The Pennsylvania State University. The Graduate School. College of Agricultural Sciences REACTION MECHANISMS OF TRANSITION METALS WITH

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1 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 by Gal Y. Kreitman 2016 Gal Y. Kreitman Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2016

2 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 ii

3 ABSTRACT Sulfidic off-odors due to hydrogen sulfide (H 2S) 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 H 2S and thiol compounds under wine-like conditions. Adding Cu(II) sulfate to air saturated model wine containing H 2S, cysteine (Cys), 6-sulfanylhexan-1-ol (6SH), or 3-sulfanylhexan-1-ol (3SH) led to a rapid formation of ~1.4:1 H 2S:Cu and ~2:1 thiol:cu complexes. This resulted in the oxidation of H 2S and thiols, and reduction of Cu(II) to Cu(I) without oxygen uptake. Both H 2S and thiols resulted in the formation of Cu(I)-SR complexes, and subsequent reactions with oxygen led to the oxidation of H 2S 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 H 2S 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 H 2S and thiols (Cys, 6SH, and 3SH) under wine-like conditions. H 2S 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 iii

4 reduced by H 2S 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 H 2S 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 H 2S oxidation in aquatic systems. Thus, the interaction of manganese with iron and copper was investigated in relation to thiol and H 2S 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. H 2S 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 H 2S 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 H 2S and methanethiol (MeSH, 100 µg/l each), Cu(II)-fining resulted in the generation of MeSHglutathione 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 (SO 2), ascorbic acid, and Cys to promote the catalytic scission of diethyl disulfide (DEDS). I found that the combination of SO 2 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 iv

5 reducing agent) and bathocuproine disulfonic acid (a chelator). The addition of the reagents successfully released H 2S 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 H 2S and thiols with Cu, Fe, and Mn under wine-like conditions. I showed that Cu(II) was readily reduced by H 2S and thiols, and that this complex remained redox active and reduced oxygen. The reaction of Cu with H 2S 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 H 2S and thiols provided a foundation for future strategies to better control sulfidic off-odors in wine. v

6 TABLE OF CONTENTS LIST OF FIGURES... x LIST OF TABLES... xv ACKNOWLEDGEMENTS... xvii Chapter 1 Literature Review Introduction Metal-catalyzed redox reactions Copper Copper fining Redox cycling of copper Iron Manganese Other transition metals Release of metal sulfide and metal thiol complexes Thiol/disulfide couple Occurrence and oxidation of disulfides Thiol-disulfide interchange Sulfitolysis Metal catalyzed disulfide scission Ascorbic acid Reactions of sulfhydryls with organic wine constituents Thioester hydrolysis Strecker degradation of amino acids Further reactions of sulfur containing compounds Research overview, significance, and hypotheses Chapter 2 Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model Wine. Part 1: Copper Catalyzed Oxidation ABSTRACT INTRODUCTION vi

7 2.3 MATERIALS AND METHODS Chemicals Model wine experiments Determination of oxygen consumption Cu-complex formation and dissolution Spectrophotometric measurements of thiols and H 2S Spectrophotometric measurement of Cu(I)-BCDA HPLC analyses of thiols and H 2S HPLC analysis of catechols HPLC analysis of acetaldehyde Copper determination EPR analysis RESULTS DISCUSSION Cu reduction and complex formation Disulfide formation Oxidation of the Cu(I)-complex Acknowledgments Chapter 3 Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model Wine. Part 2: Iron and Copper Catalyzed Oxidation ABSTRACT INTRODUCTION MATERIALS AND METHODS Chemicals Model Wine Experiments Determination of oxygen consumption Spectrophotometric measurements HPLC Analyses RESULTS AND DISCUSSION Reaction of Fe(III) with H 2S and thiols in model wine Fe(III) reduction by thiols and H 2S Fe(II) oxidation and oxygen consumption vii

8 3.4.4 Fe(III) and Cu(II) reduction by thiols and H 2S Fe(II)/Cu(I) oxidation, oxygen consumption, and acetaldehyde formation Reaction of Fe(III)/Cu(II) with H 2S in combination with thiols in model wine Formation of mixed organic polysulfanes 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 ABSTRACT INTRODUCTION MATERIALS AND METHODS Chemicals Model Wine Experiments Determination of oxygen consumption Spectrophotometric measurements HPLC Analyses RESULTS AND DISCUSSION Reaction of Cys with Mn Reaction of Cys with Mn+Fe Reaction of Cys with Mn+Fe+Cu Reaction of 6SH Reaction of H 2S CONCLUSIONS Chapter 5 Investigating Volatile Sulfur Compound Precursors and Practical Applications ABSTRACT INTRODUCTION MATERIALS AND METHODS Materials Preparation of model wine and real wine samples Disulfide and polysulfane generation Disulfide scission by Cu(II) and bathocuproine disulfonic acid viii

9 Diethyl disulfide scission in the presence of metals and reducing agents Release and reduction of bound VSCs Methods of analysis HPLC GC UV-Vis RESULTS AND DISCUSSION Disulfide and polysulfane generation Disulfide scission Reactivity of diethyl disulfide Predicting a wine s ability to exhibit reductive off-odors Chapter 6 Conclusions and Recommendations for Future Work Summary Future Work Interaction of H 2S and Thiols with Zinc Interaction of reducing agents and disulfides Using alternative treatments to Cu(II) fining Concluding Remarks REFERENCES Appendix A. Supplementary information for Chapter Appendix B: Supplementary information for Chapter Appendix C. Supplementary information for Chapter Appendix D. Supplementary information for Chapter Appendix E. Preliminary studies using Cu(II) sulfate alternatives for the control sulfidic odors in wine ix

10 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 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 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 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 Figure 1.5. Reaction of thiols with Cu(II) to produce disulfides without free radical generation Figure 1.6. Reaction mechanism of thiol-disulfide interchange via trisulfide like transition state to generate a new disulfide and corresponding thiol Figure 1.7. Example of transition metal assisted thiol-disulfide interchange resulting in the generation of a new Cu(I)-SR complex Figure 1.8. Sulfitolysis followed by acid-catalyzed cleavage of an organic thiosulfate Figure 1.9. Concurrent electrophilic and nucleophilic assisted disulfide bond scission Figure Reversible reactions of aldehydes with bisulfite in wine to generate hydroxyalkylsulfonates or with thiols to generate hemithioacetals and thioacetals Figure 2.1. Removal of H 2S by addition of Cu(II) and formation of insoluble CuS Figure 2.2. H 2S and thiols used throughout this study Figure 2.3. Loss of thiol/h 2S by Ellman s assay in air saturated model wine upon addition of Cu(II) (50 µm) to 6SH, H 2S, Cys (300 µm) and Cu(II) (100 µm) to 3SH (300 µm). Error bars indicate standard deviation of triplicate treatments Figure 2.4. Reaction of Cu(II) in (a) model wine and treatments containing (b) 3SH, (c) 6SH, (d) Cys, and (e) H 2S, showing (A) loss of electron paramagnetic resonance (EPR) active Cu(II) (0.5 mm) signal in model wine after mixing with the respective x

11 thiol/h 2S treatments (1.5 mm), and (B) UV-spectra of the thiols/h 2S (300 μm) in model wine after mixing with Cu(II) (50 μm) 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, H 2S, 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 Figure 2.6. Loss of H 2S and Cys in air saturated model wine upon adding Cu(II) (100 µm) to H 2S (~100 µm) in combination with Cys (~400 µm). Error bars indicate standard deviation of triplicate treatments Figure 2.7. O 2 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 Figure 2.8. O 2 consumption in air saturated model wine upon addition of Cu(II) (50 µm) to 6SH, H 2S, and Cys (300 µm), and addition of Cu(II) (100 µm) to 3SH (300 µm). Error bars indicate standard deviation of triplicate treatments Figure 2.9. Acetaldehyde produced in air saturated model wine upon addition of Cu(II) (50 µm) to 6SH, H 2S, and Cys (300 µm), and addition of Cu(II) (100 µm) to 3SH (300 µm). Error bars indicate standard deviation of triplicate treatments Figure Proposed mechanism for initial reaction of thiols with Cu(II) and Cu(I)-thiol complex formation. Only the thiol ligands are shown Figure Proposed thiyl radical formation and subsequent scavenging with 4-MeC and DMPO Figure Four electron steps in the reduction of O 2 to H 2O via the hydroperoxyl radical, hydrogen peroxide and the hydroxyl radical Figure Proposed Cu(I)-SH complex catalyzed two-electron reduction of O 2 to H 2O Figure Proposed Cu(I)-SH complex catalyzed two-electron reduction of H 2O 2 to H 2O Figure One-electron reduction of H 2O 2 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 Figure 3.1. Reduction of oxygen by Fe(II) to yield hydrogen peroxide without the release of hydroperoxyl radicals xi

12 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 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 Figure 3.4. Reaction of H 2S or thiols on addition of Fe(III) (200 µm) to 6SH, H 2S, Cys, or 3SH (300 µm) in air saturated model wine. (A) Consumption of H 2S or thiols; (B) %Fe(III)-tartrate based on absorbance at 336 nm; (C) O 2 consumption. Error bars indicate standard deviation of triplicate treatments Figure 3.5. Reaction of H 2S or thiols on addition of Fe(III) (200 µm) and Cu(II) (50 µm) to H 2S, 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 H 2S or thiols; (C) O 2 consumption; (D) AC generation. Error bars indicate standard deviation of triplicate treatments Figure 3.6. Proposed mechanism demonstrating initial Cu(II) reduction by thiols and H 2S 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 H 2O 2 is depicted in Figure Figure 3.7. Total thiol and H 2S loss on addition of Fe(III) (200 µm) and Cu(II) (50 µm) to (A) 6SH (300 µm) + H 2S (100 µm); (B) 3SH (300 µm) + H 2S (100 µm); (C) Cys (300 µm) + H 2S (100 µm); (D) Fe(III) (100 µm) and Cu(II) (25 µm) to Cys (300 µm) + H 2S (50 µm) to air saturated model wine. Error bars indicate standard deviation of triplicate treatments Figure 3.8. Total concentrations of Fe(III), Fe(II), O 2 (consumed), thiol, and AC in Cys+H 2S 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 Figure 4.1. Fe(III) initiated sulfite oxidation and subsequent Mn-catalyzed radical chain reaction resulting in sulfite oxidation and sulfate generation Figure 4.2. Reaction of Mn(II) with Fe(III)-superoxo complex to generate Mn(III) and H 2O 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) O 2 consumption, (C) acetaldehyde generation, and (D) %Fe(III)-tartrate based on absorbance at 336 nm. Error bars indicate standard deviation of triplicate treatments 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 xii

13 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) O 2 consumption, (C) acetaldehyde generation, and (D) %Fe(III)-tartrate based on absorbance at 336 nm. Error bars indicate standard deviation of triplicate treatments Figure 4.6. Reaction of H 2S (150 or 200 μm) with Mn(II) (100 μm), Fe(III) (100 μm), and Cu(II) (25 μm) in air saturated model wine. (A) H 2S consumption, (B) O 2 consumption, (C) acetaldehyde generation, and (D) %Fe(III)-tartrate based on absorbance at 336 nm. Error bars indicate standard deviation of triplicate treatments 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 Figure 5.2. Reduction of disulfides in the presence of TCEP Figure A.1. Fragmentation pattern of Cys-bimane Figure A.2. Fragmentation pattern of sulfide-dibimane Figure A.3. Chromatographic profile of combined MRM spectra. Rt 7.97 min Cysbimane (m/z ); min sulfide-dibimane (m/z ); min 6SH-bimane (m/z ) Figure B.1. HPLC chromatogram with detection at 210 nm showing organic polysulfanes (identified by MS) obtained from reaction of 6SH (300 µm and H 2S 100 µm) with Fe(III) (200 µm) and Cu(II) (50 µm) Figure B.2. Fragmentation pattern of organic polysulfanes shown in Figure S Figure B.3. ESI- mass spectrum of S 5-bimane obtained from reaction of H 2S (300 µm) with Fe(III) (200 µm) and Cu(II) (50 µm) followed by MBB derivatization 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) 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 Figure C.2. Lack of peaks for the Mn+Fe+Cu system after 144 hr Figure D.1. Identified Cys-polysulfanes by LC-QTOF after reacting Cys (500 µm) and H 2S (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 xiii

14 Figure D.2. Identified GSH-polysulfanes by LC-QTOF after reacting GSH (500 µm) and H 2S (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 Figure D.3. Identified mixed Cys-MeSH disulfide and polysulfanes by LC-QTOF after reacting Cys (500 µm), H 2S (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 Figure D.4. Identified mixed GSH-MeSH disulfide and polysulfanes by LC-QTOF after reacting GSH (500 µm), H 2S (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 xiv

15 LIST OF TABLES Table 1.1. Odor descriptors and thresholds for volatile sulfur compounds in wine Table 1.2. Occurrence and oxidation states of various sulfur species which may be present in wine 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 Luther 75 and sources within Table 1.4. Calculated solubilities of metal sulfides at 25 C, atm total pressure, and ph 7 in pure water. Values adapted from Ricard and Luther Table 1.5. Diagnostic test and sensory screening of sulfidic odors in wine utilizing copper, cadmium, and ascorbic acid Table 5.1. Treatment addition to anaerobic model wine containing 50 µg/l diethyl disulfide Table 5.2. Cys-polysulfanes identified by LC-QTOF after reacting Cys (500 µm) and H 2S (250 µm) with Fe(III) (100 µm) and Cu(II) (50 µm) in air saturated model wine Table 5.3. GSH-polysulfanes identified by LC-QTOF after reacting GSH (500 µm) and H 2S (250 µm) with Fe(III) (100 µm) and Cu(II) (50 µm) in air saturated model wine Table 5.4. Mixed Cys-MeSH disulfide and polysulfanes identified by LC-QTOF after reacting Cys (500 µm), H 2S (250 µm), and MeSH (250 µm) with Fe(III) (100 µm) and Cu(II) (50 µm) in air saturated model wine Table 5.5. Mixed GSH-MeSH disulfide and polysulfanes identified by LC-QTOF after reacting GSH (500 µm), H 2S (250 µm), and MeSH (250 µm) with Fe(III) (100 µm) and Cu(II) (50 µm) in air saturated model wine Table 5.6. Identified mixed GSH-MeSH disulfide and polysulfanes in white wine spiked at various concentrations of H 2S and MeSH by LC-QTOF Table 5.7. Decrease in DEDS concentration over time with respective treatments.* Table 5.8. Peak area for each corresponding compound after addition of treatments in air saturated model wine Table 5.9. Peak area for H 2S after addition of treatments in anaerobic model wine xv

16 Table 5.10: Concentrations of H 2S 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 Table E.1. Observations for H 2S. *relative to control Table E.2. Observations for EtSH. *relative to control xvi

17 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. xvii

18 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 (H 2S), 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 H 2S and other organic sulfur compounds in the final wine H 2S is produced as a byproduct during normal yeast metabolism 1

19 via the sulfate reduction pathway, in which H 2S acts as an intermediate in sulfur-containing amino acid biosynthesis. 17 The production of excess H 2S 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 EtSH 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 µg/l 30 Methanethiol Cabbage, sewage µg/l 31 Ethanethiol Onion, rubber, fecal 1.1 µg/l 27 Dimethylsulfide Cabbage, asparagus, corn, 25 µg/l 27 blackcurrant Dimethyldisulfide Cooked cabbage, sulfurous, onion 29 µg/l 27 Diethyldisulfide Onion, garlic, rubber 4.3 µg/l 27 Methylthioacetate Sulfurous, cheesy 50 µg/l 32 Ethylthioacetate Cabbage, cauliflower 10 µg/l 32 4-Methyl-4-sulfanylpentan-2- Box tree, guava, cat urine 3.3 ng/l 33 one 3-Sulfanylhexan-1-ol Passionfruit, grapefruit 60 ng/l 5 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 2

20 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 O 2 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 H 2S and MeSH above their odor detection threshold in wine may occur when O 2 is limited and can result in consumer rejection of the wine. 37,41 It appears that an intricate balance of O 2 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 O 2 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 O 2 concentration, and the presence of 3

21 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 H 2S, 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, oxidation of RSH Mild oxidant, can be further oxidized Organic RSS nsr -1,0,-1 Oxidation of RSH and Mildly oxidizing polysulfanes H 2S Elemental sulfur S 8 0 Pesticide residue, oxidation of H 2S Very weak oxidant, can be reduced by RSH Sulfenic acid RSOH 0 Transient Condenses to disulfide Sulfinic acid RSO 2H +2 Oxidation product of Adds to quinones RSH Sulfonic acid RSO 3H +4 Oxidation product of Unreactive RSH Sulfite - HSO 3 +4 Yeast byproduct, Reducing agent, antioxidant winemaking additions Sulfate 2- SO 4 +6 Sulfite oxidation, Unreactive yeast and grapes Thiosulfate - RSSO 3-1,+4 Sulfitolysis of disulfides 43,44 Hydrolyze to sulfate and free thiol Thiosulfinate +1,-1 Unknown Oxidizing Thiosulfonate +3, -1 Unknown Oxidizing Sulfinylsulfone +3, +1 Unknown Oxidizing 4

22 Disulfone +3, +3 Unknown Oxidizing Thioethers, RSR -2 Dimethylsulfide, dialkylsulfides thioesters, etc. Sulfoxide +2 dimethylsulfoxide 45 Sulfone +4 dimethylsulfone 46 Metal sulfides M ns n varies Various complexes with first row transition metals Reducing, oxidizing, or inert The generation of H 2S and MeSH have been implicated as the compounds responsible for post-bottling reduction which occurs when O 2 ingress is low In recent years, numerous studies attempted to identify precursors and conditions needed for the generation undesirable sulfidic offodors. 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. 5

23 1.2. Metal-catalyzed redox reactions Transition metals are well known to catalyze redox reactions in wine. 51,52 Under wine conditions, O 2 is reduced to H 2O in a 4-electron step manner in the presence of transition metals, 53 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 dictated by the rate of O 2 ingress. 57 O 2 is stable in its triplet ground state (i.e., 3 O 2) and its direct reaction with organic compounds (singlet state) is spin forbidden; however, O 2 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 O 2 to H 2O 2 without the release of hydroperoxyl radicals or oxidation of catechols (Figure 1.1). 55,58 Once H 2O 2 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 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 O 2 reduction process, transition metals are oxidized and can subsequently oxidize polyphenols or sulfhydryls. The quinones that result from polyphenol oxidation can 6

24 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) 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 reaction forwards. 54,66 It appears that the relationship between sulfhydryls and O 2 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 O 2, 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 H 2S 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 H 2S, which can react with transition metals, and upon further rearrangment, may result in crystal structure formation and 7

25 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 H 2S, 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 H 2S 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 Luther 75 and sources within Metal Complex Log K Mn(II) [MnHS] Fe(II) [FeHS] Co(II) [CoHS] Ni(II) [NiHS] Cu(II)* [CuHS] [CuS] Cu(I) [CuHS] Zn(II) [ZnHS] [ZnS] Ag(I) [AgHS] [AgS] Au(I) [AuHS] *Cu(II) likely reduced to Cu(I) to some extent during analysis. 8

26 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 Table 1.4. Calculated solubilities of metal sulfides at 25 C, atm total pressure, and ph 7 in pure water. Values adapted from Ricard and Luther 75 Metal sulfide Solubility (mg/l) MnS FeS CoS NiS CuS ZnS AgS AuS 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 O 2 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 9

27 > 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 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 mg/l, but may be as high as 10 mg/l 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 many decades. 2,41,90 Sulfidic off-odors are typically attributed to H 2S (and thiols such as MeSH) and it is generally assumed that reacting Cu with H 2S would result in formation and complete precipitation (and removal) of CuS, due to its low solubility product ( 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

28 of removing CuS from wine, even with filtration. 91 In fresh and saltwater it has been shown that the reaction of H 2S 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 These nonvolatile thiols would be in large molar excess to the exogenous Cu (3 6 µm) used in a fining operation, and would far exceed the concentration of H 2S (ca. 300 nm) 30 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 Redox cycling of copper Trace concentrations of Cu are now known to act synergistically with Fe in mediating nonenzymatic wine oxidation reactions, particularly by accelerating oxygen consumption and polyphenol oxidation. 52 As described above, polyphenol oxidation generates quinones which may 11

29 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 H 2S, 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 H 2S 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 ,103 Similarly, under biogeochemical conditions, H 2S reduces Cu(II) to Cu(I) during Cu 3S 3 ring formation, and these species remain in solution as polynuclear nanoclusters 72. The relevance of these reactions and their redox activity is thoroughly investigated in Chapter 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 nonenzymatic 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, O 2 will be reduced to H 2O 2 more readily. A relatively low Fe(III)/Fe(II) reduction potential will also facilitate the reduction of H 2O 2 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

30 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 tartrate 106 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. H 2S 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 H 2S, and FeS does not exhibit odors associated with H 2S. 80,101 The binding of H 2S is likely to form subunits of Fe 2S 2 similar to mackinawite structure, however, under acidic conditions it does not appear to be sufficiently stable to aggregate as a solid. 112,113 Furthermore, FeS clusters are reactive in the presence of O 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 reaction rates with some sulfhydryls, particularly H 2S, is very high. 97 Although Fe(III)-catalyzed 13

31 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 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. 14

32 1.2.4 Other transition metals Zinc concentrations average between mg/l and can exceed 1 mg/l, as such it may be present at comparable concentrations to Cu in wine. 114 Zn has been shown to effect H 2S 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 H 2S is comparable to Cu, as it has a high stability constant ( ) and low solubility ( mg/l). 34 Similarly to Cu, it forms a Zn 3S 3 ring structure that further condenses to Zn 4S 6 under aquatic conditions. 119 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 H 2S, and formation of a relatively stable complex that effectively renders the sulfhydryl group unavailable for reaction (or volatilization). The binding of H 2S to Zn(II) has been demonstrated in synthetic wine solutions and beer. 80,118 Furthermore, the generation of H 2S was positively correlated with Zn(II), 70 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 H 2S 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 H 2S in the wine prior to bottling. 120 Therefore higher Zn concentrations may result in lower H 2S 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. 15

33 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 O 2. 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 due to H 2S and MeSH, especially when O 2 ingress is low. 48,57,70 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 H 2S and MeSH is the dissociation of bound metal species. 81,101 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 solubilize Cu to generate the corresponding CuCl 3 2- and CuCl 4 3- complexes, 121,122 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 H 2S 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 H 2S and MeSH. The formation of soluble CuS nanoclusters is likely a major contributing factor for the subsequent release of H 2S and MeSH. Zn(II) reacts in a similar fashion to Cu and is also important for binding of H 2S. Fe(II) has been shown to have some ability at binding to H 2S, although as described above (section 1.2.2), it forms 16

34 a different metal sulfide complex likely consisting of Fe 2S 2. The binding of H 2S 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 H 2S and MeSH. Studies in which H 2S release was monitored in wine have indicated that during an anoxic 18 month aging period of a wine, free H 2S increased with time while total H 2S concentration remained unchanged. 101 One hypothesis is that polyphenolic compounds may reduce the CuS complex to release free H 2S and Cu(0), 101 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 H 2S and MeSH release could be attributed to the dissociation of metal sulfide complexes, it has been shown that up to 42% and 76% of H 2S 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. 17

35 1.3 Thiol/disulfide couple In general, reduced sulfur species (with S 2-, 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 (S 1- ), elemental sulfur (S 0 ), sulfoxides (S 2+ ) and sulfite (S 4+ ), are naturally occurring and are present post-fermentation in wine, and their chemical reduction post-bottling can result in the appearance of undesirable sulfidic offodors 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 O 2 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, In general, the majority of disulfides are formed during yeast metabolism 21,129 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, Occurrence and oxidation of disulfides Sulfhydryls cannot be directly oxidized by O 2 due to Pauli s exclusion principle and require transition metals to facilitate oxidation reactions. They can however, be oxidized by two-electron oxidants such as H 2O 2 to yield a sulfenic acid (RSOH) and water (Figure 1.3A). 131 Sulfenic acids are transient species that can condense with thiols to form disulfides (Figure 1.3B). 131,132 However, the initial reaction with H 2O 2 is relatively slow under wine conditions and will likely be 18

36 outcompeted by sulfite to form sulfate (Figure 1.3C). 133 As such, the oxidation of thiols by H 2O 2 is most likely of little relevance in wine. 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. 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 radicals 67,68 (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

37 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. 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 H 2S, 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 H 2S, MeSH, and EtSH upon their reduction during anoxic storage. In the presence of H 2S, oxidation of H 2S and thiols may result in the insertion of sulfur into disulfides and subsequent formation of polysulfanes. In model solutions containing 20% ethanol, H 2S was shown to react with MeSH and EtSH in the presence of Cu(II) to form mixed diand 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)

38 Alternatively, H 2S 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 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 S N2 mechanism with a trisulfide-like transition state complex and delocalized negative charge (Figure 1.6). 131, 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 pk a, and these reactions typically proceed at or above physiological ph. The pk a of cysteine s and glutathione s respective thiol groups are ca. ~8-9, whereas simpler thiols are closer to However, due to the linear-free energy relationship, increasing pk a 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 21

39 GSH, which would generate a mixed disulfide and release of EtSH and MeSH. While the pk a 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 pk a 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 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 SO 2 and 50 µg/l DEDS. 22

40 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 its fully deprotonated SO 2-3 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 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. Due to its higher pk a, EtSH is a better leaving group than GSH. 131 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 H 2S, MeSH, and EtSH due to hydrolysis of the corresponding organic thiosulfates. It may be that sulfitolysis is 23

41 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 Metal catalyzed disulfide scission Transition metals may play a role in assisting thiol-disulfide interchange and sulfitolysis (Sections 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 24

42 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 Cucontaining 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 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 H 2S may be significant. The release of H 2S from elemental sulfur has been previously shown in wine, 155 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. 25

43 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. 26

44 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) + Cu(II) (0.2 g/l) Sulfidic compound Presence of Odor gone Odor gone Odor gone H 2S sulfidic off-odors Odor gone No change Odor gone Thiols Odor gone Slight Odor gone H 2S 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 Ascorbic acid s first ionizable proton has a pk a 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 ~ 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 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 It has been suggested that in the ascorbic acid/copper system, Cu(I) drives the reduction of disulfides. 161,164 27

45 Ascorbic acid also efficiently scavenges O 2 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 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 H 2S, 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 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 H 2S adduct of 4-methylcatechol (4-methyl-5-sulfanylcatechol) demonstrated that the release of H 2S occurs at ph 6 in the presence of reducing agents. 155 Given that catechol-h 2S adducts can exist in equilibrium with the catechol and H 2S, it is possible that reducing conditions would result in H 2S when O 2 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

46 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 of H 2S may result in its reaction with multiple aldehydes. 171 Figure 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,βunsaturated 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 which may later decompose in the dough. 172 The equilibrium of H 2S 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 29

47 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, thiolthioester 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 H 2S, 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 H 2S may yield potent polysulfanes, dimethyl trisulfane and tetrasulfane, which have detection thresholds of 100 ng/l and 60 ng/l, respectively. 1,179 Reaction of H 2S with benzaldehyde generates benzyl mercaptan, which has a smoky odor, 180 whereas reaction with furfural generates furfurylthiol that is reminiscent of 30

48 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 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

49 The objectives needed to achieve the aims of this research are to: 1. Elucidate the oxidation mechanism of H 2S 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 32

50 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, ABSTRACT Sulfidic off-odors due to hydrogen sulfide (H 2S) 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 H 2S and thiol compounds (RSH) under wine-like conditions. Copper complex formation was monitored along with H 2S, thiol, oxygen, and acetaldehyde concentrations after addition of Cu(II) (50 or 100 μm) to air saturated model wine solutions containing H 2S, cysteine, 6-sulfanylhexan-1-ol, or 3-sulfanylhexan-1-ol (300 μm each). The presence of H 2S and thiols in excess to Cu(II) led to the rapid formation of ~1.4:1 H 2S:Cu and ~2:1 thiol:cu complexes, resulting in the oxidation of H 2S and thiols, and reduction of Cu(II) to Cu(I) which reacted with oxygen. H 2S was observed to initially oxidize rather than form insoluble copper sulfide. The proposed reaction mechanisms provide an insight into the extent to which H 2S can be selectively removed in the presence of thiols in wine. 33

51 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 H 2S, 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 assumed to remove H 2S by forming a highly insoluble colloidal CuS precipitate (Figure 2.1), 90,167 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, 34

52 while the average concentration of glutathione (GSH) is reported to be ca. 40 µm in wines made from Sauvignon blanc 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 H 2S (ca. 300 nm) 30 when copper fining is considered. Furthermore, a recent study by Clark et al. 91 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 H 2S 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 that are able to produce H 2S and MeSH. 50,57 The formation of H 2S from the Strecker degradation of Cys has been previously reported, 178 while some have suggested that H 2S 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 H 2S 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

53 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 H 2S and thiol oxidation under wine conditions. 2.3 MATERIALS AND METHODS 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. 36

54 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 H 2S 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 H 2S and thiol compounds, and Cu(II). H 2S, Cys, 6SH, or 3SH were added to air saturated model wine (1 L, 300 μm) followed by thorough mixing. Cu(II) was added to H 2S, Cys, and 6SH (50 μm) or 3SH (100 μm) and thoroughly mixed. For mixed H 2S and Cys system, H 2S (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. H 2S and thiols used throughout this study. 37

55 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) 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 O 2 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. 38

56 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% H 2), ~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, 6SHdisulfide, and Cu(I) concentrations were measured as described below Spectrophotometric measurements of thiols and H 2S UV-vis spectra were recorded on an Agilent 8453 UV-Vis spectrophotometer (Agilent, Santa Clara, CA). Determination of Cu binding to H 2S and thiols was determined by measurement over nm. The concentration of H 2S, 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 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 39

57 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 HPLC analyses of thiols and H 2S MBB derivatization was used to determine each H 2S 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. 40

58 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 , cysteine-bimane was monitored at m/z , and the internal standard 6SH-bimane was monitored at m/z 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. 41

59 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 ) HPLC analysis of acetaldehyde Acetaldehyde was measured in model wine treatment solutions as its 2,4- dinitrophenylhydrazone (DNPH) derivative by HPLC as described previously 67 with the following modification: the sample was centrifuged at g at 4 C for 10 min. The supernatant was then transferred to an HPLC vial for further analysis 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 42

60 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 nm 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 H 2S 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 H 2S, 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 H 2S Cu(II) addition resulted in an immediate uptake of ~1.4 (72 µm) mole equivalents of H 2S, 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

61 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). Figure 2.3. Loss of thiol/h 2S by Ellman s assay in air saturated model wine upon addition of Cu(II) (50 µm) to 6SH, H 2S, Cys (300 µm) and Cu(II) (100 µm) to 3SH (300 µm). Error bars indicate standard deviation of triplicate treatments. EPR analysis showed that Cu(II) was immediately reduced to Cu(I) due to loss of paramagnetic Cu(II) signal by Cys, 6SH and H 2S; 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 nm by the addition of H 2S 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

62 Figure 2.4. Reaction of Cu(II) in (a) model wine and treatments containing (b) 3SH, (c) 6SH, (d) Cys, and (e) H 2S, showing (A) loss of electron paramagnetic resonance (EPR) active Cu(II) (0.5 mm) signal in model wine after mixing with the respective thiol/h 2S treatments (1.5 mm), and (B) UV-spectra of the thiols/h 2S (300 μm) in model wine after mixing with Cu(II) (50 μm). The addition of Cu(II) to H 2S 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 H 2S 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). 45

63 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, H 2S, 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. 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 chelator 188 ). 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 H 2S (75 µm) and Cys (468 µm) were added together to model wine in the presence of Cu(II), ca. 53 and 135 µm of H 2S 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 H 2S + Cys:Cu(II). Subsequent reaction resulted in complete loss of H 2S 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 H 2S alone. 46

64 Figure 2.6. Loss of H 2S and Cys in air saturated model wine upon adding Cu(II) (100 µm) to H 2S (~100 µm) in combination with Cys (~400 µm). Error bars indicate standard deviation of triplicate treatments. 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 O 2 was consumed in this reaction (Figure 2.7), giving an O 2:thiol molar reaction ratio of ~1:

65 Figure 2.7. O 2 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 H 2S and thiol treatments, as well as a combination treatment consisting of Cys+H 2S (Figure 2.8). Minimal O 2 uptake (<5 µm in all treatments) was observed within the first 30 min of the reaction. During the course of the experiments, H 2S had the highest O 2 consumption (175 ± 9 µm), followed by 6SH and Cys, which showed similar O 2 consumption patterns (76 ± 6 and 66 ± 6 µm, respectively), and lastly 3SH, which consumed the least O 2 (23 ± 1 µm). The treatment containing both Cys and H 2S resulted in an O 2 consumption of 117 ± 5.2 µm. Separately H 2S or Cys were oxidized in the 48

66 presence of Cu(II) and excess 4-MeC and monitored over time. The rate of O 2 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). Figure 2.8. O 2 consumption in air saturated model wine upon addition of Cu(II) (50 µm) to 6SH, H 2S, and Cys (300 µm), and addition of Cu(II) (100 µm) to 3SH (300 µm). Error bars indicate standard deviation of triplicate treatments. Complementing the range of measurements described above, acetaldehyde (AC) generation was monitored over time (Figure 2.9). At the end of the experiment, the H 2S 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 + H 2S yielded an AC concentration of 54 ± 3 µm. 49

67 Figure 2.9. Acetaldehyde produced in air saturated model wine upon addition of Cu(II) (50 µm) to 6SH, H 2S, and Cys (300 µm), and addition of Cu(II) (100 µm) to 3SH (300 µm). Error bars indicate standard deviation of triplicate treatments. 2.5 DISCUSSION 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). H 2S is proposed to react similarly with the formation of an initial complex, which could be Cu 3S 3, as discussed below. 50

68 Figure 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 H 2S 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 H 2S 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 1 H-NMR spectrum of a 1:2 mixture of Cu(II):GSH in H 2O-D 2O (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 H 2S 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

69 the addition of H 2S 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 H 2Scontaining 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 [CuS 1.2], 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 H 2S 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 Cu 3S 3 ring systems that adopt a chair-like conformation. 72 As discussed above, these polynuclear nanoclusters are sufficiently small to behave 52

70 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 [Cu 4S 5] - 4 and [Cu 4S 6] -4 polynuclear nanoclusters 72 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, although the presence of O 2 at various concentrations has been shown to alter the rate of reaction. 192 The presence of excess H 2S 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 H 2S 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 H 2S 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 H 2S for Cu coordination. When H 2S and Cys were added in combination in the presence of Cu(II), a 2:1 binding ratio of H 2S + Cys:Cu(II) was still observed (Figure 2.6). Cu(II) binds rapidly to H 2S and relatively more strongly than Cys, which is a benefit for winemakers wanting to remove H 2S. 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 H 2S 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 for the apparent lack of a precipitate when Cu(II) is added to wine in order to remove H 2S

71 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 O 2 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). O 2 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 O 2 uptake, giving an O 2: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 54

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

73 2.5.3 Oxidation of the Cu(I)-complex Oxygen consumption was determined as Cu-mediated H 2S 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 O 2, resulting in a 1:2.6 O 2: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 O 2 (66 µm), giving a ~1:4.5 O 2:Cys molar reaction ratio. H 2S (284 µm) also reacted completely but resulted in much greater O 2 consumption, affording an O 2:H 2S molar reaction ratio of ~1:1.6. This can be explained on the basis that H 2S is capable of being oxidized to ground state S 0, effectively reducing two equivalents of Cu(II). It is also possible for H 2S 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 H 2S 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 O 2 was reduced directly to hydrogen peroxide (H 2O 2) 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). 56

74 Similarly, it has previously been concluded that the Fe(II) reduction of O 2 to H 2O 2 in model wine also proceeds without the release of hydroperoxyl radicals or oxidation of catechols. 58 Figure Four electron steps in the reduction of O 2 to H 2O via the hydroperoxyl radical, hydrogen peroxide and the hydroxyl radical. Figure Proposed Cu(I)-SH complex catalyzed two-electron reduction of O 2 to H 2O 2. Previous studies of the copper-catalyzed H 2O 2 oxidation of Cys similarly failed to detect hydroxyl radicals, and it was suggested that H 2O 2 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 O 2:thiol would result, with all four electrons being derived from the thiol to reduce O 2 to two equivalents of H 2O (Figures 2.13 and 2.14). If H 2O 2 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 O 2 concentrations, resulting in a 1:1 molar ratio of O 2:AC. However, the presence of O 2 in the system 57

75 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 O 2:AC molar ratio and a 1:3 O 2:thiol molar reaction ratio, with three electrons being provided by RSH, one electron being provided by ethanol, and O 2 accepting four electrons. Figure Proposed Cu(I)-SH complex catalyzed two-electron reduction of H 2O 2 to H 2O. 58

76 Figure One-electron reduction of H 2O 2 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. H 2S oxidation produced the most AC (Figure 2.9), and with an O 2:AC molar ratio of 2.2:1, oxidation could have proceeded mainly as shown in Figure This uptake of O 2 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 O 2:AC molar ratio of 2.5:1, while that of Cys+H 2S resulted in a ratio of 2.1:1. The O 2: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 H 2S and thiol oxidation in model wine, albeit to varying degrees. In conclusion, we show that Cu(II) is reduced by H 2S and thiols in air saturated model wine, while thiols, which are present in relative excess to added Cu(II), as well as H 2S, are oxidized. These studies were conducted at initial aerial O 2 saturation in order to follow the oxidative 59

77 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 H 2S 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 H 2S 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 O 2 is introduced as a result of racking or filtration. 60

78 2.6 Acknowledgments The authors thank Alexey Silakov from the Department of Chemistry at The Pennsylvania State University for his assistance with EPR analysis. 61

79 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, ABSTRACT Sulfidic off-odors arising during wine production are frequently removed by Cu(II) fining. In Part 1 of this study, the reaction of H 2S 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 nonenzymatic wine oxidation. The interaction of these two metals in the oxidation of H 2S and thiols (cysteine, 3-sulfanylhexan-1-ol, and 6-sulfanylhexan-1-ol) was therefore examined under wine-like conditions. H 2S 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 H 2S and thiols, oxygen, and acetaldehyde were monitored over time. H 2S 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 H 2S 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. 62

80 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 copper (Cu). 51,52 During this oxidation process, O 2 can be reduced to water in four discrete oneelectron steps, 51 resulting in the formation of reactive intermediate oxygen species 53 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 H 2O 2 from O 2 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 H 2O 2 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 forming the intermediate 1-hydroxyethyl radical (1-HER). 60 In low O 2 concentrations, 1-HER will be oxidized by Fe(III) to yield acetaldehyde (AC); however, at higher O 2 concentrations, O 2 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. 63

81 Figure 3.1. Reduction of oxygen by Fe(II) to yield hydrogen peroxide without the release of hydroperoxyl radicals. 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. 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 sulfite 54 or undergo Michael-type addition reactions with sulfite or thiols 96,97, 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- sulfanylhexan-1-ol (3SH), or a beneficial effect by reacting with hydrogen sulfide (H 2S). 54,112 The 64

82 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 O 2, 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. 65

83 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 H 2S 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 examined the reaction of H 2S with Cu(II), 91 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 synergistically in the oxidation of H 2S and thiols. As noted previously 8, the concentration of thiols, such as glutathione and cysteine analogues, far exceeds that of H 2S that at likely to occur in wine. The oxidation of H 2S 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 Chemicals L-Cysteine (Cys), monobromobimane (MBB), 6-sulfanylhexan-1-ol (6SH), and diethylenetriaminepentaacetic acid (DTPA) were obtained from Sigma-Aldrich (St. Louis, 66

84 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 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 H 2S 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. H 2S, 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 H 2S 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 H 2S, 6SH, or 3SH solutions. For Cys experiments, Fe(III) (100 μm) 67

85 and Cu(II) (25 μm) were consecutively added and mixed thoroughly. For thiol experiments in combination with H 2S and Fe/Cu, H 2S was added to the thiol treatment and mixed prior to the addition of metal stock solutions. H 2S (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, H 2S (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 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 O 2 P6000 meter (Nomacorc LLC, Zublon, NC). Further details were reported in Part 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

86 For H 2S, Cys, 6SH, and 3SH, total concentration was analyzed using Ellman s assay. Further details were reported in Part HPLC Analyses For the mixed H 2S and thiol treatments, MBB derivatization and analysis of thiol concentration was performed using negative electrospray ionization (ESI-) HPLC-MS/MS as described in Part The mass transition of sulfide-dibimane was monitored at m/z , Cysbimane was monitored at m/z , 3SH-bimane at m/z and the internal standard 6SH-bimane was monitored at m/z External standard curves prepared for sulfidedibimane, Cys-bimane, and 3SH-bimane were normalized to the 6SH-bimane internal standard. In the case of 6SH/H 2S 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 ( μm) by HPLC as described in Part 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 SH 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 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 69

87 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. 70

88 3.4 RESULTS AND DISCUSSION Reaction of Fe(III) with H 2S 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) H 2S, 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 of thiols and 1.4 equivalents of H 2S were immediately consumed (i.e. within 5 min), 55 there was no initial uptake of these substances when Fe(III) was added (Figure 3.4A). In the case of H 2S, 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 O 2 was consumed (Figures 3.4B and 3.4C). A total of 262 µm of H 2S 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. 71

89 Figure 3.4. Reaction of H 2S or thiols on addition of Fe(III) (200 µm) to 6SH, H 2S, Cys, or 3SH (300 µm) in air saturated model wine. (A) Consumption of H 2S or thiols; (B) %Fe(III)-tartrate based on absorbance at 336 nm; (C) O 2 consumption. Error bars indicate standard deviation of triplicate treatments. 72

90 3.4.2 Fe(III) reduction by thiols and H 2S 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 H 2S treatments in model wine (Figure 3.4B). For the H 2S 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 H 2S. Minimal Fe(III) reduction was observed in experiments involving 6SH and 3SH, which was matched by minimal thiol and O 2 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. 73

91 This behavior is quite different from that of Cu(II), which was very rapidly reduced to Cu(I) by thiols and H 2S in model wine. 55 Notably, H 2S behaves differently than thiols, as it is capable of reducing Fe(III) in the presence of tartaric acid (Figure 3.4B). Fe(II) can bind H 2S to yield [Fe-H 2S] 2+ which would deprotonate to yield FeS in the form of a [Fe 2S 2] n mackinawite to drive the reaction forward. 34 Under acidic conditions, FeS aggregates to form metastable nanoparticles (<150 Fe 2S 2 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 approximately fold higher than CuS. 75 Tartaric acid should also prevent H 2S coordination, but the ligated acid does not limit the ability of H 2S to reduce Fe(III), in contrast to what occurs with 6SH and 3SH. Recent work suggests that H 2S can remain bound to Fe(II), causing loss of its free sulfhydryl functionality and aroma associated with H 2S. 80, 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 H 2S, and that of Fe(II) reoxidation by O 2. 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 O 2 by a concerted 2-electron mechanism, yielding a Fe(III)-dioxygen complex that directly hydrolyzes to H 2O 2 without release of hydroperoxyl radicals. H 2O 2 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 H 2S to O 2. Based on the overall sequence of reactions, it would be 74

92 expected that 3 electrons would come from thiols or H 2S and 1 electron from ethanol to reduce O 2 to water. Consequently, it would be expected that the O 2:thiol molar reaction ratio would be 1:3, and the O 2:H 2S ratio would be 1:1.5 as H 2S is capable of reducing 2 equivalents of Fe(III) as it is oxidized to ground state sulfur. 73 The treatment containing H 2S resulted in the greatest uptake of O 2 in the presence of Fe(III). Of the 262 µm H 2S that reacted (Figure 3.4A), 135 µm of O 2 was consumed (Figure 3.4C), giving a 1:1.9 O 2:H 2S 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 H 2S. Subtracting that amount from total reacted H 2S would give ~196 µm uptake corresponding to the 135 µm O 2 uptake, thus lowering the O 2:H 2S 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 O 2 (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 O 2 uptake, giving a O 2: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 O 2: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 H 2S 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 H 2S system. In a previous study in which the Fenton reaction was investigated in model wine with iron only, up to 90% of 1-HER 75

93 radical was intercepted by thiol-containing compounds, the resulting thiyl radical likely then quickly dimerizing to yield a disulfide Fe(III) and Cu(II) reduction by thiols and H 2S The interaction of iron and copper plays an important synergistic role in wine oxidation, and it was important to investigate whether these metals impact H 2S 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

94 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 O 2 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. H 2S 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 H 2S 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 H 2S remained (308 µm consumed) at ~48 h, after which Fe(II) re-oxidized to Fe(III) in the presence of O 2 (Figures 3.5A and 3.5B). 77

95 Figure 3.5. Reaction of H 2S or thiols on addition of Fe(III) (200 µm) and Cu(II) (50 µm) to H 2S, 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 H 2S or thiols; (C) O 2 consumption; (D) AC generation. Error bars indicate standard deviation of triplicate treatments. Figure 3.6. Proposed mechanism demonstrating initial Cu(II) reduction by thiols and H 2S 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 H 2O 2 is depicted in Figure 2. 78

96 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 O 2 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 O 2 (Figure 3.3), markedly increasing the rate of Cu(I) oxidation. The degree of consumption of H 2S with copper determined previously 55 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)-H 2S complex was similar to that of the Fe(II)-tartrate complex. O 2 consumption was monitored as thiol and H 2S oxidation proceeded (Figure 3.5C). In the H 2S system, around 46% (92 µm) of iron remained reduced after 120 hr (Figure 3.5A), which would require 46 µm of H 2S. As a result, 262 µm of H 2S would be left to react with 160 µm of O 2 consumed, giving a ~1:1.6 O 2:H 2S 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 O 2, giving a 1:2.6 O 2:Cys ratio. Applying the same reasoning, 223 µm of 6SH and 217 µm of 3SH reacted with an O 2 consumption of 106 µm and 82 µm, respectively. This afforded a ~1:2.1 O 2:RSH molar ratio in the 6SH system and ~1:2.6 in the 3SH system. As with H 2S, 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 O 2 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 O 2 and thiol or H 2S consumed may indicate that both iron and copper behave in the same mechanistic manner with respect to O 2. 79

97 The ~1:3 O 2:RSH molar reaction ratio observed in the Cys and 3SH systems is indicative of a combination of 2-electron reduction of O 2 to H 2O 2, as well as the 1-electron reduction of H 2O 2 to hydroxyl radicals and subsequent one electron ethanol oxidation (i.e., Figures 3.1 and 3.2). The H 2S treatment resulted in the generation of 100 µm AC, whereas the Cys treatment resulted in 60 µm AC, giving O 2 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 O 2: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 H 2S system (Figure 3.2). Furthermore, reduction of Fe(III) by 1-HER generates Fe(II) that subsequently react with O 2, explaining why the molar ratios for the 6SH system, as well as 3SH and Cys, were lower than 1:3 O 2:RSH Reaction of Fe(III)/Cu(II) with H 2S in combination with thiols in model wine Under normal conditions, the concentration of H 2S 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) ,185,198 Therefore, to better model a real wine situation, the oxidation of H 2S 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 H 2S was targeted to be double that of the Cu(II) concentration that was established in the model wine, based on the initial 2:1 H 2S: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

98 wine conditions. This observation may explain why precipitates are not observed when Cu(II) is added to wine containing H 2S. The reduction of Cu(II) also explains the absence of the highly insoluble Cu(II)S, which may have been expected to form. 91 Compared to H 2S, the three thiols were present in large molar excess, but H 2S was still quickly oxidized, with at least 60% of free H 2S removed within 5 min in all treatments (Figures 3.7A-D). By 24 h, there was virtually no H 2S remaining in the four experiments, and even after all free H 2S was depleted, the remaining free thiol continued to oxidize without precipitation of a copper-complex. Figure 3.7. Total thiol and H 2S loss on addition of Fe(III) (200 µm) and Cu(II) (50 µm) to (A) 6SH (300 µm) + H 2S (100 µm); (B) 3SH (300 µm) + H 2S (100 µm); (C) Cys (300 µm) + H 2S (100 µm); (D) Fe(III) (100 µm) and Cu(II) (25 µm) to Cys (300 µm) + H 2S (50 µm) to air saturated model wine. Error bars indicate standard deviation of triplicate treatments. The Cys+H 2S 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, O 2 consumption, thiol consumption, and AC generation were measured to further examine the 81

99 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 H 2S and Cys to be oxidized within 2 h whereas the low metal treatment required 24 h. The total combined Cys+H 2S consumption was 302 and 326 µm for the high and low treatments, respectively, with corresponding total O 2 consumption of 132 and 138 µm for high and low treatments. This resulted in approximately the same molar reaction ratios, at ~1:2.3 O 2:Cys+H 2S, irrespective of metal concentration, and was intermediate between the expected 1:3 ratio for Cys and 1:1.5 ratio for H 2S. 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:O 2 ratio in the high metal system and a 1:2 AC:O 2 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 O 2 (Figure 3.3). Figure 3.8. Total concentrations of Fe(III), Fe(II), O 2 (consumed), thiol, and AC in Cys+H 2S 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

100 3.4.7 Formation of mixed organic polysulfanes When H 2S 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 H 2S. Similar results were obtained with H 2S 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 H 2S, methanethiol, and ethanethiol. 100 When H 2S was oxidized alone, MBB derivatization followed by HPLC-MS analysis indicated the presence of up to S 5-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 H 2S 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 H 2S and thiols. Accordingly, the presence of H 2S 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 H 2S 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. 83

101 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 H 2S 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 O 2 was introduced as a result of racking, filtration, or bottling. Copper fining quickly oxidizes H 2S, 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

102 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 H 2S oxidation in aquatic systems. It was therefore of interest to investigate the interaction of manganese with iron and copper toward catalyzing thiol and H 2S oxidation under wine-like conditions. Sulfhydryl compounds (cysteine, 6-sulfanylhexan-1-ol, and H 2S) 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. H 2S 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 world 114,199 ). Mn has been reported to catalyze browning in sherry wine in combination with iron, 200 increase acetaldehyde production in 85

103 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 O 2, H 2O 2, 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 of SO 2 autoxidation. 201 Its catalytic action is initiated by traces of Fe(III), which oxidizes SO 2 to the sulfite radical (SO - 3 ), which in turn reacts with O 2 to produce the peroxomonosulfate radical (SO - 5 ), It is proposed that this strongly oxidizing radical oxidizes Mn(II) to Mn(III), which allows the Mn catalyzed process to proceed (Figure 4.1). 116 The generated Fe(II) is able to react with O 2 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. 86

104 Fe(II) reacts with O 2 forming an intermediate Fe(III)-superoxo complex. 58 The reduction of the complex is inhibited by the presence of Fe(III) as it competes with Fe(II) to generate H 2O 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 H 2O 2. Under aquatic environments, the reaction of organic thiols and H 2S 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 H 2S and its interaction with Fe and Cu in wine conditions. 4.3 MATERIALS AND METHODS 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- 87

105 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 H 2S) 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 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 H 2S 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 H 2S, 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 H 2S, 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

106 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 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 O 2 P6000 meter (Nomacorc LLC, Zublon, NC). Initial O 2 concentrations ranged from mg/l. Further details were reported in Chapter 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 H 2S, Cys, 6SH, and 3SH, total concentration was analyzed using Ellman s assay. Further details were reported in Chapter 2. 89

107 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 ( 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 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 RESULTS AND DISCUSSION 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 O 2 (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 90

108 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 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) O 2 consumption, (C) acetaldehyde generation, and (D) %Fe(III)-tartrate based on absorbance at 336 nm. Error bars indicate standard deviation of triplicate treatments. 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 MnO 2 mediated thiol oxidation and dissolution of the polymeric complex suggest that oxygen in MnO 2 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, 91

109 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 O 2 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) O 2. After approximately 120 h, 118 µm of Cys were consumed along with 208 µm O 2, giving a O 2:Cys molar ratio of ~1.8:1. This overall molar ratio suggests that a large amount of O 2 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 O 2 in the system would favor the formation of a thiol peroxyl radical (RSOO ). 203,204 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 generation of the sulfonyl radical (RSO 2 ). 203,204 This radical can also rapidly react with O 2 to generate the sulfonyl peroxyl radical (RSO 2OO ), which is a very strong oxidant, 203,204 and could oxidize Mn(II). The sulfonyl peroxide (RSO 2OOH) would be generated, which could undergo Fenton-like reaction to yield the sulfonic acid (RSO 3H) and hydroxyl radicals (HO ). Previous work demonstrated that H 2O 2 is not a sufficiently strong oxidant to oxidize Mn(II), 116 however, RSO 2OO 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). 92

110 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 Mnmediated 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 O 2 as discussed above. The possibility that Cys coordinates with Mn(II) to catalyze the reduction of O 2 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 O 2 and eventually yield a sulfonic acid (see results for 6SH). Along with the O 2 consumption, AC was generated (Figure 4.1C), and with 184 µm generated it gives an AC:O 2 ratio of 1:1.1. The above mechanism (Figure 4.4) would indicate a 1:2 93

111 AC:O 2 reaction ratio, but it is possible that AC could also be formed by the oxidation of ethanol by the sulfonyl peroxyl radical (RSO 2OO ), which is a strong hydrogen abstractor. 203 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 O 2 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 Mncatalyzed reaction products was therefore not undertaken 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 O 2 was consumed along with 118 µm Cys, giving a total of 1.7:1 O 2:Cys molar reaction ratio. This again would suggest that O 2 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 O 2 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

112 which gave a molar ratio of 1.33:1 AC:O 2. This could be attributed to Fe(III) oxidizing 1-HER to AC directly, especially when O 2 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 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 O 2 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 O 2 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 O 2 consumed to give ~1:2.5 O 2:Cys molar ratio. This ratio was slightly lower but consistent with that of Fe+Cu system, which resulted in a ~1:2.7 O 2:Cys molar ratio (Chapter 3). A total of 46 μm of AC was generated (Figure 4.3C), giving a AC:O 2 molar ratio of ~1.6:1, which was lower than the ~2:1 ratio observed in the Fe+Cu system alone. 95

113 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 O 2 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 O 2, as observed by the fast reoxidation of Fe(II) to Fe(III) (Figure 4.3D) 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). 96

114 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) O 2 consumption, (C) acetaldehyde generation, and (D) %Fe(III)-tartrate based on absorbance at 336 nm. Error bars indicate standard deviation of triplicate treatments. Nonetheless, the reaction proceeded over time with 6SH and resulted in O 2 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 O 2 and 23 μm of 6SH were consumed over a 192 h period. This resulted in a ~1:1.6 O 2:6SH ratio, which is lower than that observed with Cys (Figure 4.3). This perhaps indicates that not as much O 2 is incorporated into the thiol. A small amount of AC (7 μm) was generated, which would correspond AC:O 2 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 O 2 and 55 μm 6SH. This resulted in a ~1:1.2 O 2:6SH ratio, which is higher than that with Mn(II) alone. AC (49 μm) was 97

115 generated to give a ~1.1:1 AC:O 2 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 reoxidized. 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 O 2 was consumed with 189 of 6SH within 72 h, giving a 1:2.9 O 2:6SH molar reaction ratio (Figure 4.5A and 4.5B). AC (58 µm) was also produced giving a ~1.1:1 AC:O 2 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 O 2 uptake relative to thiol oxidation points to the disulfide being the main product and that the O 2 is reduced to H 2O 2 to produce an equivalent of AC. Mn(II) (100 µm) alone produced a slow oxidation of 6SH (150 µm), with a 1:1.6 O 2:6SH molar reaction ratio. The reaction is accelerated by Fe(III) (100 µm) with a 1:1.2 O 2:6SH molar reaction ratio. The higher O 2 uptake relative to that of the Cu containing system (1:2.9 O 2:6SH 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 98

116 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 O 2 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 Reaction of H 2S When H 2S (150 µm) was oxidized in the presence of Mn(II) (100 µm) alone, there was no O 2 consumption or appreciable amount of H 2S 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, H 2S 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 metals to either reform H 2S or lose an electron to form elemental sulfur. 207 The reduction of Mn(III) by H 2S likely proceeds through an inner-sphere mechanism. In this process, two equivalents of Mn(III) would be reduced as H 2S is oxidized to elemental sulfur, resulting in no radical generation, 202,208 and therefore negligible O 2 consumption over time. Due to the presence of only 99

117 trace amounts of Fe, which would be capable of oxidizing Mn(II), and the fact that H 2S would not result in buildup of reactive intermediates, Mn-mediated oxidation of H 2S does not occur under the conditions described. Similarly, no AC was generated (Figure 4.6C). Figure 4.6. Reaction of H 2S (150 or 200 μm) with Mn(II) (100 μm), Fe(III) (100 μm), and Cu(II) (25 μm) in air saturated model wine. (A) H 2S consumption, (B) O 2 consumption, (C) acetaldehyde generation, and (D) %Fe(III)-tartrate based on absorbance at 336 nm. Error bars indicate standard deviation of triplicate treatments. When Fe(III) (100 µm) was added in combination to Mn(II), H 2S was slowly consumed over time, along with O 2 (Figures 4.6A and 4.6B). Presumably, the interaction occurs in the same manner as described previously with Fe(III) whereby H 2S reduces two equivalents of Fe(III) to Fe(II) with its oxidation to S 0 (Chapter 3). Over time, Fe(II)-tartrate reduces O 2, resulting in generation of H 2O 2, and subsequent Fenton reaction to generate hydroxyl radicals. Overall, 54 µm of O 2 were consumed in conjunction with 84 µm of H 2S, giving an O 2:H 2S molar ratio of ~1:1.6, 100

118 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 H 2S (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 H 2S, with approximately 58 µm consumed within 5 min. At the end of the reaction at around 120 h, there was 115 µm O 2 consumed along with 180 µm of H 2S (Figures 4.6A and 4.6B), again resulting in a ~1:1.6 O 2:H 2S molar reaction ratio. Therefore, it would appear that the addition of Mn to the H 2S system does not alter the course of the reaction. H 2S is likely oxidized to S 0 and the reduced metals are re-oxidized by O 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:O 2 molar ratio that is consistent with previous findings. 4.5 CONCLUSIONS Mn(II) was found to catalyze Cys and 6SH oxidation with high O 2 consumption relative to that of the thiol. It is proposed, therefore, that thiyl radicals are released and subsequently add O 2 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

119 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 similar for H 2S and occurs without O 2 consumption and likely generates S 0. When present, O 2 is reduced by Cu(I) or Fe(II) to produce H 2O 2, 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

120 Chapter 5 Investigating Volatile Sulfur Compound Precursors and Practical Applications 5.1 ABSTRACT The addition of Cu(II) to model systems containing H 2S 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 H 2S 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 (SO 2), ascorbic acid, and cysteine to promote the catalytic scission of diethyl disulfide (DEDS) was investigated. It was found that the combination of SO 2 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 H 2S and MeSH from red and white wines that were free of reductive faults at the time of addition. 103

121 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 H 2S and model thiols. It was found that the addition of Cu(II) oxidized thiols to disulfides and the presence of H 2S 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 H 2S 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 when O 2 ingress is limited. 47,48,186 There have been numerous studies suggesting that the most common VSCs responsible for post-bottling reductive aroma are H 2S, 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 sulfurcontaining amino acids. 71,178 Another well accepted hypothesis is the reduction of symmetrical disulfides of MeSH and ethanethiol (EtSH), which typically have 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

122 Recent work has suggested that upwards of 99% H 2S 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 O 2. Nonetheless, disulfides and polysulfanes are generated in the presence of H 2S and thiols in the initial Cu(II) fining process with no O 2 uptake. Subsequent oxidation of Cu(I)-SR upon O 2 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 offodors 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. 105

123 5.3 MATERIALS AND METHODS 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 H 2S) 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 Preparation of model wine and real wine samples Disulfide and polysulfane generation 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. Either glutathione (GSH, 500 µm) or cysteine (Cys, 500 µm) were added to model wine and mixed thoroughly. H 2S (250 µm) and/or MeSH (250 µm) were subsequently added to the 106

124 solutions to give a total of four treatments: (1) Cys+H 2S, (2) Cys+H 2S+MeSH, (3) GSH+H 2S, and (4) GSH+H 2S+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. H 2S 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 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. 107

125 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 O 2 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 (SO 2), 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 SO 2 AA Cu(II) Fe(II) T T mg/l - - T3-50 mg/l T mg/l 5 mg/l T5 12 mg/l mg/l 5 mg/l T 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) 108

126 and capped immediately based on previously described methodology in order to release metal-thiol complexes. 80 Experiments were conducted in duplicate Release and reduction of bound VSCs Initial experiments were conducted in either air saturated model wine (dissolved [O 2]: 7 8 mg/l) or in an anaerobic chamber (dissolved [O 2]: <100 µg/l). The model wine was spiked with a combination of H 2S (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. 109

127 5.3.3 Methods of analysis 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 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 110

128 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 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 Disulfide and polysulfane generation We showed that Cu(II) fining results in near immediate Cu(II) reduction along with oxidation of H 2S and thiols in Chapter 2 of this thesis. We subsequently showed that the oxidation 111

129 of 6-sulfanylhexan-1-ol (6SH) and H 2S 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 H 2S, 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 O 2. 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+H 2S. Similarly, the oxidation of the GSH+H 2S combination treatment resulted in GSH polysulfanes up to n=7 (Table 5.3). When MeSH was added along with H 2S, 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 H 2S is present, it results in the incorporation of sulfur to the disulfide, and results in generation of polysulfanes. 112

130 Table 5.2. Cys-polysulfanes identified by LC-QTOF after reacting Cys (500 µm) and H 2S (250 µm) with Fe(III) (100 µm) and Cu(II) (50 µm) in air saturated model wine. S (n) Molecular formula M+H monoisotopic mass Retention time (min) S/N ratio Intensity (ion count) 1 C 3H 7NO 2S ± C 6H 12N 2O 4S ± C 6H 12N 2O 4S ± C 6H 12N 2O 4S ± C 6H 12N 2O 4S ± C 6H 12N 2O 4S ± Table 5.3. GSH-polysulfanes identified by LC-QTOF after reacting GSH (500 µm) and H 2S (250 µm) with Fe(III) (100 µm) and Cu(II) (50 µm) in air saturated model wine. S (n) Molecular formula M+H monoisotopic mass Retention time (min) S/N ratio Intensity (ion count) 1 C 10H 17N 3O 6S ± , , , C 20H 32N 6O 12S ± , 1.49, , , , , C 20H 32N 6O 12S ± , , , C 20H 32N 6O 12S ± C 20H 32N 6O 12S ± C 20H 32N 6O 12S ± C 20H 32N 6O 12S ± Table 5.4. Mixed Cys-MeSH disulfide and polysulfanes identified by LC-QTOF after reacting Cys (500 µm), H 2S (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 mass Retention time (min) S/N ratio Intensity (ion count) 2 C 4H 9NO 2S ± C 4H 9NO 2S ± C 4H 9NO 2S ± C 4H 9NO 2S ± C 4H 9NO 2S ±

131 Table 5.5. Mixed GSH-MeSH disulfide and polysulfanes identified by LC-QTOF after reacting GSH (500 µm), H 2S (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 mass Retention time (min) S/N ratio Intensity (ion count) 2 C 11H 19N 3O 6S ± C 11H 19N 3O 6S ± C 11H 19N 3O 6S ± C 11H 19N 3O 6S ± C 11H 19N 3O 6S ± C 11H 19N 3O 6S ± C 11H 19N 3O 6S ± 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 O 2 plays an important role in the re-oxidation of the metals and in accepting electrons via metal catalysis, O 2 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 O 2 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 114

132 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 H 2S 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 H 2S 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 H 2S and MeSH by LC-QTOF. H 2S 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 Winemakers are advised to avoid and minimize O 2 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

133 O 2. The presence of O 2 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 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

134 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). 117

135 1 5 0 p H 2 C u ( I) ( M ) p H 3 p H 4 p H 5 p H 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 investigated, and it was found that low ph is associated with a lower generation of H 2S and MeSH. 71 The possibility that disulfides are cleaved at higher ph to generate H 2S 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). 118

136 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 T ± 3.0 Aa 42.1 ± 2.7 Aa 45.4 ± 0.0 Aa T ± 4.1 Aa 39.6 ± 2.4 Bab 41.2 ± 4.2 ABab T ± 2.9 Aa 36.4 ± 3.7 Bab 36.4 ± 0.6 Bbc T ± 2.9 Aa 36.4 ± 0.6 Bab 37.4 ± 0.0 Bbc T ± 1.1 Aa 35.7 ± 1.0 ABab 34.7 ± 1.5 Bbc T ± 0.8 Aa 35.1 ± 0.2 Bab 32.9 ± 0.7 Bc T ± 1.6 Aa 32.6 ± 1.0 Bb 24.4 ± 5.0 Cd T ± 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 119

137 (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 basecatalyzed 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

138 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 H 2S 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 thioldisulfide 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 The mechanism for ascorbate-mediated cleavage of the disulfide is 121

139 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

140 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 (H 2S, 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. 123

141 Table 5.8. Peak area for each corresponding compound after addition of treatments in air saturated model wine. H 2S MeSH EtSH Treatment Average peak area Recovery (%) Average peak area Recovery (%) Average peak area Recovery (%) control ± ± ± Cu(II) 0 ± ± ± 0 0 Cu+TCEP ± ± ± Cu+BCDA 0 ± ± ± 0 0 Cu+TCEP+BCDA ± ± ± Cu+TCEP+BCDA +Cys ± ± ± The addition of TCEP resulted in the release of ~70% of MeSH and EtSH, but was relatively ineffective in releasing H 2S (~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 H 2S 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

142 oxidation. In a separate experiment, large amounts of H 2S 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 H 2S as noted by smell, however, BCDA addition did not result in H 2S release. Addition of barium hydroxide to the solution after BCDA addition resulted in a fine white precipitate due to BaSO 4, suggesting that H 2S 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 H 2S. Even in the presence of excess reducing agents, it appears that O 2 interferes with the recovery of labile H 2S, and so the experiment was repeated under anaerobic conditions for H 2S (Table 5.9). Table 5.9. Peak area for H 2S after addition of treatments in anaerobic model wine. Treatment Average peak area Recovery (%) control ± Cu(II) 0 ± 0 0 Cu+TCEP 4348 ± Cu+BCDA 16.7 ± Cu+TCEP+BCDA ± Cu+TCEP+BCDA+Cys ± A markedly higher recovery was observed with TCEP in the absence of O 2, resulting in 68% recovery compared to ~2% in the presence of oxygen. For BCDA, virtually no H 2S 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 H 2S, giving a slight increase compared to TCEP alone. When Cys 125

143 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 H 2S 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 H 2S 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 H 2S (µg/l) MeSH (µg/l) H 2S (µg/l) MeSH (µg/l) H 2S (µg/l) MeSH (µg/l) control 2.50 ± ± ± ± 0.01 ND ND Cu+TCEP ± ± ± ± ± ± 0.05 BCDA Cu+TCEP+ BCDA+Cys ± ± ± ± ± ± 0.03 RW1 RW2 RW3 H 2S (µg/l) MeSH (µg/l) H 2S (µg/l) MeSH (µg/l) H 2S (µg/l) MeSH (µg/l) control ND ND ND ND 2.22 ± 0.01 ND Cu+TCEP ± ± ± ± ± ± 0 BCDA Cu+TCEP+ BCDA+Cys ± ± ± ± ± ± 0.09 EtSH was not detected in any of the samples before or after the addition of the reagents, however free H 2S and MeSH ranged from undetectable concentrations to 2.50 µg/l and 2.57 µg/l, respectively. In all cases, H 2S and MeSH were released in the wines above their reported threshold upon the addition of the test reagents. H 2S release ranged from 5.19 to µg/l, and MeSH ranged from 2.33 to 8.37 µg/l, with an outlier at µg/l. These concentrations were consistent with the study reported by Franco-Luesma and Ferreira, and were consistent with the fact that over 50% of MeSH and 90% of H 2S are bound. 80 It appears that the addition of Cys improved recovery 126

144 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 H 2S 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 127

145 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. 128

146 Chapter 6 Conclusions and Recommendations for Future Work 6.1 Summary In this dissertation, I examined the interaction of transition metals with H 2S 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 H 2S 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 H 2S and thiols, the oxidation of H 2S 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 H 2S 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 H 2S 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 H 2S and 6SH were oxidized in the presence of Fe and Cu, I was able to detect polysulfanes up to 5 linking sulfur atoms. 129

147 I had also investigated the effect of manganese at catalyzing thiol and H 2S 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 Cudriven 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 H 2S 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 (SO 2, 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 H 2S 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 Interaction of H 2S 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 H 2S in wine and beer, but whether it effects overall redox reactions in wine need to be further investigated. 130

148 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 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 H 2S 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 H 2S and 131

149 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 H 2S 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. 132

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174 Appendix A. Supplementary information for Chapter % m/z Figure A.1. Fragmentation pattern of Cys-bimane. 157

175 % m/z Figure A.2. Fragmentation pattern of sulfide-dibimane. 158

176 % Time Figure A.3. Chromatographic profile of combined MRM spectra. Rt 7.97 min Cys-bimane (m/z ); min sulfide-dibimane (m/z ); min 6SH-bimane (m/z ). 159

177 Appendix B: Supplementary information for Chapter n = 2 n = 3 % n = 4 n = 5 0 Time Figure B.1. HPLC chromatogram with detection at 210 nm showing organic polysulfanes (identified by MS) obtained from reaction of 6SH (300 µm and H 2S 100 µm) with Fe(III) (200 µm) and Cu(II) (50 µm). 160

178 n = 5 % % n = n = 3 % n = 2 % m/z Figure B.2. Fragmentation pattern of organic polysulfanes shown in Figure S

179 Figure B.3. ESI- mass spectrum of S 5-bimane obtained from reaction of H 2S (300 µm) with Fe(III) (200 µm) and Cu(II) (50 µm) followed by MBB derivatization. 162