes shown in Figure S1.

161

Figure B.3. ESI- mass spectrum of S5-bimane obtained from reaction of H2S (300 µM) with Fe(III) (200 µM) and Cu(II) (50 µM) followed by MBB derivatization.

162

Appendix C. Supplementary information for Chapter 4 181>81 sulfoante fragmentation in 6SH-Fe/Cu(+Mn) GYK160408_5 MRM of 1 Channel ES- 6.69 7.23 7.84 8.52 9.56 TIC 100 3.66 5.45 8.15 9.68 0.73 1.49 2.64 2.83 4.89 452

0 Time 2.00 4.00 6.00 8.00 10.00 181>81 sulfoante fragmentation GYK160408_4 MRM of 1 Channel ES- 1.87 100 1.76 TIC 1.97 1.62e3 2.02 2.17 % 0.26 2.32 0.40 1.36 7.68 8.84 9.49 9.70 9.96

0 Time 2.00 4.00 6.00 8.00 10.00

Figure C.1. LC-MS/MS monitoring fragmentation of 6SH-sulfonic acid (181>81 m/z) during the oxidation of 6SH in the presence of (top) Fe(III), Cu(II), and Mn(II) or (bottom) Fe(III) and Mn(II).

163

6SH+MN+FE GYK160506_4 Scan ES- 8.90 100 329 5.72e5 8.85 8.97 10.07 9.04 % 9.479.55 9.78 8.32 9.11 9.94 7.75 7.96 8.16 8.54 8.58 0 Time 7.50 8.00 8.50 9.00 9.50 10.00 6SH+MN+FE GYK160506_6 Scan ES+ 9.02 100 315 6.98e6

% 9.60 9.89 8.89 9.29 9.48 10.0310.08 7.70 7.87 8.08 8.25 8.52 0 7.50 8.00 8.50 9.00 9.50 10.00 GYK160506_6 Scan ES+ 8.85 100 299 8.89 5.29e6 8.91 10.08

% 9.00 9.98 8.75 9.339.37 9.61 9.77 7.69 7.91 8.16 8.398.54 0 7.50 8.00 8.50 9.00 9.50 10.00 GYK160506_6 Scan ES+ 8.56 100 283 5.54e7

% 9.60 0 7.50 8.00 8.50 9.00 9.50 10.00 GYK160506_6 Scan ES+ 9.70 100 267 4.42e7

0 Time 7.50 8.00 8.50 9.00 9.50 10.00

Figure C.2. Peak corresponding to 6SH-disulfide, thiol-sulfinate, thiol-sulfonate, sulfinyl-sulfone, and α-disulfone in 6SH oxidation by Fe(III) and Mn(II) after ~190 hr.

164

6SH+MN+FE+CU GYK160506_9 Scan ES+ 10.07 100 10.01 315 1.61e6 9.20 9.359.54 9.59 9.86

% 8.128.33 8.63 8.80 8.88 7.697.83 8.00 0 7.50 8.00 8.50 9.00 9.50 10.00 GYK160506_9 Scan ES+ 10.08 100 10.03 299 9.94 1.49e6 9.71 9.75 8.898.96 9.27 9.44

% 8.67 7.75 7.84 8.09 8.32 8.74 0 7.50 8.00 8.50 9.00 9.50 10.00 GYK160506_9 Scan ES+ 8.55 100 283 1.13e7 8.66 10.07 % 9.99 7.97 8.77 9.22 9.42 9.489.51 7.66 7.87 8.26 8.42 8.91 0 7.50 8.00 8.50 9.00 9.50 10.00 GYK160506_9 Scan ES+ 9.69 100 9.72 267 1.13e8

0 Time 7.50 8.00 8.50 9.00 9.50 10.00

Figure C.2. Lack of peaks for the Mn+Fe+Cu system after 144 hr

165

Appendix D. Supplementary information for Chapter 5

S (n) Molecular M+H monoisotopic Retention time S/N Intensity (AU) formula mass (min) ratio 1 C3H7NO2S 122.027 ± 0.005 0.99 1027.4 52270

2 C6H12N2O4S2 241.031 ± 0.005 0.99 6820.7 685100

3 C6H12N2O4S3 273.003 ± 0.005 0.99 3737.2 319400

4 C6H12N2O4S4 304.975 ± 0.005 1.22 39805.8 190900

5 C6H12N2O4S5 336.947 ± 0.005 2.38 203.6 9045

6 C6H12N2O4S6 368.919 ± 0.005 3.41 47.4 612.2

166

S (n) Molecular M+H monoisotopic Retention S/N ratio Intensity (AU) formula mass time (min) 1 C10H17N3O6S 308.091 ± 0.005 1.28,1.42 4650, 1855 2308000, 1180000

5 C20H32N6O12S5 709.075 ± 0.005 4.25 1150.6 28550

6 C20H32N6O12S6 741.043 ± 0.005 5.1 161.4 1513

167

S (n) Molecular M+H monoisotopic Retention S/N ratio Intensity formula mass time (min) (AU) 2 C4H9NO2S2 168.015 ± 0.005 1.48 2683.8 201400

3 C4H9NO2S3 199.987 ± 0.005 3.1 3843.1 134200

4 C4H9NO2S4 231.959 ± 0.005 4.68 1154.5 31140

5 C4H9NO2S5 263.931 ± 0.005 6.27 805.5 6398

6 C4H9NO2S6 295.903 ± 0.005 7.75 146.7 915

168

S (n) Molecular formula M+H monoisotopic Retention time S/N ratio Intensity mass (min) (AU) 2 C11H19N3O6S2 354.079 ± 0.005 3.32 68539.5 3601000

3 C11H19N3O6S3 386.051 ± 0.005 4.69 36202.7 2277000

4 C11H19N3O6S4 418.023 ± 0.005 6.03 19465.6 703200

5 C11H19N3O6S5 449.995 ± 0.005 7.33 5645.9 120200

6 C11H19N3O6S6 481.967 ± 0.005 8.48 13701.3 17660

7 C11H19N3O6S7 513.939 ± 0.005 9.5 1293.9 2361

8 C11H19N3O6S8 545.911 ± 0.005 10.47 40 337

169

Appendix E. Preliminary studies using Cu(II) sulfate alternatives for the control sulfidic odors in wine

The use of PDMS-encapsulated copper particles as an alternative to copper fining was investigated. Given the importance that disulfides, polysulfanes and, and metal-thiol complexes play with respect to wine quality, an alternative to copper fining has been investigated using a variety of bound copper particles. If the copper fining process could be conducted without the risk of leaving residual copper and sulfidic odor precursors was available, the potential for post-bottling generation of sulfidic off-odors could be dramatically decreased. It was found that the use of certain encapsulated materials was more effective at removing H2S and EtSH and their oxidized precursor compounds compared to traditional Cu(II) sulfate additions.

Methodology. Various copper and silver particles were encapsulated in a thin PDMS film and kindly donated by Martin Schmitt. The PDMS film treatments were placed inside a 300 mL

B.O.D. bottle prior to the experiment and allowed to equilibrate in the anaerobic chamber overnight. Model wine was spiked with H2S (50 µg/L) and EtSH (50 µg/L), and then immediately transferred to B.O.D. bottles containing the PDMS film treatment. For every experiment, a control sample was included which was transferred to a B.O.D. bottle containing no PDMS film treatments.

After 24 hours, one 10 mL sample aliquot was transferred to a 20 mL amber GC vials and capped immediately to determine free H2S and EtSH in solution. A separate 10 mL aliquot was spiked with

TCEP (1 mM), BCDA (1 mM), and Cys (1 mM) for determination of residual bound forms of H2S and EtSH. The experiments were conducted in duplicate in an anaerobic chamber.

Bound Cu-particles. The use of copper fining may result in the generation of sulfhydryl metal complexes and disulfides which can be subsequently reduced post-bottling and cause chemical spoilage of the wine. There are numerous downsides to the use of Cu(II) salts, but they

170

are extremely effective in the removal of free thiol functionality. For these reasons, the use of Cu(II) as physically or chemically bound forms could be an effective way of removing thiols without the introduction of unwanted precursors for VSCs.

A variety of PDMS encapsulated copper and silver particles were explored in the experiments described in this section. PDMS was used here to provide a barrier that both prevents the migration of metal particles into the wine while also allowing for the migration of VSCs into the capsule. Once the sulfhydryl-containing compound reacts with the metal (i.e., Cu or Ag), it should become physically immobilized as the metal complex forms. The release of the resulting disulfide from the PDMS film is less likely due to the higher molecular weight of the compound needing to transport out of the PDMS film, furthermore, the compounds may potentially scalp into the PDMS material. The results for removal and regeneration of H2S and EtSH are described below

(Tables E.1 and E.2).

Table E.1. Observations for H2S. *relative to control Treatment % removal* % removal after "reduction"* Relative % regenerated PDMS 8.8 3.2 8.2 -1.0 6.7 0.0 (negative control) Cu sulfate 100.0 100.0 50.0 61.2 50.0 38.8 (positive control) Cu powder 91.1 67.4 62.2 56.3 31.7 16.6 CuIDA 76.5 100.0 46.6 78.1 39.0 21.9 immobilized 99.4 100.0 72.3 75.5 27.2 24.5 CuIDA Cu foil 42.9 34.3 40.8 31.1 5.1 9.2 Cu oxide 100.0 92.3 64.9 67.7 35.1 26.7 Cu stearate 84.3 99.4 55.2 67.3 34.5 32.3 Ag powder 38.9 29.0 37.1 25.8 4.7 11.0 Ag acetate 100.0 98.3 40.1 42.1 59.9 57.2 Ag 98.6 93.5 72.9 79.3 26.1 15.1 encapsulated Ag stearate 22.4 36.1 19.4 29.2 13.7 19.3

171

Table E.2. Observations for EtSH. *relative to control Treatment % removal* % removal after "reduction"* Relative % regenerated PDMS 17.5 8.8 13.1 6.3 25.0 27.7 (negative control) Cu sulfate 100.0 100.0 20.3 15.0 79.7 85.0 (positive control) Cu powder 84.0 68.1 61.0 55.7 27.5 18.1 CuIDA 74.9 100.0 40.6 60.8 45.8 39.2 immobilized 90.4 100.0 66.7 62.1 26.2 37.9 CuIDA Cu foil 67.3 62.9 64.0 56.4 4.9 10.2 Cu oxide 100.0 86.2 69.8 49.9 30.2 42.1 Cu stearate 84.6 97.1 63.3 67.2 25.2 30.7 Ag powder 70.3 57.7 68.4 53.5 2.6 7.4 Ag acetate 100.0 93.9 68.9 64.7 31.1 31.0 Ag 100.0 93.7 87.0 79.0 13.0 15.8 encapsulated Ag stearate 47.7 61.1 44.4 56.3 6.9 7.8

All treatments were more effective with respect to removing H2S and EtSH compared to the PDMS film negative control, although some scalping by the PDMS material was observed.

None of the treatments except Cu(II) sulfate resulted in consistent 100% removal of H2S and EtSH, but the immobilized CuIDA, Cu oxide, Ag acetate, and encapsulated Ag were very effective.

However, after forcing the reduction of the model wine (see section 5.4.4), Cu sulfate had the most

H2S and EtSH regenerated compared to all treatments (except for Ag acetate, for unknown reasons).

Some of the treatments varied widely between the two experimental replicates, which may be due to holes in some of the PDMS sachets.

Some compromises will have to be made such that a complete removal of VSCs can occur within a reasonable time frame in a winery, but the treatment must also result in the least disulfides and metal-thiols after use. A few of the treatments were particularly effective at preventing accumulation of either disulfides and/or metal-bound VSCs (Cu foil, Ag powder, Ag stearate) but

172

also reacted slowly, resulting in incomplete removal of the VSCs within 24 hours. The immobilized

CuIDA and encapsulated Ag cation exchange (and perhaps the Cu oxide) resulted in almost complete removal of VSCs with less generation after 'reduction' compared to copper sulfate.

Although these results are preliminary in nature, they may provide a useful alternative for copper fining to limit the negative aspects associated with it. Further analysis is needed to measure residual free metal ions in solution.

173

Vita

Gal Y. Kreitman

Education Ph.D. Food Science, The Pennsylvania State University, University Park, PA, 2016 M.S. Food Science, The Pennsylvania State University, University Park, PA, 2013 B.S. Food Science, The Pennsylvania State University, University Park, PA, 2011

Publications Kreitman, G.Y.; Danilewicz, J.C.; Jeffery, D.W.; Elias, R.J. Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model Wine. Part 1: Copper Catalyzed Oxidation. J. Agric. Food Chem. 2016, 64, 4095-4104. Kreitman, G.Y.; Danilewicz, J.C.; Jeffery, D.W.; Elias, R.J. Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model Wine. Part 2: Iron- and Copper- Catalyzed Oxidation. J. Agric. Food Chem. 2016, 64, 4105-4113. Kreitman, G.Y., Cantu, A., Waterhouse, A.L., Elias, R.J. Effect of Metal Chelators on the Oxidative Stability of Model Wine. J. Agric. Food Chem. 2013, 61, 9480–9487. Kreitman, G.Y., Laurie, V.F., Elias, R.J. Investigation of ethyl radical quenching by phenolics and thiols in model wine. J. Agric. Food Chem. 2013, 61, 685–92.

Presentations Kreitman G.Y., Elias R.J. What’s that smell?! Predicting Reductive Aroma in Wine (invited talk). PA Wine Marketing and Research Board Symposium, State College, PA, 2016. Oral Presentation Kreitman G.Y., Danilewicz J.C., Elias R.J. A Mechanistic Investigation of Copper-Mediated Oxidation of Thiols in Model Wine. 66th Annual Meeting of the American Society for Enology and Viticulture, Portland, OR. 2015. Poster Presentation. Kreitman G.Y. and Elias R.J. The Role of Copper in the Evolution of Sulfur Compounds in Wine (invited talk). PA Wine Marketing and Research Board Symposium, State College, PA, 2015. Oral Presentation Kreitman G.Y., Cantu A., Waterhouse A.L., Elias R.J. Controlling oxidation of model wine using metal chelators. 65th Annual Meeting of the American Society for Enology and Viticulture, Austin, TX. 2014. Oral Presentation. Kreitman G.Y., Elias R.J. Oxidative loss of thiols in model wine solution by 1-hydroxyethyl radicals (invited talk). 244th National Meeting & Exposition of the American Chemical Society, Philadelphia, PA. 2012. Oral Presentation.

Awards PA Wine Marketing and Research Program Grant Recipient (2015, 2016) American Wine Society Educational Foundation Scholarship (2015) American Society for Enology and Viticulture (2014, 2015) Penn State College of Agricultural Sciences Competitive Grants Winner (2014)