reaction mechanisms of transition metals with …

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

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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

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ABSTRACT

Sulfidic off-odors due to hydrogen sulfide (H2S) and low molecular weight thiols are

commonly encountered in wine production. These odors are a serious quality issue in wine and

may result in consumer rejection. Therefore, sulfidic off-odors are generally controlled prior to

bottling, and are frequently removed by the process of Cu(II) fining – a process that remains poorly

understood. Cu(II) is effective at binding with sulfhydryl functionalities and forming nonvolatile

complexes thereby removing aroma associated with the compound. However, this technique leaves

residual copper in the wine which catalyzes non-enzymatic wine oxidations. Furthermore, elevated

copper concentrations are usually associated with increased sulfidic off-odors under anaerobic

aging conditions.

In this work, I elucidated the underlying mechanisms by which Cu(II) interacts with H2S

and thiol compounds under wine-like conditions. Adding Cu(II) sulfate to air saturated model wine

containing H2S, cysteine (Cys), 6-sulfanylhexan-1-ol (6SH), or 3-sulfanylhexan-1-ol (3SH) led to

a rapid formation of ~1.4:1 H2S:Cu and ~2:1 thiol:Cu complexes. This resulted in the oxidation of

H2S and thiols, and reduction of Cu(II) to Cu(I) without oxygen uptake. Both H2S and thiols

resulted in the formation of Cu(I)-SR complexes, and subsequent reactions with oxygen led to the

oxidation of H2S rather than the formation of insoluble copper sulfide, which has been previously

assumed. The proposed reaction mechanisms provide an insight into the extent to which H2S can

be selectively removed in the presence of thiols in wine.

The interaction of iron and copper is also known to play an important synergistic role in

mediating non-enzymatic wine oxidation. Therefore, I assessed the interaction of these two metals

in the oxidation of H2S and thiols (Cys, 6SH, and 3SH) under wine-like conditions. H2S and thiols

were shown to be slowly oxidized in the presence of Fe(III) alone, and were not bound to Fe(III)

under model wine conditions. However, Cu(II) added to model wine containing Fe(III) was quickly

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

Oxidation of Fe(II) in the presence of oxygen regenerated Fe(III) and completed the iron redox

cycle. This work clearly demonstrated a synergistic effect between Fe and Cu during the oxidation

of H2S and thiols. In addition, sulfur-derived oxidation products were observed, and the formation

of organic polysulfanes was demonstrated for the first time under wine-like conditions.

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

Furthermore, manganese is known to have a catalytic activity at mediating thiol and H2S oxidation

in aquatic systems. Thus, the interaction of manganese with iron and copper was investigated in

relation to thiol and H2S oxidation in model wine. The reaction of thiols with Mn alone or in

combination with Fe resulted in radical chain reaction paired with large oxygen uptake and

generation of sulfur oxyanions. H2S did not generate free thiyl radicals, and had minimal interaction

with Mn(II). When Cu(II) was introduced, Cu-mediated oxidation dominated in all treatments and

Mn-mediated radical reaction was limited. Mn demonstrated a different reaction mechanism with

thiols compared to Cu and Fe, and may generate transient thiyl radicals during wine oxidation.

Demonstrating that Cu(II) addition to model systems containing H2S and thiols resulted in

the generation of polysulfanes led to an investigation of the formation of mixed disulfides and

polysulfanes in model and white wine samples. I found that at relatively low concentrations of H2S

and methanethiol (MeSH, 100 µg/L each), Cu(II)-fining resulted in the generation of MeSH-

glutathione disulfide and trisulfane in white wine. The reduction of the resulting nonvolatile

disulfides may then play a role in the generation of undesirable sulfidic off-odors. Therefore,the

ability of Fe and Cu in combination of bisulfite (SO2), ascorbic acid, and Cys to promote the

catalytic scission of diethyl disulfide (DEDS). I found that the combination of SO2 along with Fe

and Cu depleted more DEDS than the other treatments. Furthermore, a method for releasing volatile

sulfur compounds from their precursors was investigated using tris(2-carboxyethyl)phosphine (a

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reducing agent) and bathocuproine disulfonic acid (a chelator). The addition of the reagents

successfully released H2S and MeSH from red and white wines that were free of reductive faults at

the time of addition.

I have demonstrated the underlying reaction mechanisms of H2S and thiols with Cu, Fe,

and Mn under wine-like conditions. I showed that Cu(II) was readily reduced by H2S and thiols,

and that this complex remained redox active and reduced oxygen. The reaction of Cu with H2S

and thiols is further accelerated by the presence of Fe and Mn. While the initial Cu(II) fining

process removed volatile sulfhydryl compounds, it generated disulfides, polysulfanes, and Cu(I)-

SR complexes that remain in the wine. I showed that disulfide scission is accelerated by the

presence of metals and reducing agents under wine conditions. Furthermore, I provided a strategy

to quickly reduce or dissociate disulfides, polysulfanes, and metal complexes for the release of

volatile sulfur compounds in both red and white wines. This can be used by winemakers to

predict a wine’s potential to exhibit sulfidic odors and take further action. Overall, a better

understanding of the underlying reaction mechanisms with H2S and thiols provided a foundation

for future strategies to better control sulfidic off-odors in wine.

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TABLE OF CONTENTS

LIST OF FIGURES .................................................................................................................... x

LIST OF TABLES .................................................................................................................... xv

ACKNOWLEDGEMENTS .................................................................................................... xvii

Chapter 1 Literature Review....................................................................................................... 1

1.1 Introduction .............................................................................................................. 1

1.2 Metal-catalyzed redox reactions ................................................................................ 6

1.2.1 Copper ......................................................................................................... 10

1.2.1.1 Copper fining ............................................................................................ 10

1.2.1.2 Redox cycling of copper ........................................................................ 11

1.2.2 Iron .............................................................................................................. 12

1.2.3 Manganese ................................................................................................... 14

1.2.4 Other transition metals ................................................................................. 15

1.2.5 Release of metal sulfide and metal thiol complexes ...................................... 16

1.3 Thiol/disulfide couple ............................................................................................. 18

1.3.1 Occurrence and oxidation of disulfides ......................................................... 18

1.3.2 Thiol-disulfide interchange ........................................................................... 21

1.3.3 Sulfitolysis ................................................................................................... 22

1.3.4 Metal catalyzed disulfide scission ........................................................................ 24

1.3.5 Ascorbic acid ............................................................................................... 26

1.4 Reactions of sulfhydryls with organic wine constituents .......................................... 28

1.5 Thioester hydrolysis................................................................................................ 29

1.6 Strecker degradation of amino acids ........................................................................ 30

1.7 Further reactions of sulfur containing compounds ................................................... 30

1.8 Research overview, significance, and hypotheses .................................................... 31

Chapter 2 Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model Wine.

Part 1: Copper Catalyzed Oxidation. ......................................................................................... 33

2.1 ABSTRACT ........................................................................................................... 33

2.2 INTRODUCTION .................................................................................................. 34

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2.3 MATERIALS AND METHODS ............................................................................ 36

2.3.1 Chemicals .................................................................................................... 36

2.3.2 Model wine experiments .............................................................................. 37

2.3.3 Determination of oxygen consumption ......................................................... 38

2.3.4 Cu-complex formation and dissolution ......................................................... 39

2.3.5 Spectrophotometric measurements of thiols and H2S .................................... 39

2.3.6 Spectrophotometric measurement of Cu(I)-BCDA ........................................ 39

2.3.7 HPLC analyses of thiols and H2S.................................................................. 40

2.3.8 HPLC analysis of catechols .......................................................................... 42

2.3.9 HPLC analysis of acetaldehyde .................................................................... 42

2.3.10 Copper determination ................................................................................. 42

2.3.11 EPR analysis .............................................................................................. 43

2.4 RESULTS .............................................................................................................. 43

2.5 DISCUSSION ........................................................................................................ 50

2.5.1 Cu reduction and complex formation ............................................................ 50

2.5.2 Disulfide formation ...................................................................................... 54

2.5.3 Oxidation of the Cu(I)-complex .................................................................... 56

2.6 Acknowledgments .................................................................................................. 61


Part 2: Iron and Copper Catalyzed Oxidation............................................................................. 62

3.1 ABSTRACT ........................................................................................................... 62

3.2 INTRODUCTION .................................................................................................. 63


3.3.1 Chemicals .................................................................................................... 66

3.3.2 Model Wine Experiments ............................................................................. 67


3.3.4 Spectrophotometric measurements ............................................................... 68

3.3.5 HPLC Analyses............................................................................................ 69

3.4 RESULTS AND DISCUSSION .............................................................................. 71

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

3.4.2 Fe(III) reduction by thiols and H2S ............................................................... 73

3.4.3 Fe(II) oxidation and oxygen consumption ..................................................... 74

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3.4.4 Fe(III) and Cu(II) reduction by thiols and H2S .............................................. 76

3.4.5 Fe(II)/Cu(I) oxidation, oxygen consumption, and acetaldehyde formation..... 79

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

......................................................................................................................................... 80

3.4.7 Formation of mixed organic polysulfanes ..................................................... 83


Part 3: Manganese Catalyzed Oxidation and Interaction with Iron and Copper........................... 85

4.1 ABSTRACT ........................................................................................................... 85

4.2 INTRODUCTION .................................................................................................. 85


4.3.1 Chemicals .................................................................................................... 87

4.3.2 Model Wine Experiments ............................................................................. 88


4.3.4 Spectrophotometric measurements ............................................................... 89

4.3.5 HPLC Analyses............................................................................................ 90

4.4 RESULTS AND DISCUSSION .............................................................................. 90

4.4.1 Reaction of Cys with Mn .............................................................................. 90

4.4.2 Reaction of Cys with Mn+Fe ........................................................................ 94

4.4.3 Reaction of Cys with Mn+Fe+Cu ................................................................. 95

4.4.4 Reaction of 6SH ........................................................................................... 96

4.4.5 Reaction of H2S............................................................................................ 99

4.5 CONCLUSIONS .................................................................................................. 101

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

5.1 ABSTRACT ......................................................................................................... 103

5.2 INTRODUCTION ................................................................................................ 104

5.3 MATERIALS AND METHODS .......................................................................... 106

5.3.1 Materials .................................................................................................... 106

5.3.2 Preparation of model wine and real wine samples ....................................... 106

5.3.2.1 Disulfide and polysulfane generation ................................................... 106

5.3.2.2 Disulfide scission by Cu(II) and bathocuproine disulfonic acid ............ 107

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

.................................................................................................................................... 108

5.3.2.4 Release and reduction of bound VSCs ................................................. 109

5.3.3 Methods of analysis.................................................................................... 110

5.3.3.1 HPLC .................................................................................................. 110

5.3.3.2 GC ...................................................................................................... 110

5.3.3.3 UV-Vis ............................................................................................... 111

5.4 RESULTS AND DISCUSSION ............................................................................ 111

5.4.1 Disulfide and polysulfane generation .......................................................... 111

5.4.2 Disulfide scission ....................................................................................... 116

5.4.3 Reactivity of diethyl disulfide..................................................................... 119

5.4.4 Predicting a wine’s ability to exhibit reductive off-odors ............................ 123

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

6.1 Summary .............................................................................................................. 129

6.2 Future Work ......................................................................................................... 130

6.2.1 Interaction of H2S and Thiols with Zinc ...................................................... 130

6.2.2 Interaction of reducing agents and disulfides .............................................. 131

6.2.3 Using alternative treatments to Cu(II) fining ............................................... 131

6.3 Concluding Remarks ............................................................................................ 131

REFERENCES ....................................................................................................................... 133

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

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

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

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

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

wine ........................................................................................................................................ 170

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LIST OF FIGURES

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

wine. ........................................................................................................................... 6

Figure 1.2. Oxidation of o-catechol to o-quinone in the presence of Fe(III) and

subsequent Michael type addition reaction of sulfhydryl to give a catechol-thiol adduct. ........................................................................................................................ 7

Figure 1.3. Proposed reaction mechanism of hydrogen peroxide thiols to generate

sulfenic acid (A) which subsequently reacts with thiol to generate disulfide (B). Bisulfite will react with hydrogen peroxide to generate sulfuric acid, which will exist

as sulfate in wine. ........................................................................................................ 19

Figure 1.4. (A) Generation of thiyl radical under wine conditions by a one electron oxidant and subsequent (B) dimerization to a disulfide, or (C) reaction with oxygen

to generate disulfide anion radical followed by (D) disproportionation to disulfide

and peroxyl radical. Alternatively, (E) the thiyl radical can be scavenged by a

catechol moiety. .......................................................................................................... 20

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

generation. .................................................................................................................. 21

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

Figure 1.7. Example of transition metal assisted thiol-disulfide interchange resulting in

the generation of a new Cu(I)-SR complex. ................................................................. 22

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

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

Figure 1.10. Reversible reactions of aldehydes with bisulfite in wine to generate

hydroxyalkylsulfonates or with thiols to generate hemithioacetals and thioacetals. ....... 29

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

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

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

(300 µM). Error bars indicate standard deviation of triplicate treatments. ..................... 44

Figure 2.4. Reaction of Cu(II) in (a) model wine and treatments containing (b) 3SH, (c)

6SH, (d) Cys, and (e) H2S, showing (A) loss of electron paramagnetic resonance (EPR) active Cu(II) (0.5 mM) signal in model wine after mixing with the respective

xi

thiol/H2S treatments (1.5 mM), and (B) UV-spectra of the thiols/H2S (300 μM) in

model wine after mixing with Cu(II) (50 μM). ............................................................. 45

Figure 2.5. (A) UV-Vis spectra over time of air saturated model wine after addition of

6SH (300 uM) and Cu(II) (50 uM) in model wine. Removal of the Cu(I) complex by filtration. (B) Cu concentration after filtration after having added 6SH, H2S, Cys

(300 µM) to Cu(II) (50 µM) and 3SH (300 µM) to Cu(II) (100 µM) at each

respective time point. Error bars indicate standard deviation of triplicate treatments. .... 46

Figure 2.6. Loss of H2S and Cys in air saturated model wine upon adding Cu(II) (100

µM) to H2S (~100 µM) in combination with Cys (~400 µM). Error bars indicate

standard deviation of triplicate treatments. ................................................................... 47

Figure 2.7. O2 and 6SH consumption, and 6SH-disulfide formation in air saturated model

wine containing 240 μM 6SH and 50 μM Cu(II). Error bars indicate standard

deviation of triplicate treatments. ................................................................................. 48

Figure 2.8. O2 consumption in air saturated model wine upon addition of Cu(II) (50 µM) to 6SH, H2S, and Cys (300 µM), and addition of Cu(II) (100 µM) to 3SH (300 µM).

Error bars indicate standard deviation of triplicate treatments. ...................................... 49

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

(300 µM). Error bars indicate standard deviation of triplicate treatments. ..................... 50

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

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

and DMPO. ................................................................................................................. 55

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

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

H2O2. ........................................................................................................................... 57

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

H2O. ............................................................................................................................ 58

Figure 2.15. One-electron reduction of H2O2 to produce hydroxyl radicals, and the

oxidation of ethanol by the Fenton reaction to form 1-hydroxyethyl radicals. 1-hydroxyethyl radicals are oxidized by oxygen and subsequently reduced by metals to

yield acetaldehyde. ...................................................................................................... 59

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

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Figure 3.2. Reduction of hydrogen peroxide to produce hydroxyl radicals by the Fenton

reaction and subsequent formation of the 1-hydroxyethyl radical. 1-hydroxyethyl

radical is further oxidized by oxygen or Fe(III) to eventually yield acetaldehyde. ......... 64

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

Figure 3.4. Reaction of H2S or thiols on addition of Fe(III) (200 µM) to 6SH, H2S, Cys,

or 3SH (300 µM) in air saturated model wine. (A) Consumption of H2S or thiols; (B) %Fe(III)-tartrate based on absorbance at 336 nm; (C) O2 consumption. Error bars

indicate standard deviation of triplicate treatments. ...................................................... 72

Figure 3.5. Reaction of H2S or thiols on addition of Fe(III) (200 µM) and Cu(II) (50 µM) to H2S, 6SH, 3SH (300 µM), and Fe(III) (100 µM) and Cu(II) (25 µM) to Cys (300

µM) to air saturated model wine. (A) %Fe(III)-tartrate based on absorbance at 336

nm; (B) Consumption of H2S or thiols; (C) O2 consumption; (D) AC generation.

Error bars indicate standard deviation of triplicate treatments. ...................................... 78

Figure 3.6. Proposed mechanism demonstrating initial Cu(II) reduction by thiols and H2S

to yield Cu(I)-SR complex and subsequent oxidation of the complex by Fe(III).

Fe(II) then reduces oxygen to hydrogen peroxide. Subsequent reaction of H2O2 is depicted in Figure 2. .................................................................................................... 78

Figure 3.7. Total thiol and H2S loss on addition of Fe(III) (200 µM) and Cu(II) (50 µM)

to (A) 6SH (300 µM) + H2S (100 µM); (B) 3SH (300 µM) + H2S (100 µM); (C) Cys (300 µM) + H2S (100 µM); (D) Fe(III) (100 µM) and Cu(II) (25 µM) to Cys (300

µM) + H2S (50 µM) to air saturated model wine. Error bars indicate standard

deviation of triplicate treatments. ................................................................................. 81

Figure 3.8. Total concentrations of Fe(III), Fe(II), O2 (consumed), thiol, and AC in Cys+H2S treatment containing low and high metal concentration. (A) Low Fe (100

µM) and Cu (25 µM), (B) High Fe (200 µM) and Cu (50 µM). Error bars indicate

standard deviation of triplicate treatments. ................................................................... 82

Figure 4.1. Fe(III) initiated sulfite oxidation and subsequent Mn-catalyzed radical chain

reaction resulting in sulfite oxidation and sulfate generation. ....................................... 86

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

H2O2. ........................................................................................................................... 87

Figure 4.3. Reaction of Cys (150 or 200 μM) with Mn(II) (100 μM), Fe(III) (100 μM),

and Cu(II) (25 μM) in air saturated model wine. (A) Cysteine consumption, (B) O2

consumption, (C) acetaldehyde generation, and (D) %Fe(III)-tartrate based on absorbance at 336 nm. Error bars indicate standard deviation of triplicate treatments. .. 91

Figure 4.4. Proposed mechanism of Mn(III)-catalyzed radical chain reactions of thiols in

air saturated model wine resulting in thiyl radical intermediates which subsequently oxygen and ethanol. ..................................................................................................... 93

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Figure 4.5: Reaction of 6SH (150 or 200 μM) with Mn(II) (100 μM), Fe(III) (100 μM),

and Cu(II) (25 μM) in air saturated model wine. (A) 6SH consumption, (B) O2

consumption, (C) acetaldehyde generation, and (D) %Fe(III)-tartrate based on

absorbance at 336 nm. Error bars indicate standard deviation of triplicate treatments. .. 97

Figure 4.6. Reaction of H2S (150 or 200 μM) with Mn(II) (100 μM), Fe(III) (100 μM),

and Cu(II) (25 μM) in air saturated model wine. (A) H2S consumption, (B) O2

consumption, (C) acetaldehyde generation, and (D) %Fe(III)-tartrate based on absorbance at 336 nm. Error bars indicate standard deviation of triplicate treatments. .. 100

Figure 5.1. Cu(I)-BCDA generation over time in the presence of cystine (400 µM),

Cu(II) (100 µM), and BCDA (1 mM) in air saturated model wine over different pH values. ......................................................................................................................... 118

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

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

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

Figure A.3. Chromatographic profile of combined MRM spectra. Rt 7.97 min – Cys-

bimane (m/z 310→223); 12.59 min – sulfide-dibimane (m/z 413→191); 13.63 min –

6SH-bimane (m/z 323→222). ...................................................................................... 159

Figure B.1. HPLC chromatogram with detection at 210 nm showing organic

polysulfanes (identified by MS) obtained from reaction of 6SH (300 µM and H2S

100 µM) with Fe(III) (200 µM) and Cu(II) (50 µM)..................................................... 160

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

Figure B.3. ESI- mass spectrum of S5-bimane obtained from reaction of H2S (300 µM)

with Fe(III) (200 µM) and Cu(II) (50 µM) followed by MBB derivatization. ............... 162

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

(bottom) Fe(III) and Mn(II). ........................................................................................ 163

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

hr. ............................................................................................................................... 164

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

Figure D.1. Identified Cys-polysulfanes by LC-QTOF after reacting Cys (500 µM) and H2S (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model

wine. The insert shows the maximum abundance based on percent of each given

mass. ........................................................................................................................... 166

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Figure D.2. Identified GSH-polysulfanes by LC-QTOF after reacting GSH (500 µM) and

H2S (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model

wine. The insert shows the maximum abundance based on percent of each given

mass. ........................................................................................................................... 167

Figure D.3. Identified mixed Cys-MeSH disulfide and polysulfanes by LC-QTOF after

reacting Cys (500 µM), H2S (250 µM), and MeSH (250 µM) with Fe(III) (100 µM)

and Cu(II) (50 µM) in air saturated model wine. The insert shows the maximum abundance based on percent of each given mass........................................................... 168

Figure D.4. Identified mixed GSH-MeSH disulfide and polysulfanes by LC-QTOF after

reacting GSH (500 µM), H2S (250 µM), and MeSH (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model wine. The insert shows the maximum

abundance based on percent of each given mass........................................................... 169

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LIST OF TABLES

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

Table 1.2. Occurrence and oxidation states of various sulfur species which may be

present in wine. ........................................................................................................... 4

Table 1.3. Experimental stability constants (log K) for metal sulfides at 25 °C in water

with ionic strength of 0.7 at pH 7. Values adapted from Ricard and Luther75 and sources within.82–85 ...................................................................................................... 8

Table 1.4. Calculated solubilities of metal sulfides at 25 °C, 1.013 atm total pressure, and

pH 7 in pure water. Values adapted from Ricard and Luther75 ...................................... 9

Table 1.5. Diagnostic test and sensory screening of sulfidic odors in wine utilizing

copper, cadmium, and ascorbic acid. ............................................................................ 27

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

Table 5.2. Cys-polysulfanes identified by LC-QTOF after reacting Cys (500 µM) and


wine. ........................................................................................................................... 113

Table 5.3. GSH-polysulfanes identified by LC-QTOF after reacting GSH (500 µM) and


wine. ........................................................................................................................... 113

Table 5.4. Mixed Cys-MeSH disulfide and polysulfanes identified by LC-QTOF after

reacting Cys (500 µM), H2S (250 µM), and MeSH (250 µM) with Fe(III) (100 µM)

and Cu(II) (50 µM) in air saturated model wine. .......................................................... 113

Table 5.5. Mixed GSH-MeSH disulfide and polysulfanes identified by LC-QTOF after

reacting GSH (500 µM), H2S (250 µM), and MeSH (250 µM) with Fe(III) (100 µM)

and Cu(II) (50 µM) in air saturated model wine. .......................................................... 114

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

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

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

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

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Table 5.10: Concentrations of H2S and MeSH in three PA white wines and three PA red

wines before and after addition of treatment reagents. None of the wines released

detectable amounts of EtSH before or after the kit was used. ........................................ 126

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

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

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ACKNOWLEDGEMENTS

I am very grateful to my advisor, Dr. Ryan Elias, for providing me the opportunity to

undertake this research project under his guidance. Ryan was supportive of my ideas and provided

me with the freedom to fully explore my research interests. I thank my committee members, Dr.

Josh Lambert, Dr. John Coupland, and Dr. Michela Centinari for their guidance. Their knowledge

on aspects outside of wine chemistry helped me realize a larger context to my work.

I am deeply indebted to Dr. John Danilewicz for continually guiding me throughout my

research project. John has been giving me stimulating suggestions and encouraged me throughout

my PhD. I greatly appreciate John’s feedback and I believe he helped tremendously in my growth

as a scientist.

I also want to thank Dr. David Jeffery for serving on my committee. Dave provided me

with the opportunity to work with him in Adelaide, which ultimately led to the conception of this

project. Dave’s expertise in wine chemistry and his critiques had greatly improved my

communication skills as a scientist.

I would like to thank the Department of Food Science for providing salary and tuition

support. I would also like to thank the Pennsylvania Wine Research and Marketing Board for

providing some funding support for this project.

I thank all my lab mates and classmates for being supportive of me. They have taught me

many valuable skills and helped me develop as a scientist. They have made my experiences here

much more enjoyable by being great friends socially and academically. My family and friends at

home have always provided love and support, and for that, I am eternally grateful.

1

Chapter 1

Literature Review

1.1 Introduction

Volatile sulfur containing compounds (VSCs) are a group of aroma compounds that have

a tremendous impact on the sensory quality of wine.1–4 Typically, VSCs have low odor detection

thresholds and, depending on their chemical structures, can have beneficial or detrimental effects

on the sensory quality of wine. In general, VSCs containing the sulfhydryl (-SH) functionality have

lower detection thresholds than other forms and are commonly responsible for sulfurous aromas in

wine. However, disulfides, thioethers, and thioesters have important contributions to overall wine

aroma as well.

Sulfur-containing compounds such as 3-sulfanylhexan-1-ol (3SH) and 4-methyl-4-

sulfanylpentan-2-one (4MSP) contribute to pleasant aromas in wine, such as grapefruit,

passionfruit, and blackcurrant.5–7 The yeast generates these compounds by cleaving 3SH and 4MSP

from odorless precursors in the must.8,9 These compounds are often referred to as varietal thiols as

they typify certain grape varieties (e.g. Sauvignon Blanc) and have aroma detection thresholds at

nanogram-per-liter concentrations (Table 1.1).7,10,11 On the other hand, fermentative VSCs such as

hydrogen sulfide (H2S), methanethiol (MeSH), and ethanethiol (EtSH) are considered defects as

they contribute to “reductive” sulfidic off-odors that are associated with rotten egg, sewage, and

burnt rubber (Table 1.1). The alcoholic fermentation process of juice or must to wine by the yeast

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

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

2

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

acid biosynthesis.17 The production of excess H2S depends on the fermentation and nutrition

conditions, as well as yeast strain, and can lead to the formation of other VSCs such as MeSH and

EtSH17–21 as well as dimethylsulfide (DMS) and dimethyl disulfide (DMDS), which are reminiscent

of rotten cabbage or canned vegetables.1,22,21 Wine yeast can also form thioacetates by enzymatic

action.17,23 These VSCs have relatively low detection thresholds (i.e. low microgram-per-liter)

(Table 1.1), and have a negative effect on wine quality.1,24–28 DMS may positively impact the

bouquet of the wine at subthreshold concentrations, although this is generally not the case.1,29 In

depth examination of the flavor impact of VSCs in wines, associated aromas, and detection

thresholds are outside of the scope of this review, and have been thoroughly reviewed

elsewhere.1,6,22

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

Compound Odor descriptor Odor detection threshold

Hydrogen sulfide Rotten egg 1.1 – 1.6 µg/L30

Methanethiol Cabbage, sewage 1.8 – 3.1 µg/L31

Ethanethiol Onion, rubber, fecal 1.1 µg/L27

Dimethylsulfide Cabbage, asparagus, corn, blackcurrant

25 µg/L27

Dimethyldisulfide Cooked cabbage, sulfurous, onion 29 µg/L27

Diethyldisulfide Onion, garlic, rubber 4.3 µg/L27 Methylthioacetate Sulfurous, cheesy 50 µg/L32

Ethylthioacetate Cabbage, cauliflower 10 µg/L32

4-Methyl-4-sulfanylpentan-2-

one

Box tree, guava, cat urine 3.3 ng/L33

3-Sulfanylhexan-1-ol Passionfruit, grapefruit 60 ng/L5

Many of the sulfur compounds occurring in wine due to viticultural practices and

subsequent yeast fermentation remain redox-active in wine during aging, where they are able to

participate in one- and two-electron transfer, radical processes, and exchange reactions. Many of

these compounds, particularly species containing sulfhydryl moieties, can also bind to metals and

result in a range of metal complexes that are commonly found in biological and geochemical

3

systems.34,35 Indeed, sulfur plays an important in vivo role in redox systems that is critical for all

organisms (e.g. plants, bacteria, fungi, yeast).35,36 As such, the presence of these various sulfur

compounds in wine is a combination of overall grape and yeast metabolism. The major changes

occurring during grape maturation and grape juice/must fermentation are due to enzymatic

processes that have been (and remain) the focus of much research with the ultimate goal of

predicting and improving wine quality.4,37 However, once a finished wine is bottled, enzymatic

action ceases yet subsequent non-enzymatic chemical reactions may result in nuanced aroma

changes over time.

Many non-enzymatic wine oxidation reactions in wine occur due to oxygen, and can result

in loss of pleasant fruity aromas containing sulfhydryl functionality (e.g. 3SH and 4MSP)38 and the

generation of various undesirable aldehydes that derive from ethanol, organic acids, and sugars in

wine.39 To avoid excessive wine oxidation, modern winemakers take great care to minimize oxygen

exposure throughout the winemaking process.40 Unfortunately, the increasing use of reductive

winemaking (i.e. minimizing O2 exposure) and use of low oxygen transmission rate (OTR) closures

in recent years has made post-bottling generation of sulfidic off-odors more common. The

generation of H2S and MeSH above their odor detection threshold in wine may occur when O2 is

limited and can result in consumer rejection of the wine.37,41 It appears that an intricate balance of

O2 ingress through the wine’s packaging system (e.g., its closure) is needed to prevent wine

spoilage due to either oxidation or reduction; however, no model currently exists that can accurately

predict what such an O2 balance should be based on a given wine’s chemical composition, its

closure type, the environmental conditions to which it is exposed, and its time in-bottle.

Sulfur-containing compounds can possess various oxidation states and can remain redox

active in wine. These species can have either reducing or oxidizing capacity which is influenced by

factors such as the overall redox state of the wine, dissolved O2 concentration, and the presence of

4

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

Oxidation State

Occurrence Reactivity

Sulfhydryl H2S, RSH -2 Grapes and yeast

metabolism

Reducing agent

Thiyl radical -1 Transient Reducing or oxidizing, can

dimerize to RSSR

Perthiol RSSH -1 Reduction of

polysulfanes

Strongly reducing

Disulfide RSSR -1 Naturally present,

oxidation of RSH

Mild oxidant, can be further

oxidized

Organic polysulfanes

RSSnSR -1,0,-1 Oxidation of RSH and H2S

Mildly oxidizing

Elemental sulfur S8 0 Pesticide residue,

oxidation of H2S

Very weak oxidant, can be

reduced by RSH Sulfenic acid RSOH 0 Transient Condenses to disulfide

Sulfinic acid RSO2H +2 Oxidation product of

RSH

Adds to quinones

Sulfonic acid RSO3H +4 Oxidation product of RSH

Unreactive

Sulfite HSO3- +4 Yeast byproduct,

winemaking additions

Reducing agent, antioxidant

Sulfate SO42- +6 Sulfite oxidation,

yeast and grapes

Unreactive

Thiosulfate RSSO3- -1,+4 Sulfitolysis of

disulfides43,44

Hydrolyze to sulfate and

free thiol

Thiosulfinate

+1,-1 Unknown Oxidizing

Thiosulfonate

+3, -1 Unknown Oxidizing

Sulfinylsulfone

+3, +1 Unknown Oxidizing

5

Disulfone

+3, +3 Unknown Oxidizing

Thioethers,

dialkylsulfides

RSR -2 Dimethylsulfide,

thioesters, etc.

Sulfoxide

+2 dimethylsulfoxide45

Sulfone

+4 dimethylsulfone46

Metal sulfides MnSn varies Various complexes with first row

transition metals

Reducing, oxidizing, or inert

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

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

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

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

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

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

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

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

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

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

redox chemistry underlying the reactions between these sulfhydrdryl compounds and transition

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

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

proposed relevance of previous research on sulfur chemistry within physiological and

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

6

1.2. Metal-catalyzed redox reactions

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

conditions, O2 is reduced to H2O 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 O2 ingress.57 O2 is stable in its triplet ground state (i.e., 3O2) and its direct

reaction with organic compounds (singlet state) is spin forbidden; however, O2 can be reduced by

transition metals prior to its reaction with wine constituents. It has recently been argued that Fe(II)

and Cu(I) can mediate the concerted reduction of O2 to H2O2 without the release of hydroperoxyl

radicals or oxidation of catechols (Figure 1.1).55,58 Once H2O2 is generated it may undergo

reduction via Fenton reaction involving Fe(II) (or other reduced metals) to generate hydroxyl

radicals (HO·).51,59 The highly reactive hydroxyl radical reacts at diffusion limiting rates with

organic compounds in proportion to their concentrion. As ethanol is the most abudant organic

species in wine (ca. 2 M), it has been shown to be the most likely target of hydroxyl radicals in

wine. This reaction results in ethanol oxidation and the formation of the intermediate 1-

hydroxyethyl radical (1-HER) which can subsequently be oxidized to acetaldehyde.59–61

Figure 1.1. Proposed reaction mechanism of Fe(II) with oxygen to produce hydrogen peroxide,

followed by Fenton reaction and oxidation of ethanol to acetaldehyde in wine.

During the O2 reduction process, transition metals are oxidized and can subsequently

oxidize polyphenols or sulfhydryls. The quinones that result from polyphenol oxidation can

7

undergo Michael-type addition reaction with sulfhydryls, resulting in another mechanism for the

loss of aroma through binding of the sulfhydryl functionality (Figure 1.2).62–64 The presence of

transitions metals is needed to drive this reaction forward,65 and it has been shown that the presence

of nucleophiles, such as sulfhydryls, can drastically increases the rate of reaction as it drives the

reaction forwards.54,66 It appears that the relationship between sulfhydryls and O2 is facilitated by

redox cycling of transition metals (especially Fe and Cu), but some studies indicate that radical

intermediates, such as 1-HER, may react directly with thiols.67,68

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

Clearly transition metals play a critical role in mediating wine oxidation, and many

oxidation intermediates may result in loss of sulfhydryl compounds. Ribéreau-Gayon showed that

the rate of oxidation could be slowed and eventually stopped in wine by the removal of iron and

copper with potassium ferrocyanide.69 This was more recently confirmed in another study by

Danilewicz and Wallbridge.65

On the other hand, in the absence of O2, VSCs that contribute to reductive sulfidic odors

can accumulate, particularly in the presence of transition metals.48,50,70,71 The formation of sulfidic

odors is attributed to H2S and MeSH, but the mechanism for their formation and involvement of

transition metals remains poorly understood.

In addition to their redox cycling capability, transition metals and sulfhydryls are also

capable of forming ionic bonds. This is especially important in the case of H2S, which can react

with transition metals, and upon further rearrangment, may result in crystal structure formation and

8

subsequent mineral precipitation.72,73 The ability of sulfhydryls to both dissociate bulk minerals and

generate metal-sulfide structures has been heavily studied in geochemical processes.34,74–78 Some

of these metal sulfide structures are relatively inert, wheras others remain redox active and can

effectively behave as aqueous species.73 It is relatively well known that the majority of sulfide (over

90%) in bodies of water is complexed to copper, iron, and zinc.79 The importance of these

complexes in the context of wine chemistry remains poorly understood, but has piqued interest in

recent years.80,81

The stability constants for metal sulfide complexes of wine relevant transition metals are

reported in Table 1.3. Generally speaking, the larger the stability constant, the more likely it is for

the transition metal to bind with H2S, and potentially with thiol compounds too. These values are

reported for sea water conditions but this information may still be applicable to wine. For example,

log K values for Cu(I), Cu(II), and Zn(II) are higher than Fe(II) and Mn(II), and this is consistent

with recent studies in wine showing Cu and Zn species correlate with H2S concentrations moreso

than Fe and Mn.70,80

Table 1.3. Experimental stability constants (log K) for metal sulfides at 25 °C in water with ionic

strength of 0.7 at pH 7. Values adapted from Ricard and Luther75 and sources within.82–85

*Cu(II) likely reduced to Cu(I) to some extent during analysis.

Metal Complex Log K

Mn(II) [MnHS]+ 4.5 Fe(II) [FeHS]+ 5.4

Co(II) [CoHS]+ 5.5

Ni(II) [NiHS]+ 5.0 Cu(II)* [CuHS]+ 6.5

[CuS]0 11.2

Cu(I) [CuHS]0 12.1

Zn(II) [ZnHS]+ 6.1 [ZnS]0 11.7

Ag(I) [AgHS]0 11.2

[AgS]- 22.8 Au(I) [AuHS]0 24.5

9

Furthermore, the solubilities of the metal sulfides are reported in Table 1.4. These values

are calculated for pure water and may give an indication of the general solubilities of some metal

sulfides under wine conditions. For example, as can be seen from this table, CuS and ZnS are

predicted to be considerably less soluble than FeS and MnS. However, there are limitations to this

table as it does not consider other wine constituents (e.g. organic acids, polyphenols, thiols) which

may limit the formation of metal sulfide solids. Futhermore, metastable metal sulfide clusters may

be kinetically significant in wine and have higher solubilities compared to their more stable solid

forms.75 The misconception that the comlexes are virtually insoluble is especially important in

copper fining, where CuS is reported to have an exceedingly low solubility, yet is not readily

formed in wine. This is discussed further in Section 1.2.1.1.

Table 1.4. Calculated solubilities of metal sulfides at 25 °C, 1.013 atm total pressure, and pH 7 in

pure water. Values adapted from Ricard and Luther75

Metal sulfide Solubility (mg/L)

MnS 6×100 FeS 6×10-2

CoS 5×10-3

NiS 2×10-5

CuS 3×10-14 ZnS 8×10-9

AgS 2×10-14

AuS 2×10-27

The importance of transition metals in wine with respect to the loss and formation of

sulfhydryl compounds is two-fold. One is the ability of the metals to redox cycle sulfur, and the

other is forming ionic bonds and corresponding metal sulfides and metal thiol complexes. Catalytic

oxidation of organic thiols by O2 in the presence of metals was investigated in borate-phosphate

buffer at a wide range (pH 2 – 13) where it was found to follow the trend of Cu > Mn > Fe > Ni >>

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

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

10

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

wine-relevant pH.

The nature of redox reactions and ionic bonding under wine conditions remains poorly

understood; however, it is critical to understand these reactions in order to better control and predict

sulfhydryl compound loss and regeneration in wine. The importance of some first row transition

metals and their relevance to wine is elaborated in the following sections.

1.2.1 Copper

Cu is naturally present in grapes, and Cu based fungicide treatments in the vineyard may

cause carryover into the juice;87 however, the concentration of Cu is known to decrease during

fermentation due to Cu adsorption and removal by yeast cells.88,89 The major source of Cu in

finished, packaged wine is the intentional addition of Cu salts during the process known as Cu

fining. The legal limits globally for Cu in finished wine generally vary between 0.5 – 1 mg/L, but

may be as high as 10 mg/L.90

1.2.1.1 Copper fining

The accumulation of sulfidic off-odors is a common problem in wine production, and the

addition of Cu(II) salts for their removal has been used as a standard procedure in winemaking for

many decades.2,41,90 Sulfidic off-odors are typically attributed to H2S (and thiols such as MeSH)

and it is generally assumed that reacting Cu with H2S would result in formation and complete

precipitation (and removal) of CuS, due to its low solubility product (3×10-14 mg/L, Table 1.4).

However, it has been noted that this precipitate is not always formed and that tartaric acid might

inhibit the aggregation of CuS.71,90,91 A recent study Clark et al. demonstrated the practical difficulty

11

of removing CuS from wine, even with filtration.91 In fresh and saltwater it has been shown that

the reaction of H2S with Cu results in CuS nanoclusters that effectively behave as soluble species.

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

chemically inert.72

It has been suggested that other agents, such as nonvolatile thiols, could interfere with

precipitation during the fining process by competing for Cu(II).55,56,91 For example, the average

combined concentration of cysteine (Cys), N-acetylcysteine and homocysteine was reported to be

ca. 20 µM in a survey of white wines, while the average concentration of glutathione (GSH) was

reported to be ca. 40 µM in wines made from Sauvignon blanc grapes.92–95 These nonvolatile thiols

would be in large molar excess to the exogenous Cu (3–6 µM) used in a fining operation, and would

far exceed the concentration of H2S (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.

1.2.1.2 Redox cycling of copper

Trace concentrations of Cu are now known to act synergistically with Fe in mediating non-

enzymatic wine oxidation reactions, particularly by accelerating oxygen consumption and

polyphenol oxidation.52 As described above, polyphenol oxidation generates quinones which may

12

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

1.2).38,64,96–98 Furthermore, the importance of Cu(II) in bridging reactions involving catechin with

glyoxylic acid with a quinone intermediate has been demonstrated.99

Surprisingly, limited research has been conducted under wine conditions that focuses on

the direct interaction of Cu with sulfhydryl compounds. When H2S, MeSH, and EtSH were oxidized

in model brandy by Cu(II), the formation of mixed disulfides and trisulfanes was observed.100

Recent work by Franco-Luesma and Ferreira found that virtually all H2S is bound when Cu(II) is

added, forming an inert Cu(II)S complex that remains in solution and is resistant to aerial

oxidation.80,81,101 However, biologically relevant thiols have been shown to readily reduce Cu(II) to

Cu(I) with their concomitant oxidation to disulfides at pH 7.4.102,103 Similarly, under

biogeochemical conditions, H2S reduces Cu(II) to Cu(I) during Cu3S3 ring formation, and these

species remain in solution as polynuclear nanoclusters72. The relevance of these reactions and their

redox activity is thoroughly investigated in Chapter 2.

1.2.2 Iron

Fe has been focused on heavily by wine chemists because it mediates many wine oxidation

reactions involving oxygen, polyphenols, and sulfite (Figures 1.1 and 1.2). The overall rate of non-

enzymatic wine oxidation is highly dependent on the reduction potential of the Fe(III)/Fe(II)

couple, which is lowered by tartaric acid.51,58,59,104 The lower the reduction potential, the greater the

reducing power; therefore, if the reduction potential of the Fe(III)/Fe(II) couple is low, O2 will be

reduced to H2O2 more readily. A relatively low Fe(III)/Fe(II) reduction potential will also facilitate

the reduction of H2O2 to hydroxyl radicals via the Fenton reaction (Figure 1.1). When Fe(II) is

oxidized, the Fe(III) formed is quickly reduced back to Fe(II) in the presence of sulfite and

phenolics, both which are abundant in wine.59 Fe speciation in wine has been examined and it has

13

been suggested that the majority of free Fe is present as Fe(II),59,105 although Fe remains bound to

the organic fraction of wine such as tartrate106 and tannins.107 Fe(II) is the major species of Fe in

wine due to wine’s low pH and abundance of phenolics, which has been recently confirmed in a

variety of wines.108

Although Fe has been shown to play an important role in the generation of reactive

intermediates that are subsequently capable of reacting with sulfhydryl compounds in wine, the

amount of research that focuses on the direct reaction of Fe with sulfhydryl compounds is sparse.

It has been proposed that the oxidation of thiols by Fe(III) may be radical-mediated with the

generation of disulfides.54 Studies performed with GSH in a range of pH conditions (3-7) have

shown that Fe(II) is spontaneously produced when GSH is added to Fe(III).109,110 The same has

been shown with Cys at low pH, as the Fe(III)-Cys complex is unstable and quickly reacts to yield

Fe(II) and cystine.111 After the reduction of Fe(III) to Fe(II), GSH and Cys appear to be coordinated

with the carboxylate group under wine’s acidic conditions (pH<4), and not the sulfhydryl group.

109,110 Therefore, under wine conditions it is unlikely that the sulfhydryl compounds remain bound

to Fe(II) due to competition by excess tartaric acid as the dominant ligand, which is addressed

directly in Chapter 3. H2S may behave differently than thiols and remain bound to Fe(II) to some

degree. It has been shown that Fe(II) can form a complex with H2S, and FeS does not exhibit odors

associated with H2S.80,101 The binding of H2S is likely to form subunits of Fe2S2 similar to

mackinawite structure, however, under acidic conditions it does not appear to be sufficiently stable

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

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 H2S, is very high.97 Although Fe(III)-catalyzed

14

oxidation of suldhydryls is possible,54 it is unlikely this reaction will occur to a considerable degree

relative to other chemical reactions (e.g. Figure 1.2) that may occur under real wine conditions.

1.2.3 Manganese

Mn is typically present in wines at concentrations that are comparable to Fe,114 and has

been suggested to play an important role in non-enzymatic wine oxidation. Cacho et al. showed

that Mn, along with Fe, affected the rate of non-enzymatic oxidation in white wine.115 The presence

of Mn resulted in elevated acetaldehyde concentrations, suggesting the ability of Mn to catalyze

Fenton-like reactions in wine (Figure 1.1).115 The exact mechanism for reaction of Mn in wine

conditions remains poorly understood, but it may behave in a similar manner to Fe.

Recent work has investigated the Mn(II)-mediated oxidation of polyphenols and sulfite in

wine. The Mn(III)/Mn(II) couple has a high reduction potential and is difficult to redox cycle under

wine conditions. However, once Mn(III) is formed, presumably due to interaction with Fe-superoxo

complex, it is capable of oxidizing wine constituents.116 In a system without polyphenols, Mn(III)

has been shown to initiate radical chain reaction with sulfites.116

Based on work in non-wine model systems, it would appear that sulfhydryls are more

susceptible to oxidation by Mn than Fe.86 It was recently reported that Mn was responsible for the

oxidative degradation of MeSH.117 Mn(III) may be more selective towards sulfhydryl compared to

other wine constituents, and promote their oxidation. This mechanism is investigated further in

Chapter 4.

15

1.2.4 Other transition metals

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

may be present at comparable concentrations to Cu in wine.114 Zn has been shown to effect H2S

and MeSH concentrations in beer and wine.70,80,118 However, unlike the other metals described

above, Zn(II) does not redox cycle and is unlikely to have an effect on rate of oxidation reactions

in wine, but needs to be investigated further. Nonetheless, Zn(II) binding with H2S is comparable

to Cu, as it has a high stability constant (1×10-13) and low solubility (8×10-9 mg/L).34 Similarly to

Cu, it forms a Zn3S3 ring structure that further condenses to Zn4S6 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 H2S, and formation of a relatively stable complex that effectively renders

the sulfhydryl group unavailable for reaction (or volatilization).

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

beer.80,118 Furthermore, the generation of H2S 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 H2S production,

which may not necessarily be due to post-bottling chemical reactions,81 but rather that low Zn

concentrations resulted in sluggish fermentations which generated more H2S in the wine prior to

bottling.120 Therefore higher Zn concentrations may result in lower H2S production during

fermentation, but this needs to be investigated further.

Other first row transition metals including chromium, cobalt, and nickel are less understood

under wine conditions. While they have catalytic abilities and binding affinities with sulfhydryls,

these metals are generally present at concentrations far below 0.1 mg/L. Due to their low natural

abundance they may be of lesser importance compared to the transition metals discussed above.

16

1.2.5 Release of metal sulfide and metal thiol complexes

Transition metal catalyzed wine oxidation has been fairly well studied in recent years. As

described above, elevated concentrations of any transition metals cause a decrease in sulfhydryl

concentration in the presence of O2. Although the mechanisms by which these metals promote wine

oxidation have been elucidated to varying degrees, the most abundant oxidation products arising

from metal-catalyzed reactions are disulfides, catechol-thiol adducts, and metal complexes. The

reduction and dissociation of these compounds has been hypothesized to generate sulfidic off-odors

due to H2S and MeSH, especially when O2 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

H2S 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 CuCl32- and CuCl4

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 H2S and MeSH, respectively, are effectively bound to the metals under wine

conditions.80,101

Of the first row transition metals present in wine, Cu is the one that binds most strongly to

sulfhydryls (Table 1.3). Perhaps counterintuitively though, elevated Cu concentrations in a finished

wine are associated with higher generation of H2S and MeSH. The formation of soluble CuS

nanoclusters is likely a major contributing factor for the subsequent release of H2S and MeSH.

Zn(II) reacts in a similar fashion to Cu and is also important for binding of H2S. Fe(II) has been

shown to have some ability at binding to H2S, although as described above (section 1.2.2), it forms

17

a different metal sulfide complex likely consisting of Fe2S2. The binding of H2S and MeSH correlate

with the stability constants of the corresponding metal sulfides (Table 1.3).

Given that metal sulfides are non-volatile and therefore odorless, a wine may appear free

of faults until the complexes dissociate. Further research is needed to understand what drives these

dissociation reactions, but it is clear that anaerobic conditions are the key driving force for the

dissociation and release of H2S and MeSH. Studies in which H2S release was monitored in wine

have indicated that during an anoxic 18 month aging period of a wine, free H2S increased with time

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

compounds may reduce the CuS complex to release free H2S 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 H2S and MeSH release could be attributed to the dissociation

of metal sulfide complexes, it has been shown that up to 42% and 76% of H2S and MeSH,

respectively, are generated due to de novo formation.81 There are several hypotheses for the

generation mechanisms of these sulfidic compounds, and these are discussed in depth in the

following sections.

18

1.3 Thiol/disulfide couple

In general, reduced sulfur species (with S2-, Table 1.2) have considerably lower detection

thresholds than their corresponding oxidized species, and thus have a greater impact on overall

wine aroma. Several of these oxidized species including disulfides (S1-), elemental sulfur (S0),

sulfoxides (S2+) and sulfite (S4+), are naturally occurring and are present post-fermentation in wine,

and their chemical reduction post-bottling can result in the appearance of undesirable sulfidic off-

odors in wine previously deemed to be free of apparent faults.

Winemakers are advised to avoid aerating their wines or utilizing Cu fining in the presence

of O2 as it may result in the generation of disulfides that can be subsequently reduced, thus

adversely affecting wine quality.43,124,125 The implication of disulfides on wine reduction has been

commonly referred to and accepted in enology text books. However, the generation of symmetrical

disulfides from MeSH and EtSH (that is, DMDS and DEDS, respectively) are rarely observed, if

ever, post-fermentation.49,126–128 In general, the majority of disulfides are formed during yeast

metabolism21,129 although there is some evidence for the generation of disulfides and polysulfanes

under wine and model wine conditions during Cu(II) addition and subsequent aging.55,100,130

1.3.1 Occurrence and oxidation of disulfides

Sulfhydryls cannot be directly oxidized by O2 due to Pauli’s exclusion principle and require

transition metals to facilitate oxidation reactions. They can however, be oxidized by two-electron

oxidants such as H2O2 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 H2O2 is relatively slow under wine conditions and will likely be

19

outcompeted by sulfite to form sulfate (Figure 1.3C).133 As such, the oxidation of thiols by H2O2

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

radicals67,68 (Figure 1.4B) or reaction of thiyl radical with a thiol to form the disulfide anion radical,

which further reacts with oxygen to yield a disulfide and peroxyl radical (Figures 1.4C and

1.4D).54,131,134 However, wine contains an excess of polyphenolics containing the catechol and

galloyl moieties that will quickly scavenge the thiyl radical (Figure 1.4E).67 Alternatively, the thiyl

radical may further react with α,β-unsaturated side chains.135

20

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 H2S, MeSH, and EtSH

would be present at concentrations below 100 nM, whereas Cys and its analogues may be present

at concentrations up to 0.1 mM. Therefore, it is likely that mixed disulfides and polysulfanes with

S-containing amino acids would be generated rather than DMDS and DEDS. These effectively non-

volatile disulfides may result in release of H2S, MeSH, and EtSH upon their reduction during anoxic

storage. In the presence of H2S, oxidation of H2S and thiols may result in the insertion of sulfur

into disulfides and subsequent formation of polysulfanes. In model solutions containing 20%

ethanol, H2S was shown to react with MeSH and EtSH in the presence of Cu(II) to form mixed di-

and trisulfanes.100 It has been suggested that this is formed with the generation of a perthiol (RSSH)

intermediate followed by oxidation in the presence of a thiols to generate the trisulfane (RSSSR).100

21

Alternatively, H2S is oxidized to elemental sulfur followed by its insertion into the disulfide to

generate the trisulfane.136

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

1.3.2 Thiol-disulfide interchange

Thiol-disulfide interchange reactions are biologically important, and have been studied

extensively as they are responsible for intracellular redox homeostasis, and play a critical roles in

antioxidant defense and redox regulation of cell signaling in vivo.137 These interchange reactions

involve a nucleophilic substitution of a free thiol with a thiol from the disulfide. The reaction

follows a one-step SN2 mechanism with a trisulfide-like transition state complex and delocalized

negative charge (Figure 1.6).131,138–141

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

In the above describe reaction, the thiolate anion serves as a nucleophile because it is a

stronger nucleophile than its corresponding thiol. The nucelophilicty of a thiol is inversely

dependent upon its pKa, and these reactions typically proceed at or above physiological pH. The

pKa of cysteine’s and glutathione’s respective thiol groups are ca. ~8-9, whereas simpler thiols are

closer to 10.142 However, due to the linear-free energy relationship, increasing pKa is directly

correlated with thiol nucleophlicity.131

If the interchange reaction were to proceed in wine, DMDS or DEDS would potentially

undergo thiol-disulfide interchange with the abundant concentrations of Cys (or its analogs) and

22

GSH, which would generate a mixed disulfide and release of EtSH and MeSH. While the pKa is

higher for EtSH and MeSH, they make a better leaving group due to their higher linear free energy.

Furthermore, concentrations may play a role in driving the reaction,139 and Cys and GSH are present

in molar excess compared to DMDS and DEDS. However, given the pH of wine is well below the

pKa of thiols, the unassisted reaction is prohibitively slow.

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

Recent work has shown that phosphine Au(I) thiolate complexes accelerated thiol-disulfide

interchange reactions.143 Although phosphine is a strongly electron withdrawing group, a similar

pathway may occur by Cu(I) or Zn(II) thiolate complex. Because of the abundance of transition

metals in wine, these reactions, and their potential relevance to wine thiol phenomena, should be

the topic of future research.

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

1.3.3 Sulfitolysis

Sulfitolysis works in a similar manner to thiol-disulfide interchange wherein sulfite

substitutes one of the thiols of a disulfide and forms an organic thiosulfate, also known as Bunte

salt (Figure 1.8).144 The organic thiosulfate may then undergo acid-catalyzed scission over time to

yield the other thiol that was present in the original disulfide. This reaction was initially proposed

by Bobet et al. to be feasible under wine conditions.43 However, results from their study indicate

that the release of EtSH to reach above threshold concentrations would require over 2 years with

30 mg/L free SO2 and 50 µg/L DEDS.

23

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 SO32- form would dominate, and like thiol-disulfide interchange this reaction

appears to be driven by higher pH. The reaction comes to completion in a matter of hours at pH

7.2, but would take years to detect any differences at pH 3.5.43 In contrast, the acid-catalyzed

cleavage of the thiosulfate would be expected to be much faster at wine pH compared to the initial

bisulfite substitution (Figure 1.8).

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

GSH disulfide and cystine (i.e., the disulfide of cysteine).44 However, unlike the slow sulfitolysis

of DEDS, GSH disulfide was shown to react with sulfite to generate detectable concentrations of

free GSH and GSH S-sulfonate in a matter of hours. Furthermore, GSH disulfide was not detectable

in wines, but GSH S-sulfonate was detectable, which would suggest that the acid-catalyzed

hydrolysis of GSH S-sulfonate is not as fast as the initial sulfite substitution.

Due to its higher pKa, 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 H2S, MeSH, and

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

24

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

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

could drive such reactions.

1.3.4 Metal catalyzed disulfide scission

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

(Sections 1.3.2 and 1.3.3). This reaction may proceed because of the metal’s ability to catalyze

electrophilic and nucleophilic reactions of the disulfide bond (Figure 1.9).144 The binding of an

electrophilic species (e.g. oxidized metals) makes one sulfur on the disulfide a better leaving group,

facilitating its subsequent displacement by nucleophilic attack of the other sulfur moiety.144 This

may be sufficient in cleaving the disulfide in the presence of wine nucleophiles including bisulfite,

ascorbic acid, and perhaps polyphenolic compounds. A reduced metal can also bind to a thiol, as is

the case with Cu(I)-SR, effectively making the thiol more nucleophilic (Figure 1.7). This will be

more prevalent if the metal is simultaneously bound to an electron withdrawing group.143 Cobalt

has been implicated in metal-assisted nucleophilic cleavage of disulfides.145

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

It appears that metals may play a role in both oxidative and reductive cleavage of disulfides,

consistent with studies investigating DMDS and DEDS in wine that have demonstrated that

concentrations of the disulfides decrease over time regardless of anaerobic or aerobic

25

conditions.49,117 It is likely that both reductive and oxidative cleavage mechanisms could occur, but

would depend on the redox status of the wine.

In a study investigating disulfide bonds in wheat proteins, the combination of Mn and Cu-

containing proteins (Cu(I) in particular) was found to be responsible for the reduction of the

disulfide bond.146 In hydro(solvo)thermal conditions, the addition of transition metals including

Cu(II), Cu(I), Ni(II), Co(II), and Mn(II) to a disulfide resulted in the generation of multiple reaction

products including the corresponding free thiols, trisulfides, and even new thiols, and generally

with the corresponding metal-sulfur cluster coordination.147–150 Although these reactions are

generally carried out under extreme conditions, they have been shown to also occur at room

temperature.145,151 In some experiments, the cleavage of cystamine in the presence of Cu(II) was

nearly instantaneous with water as the nucleophile.152,153

In general, the reactions described above are base-catalyzed, as the anionic form of water,

thiols, and sulfite are much stronger nucleophiles that drive the reaction forward. However, the

combination of both metal-assisted electrophilic and metal-assisted nucleophilic reactions may

drastically accelerate the rates, which would be faster than the predicted year-long disulfide scission

under simple model wine conditions.43

The interaction of polysulfanes may further drive metal-catalyzed scission reactions

forward. The binding energy generally increases as the S-chain gets longer, and the maximum

coordination number also increases corresponding with the number of S-atoms.154 Therefore, the

interaction of polysulfanes with transition metals and possible release of H2S may be significant.

The release of H2S 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.

26

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.

27

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

sulfidic off-odors

Odor gone Odor gone Odor gone H2S

Odor gone No change Odor gone Thiols

Odor gone Slight

improvement

Odor gone H2S and thiols

No change No change Odor gone Disulfides

No change No change No change Dimethyl

sulfide

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

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

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

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

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

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

form.156

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

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

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

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

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

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

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

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

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

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

28

Ascorbic acid also efficiently scavenges O2 by accelerating its reduction, and it promotes

the anoxic conditions in bottled wine which are generally associated with release of VSCs. It is also

possible that ascorbic acid plays a role in reducing metal sulfide complexes. Further studies should

be conducted to decipher the mechanism of VSC generation as it relates to ascorbic acid.

1.4. Reactions of sulfhydryls with organic wine constituents

The reaction of sulfhydryls with organic compounds in wine results in C-S bond formation,

and depending on the compound, may create a new aroma-active compounds or become non-

volatile and therefore eliminate the odor. Sulfhydryl compounds are nucleophilic species,

especially H2S, and may react with electrophilic compounds in either reversible or non-reversible

reactions. Wine contains a host of electrophilic compounds for such reactions, including quinones

and aldehydes.

There is abundant research in wine showing the formation of catechol-thiol adducts during

the wine oxidation process.62–64 These are formed by the reaction of thiol and quinone via a

Michael-type addition reaction, as shown in Figure 1.2. Given that the catechol-thiol adduct is non-

volatile, it effectively causes loss of aroma associated with the compound. The reaction is

reversible, but whether this can be driven backward remains poorly understood. Preliminary results

involving the H2S adduct of 4-methylcatechol (4-methyl-5-sulfanylcatechol) demonstrated that the

release of H2S occurs at pH 6 in the presence of reducing agents.155 Given that catechol-H2S adducts

can exist in equilibrium with the catechol and H2S, it is possible that reducing conditions would

result in H2S when O2 is limited.

It is well known that sulfite can react reversibly with aldehydes, forming a strong covalent

bond (Figure 1.10).166,167 Reaction of sulfhydryls with aldehydes may also occur, resulting in

29

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 H2S may result in its reaction with multiple aldehydes.171

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

Wines contain abundant amounts of hydroxycinnamic acids bearing the electrophilic α,β-

unsaturated carboxylic side chain, and their reversible reactions with sulfhydryls may be relevant

in wine. Bouzanquet et al. have demonstrated an irreversible GSH-hydroxycinnamic acid product

under wine conditions which involve free radicals.135 Another group investigated the reaction of

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

which may later decompose in the dough.172 The equilibrium of H2S and thiols with the

hydroxycinnamic acids may exist under wine conditions, but would need to be investigated further.

1.5 Thioester hydrolysis

Thioacetates are present in wine and are primarily generated by yeast during primary

alcoholic fermentation. The formation of thioacetates is thermodynamically unfavorable and

therefore unlikely to form without enzymatic action. However, thioesters can be hydrolyzed to their

corresponding thiols at low pH, and given the lower detection threshold of thiols released, this may

30

have a significant impact on a wine’s aroma.173 The thioacetates of MeSH and EtSH have been

observed in wines, and their hydrolysis could be an explanation for their release, however, there

have been no studies showing conclusive evidence for their cleavage. On the other hand, thiol-

thioester exchange may also have implications with respect to the generation of VSCs;174 for

example, sulfite may react with methyl thioacetate to generate the corresponding sulfonate, with

the release of MeSH.

1.6 Strecker degradation of amino acids

Strecker degradation of amino acids is known to occur in the presence of a dicarbonyl

compound. It was first suggested that an o-quinone can play this role in tea leaves,175 and has since

been shown to occur in synthetic solution and model wine.176,177 It has been demonstrated that Cys

can generate H2S, and formation of MeSH from methional and methionine was also reported under

wine-like conditions.178 Recent work supports the idea that methionine is one of the most important

precursors for the formation of MeSH post-fermentation.117 These reactions are non-reversible, and

transition metals play an important role in generating the o-quinone as the starting reactant for

Strecker degradation compounds.

1.7 Further reactions of sulfur containing compounds

There are likely numerous yet-to-be identified sulfur-containing compounds in wine that

may further contribute to wine aroma. Oxidation of MeSH in the presence of H2S may yield potent

polysulfanes, dimethyl trisulfane and tetrasulfane, which have detection thresholds of 100 ng/L and

60 ng/L, respectively.1,179 Reaction of H2S with benzaldehyde generates benzyl mercaptan, which

has a smoky odor,180 whereas reaction with furfural generates furfurylthiol that is reminiscent of

31

roasted coffee.181 In food systems other than wine, sulfur compounds with extremely low threshold

have been identified; for example, (S)-1-p-menthene-8-thiol (grapefruit mercaptan) has an odor

threshold of 6.6×10-6 ng/L in air. Furthermore, modification of grapefruit mercaptan structure by

changing the location of the sulfur atom resulted in unique odors described as sulfury, rubber-like,

burned, soapy, and mushroom-like.182 Some of these compounds would generally be considered as

defects in food and beverages. The occurrence of sulfur compounds may be specific for certain

wine styles, and the contribution of unidentified compounds may be important in explaining the

phenomenon of ‘reduction’ of certain wines.

1.8 Research overview, significance, and hypotheses

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

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

Because wine is an important agricultural commodity, wine quality and long shelf life are crucial

for consumers. The generation of reductive sulfidic off-odors is not an uncommon fault in wines,

reportedly accounting for 25% of faults in wine shows.184 The presence of sulfidic off-odors in

wine can adversely affect sales and brand image with consumers.

The overall aim of this thesis is to elucidate some key mechanisms that govern the redox

cycling of sulfhydryl compounds in the presence of transition metals in wine. VSCs are amongst

the most important aroma compounds in wine, as they can either contribute pleasant varietal aromas

or deleterious sulfidic off-odors, depending on their structures. I hypothesize that the decline of

these compounds in wine is linked to oxidation reactions mediated by transition metals.

Furthermore, I hypothesize that the reappearance of unwanted sulfidic off-odors is linked to the

reduction of disulfides, polysulfanes, and metal sulfide complexes, which is also mediated by

transition metals.

32

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

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

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

copper, iron, and manganese

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

compounds

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

perspective

33

Chapter 2

Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model

Wine. Part 1: Copper Catalyzed Oxidation.

Published as:

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

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

Chem. 2016, 64, 4095-4104.

2.1 ABSTRACT

Sulfidic off-odors due to hydrogen sulfide (H2S) and low molecular weight thiols are commonly

encountered in wine production. These odors are usually removed by the process of Cu(II) fining

– a process that remains poorly understood. The present study aims to elucidate the underlying

mechanisms by which Cu(II) interacts with H2S and thiol compounds (RSH) under wine-like

conditions. Copper complex formation was monitored along with H2S, thiol, oxygen, and

acetaldehyde concentrations after addition of Cu(II) (50 or 100 μM) to air saturated model wine

solutions containing H2S, cysteine, 6-sulfanylhexan-1-ol, or 3-sulfanylhexan-1-ol (300 μM each).

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

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

Cu(I) which reacted with oxygen. H2S was observed to initially oxidize rather than form insoluble

copper sulfide. The proposed reaction mechanisms provide an insight into the extent to which H2S

can be selectively removed in the presence of thiols in wine.

34

2.2 INTRODUCTION

Volatile sulfur containing compounds (VSCs) have a major impact on the sensory quality

of wine.1–3 Typically, VSCs have exceedingly low aroma detection thresholds (i.e., μg/L to ng/L)

and, depending on their structure, can have beneficial or deleterious effects with respect to

consumer acceptance. Grape-derived varietal thiols, such as 3-sulfanylhexan-1-ol (3SH), 3-

sulfanylhexyl acetate (3SHA), and 4-methyl-4-sulfanypentan-2-one (4MSP), contribute pleasant

aromas (e.g., grapefruit, passionfruit, and blackcurrant).5–7 On the other hand, the production of

fermentation-related VSCs, such as H2S, methanethiol (MeSH), and ethanethiol (EtSH), can result

in the development of undesirable odors, often described as rotten egg, putrefaction, sewage and

burnt rubber, that are obviously detrimental to wine quality.1,41,185 These odors are generally most

evident at low oxygen concentrations and are described to be sulfidic off-odors. Wines that display

such odors are described as having reductive character.

The accumulation of sulfidic off-odors is a common problem for winemakers and is usually

remedied by splash racking in order to volatilize and/or oxidize VSCs or, classically, by the use of

copper fining.2,41,90 In this latter practice, Cu(II) is added as its sulfate or citrate salt whereby it is

assumed to remove H2S 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,

35

while the average concentration of glutathione (GSH) is reported to be ca. 40 µM in wines made

from Sauvignon blanc.92–95 These nonvolatile thiols would be in large molar excess to the

exogenous copper (3–6 µM) used in a fining operation, and would far exceed the concentration of

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

After bottling, the concentration of sulfidic off-odors can increase, especially under

reductive conditions when oxygen exposure is limited such as when screw cap closures are

used.47,48,186 Although the causative mechanism remains unclear, wine appears to contain precursors

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

of Cys has been previously reported,178 while some have suggested that H2S may be formed by the

direct reduction of sulfate or sulfite.47 It has also been shown that thiols can be reversibly bound by

iron and copper,80,81 and that wines containing higher copper concentrations can accumulate sulfidic

off-odors during bottle aging.48,70 While transition metals are known to be essential for catalyzing

oxidation reactions in wine,51 Cu, Fe, Mn, Zn, and Al have more recently been shown to

synergistically affect the evolution of VSCs under anaerobic storage conditions.70

In order to understand how wines develop sulfidic off-odors during storage, it is essential

to understand how H2S and thiols react in the presence of oxygen and transition metals prior to

bottling. The identification of reaction products may then allow potentially troublesome precursors

36

to be targeted. Recent studies in this area have advanced our general mechanistic understanding of

iron-catalyzed wine oxidation; however, the role of copper remains poorly understood. The goal of

this present study is to determine the underlying mechanism of Cu-catalyzed H2S and thiol

oxidation under wine conditions.

2.3 MATERIALS AND METHODS

2.3.1 Chemicals

4-Methylcatechol (4-MeC), L-cysteine (Cys), monobromobimane (MBB), 5,5-

dimethyl-1-pyrroline N-oxide (DMPO), bathocuproinedisulfonic acid (BCDA) disodium

salt, 6-sulfanylhexan-1-ol (6SH), and diethylenetriaminepentaacetic acid (DTPA) were

obtained from Sigma-Aldrich (St. Louis, MO). 2,4-Dinitrophenylhydrazine (DNPH) was

purchased from MCB laboratory chemicals (Norwood, OH) and L-tartaric acid, 3SH, and

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

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

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

(as a source of H2S) was purchased from Acros Organics (Geel, Belgium). Water was

purified through a Millipore Q-Plus system (Milipore Corp., Bedford, MA). All other

chemicals and solvents were of analytical or HPLC grade, and solutions were prepared

volumetrically, with the balance made up with Milli-Q water unless specified otherwise.

37

2.3.2 Model wine experiments

Model wine was prepared by dissolving tartaric acid (5 g/L) in water, followed by the

addition of ethanol to yield a final concentration of 12% v/v. The solution was adjusted to pH 3.6

with sodium hydroxide (10 M) and brought to volume with water. For H2S and Cys, an aqueous

stock solution of each (0.5 M) was freshly prepared, whereas 6SH and 3SH were added directly by

syringe during experimentation (Figure 2.2). An aqueous stock solution of Cu(II) sulfate (0.1 M)

was prepared freshly. In certain experiments, 4-MeC (1 mM) was added prior to the addition of

H2S and thiol compounds, and Cu(II). H2S, Cys, 6SH, or 3SH were added to air saturated model

wine (1 L, 300 μM) followed by thorough mixing. Cu(II) was added to H2S, Cys, and 6SH (50 μM)

or 3SH (100 μM) and thoroughly mixed. For mixed H2S and Cys system, H2S (100 µM) and Cys

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

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

Oxygen Demand (B.O.D.) bottles (Wheaton, Millville, NJ), allowing the solution to overflow, and

bottles were capped immediately with ground glass stoppers, thereby eliminating headspace. The

glass reservoir of the B.O.D. bottles was topped off with water daily. The bottles were stored in the

dark at ambient temperature. One B.O.D. bottle was sacrificed per time point per replicate and used

for further analyses. All experiments were conducted in triplicate and had their own series of

sacrificial bottles.

Figure 2.2. H2S and thiols used throughout this study.

38

For experiments focusing on 6SH-disulfide formation, one experiment was

prepared as described above and followed over time. For additional experiments for

deciphering immediate disulfide generation, model wine (3 mL) containing 6SH (600 μM)

in a glass test tube was deoxygenated for 2 min under argon with stirring. After sparging,

Cu(II) was added at varying concentrations (50, 100, or 200 µM) under argon and reacted

with stirring for 5 minutes. The solution was then immediately analyzed to determine 6SH

and 6SH-disulfide concentrations (described below). In experiments involving 4-MeC or

DMPO, these compounds were dissolved directly into model wine to achieve a final

concentration of 1 mM prior to addition of Cu(II) (100 µM).

2.3.3 Determination of oxygen consumption

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

LLC, Zublon, NC) were filled with air saturated model wine for a minimum of 2 hours to allow the

oxidots to equilibrate. One B.O.D. bottle was used as a model wine control (i.e., did not contain a

treatment) and two other bottles were used as technical duplicates to determine oxygen

concentration for each treatment replicate (3 treatment replicates total). Thus, immediately after the

addition of Cu(II) solution, the model wine used for equilibration was discarded and the respective

treatment solution was instantly transferred into the bottles. Oxygen readings were taken per time

point using NomaSense O2 P6000 meter (Nomacorc LLC, Zublon, NC), and data were normalized

to the model wine reference sample. Starting oxygen concentrations were approximately 7 mg/L

(~220 µM) in all solutions.

39

2.3.4 Cu-complex formation and dissolution

6SH-Cu(I) complex was prepared by adding Cu(II) (100 µM) to model wine (1 L)

containing 6SH (400 µM). The immediately formed precipitate was vacuum filtered with a 0.45

µm nylon membrane (Wheaton, Millville, NJ), washed with water followed by ethyl acetate in

order to remove residual disulfide, and dried under vacuum. In an anaerobic chamber (95% Ar, 5%

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

This mixture was stirred for approximately 30 min until all of the solid dissolved. 6SH, 6SH-

disulfide, and Cu(I) concentrations were measured as described below.

2.3.5 Spectrophotometric measurements of thiols and H2S

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

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

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

(DTNB).187 An aliquot of sample (100 μL) diluted with model wine (900 μL) was treated with a

solution of DTNB (400 μL, 2 mM) in phosphate buffer (10 mM, pH 7.0) followed by addition of

TRIS-phosphate buffer (100 μL, 1 M, pH 8.1). The mixture was left at ambient temperature for 30

min before the absorbance was measured at 412 nm against a blank consisting of model wine,

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

2.3.6 Spectrophotometric measurement of Cu(I)-BCDA

Cu(I) concentration was analyzed using the BCDA assay.188 Treatment and standard

solutions consisted of excess Cys (5 mM) to ensure Cu(I) remained in its reduced state. An external

40

standard curve of the Cu(I)-BCDA complex was prepared in model wine, and absorbance values

were recorded at 484 nm against a model wine blank.

2.3.7 HPLC analyses of thiols and H2S

MBB derivatization was used to determine each H2S and Cys concentrations in the mixed

system based on a modification of a previous method.189 MBB reagent (40 mM) was prepared

anaerobically by dissolving the solid in acetonitrile. Aliquots of the reagent were stored at -80 °C.

Briefly, a sample aliquot (70 μL) was mixed with an equal volume of TRIS-HCl buffer (100 mM)

containing DTPA (0.1 mM) at pH 9.5, followed by the immediate addition of MBB (10 μL; 40

mM). The reaction was allowed to proceed aerobically at room temperature in the dark for 30 min

before the addition of sulfuric acid (50 μL, 200 mM) and 6SH-bimane internal standard (50 μL).

6SH-bimane was prepared following a sulfide-dibimane synthesis described previously.189 Samples

were filtered through PTFE syringe tip filters (0.45 μm, 13 mm filter diameter; AcrodiscTM, Ann

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

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

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

(Milford, MA) that was operated with MassLynx software. Bimane adducts were separated

on a ZORBAX Eclipse Plus C18 column (2.1 x 150 mm, 5 μm) with a guard column of the

same material at a flow rate of 0.2 mL/min with mobile phases consisting of 0.1% v/v

formic acid (A) and 0.1% v/v formic acid in acetonitrile (B) and a linear gradient according

to the following program: 0 min, 2% B; 9 min, 50% B; 14 min, 100% B; 18 min, 100% B;

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

41

Detection of bimane adducts was performed using negative ion electrospray ionization

(ESI-) with multiple reaction monitoring (MRM) (Figures A.1-A.3). The ESI capillary spray

voltage was set to 4 kV, the sample cone voltage was set to 25 V, and the source temperature was

120 °C. The desolvation gas flow was 450 L/h and collision energy was set to 20 eV. The mass

transition of sulfide-dibimane was monitored at m/z 413→191, cysteine-bimane was monitored at

m/z 310→223, and the internal standard 6SH-bimane was monitored at m/z 323.2→222.2. An

external standard curve was prepared for sulfide-dibimane and Cys-bimane and data were

normalized to the 6SH-bimane internal standard.

For experiments involving 6SH and its disulfide, quantitative analysis was

performed using the HPLC system described above and UV detection at 210 nm with

external standard calibration curves. Separation was achieved at a flow rate of 0.2 mL/min

with mobile phases consisting of 0.1% v/v formic acid (A) and 0.1% v/v formic acid in

acetonitrile (B) and a linear gradient according to the following program: 0 min, 5% B; 20

min, 95% B; 28 min, 95% B; 28.1 min, 5% B; 38 min, 5% B.

For experiments involving dissolution of 6SH-Cu complex with BCDA, the same

chromatographic conditions described for 6SH and its disulfide were followed. However,

the BCDA peak could not be resolved from that of 6SH at 210 nm, therefore detection of

6SH was performed using ESI+ with selective ion monitoring (SIM) at m/z 135 with an

external calibration curve. The ESI capillary spray voltage was set at 4 kV, the sample cone

voltage was set to 25 V and the source temperature was 120 °C. The desolvation gas flow

was 650 L/h.

42

2.3.8 HPLC analysis of catechols

For experiments containing 4-MeC, quantitative analysis was performed with the HPLC

system described above and UV detection at 280 nm with an external standard calibration curve.

4-MeC was separated on an Ultra Aromax column (2.1 x 150 mm, 5 μm) with a guard column of

the same material at a flow rate of 0.2 mL/min with mobile phases consisting of 0.1% v/v formic

acid (A) and 0.1% v/v formic acid in acetonitrile (B) and a linear gradient according to the following

program: 0 min, 30% B; 3 min, 30% B; 12 min, 100% B; 20 min, 100% B; 20.1 min, 30% B; 25

min, 30% B. The putative formation of oxidation products including catechol-thiol adducts and

condensed units was monitored both at 280 nm and with negative ion ESI-MS (total ion

chromatogram m/z 100-1000).

2.3.9 HPLC analysis of acetaldehyde

Acetaldehyde was measured in model wine treatment solutions as its 2,4-

dinitrophenylhydrazone (DNPH) derivative by HPLC as described previously67 with the following

modification: the sample was centrifuged at 15000 × g at 4 °C for 10 min. The supernatant was

then transferred to an HPLC vial for further analysis.

2.3.10 Copper determination

For each given time point, samples were mixed in B.O.D. bottles and then filtered through a 0.45

um PTFE syringe filter. The resulting filtrate (5 mL) was digested by the addition of 30% hydrogen

peroxide (3 mL) and sulfuric acid (100 μL) based on modification of previous reported

methodology.190 The samples were heated in a convection oven at 110 °C overnight before being

43

reconstituted to 5 mL with 0.1 M nitric acid. Samples were analyzed by inductively coupled plasma

optical emission spectroscopy (Agilent 700 Series, Santa Clara, CA) using a vertically aligned torch

and with monitoring at 324.7 nm.

2.3.11 EPR analysis

Loss of the electron paramagnetic resonance (EPR) signal for active Cu(II) (0.5 mM) in

model wine was monitored after the metal solution was mixed with the respective H2S and thiol

treatments (1.5 mM). Samples were transferred to a cuvette and snap frozen in liquid nitrogen.

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

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

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

Instruments, Abingdon, UK). Data acquisition and control of experimental parameters were

performed using the EWWIN 2012 software package. Instrument settings were as follows:

temperature, 100 K; microwave power, 2 mW; modulation frequency, 9480 MHz; modulation

amplitude, 20 dB; scan range, 2000 G.

2.4 RESULTS

The reactivity of Cu(II) with H2S, which is the primary target of Cu fining, and the

following three thiols was investigated under wine conditions (Figure 2.2): (1) Cys, which also

represented homo-Cys and Cys derivatives, (2) 6SH to represent primary thiols, and (3) 3SH to

represent secondary thiols. With H2S Cu(II) addition resulted in an immediate uptake of ~1.4 (72

µM) mole equivalents of H2S, the remainder was then fully consumed within 72 h. However, with

the thiols, the immediate uptake increased to approximately two equivalents (Figure 2.3), with

44

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/H2S by Ellman’s assay in air saturated model wine upon addition of Cu(II)

(50 µM) to 6SH, H2S, Cys (300 µM) and Cu(II) (100 µM) to 3SH (300 µM). Error bars indicate

standard deviation of triplicate treatments.

EPR analysis showed that Cu(II) was immediately reduced to Cu(I) due to loss of

paramagnetic Cu(II) signal by Cys, 6SH and H2S; again, 3SH reacted more slowly (Figure 2.4A),

with Cu(II) reduction being complete after 2 h (data not shown). The apparent formation of a Cu(I)

complex was observed by UV spectroscopy (Figure 2.4B). Absorbance increased markedly from

200-400 nm by the addition of H2S and Cys to model wine containing Cu(II), but did not produce

a distinct absorbance maximum above 220 nm. In contrast, 6SH showed a maximum at 353 nm,

and 3SH had absorbance maxima at 282 and 311 nm (Figure 2.4B).

45

Figure 2.4. Reaction of Cu(II) in (a) model wine and treatments containing (b) 3SH, (c) 6SH, (d)

Cys, and (e) H2S, showing (A) loss of electron paramagnetic resonance (EPR) active Cu(II) (0.5

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

The addition of Cu(II) to H2S in model wine resulted in a clear golden colored solution that

yielded a green/black precipitate over time, whereas a haze that developed with the three thiol

treatments (Cys, 6SH, 3SH) aggregated to form a fine white/yellow precipitate. This was

particularly evident for 6SH, as essentially all the Cu(I) complex was removed by filtration (0.45

µm) from 5 to 45 min after mixing (Figure 2.5A). Filtration at earlier time points and measurement

of residual copper remaining in solution confirmed that the 6SH aggregate formed rapidly and

could be removed from solution by filtration after 5 min (Figure 2.5B). However, at the last time

point, copper had been released from the insoluble Cu(I) complex in a copper form that could not

be removed by a 0.45 µm filter. 3SH reacted in the same manner, but more slowly. For the H2S

treatment, ca. 60% of the copper was removed by filtration within 5 min and up to 24 h. After 72

h, there was a green-black precipitate. Approximately 90% of copper was then removed from

solution (Figure 2.5B).

46

Figure 2.5. (A) UV-Vis spectra over time of air saturated model wine after addition of 6SH (300

uM) and Cu(II) (50 uM) in model wine. Removal of the Cu(I) complex by filtration. (B) Cu

concentration after filtration after having added 6SH, H2S, Cys (300 µM) to Cu(II) (50 µM) and 3SH (300 µM) to Cu(II) (100 µM) at each respective time point. Error bars indicate standard

deviation of triplicate treatments.

The aggregate initially formed from the reaction between Cu(II) and 6SH on drying gave

a fine powder, which was solubilized in water containing BCDA (a Cu(I) selective chelator188). The

insoluble Cu(I)-complex dissolved as BCDA displaced the thiolate ligand, yielding 1.17 ± 0.02

mM Cu(I), as determined by UV spectrophotometry, and 1.17 ± 0.13 mM 6SH was released, as

determined by HPLC-MS, giving a ~1:1 Cu(I):6SH molar ratio with minimal disulfide formation

(data not shown).

When H2S (75 µM) and Cys (468 µM) were added together to model wine in the presence

of Cu(II), ca. 53 and 135 µM of H2S and Cys, respectively, were consumed within 5 min (Figure

2.6). Together this gives 189 µM of sulfhydryl compounds consumed with added 100 µM Cu(II)

which translates to a ~2:1 binding ratio of H2S + Cys:Cu(II). Subsequent reaction resulted in

complete loss of H2S within 40 min and Cys after 48 h. While a visible precipitate was observed at

the end of the reaction (74 h), it was not observed to the same extent as was the case with H2S

alone.

47

Figure 2.6. Loss of H2S and Cys in air saturated model wine upon adding Cu(II) (100 µM) to H2S

(~100 µM) in combination with Cys (~400 µM). Error bars indicate standard deviation of triplicate treatments.

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

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

3.6 µM, respectively (data not shown). In addition, the oxidation of 6SH (240 μM), in the presence

of 50 µM Cu(II) was monitored over time in air saturated model wine (Figure 2.7). After 262 h,

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

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

~1:3.3.

48

Figure 2.7. O2 and 6SH consumption, and 6SH-disulfide formation in air saturated model wine

containing 240 μM 6SH and 50 μM Cu(II). Error bars indicate standard deviation of triplicate

treatments.

To further examine the mechanism of disulfide formation using 6SH as a model, an attempt

was made to intercept potential intermediate thiyl radicals with the o-quinone-producing 4-MeC,

and the radical trap DMPO. However, no change in disulfide formation was observed by HPLC

upon addition of Cu(II) (100 µM) to model wine containing 6SH (600 µM) and 4-MeC or DMPO

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

Oxygen consumption was also measured in model wines containing the H2S and thiol

treatments, as well as a combination treatment consisting of Cys+H2S (Figure 2.8). Minimal O2

uptake (<5 µM in all treatments) was observed within the first 30 min of the reaction. During the

course of the experiments, H2S had the highest O2 consumption (175 ± 9 µM), followed by 6SH

and Cys, which showed similar O2 consumption patterns (76 ± 6 and 66 ± 6 µM, respectively), and

lastly 3SH, which consumed the least O2 (23 ± 1 µM). The treatment containing both Cys and H2S

resulted in an O2 consumption of 117 ± 5.2 µM. Separately H2S or Cys were oxidized in the

49

presence of Cu(II) and excess 4-MeC and monitored over time. The rate of O2 consumption was

not effeceted by the presence of the catechol, and its concentration did not decrease over time.

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

shown).

Figure 2.8. O2 consumption in air saturated model wine upon addition of Cu(II) (50 µM) to 6SH,

H2S, and Cys (300 µM), and addition of Cu(II) (100 µM) to 3SH (300 µM). Error bars indicate standard deviation of triplicate treatments.

Complementing the range of measurements described above, acetaldehyde (AC)

generation was monitored over time (Figure 2.9). At the end of the experiment, the H2S containing

system had accumulated the highest concentration of AC (79 ± 2 µM), followed by 6SH with 52 ±

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

concentration of 54 ± 3 µM.

50

Figure 2.9. Acetaldehyde produced in air saturated model wine upon addition of Cu(II) (50 µM)

to 6SH, H2S, and Cys (300 µM), and addition of Cu(II) (100 µM) to 3SH (300 µM). Error bars

indicate standard deviation of triplicate treatments.

2.5 DISCUSSION

2.5.1 Cu reduction and complex formation

From the above results, it is proposed that when a thiol is added to Cu(II), Cu(II)

coordinates with two thiol moieties to give product (1, Figure 2.10). Electron transfer from sulfur

gives the Cu(I) intermediate, two of which associate to (2) allowing bond formation between the

two sulfur atoms to form the disulfide bound to Cu(I) (3), without release of free thiyl radicals. The

released Cu(I)-complex then associates to give the sparingly soluble aggregate (4). H2S is proposed

to react similarly with the formation of an initial complex, which could be Cu3S3, as discussed

below.

51

Figure 2.10. Proposed mechanism for initial reaction of thiols with Cu(II) and Cu(I)-thiol complex

formation. Only the thiol ligands are shown.

The initial binding of H2S and thiols to Cu(II) (Figure 2.3), therefore, appears to coincide

with the reduction of Cu(II) to Cu(I) as seen by the rapid loss of the cupric species’ paramagnetic

signal (Figure 2.4A). Of note is that with H2S a signal due solid Cu(II)S is not evident; furthermore,

there was no appreciable oxygen consumption within this time frame (Figure 2.8). The immediate

reduction of Cu(II) by Cys to form a Cu(I) complex has previously been demonstrated in phosphate

buffer (pH 7.4) by EPR.102 No paramagnetic Cu(II) signal was observed immediately after thiol

addition but returned as the Cu(I) was allowed to oxidize in air. In a previous study, EPR was used

to show that GSH reduced Cu(II) in the pH range of 4-7, while the 1H-NMR spectrum of a 1:2

mixture of Cu(II):GSH in H2O-D2O (pH 7.5) indicated that one GSH was coordinated to Cu(I),

while a second GSH had been oxidized to the corresponding disulfide.103 This also demonstrated

that the stoichiometry required for complete loss of the Cu(II) signal was 1:2 Cu(II):RSH. Similar

results were obtained with Cys, N-acetyl-cysteine and 2-mercaptoethanol, in which disulfide peaks

were observed in the absence of Cu(II).103 Our results obtained in model wine were consistent with

these studies, despite the large molar excess of tartaric acid, which did not appear to interfere with

H2S or thiol coordination by Cu(II).

Previous studies in phosphate buffer (pH 7.4) have shown that the Cu(I)-Cys complex has

an absorbance maximum at 260 nm with a characteristic shoulder at 300 nm.102 In the present study,

52

the addition of H2S and Cys to model wine containing Cu(II) did not produce a distinct absorbance

maximum above 220 nm, although the absorbance increased markedly (Figure 2.4B). The H2S-

containing system’s UV spectrum had an elevated baseline, which could be due to the presence of

Cu(I) complex nanoparticles, some of which are sufficiently small to behave as dissolved species

capable of absorbing energy in the UV region of the spectrum.34 In contrast, 6SH showed an

absorbance maximum of 353 nm, and 3SH had absorbance maxima at 282 and 311 nm (Figure

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

6SH (Figure 2.10) and the complex was retained on a 0.45 µm filter, causing complete loss of

absorbance in the UV region (Figure 2.5A), including that due to the Cu(II)-tartrate species (240

nm). As the Cu(I)-complex was allowed to slowly oxidize from the initial air saturation, a fraction

of Cu(II) was shown to be released back into solution as particles smaller than 0.45 μm, as was

evident by the increase in total Cu concentration at later time points (Figure 2.5B). Previous studies

using X-ray absorption spectroscopy found that the aggregated GSH-Cu(I) complex was

coordinated to three sulfur atoms with a stoichiometry of [CuS1.2], suggesting that the structure was

polymeric with a thiolate sulfur serving as a bridge.191 This complex, however, did not have a single

rigid cluster structure but was comprised of a mixture of various polymers.191 The triply-bridged

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

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

previous work.191

The reaction between H2S and Cu(II) has been shown to be different from that of thiols,

and has been studied in some detail. Initial coordination and reduction of Cu(II) to Cu(I), which is

proposed to occur by inner-sphere electron transfer, is relatively fast.72 The resulting Cu(I) complex

forms clusters composed of neutral 6-membered Cu3S3 ring systems that adopt a chair-like

conformation.72 As discussed above, these polynuclear nanoclusters are sufficiently small to behave

53

like dissolved species.34 This process is consistent with our observation of a clear golden-brown

solution in model wine, the UV-spectrum of which showed a broad increase in absorbance with an

elevated baseline (Figure 2.4B), and thus indicative of light scattering by nanoparticles. Over time,

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

4 and [Cu4S6]-4 polynuclear nanoclusters72 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 O2 at various concentrations has been shown to alter the rate of reaction.192

The presence of excess H2S may favor formation of higher order clusters and further

binding of S by Cu,72 which results in aggregation and may explain why approximately 40% of Cu

was able to be filtered from solution after mixing (Figure 2.5B). A similar effect has been

previously observed in model wine solutions when the ratio of H2S to Cu(II) exceeded 2.5:1, in

which Cu was shown to aggregate and was able to be partially filtered from solution.91 An important

consideration is that Cu(II) is typically added in excess to H2S in winemaking, which would limit

ring formation and further aggregation of the Cu(I)-complex. In addition, other thiols also present

in wine may compete with H2S for Cu coordination.

When H2S and Cys were added in combination in the presence of Cu(II), a 2:1 binding

ratio of H2S + Cys:Cu(II) was still observed (Figure 2.6). Cu(II) binds rapidly to H2S and relatively

more strongly than Cys, which is a benefit for winemakers wanting to remove H2S. While there

was a visible precipitate towards the end of the reaction, it was not observed to the same extent as

was the case with H2S alone. This could be due to the presence of Cys, which may prevent further

aggregation of the Cu(I)-complex, as organic thiols are capable of terminating the highly ordered

polymerization and condensation of the bulk metal sulfide complex.75 This process may account

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

54

2.5.2 Disulfide formation

The formation of 6SH-disulfide as a model for disulfide formation by other volatile thiols

was monitored to confirm the proposed mechanism. No appreciable uptake of O2 was observed

during the first phase of the reaction of 6SH (or any of the treatments) in which Cu(II) was reduced

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

(Figure 2.10). When Cu(II) was added to model wine containing excess 6SH at increasing

concentrations (50, 100, and 200 µM) under argon, 0.5 moles of disulfide was produced (19.7 ±

3.6, 43.4 ± 3.1, and 98.2 ± 3.6 µM, respectively) for each mole of Cu(II) that was present. One thiol

would be oxidized to yield half an equivalent of disulfide while the other would coordinate to Cu(I),

which supports our proposed mechanism (Figure 2.10). Evidently, this Cu(I)-bound thiol can be

removed from solution by filtration (0.45 μm) prior to HPLC analysis and does not react with

Ellman’s reagent, which was used to measure thiol concentration. 6SH was also oxidized in air

saturated model wine in the presence of Cu(II) and monitored over time (Figure 2.7). The entirety

of the thiol appeared to have reacted after 74 h, leaving an equimolar quantity bound to Cu(I) (50

µM). O2 uptake and disulfide formation then continued as this remaining thiol was oxidized. The

aggregate had settled over time, and the heterogeneous nature of the system likely accounts for the

slowness of the reaction. After 262 h, the reaction was complete and the 1:0.5 RSH:RSSR molar

ratio showed that the disulfide was essentially the sole product. This was paired with 69 µM of O2

uptake, giving an O2:thiol molar reaction ratio of ~1:3.3.

We further examined disulfide formation by ascertaining whether free thiyl radicals were

produced in the thiol/Cu(II) systems, as recently suggested,50 using 6SH/Cu(II) system. Wine

contains various compounds such as polyphenols that could preferentially react with radicals,

thereby preventing the formation of disulfides. Experiments were therefore conducted with 4-MeC

and 6SH in anaerobic model wine prior to addition of Cu(II); if free thiyl radicals were formed

55

under such condition, the catechol would be expected to scavenge those radicals to yield

semiquinone radicals (Figure 2.11) and ultimately o-quinones that could undergo 1,4-Michael

addition with thiols to yield a catechol-thiol adducts.96 However, this was not observed as disulfide

concentration remained unchanged and no catechol-thiol adducts were detected (data not shown).

In a separate experiment, DMPO was added to anaerobic model wine prior to Cu(II)-catalyzed 6SH

oxidation, which should have yielded DMPO-thiyl radical adducts at the expense of disulfide

formation (Figure 2.11), yet no depression in disulfide formation was observed (data not shown).

Based on the lack of evidence of thiyl radical formation in this, as well as from previous studies

conducted at physiological pH,122,193 it appears that such radicals are not produced during the initial

Cu(II) reduction. Instead, it is proposed that disulfides arise through bond formation between two

sulfur atoms in the Cu(I)(SR)2 dimer (2) without release of free thiyl radicals (Figure 2.10).

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

56

2.5.3 Oxidation of the Cu(I)-complex

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

(Figure 2.8). 3SH (307 µM) reacted slowly and incompletely up to 168 h. When 100 µM Cu(II)

was added, an equimolar concentration (i.e. 100 µM) of the thiol would have initially been oxidized

to the disulfide in the production of the Cu(I) complex, leaving 100 µM of thiol coordinated to the

Cu(I) according to our proposed mechanism (Figure 2.10). It can be estimated from the 3SH that

remained, and accounting for the 100 µM of the thiol bound to Cu(I), that ~74 µM of thiol would

have reacted to correspond to a consumption of 28 µM of O2, resulting in a 1:2.6 O2:thiol molar

reaction ratio. The presence of free 3SH indicated that all the Cu remained as Cu(I) at the end of

the reaction. In comparison, Cys (299 µM) reacted completely but consumed relatively less O2 (66

µM), giving a ~1:4.5 O2:Cys molar reaction ratio. H2S (284 µM) also reacted completely but

resulted in much greater O2 consumption, affording an O2:H2S molar reaction ratio of ~1:1.6. This

can be explained on the basis that H2S is capable of being oxidized to ground state S0, effectively

reducing two equivalents of Cu(II). It is also possible for H2S to be fully oxidized to sulfate, or to

form partially oxidized polysulfides.194

Oxygen may be reduced in four discrete one-electron steps in metal-catalyzed wine

oxidation (Figure 2.12). The possibility that hydroperoxyl radicals were generated under this

scenario was tested by oxidizing H2S or Cys in the presence of excess 4-MeC, wherein the catechol

would quench hydroperoxyl radicals to generate the o-quinone.51 However, the concentration of 4-

MeC did not change as oxidation proceeded, and formation of catechol-thiol adducts was not

observed (data not shown). Thus, it appears that hydroperoxyl radicals are not produced and so O2

was reduced directly to hydrogen peroxide (H2O2) in a two electron process. It is proposed that the

close proximity of two Cu(I) ions in aggregate (4) allows for such a process to occur (Figure 2.13).

57

Similarly, it has previously been concluded that the Fe(II) reduction of O2 to H2O2 in model wine

also proceeds without the release of hydroperoxyl radicals or oxidation of catechols.58

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

hydrogen peroxide and the hydroxyl radical.

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

Previous studies of the copper-catalyzed H2O2 oxidation of Cys similarly failed to detect

hydroxyl radicals, and it was suggested that H2O2 was also reduced in a two-electron step (Figure

2.14). However, it was proposed that at higher dilution rates, when the Cu(I) complex is less

aggregated, the usual Fenton pathway would be favored (Figure 2.15).103 Without the hydroxyl

radical, the Fenton reaction-mediated oxidation of ethanol in model wine would not occur and no

AC should be produced. Overall a 1:4 molar reaction ratio of O2:thiol would result, with all four

electrons being derived from the thiol to reduce O2 to two equivalents of H2O (Figures 2.13 and

2.14). If H2O2 was reduced in a one-electron step, hydroxyl radicals would result (Figure 2.15). As

these radicals are powerful, non-selective oxidants that react at diffusion-controlled rates, they

would be expected to react with solution components in proportion to their concentration. As the

most abundant oxidizable constituent in model wine, ethanol would serve as the likely target of

hydroxyl radical oxidation, from which 1-hydroxyethyl radicals (1-HER) would be generated.59 In

the Fe-catalyzed Fenton reaction, 1-HER would be oxidized to AC by Fe(III) at very low O2

concentrations, resulting in a 1:1 molar ratio of O2:AC. However, the presence of O2 in the system

58

would favor the formation of the 1-hydroxyethylperoxyl radical (1-HEPR).60,61 It has been

previously proposed that 1-HEPR can release the hydroperoxyl radical and form acetaldehyde;

however, the lack of 4-MeC oxidation suggests that again the hydroperoxyl radical is not formed.

Instead, it is proposed that 1-HEPR is quickly reduced in the presence of Cu(I)-complex, yielding

the corresponding peroxide (Figure 2.15).195 This peroxide may then be reduced to the alkoxyl

radical, and quickly reduced to 1,1-dihydroxyethane by the Cu(I)-complex due to its close

proximity rather than reacting with 4-MeC. 1,1-Dihydroxyethane (i.e. acetaldehyde hydrate) is then

expected to dehydrate under wine conditions to yield acetaldehyde (Figure 2.15). This route would

result in a 2:1 O2:AC molar ratio and a 1:3 O2:thiol molar reaction ratio, with three electrons being

provided by RSH, one electron being provided by ethanol, and O2 accepting four electrons.

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

59

Figure 2.15. One-electron reduction of H2O2 to produce hydroxyl radicals, and the oxidation of

ethanol by the Fenton reaction to form 1-hydroxyethyl radicals. 1-hydroxyethyl radicals are

oxidized by oxygen and subsequently reduced by metals to yield acetaldehyde.

H2S oxidation produced the most AC (Figure 2.9), and with an O2:AC molar ratio of 2.2:1,

oxidation could have proceeded mainly as shown in Figure 2.15. This uptake of O2 and production

of AC clearly showed that Cu(II) did not simply form Cu(II)S. The oxidation of Cys resulted in

lower AC formation, with an O2:AC molar ratio of 2.5:1, while that of Cys+H2S resulted in a ratio

of 2.1:1. The O2:AC molar ratios of 6SH and 3SH were 1.5:1 and 1.8:1, respectively. Cys produced

relatively less AC, and it may be inferred that the mechanisms shown in Figures 2.13 and 2.14

might operate to a greater extent, although there is some uncertainty as to the fate of AC and a

closer examination of AC production in these systems is warranted. Nonetheless, it can be

concluded that the Fenton reaction does occur during H2S and thiol oxidation in model wine, albeit

to varying degrees.

In conclusion, we show that Cu(II) is reduced by H2S and thiols in air saturated model

wine, while thiols, which are present in relative excess to added Cu(II), as well as H2S, are oxidized.

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

60

processes. These conditions are unlikely to occur during the fining process. However, it should be

noted that the reactions were followed down to ~50% and 25% air saturation. Furthermore, the

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

6SH, the Cu(I)-SR complex precipitated immediately, before any oxygen reacted. Similarly, when

the Cu(I)-6SH complex was formed under argon, quantitative yields of disulfide were obtained in

5 min.

It can therefore be concluded that if fining were conducted under anaerobic conditions, all

the Cu(II) would be quickly reduced to Cu(I) by H2S and thiols, which would be oxidized. The

present work, therefore, provides a mechanistic foundation for future studies in both model and real

wine systems, which would contain sulfite, as well as in other alcoholic beverages in which thiols

and H2S play an important role with respect to quality (e.g. beer and cider). In part 2 of this

investigation, it is shown that Cu(I) complexes react rapidly with Fe(III); as such, any Fe(III) that

remained in these conditions would be reduced to Fe(II) and Cu(I) would recycle until no Fe(III)

remained. The reaction would then stop until O2 is introduced as a result of racking or filtration.

61

2.6 Acknowledgments

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

State University for his assistance with EPR analysis.

62

Chapter 3


Wine. Part 2: Iron and Copper Catalyzed Oxidation.

Published as:


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

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

3.1 ABSTRACT

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

In Part 1 of this study, the reaction of H2S and thiols with Cu(II) was examined; however, the

interaction of iron and copper is also known to play an important synergistic role in mediating non-

enzymatic wine oxidation. The interaction of these two metals in the oxidation of H2S and thiols

(cysteine, 3-sulfanylhexan-1-ol, and 6-sulfanylhexan-1-ol) was therefore examined under wine-like

conditions. H2S and thiols (300 μM) were reacted with Fe(III) (100 or 200 μM) alone and in

combination with Cu(II) (25 or 50 μM), and concentrations of H2S and thiols, oxygen, and

acetaldehyde were monitored over time. H2S and thiols were shown to be slowly oxidized in the

presence of Fe(III) alone, and were not bound to Fe(III) under model wine conditions. However,

Cu(II) added to model wine containing Fe(III) was quickly reduced by H2S and thiols to form Cu(I)-

complexes, which then rapidly reduced Fe(III) to Fe(II). Oxidation of Fe(II) in the presence of

oxygen regenerated Fe(III) and completed the iron redox cycle. In addition, sulfur-derived

oxidation products were observed, and the formation of organic polysulfanes was demonstrated.

63

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, O2 can be reduced to water in four discrete one-

electron steps,51 resulting in the formation of reactive intermediate oxygen species53 that can

oxidize wine constituents.39,59,196 However, recently, it was proposed that under wine-like

conditions, Fe(II) reduces an intermediate Fe(III)-oxygen complex in a concerted 2-electron

reduction to produce H2O2 from O2 without the formation of an intermediate hydroperoxyl radical

(Figure 3.1).58 Similar results were obtained for the Cu(I)-mediated reduction of oxygen, where no

evidence of an intermediate hydroperoxyl radical was observed.55 In combination, these metals act

synergistically, with copper playing an important role in the overall wine oxidation process by

accelerating the reaction of Fe(II) with oxygen to regenerate Fe(III),52 presumably, copper

facilitates Fe(III)/Fe(II) redox cycling. Once H2O2 is formed, it is reduced by Fe(II) through the

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

forming the intermediate 1-hydroxyethyl radical (1-HER).60 In low O2 concentrations, 1-HER will

be oxidized by Fe(III) to yield acetaldehyde (AC); however, at higher O2 concentrations, O2 is

known to add to 1-HER to yield the 1-hydroxyethylperoxyl radical (1-HEPR) (Figure 3.2). Recent

work suggests that rather than 1-HEPR releasing AC and hydroperoxyl radicals, 1-HEPR is reduced

to the peroxide by the presence of reduced metal complexes.55 The peroxide can then undergo a

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

dihydroxyethane that dehydrates to AC.

64

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 sulfite54 or undergo Michael-type addition

reactions with sulfite or thiols96,97, effectively driving the reaction forward by consuming the

product of phenolic oxidation. Fe(III) may also interact with thiols directly, which could either have

deleterious effects by causing the oxidative loss of important aroma compounds such as 3-

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

65

presence of thiols in wine may, therefore, play an important role in mediating wine oxidation,

although the mechanism by which sulfhydryl compounds (i.e., species containing an –SH moiety)

directly interact with iron and copper in wine remains poorly understood. Such information is

important to winemakers in order for them to make informed decisions about managing oxidation

to improve wine quality.

Studies performed with glutathione (GSH) in a wine pH range (3-7) have shown that Fe(II)

is spontaneously produced when GSH is added to Fe(III) (Figure 3.3).109,110 The same has been

shown with Cys at low pH, as the Fe(III)-Cys complex is unstable and quickly reacts to yield Fe(II)

and cystine.111 Previous work has failed to provide evidence of free thiyl radical generation under

those conditions,109 and the disulfide is seemingly formed in situ before being released from the

metal complex. The resulting Fe(II) remains bound to GSSG and is only released when excess GSH

is present; however, unlike Cu(I), which coordinates strongly with thiols, Mössbauer spectroscopy

showed that Fe(II) is not bound to sulfur. It was concluded that coordination to GSSG, GSH and

also Cys occurred by interaction with carboxylate groups under acidic conditions (pH<4).109,110 As

discussed above, the Fe(II) produced can be reoxidized to Fe(III) by reacting with O2, with the

reaction markedly accelerated by copper.

Figure 3.3. Proposed mechanism for initial Fe(III) reduction by thiols showing that the resulting

Fe(II) is not coordinated to sulfur after the disulfide is formed.

66

Recent work in model systems has demonstrated that tartaric acid determines the reduction

potential of the Fe(III)/Fe(II) couple in wine,197 but it may be possible that thiols also affect that

potential. This is of particular interest to copper-containing systems, as H2S and thiols keep copper

in its reduced Cu(I) state under wine-like conditions.55 In view of the known interaction of iron and

copper in relation to wine oxidation, it is of interest to examine the effect of the metal combination

in the removal of undesirable sulfidic off-odors in comparison to copper alone. Recent work has

examined the reaction of H2S 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 H2S and thiols. As noted previously8, the concentration of thiols,

such as glutathione and cysteine analogues, far exceeds that of H2S that at likely to occur in wine.

The oxidation of H2S in the presence of greater concentrations of Cys, as a representative thiol, was

therefore investigated due to its relevance to the copper fining operation in winemaking.


3.3.1 Chemicals

L-Cysteine (Cys), monobromobimane (MBB), 6-sulfanylhexan-1-ol (6SH), and

diethylenetriaminepentaacetic acid (DTPA) were obtained from Sigma-Aldrich (St. Louis,

67

MO). 2,4-Dinitrophenylhydrazine (DNPH) was purchased from MCB laboratory

chemicals (Norwood, OH) and L-tartaric acid, 3SH, and 5,5’-dithiobis(2-nitrobenzoic acid)

(DTNB) were obtained from Alfa Aesar (Ward Hill, MA). Copper(II) sulfate pentahydrate

was purchased from EMD Chemicals (Gibbstown, NJ), TRIS hydrochloride from J.T.

Baker (Center Valley, PA), and sodium hydrosulfide hydrate (as a source of H2S) was

purchased from Acros Organics (Geel, Belgium). Iron(III) chloride hexahydrate was

purchased from Mallinckrodt Chemicals (St. Louis, MO). Water was purified through a

Millipore Q-Plus system (Milipore Corp., Bedford, MA). All other chemicals and solvents

were of analytical or HPLC grade and solutions were prepared volumetrically, with the

balance made up with Milli-Q water unless specified otherwise.

3.3.2 Model Wine Experiments



with sodium hydroxide (10 M) and brought to volume with water.

For H2S and Cys, an aqueous stock solution of each (approximately 0.5 M) were freshly

prepared, whereas 6SH and 3SH were added directly by syringe during experimentation. Aqueous

stock solutions of Cu(II) sulfate and Fe(III) chloride (0.1 M and 0.4 M, respectively) were freshly

prepared. H2S, Cys, 6SH, or 3SH were added to air saturated model wine (1 L, 300 μM) followed

by thorough mixing.

For Fe experiments, Fe(III) (200 μM) was added to all H2S and thiol treatments and

thoroughly mixed. For Fe and Cu combination experiments, Fe(III) (200 μM) and Cu(II) (50 μM)

were consecutively added to H2S, 6SH, or 3SH solutions. For Cys experiments, Fe(III) (100 μM)

68

and Cu(II) (25 μM) were consecutively added and mixed thoroughly. For thiol experiments in

combination with H2S and Fe/Cu, H2S was added to the thiol treatment and mixed prior to the

addition of metal stock solutions. H2S (100 μM), Fe(III) (200 μM), and Cu(II) (50 μM) were added

to Cys, 6SH, and 3SH. For Cys experiments with low metal concentrations, H2S (50 μM), Fe(III)

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

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


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.


Glass B.O.D. bottles were fitted with PSt3 oxidots and oxygen readings were taken per

time point using a NomaSense O2 P6000 meter (Nomacorc LLC, Zublon, NC). Further details were

reported in Part 1.55

3.3.4 Spectrophotometric measurements

UV-vis spectra of the treatments were recorded at each time point using 10 mm quartz

cuvettes (model wine blank) and measured using Agilent 8453 UV-Vis spectrophotometer

(Agilent, Santa Clara, CA). Determination of Fe(III) concentration was achieved by measurement

of absorbance at 336 nm associated with the Fe(III)-tartrate complex.58

69

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

Further details were reported in Part 1.55

3.3.5 HPLC Analyses

For the mixed H2S and thiol treatments, MBB derivatization and analysis of thiol

concentration was performed using negative electrospray ionization (ESI-) HPLC-MS/MS as

described in Part 1.55 The mass transition of sulfide-dibimane was monitored at m/z 413→191, Cys-

bimane was monitored at m/z 310→223, 3SH-bimane at m/z 323→222 and the internal standard

6SH-bimane was monitored at m/z 323→222. External standard curves prepared for sulfide-

dibimane, Cys-bimane, and 3SH-bimane were normalized to the 6SH-bimane internal standard. In

the case of 6SH/H2S combination experiment, external calibration curves were made the same day

prior to analysis and used without addition of 6SH-bimane internal standard.


dinitrophenylhydrazone (DNPH) derivative with an external standard curve (10 – 220 μM) by

HPLC as described in Part 1.55

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

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

HPLC separation parameters. Mass spectra were obtained using ESI- and full scan between

m/z 100-1000. 6SH and 3SH polysulfanes were obtained by adding H2S (100 μM), Fe(III)

(200 μM), and Cu(II) (50 μM) to 6SH or 3SH (300 μM). The organic polysulfanes were

detected by UV absorbance at 210 nm and verified using MS detection with ESI+ and full

scan between m/z 100-1000. Mobile phases consisted of 0.1% v/v formic acid (A) and 0.1%

v/v formic acid in acetonitrile (B) with a linear gradient according to the following

70

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.

71

3.4 RESULTS AND DISCUSSION

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

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

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

3SH, to represent secondary thiols; (4) H2S, as it is one of the primary targets associated with

sulfidic off-odors. Unlike the Cu(II) experiments described in Part 1, in which 2 mole equivalents

of thiols and 1.4 equivalents of H2S 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 H2S,

although there was no appreciable consumption observed within the first few hours of the

experiment, it reacted faster than the other thiol compounds, its concentration declining as Fe(III)

was reduced and O2 was consumed (Figures 3.4B and 3.4C). A total of 262 µM of H2S was

consumed after 144 h elapsed, and 192 µM of Cys was consumed after 193 h. Both 6SH and 3SH

reacted extremely slowly, with negligible losses (<15 µM) throughout the time course of the

experiments.

72

Figure 3.4. Reaction of H2S or thiols on addition of Fe(III) (200 µM) to 6SH, H2S, Cys, or 3SH (300 µM) in air saturated model wine. (A) Consumption of H2S or thiols; (B) %Fe(III)-tartrate

based on absorbance at 336 nm; (C) O2 consumption. Error bars indicate standard deviation of

triplicate treatments.

73

3.4.2 Fe(III) reduction by thiols and H2S

The Fe(III)-tartrate complex shows an absorbance maximum at 336 nm due to a d→d

electronic transition, which can be used to obtain Fe(III):Fe(II) ratios in model wine systems.58

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

Fe(III)-complex was followed by UV spectroscopy over time upon adding Fe(III) to thiol or H2S

treatments in model wine (Figure 3.4B). For the H2S treatment, Fe(III) was gradually reduced up

to a maximum of approximately 66% of Fe(II) within 96 h. For the Cys treatment, a maximum of

approximately 17% of Fe(III) was reduced to Fe(II) within 24 h, before apparently reaching an

equilibrium state wherein the rates of Fe(II) oxidation and Fe(III) reduction equalized. This

difference was consistent with a slower rate of Fe(III) reduction compared to that produced by H2S.

Minimal Fe(III) reduction was observed in experiments involving 6SH and 3SH, which was

matched by minimal thiol and O2 uptake (Figures 3.4A and 3.4C) None of the treatments showed

changes in absorbance maxima compared to Fe(III)-tartrate in model wine or resulted in the

appearance of additional peaks, which indicated that these treatments did not displace tartaric acid

from its Fe(III) complex.

Based on these results obtained in model wine (Figure 3.4B), and compared to previous

studies where GSH and Cys were shown to reduce Fe(III) in simple aqueous systems,109,110 it is

apparent that tartaric acid inhibits both the coordination of thiols with Fe(III) and its subsequent

reduction to Fe(II). Furthermore, as Fe(III) coordinates preferentially with carboxylate moieties

rather than with the thiolate function at wine pH,110 it would appear that Fe(III) remains bound to

tartaric acid. However, due to its carboxylate function, Cys can presumably compete for Fe to

displace tartrate ligands. In contrast, 6SH and 3SH, which lack a carboxylate function, are unable

to displace tartaric acid in the Fe-containing systems, which would account for their low reactivity.

74

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

thiols and H2S in model wine.55

Notably, H2S 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 H2S to yield [Fe-H2S]2+ which would

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

Under acidic conditions, FeS aggregates to form metastable nanoparticles (<150 Fe2S2 subunits)

that behave like dissolved species but will quickly dissociate under low pH conditions,75 such as

those encountered in wine. This will prevent further FeS aggregation and precipitation, and would

explain why bulk FeS formation is not observed in wine, furthermore, FeS solubility is

approximately 1012-fold higher than CuS.75 Tartaric acid should also prevent H2S coordination, but

the ligated acid does not limit the ability of H2S to reduce Fe(III), in contrast to what occurs with

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

sulfhydryl functionality and aroma associated with H2S.80,81

3.4.3 Fe(II) oxidation and oxygen consumption

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

Fe(III) reduction by thiols or H2S, and that of Fe(II) reoxidation by O2. As tartaric acid determines

the reduction potential of the Fe(III)/Fe(II) redox couple in the model system described here, it is

likely that the reoxidation of Fe(II) will proceed as described previously (Figure 3.1).58 Fe(II) is

expected to reduce O2 by a concerted 2-electron mechanism, yielding a Fe(III)-dioxygen complex

that directly hydrolyzes to H2O2 without release of hydroperoxyl radicals. H2O2 should then

undergo reduction via the Fenton reaction in the presence of Fe(II) to yield hydroxyl radicals that

will subsequently oxidize ethanol (Figures 3.1 and 3.2). Fe behaves as a redox catalyst, cycling

electrons from thiols and H2S to O2. Based on the overall sequence of reactions, it would be

75

expected that 3 electrons would come from thiols or H2S and 1 electron from ethanol to reduce O2

to water. Consequently, it would be expected that the O2:thiol molar reaction ratio would be 1:3,

and the O2:H2S ratio would be 1:1.5 as H2S is capable of reducing 2 equivalents of Fe(III) as it is

oxidized to ground state sulfur.73

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

Of the 262 µM H2S that reacted (Figure 3.4A), 135 µM of O2 was consumed (Figure 3.4C), giving

a 1:1.9 O2:H2S molar reaction ratio. However, roughly 66% of Fe(III) had also been reduced to

Fe(II) (~132 µM) (Figure 3.4B), which would have required ~66 µM of H2S. Subtracting that

amount from total reacted H2S would give ~196 µM uptake corresponding to the 135 µM O2 uptake,

thus lowering the O2:H2S molar reaction ratio to ~1:1.5, as anticipated from the proposed

mechanism (Figures 3.1 and 3.2). Fe(III) is reduced to some extent by Cys, likely in the same

manner proposed in Figure 3.3, and 192 µM Cys (Figure 3.4A) reacted to reduce Fe(III) with

subsequent consumption of 49 µM of O2 (Figure 3.4C). However, roughly 17.5% (35 µM) of Fe(II)

remained at the end of the reaction, which corresponded to 35 µM Cys uptake. Subtracting this

amount results in 157 µM Cys oxidized with the corresponding 49 µM O2 uptake, giving a O2:thiol

molar ratio of ~1:3.2, which is in agreement with the proposed mechanism. (Figures 3.1 and 3.2).

Due to the inability of 6SH and 3SH to outcompete tartaric acid to form an Fe(III) complex, the

oxidation of 6SH and 3SH was extremely slow and the O2:thiol molar reaction ratios could not be

calculated (Figures 3.4A and 3.4C).

Low concentrations of acetaldehyde (AC) (15 – 30 μM) were formed in the Cys and H2S

systems (data not shown), demonstrating that the Fenton reaction does proceed in the system

described. The formation of AC is thought to proceed as described in Figure 3.2. It was expected

that a higher concentration of acetaldehyde would be formed in the H2S system. In a previous study

in which the Fenton reaction was investigated in model wine with iron only, up to 90% of 1-HER

76

radical was intercepted by thiol-containing compounds, the resulting thiyl radical likely then

quickly dimerizing to yield a disulfide.67

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

The interaction of iron and copper plays an important synergistic role in wine oxidation,

and it was important to investigate whether these metals impact H2S and thiol oxidation. The

treatments described above were employed again, this time using a combination Cu(II) (50 µM)

and Fe(III) (200 µM). Cu(II) concentration was chosen to remain consistent with Part 1 of this

investigation, and these concentration ratios were chosen as wines typically have 5–10-fold higher

relative concentrations of iron to copper.114 In this experiment, Cys reacted rapidly and was

completely consumed within 5 min (data not shown); therefore, the concentrations of Fe(III) and

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

conveniently monitored.

In the presence of Fe(III) alone, Cys was slowly oxidized, with the reaction remaining

incomplete after 200 h (Figure 3.4A). It was also determined that Cys did not coordinate to any

significant extent to Fe(III) under the experimental conditions, with the metal center remaining

largely bound to tartaric acid (Figure 3.4B). The addition of Cu(II) markedly increased the rate of

the reaction, and Fe(III) was almost fully reduced within 5 min in the Cys system (Figure 3.5A),

as less than 5% of the absorbance at 336 nm due to Fe(III)-tartrate complex was observed. Despite

the fact that the concentration of Cu(II) and Fe(III) had to be decreased in this experiment, oxidation

of Cys (296 µM) was complete within 7 h (Figure 3.5B). It was concluded that Fe(III) was not

reduced by Cys directly but by the Cu(I)-Cys complex (Figure 3.6), which was rapidly formed.55

Given that 25 µM of Cu(II) was added initially, 25 µM of the Cu(I) complex would have been

immediately produced and then oxidized by Fe(III). Recycling of copper three further times (with

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

3.5A). At this point, the resulting Cu(II) would oxidize 25 µM of Cys to cystine, and 25 µM of Cys

would be bound in the Cu(I) complex. In total, 150 µM of Cys would be consumed when all Fe(III)

and Cu(II) were reduced, in accordance with the amount actually consumed during the initial rapid

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

3SH and 6SH were less reactive than Cys, and led to an initial ~40% reduction of Fe(III) to Fe(II),

with iron speciation reaching equilibrium at ~25% Fe(II) (Figure 3.5A). 6SH (273 µM) was fully

oxidized within 7 h whereas 3SH, as a secondary thiol, oxidized more slowly and the reaction was

incomplete at the 150 h time point (Figure 3.5B). The limiting factor for 3SH oxidation could

potentially be the rate of formation of the Cu(I)-complex due to steric hindrance of the thiol.55

However, the reaction for 3SH proceeded more quickly in the iron/copper combination treatment

compared to the systems with Fe(III) (or Cu(II)55) alone, resulting in the consumption of 267 µM

of 3SH. H2S caused a rapid and near complete reduction of Fe(III) to Fe(II) within 30 min,

corresponding to the loss of the absorbance peak at 336 nm (Figure 3.5A) along with a sharp initial

drop (~135 µM) in H2S concentration (Figure 3.5B). However, the formation of Cu(I)-complex

nanoparticles resulted in an elevated baseline, therefore the data were normalized to the baseline.55

It appears that iron remained reduced until no free H2S remained (308 µM consumed) at ~48 h,

after which Fe(II) re-oxidized to Fe(III) in the presence of O2 (Figures 3.5A and 3.5B).

78

Figure 3.5. Reaction of H2S or thiols on addition of Fe(III) (200 µM) and Cu(II) (50 µM) to H2S,

6SH, 3SH (300 µM), and Fe(III) (100 µM) and Cu(II) (25 µM) to Cys (300 µM) to air saturated

model wine. (A) %Fe(III)-tartrate based on absorbance at 336 nm; (B) Consumption of H2S or

thiols; (C) O2 consumption; (D) AC generation. Error bars indicate standard deviation of triplicate treatments.

Figure 3.6. Proposed mechanism demonstrating initial Cu(II) reduction by thiols and H2S to yield

Cu(I)-SR complex and subsequent oxidation of the complex by Fe(III). Fe(II) then reduces oxygen to hydrogen peroxide. Subsequent reaction of H2O2 is depicted in Figure 2.

79

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

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

O2 with the Cu(I)-complex; however, when iron is present, the reaction rate is dependent on the

oxidation rate of the Fe(II)-tartrate complex, which is known to be fast.197 When the two metals are

present in combination, Fe(III) rapidly oxidizes Cu(I) first (Figure 3.6) and the Fe(II) produced is

oxidized by O2 (Figure 3.3), markedly increasing the rate of Cu(I) oxidation. The degree of

consumption of H2S with copper determined previously55 was similar to that when Fe(III) was

added in combination with Cu(II) (Figure 3.5B). It would appear that, in this case, the rate of

oxidation of the Cu(I)-H2S complex was similar to that of the Fe(II)-tartrate complex.

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

H2S system, around 46% (92 µM) of iron remained reduced after 120 hr (Figure 3.5A), which

would require 46 µM of H2S. As a result, 262 µM of H2S would be left to react with 160 µM of O2

consumed, giving a ~1:1.6 O2:H2S molar reaction ratio, approximately the same as the Fe(III) or

Cu(II) treatment alone. As for the Cys treatment, roughly 12% (12 µM) of Fe remained reduced,

which would require 12 µM Cys. Therefore, 284 µM Cys reacted with 110 µM O2, giving a 1:2.6

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

O2 consumption of 106 µM and 82 µM, respectively. This afforded a ~1:2.1 O2:RSH molar ratio in

the 6SH system and ~1:2.6 in the 3SH system. As with H2S, reaction ratios were comparable to

those involving Cu(II) alone. Given that treatments involving the combination of Fe(III) and Cu(II)

resulted in quicker thiol consumption than Fe(III) alone, it would suggest that the Cu(I)-SR

aggregate reacts more slowly with O2 than with Fe(III), with the overall reaction rate being dictated

by Fe(II)-tartrate oxidation, as alluded to above. However, the similarity in the molar ratio of O2

and thiol or H2S consumed may indicate that both iron and copper behave in the same mechanistic

manner with respect to O2.

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The ~1:3 O2:RSH molar reaction ratio observed in the Cys and 3SH systems is indicative

of a combination of 2-electron reduction of O2 to H2O2, as well as the 1-electron reduction of H2O2

to hydroxyl radicals and subsequent one electron ethanol oxidation (i.e., Figures 3.1 and 3.2). The

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

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

3.5D). This was in accord with the Fenton-catalyzed wine oxidation described from Part 1,55 in

which 1-HEPR is formed and subsequently reduced by metals. However, in the case of 6SH, in

which 146 µM of AC was formed, the ratio was closer to 1:1 O2:AC, which would suggest direct

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

H2S system (Figure 3.2). Furthermore, reduction of Fe(III) by 1-HER generates Fe(II) that

subsequently react with O2, explaining why the molar ratios for the 6SH system, as well as 3SH

and Cys, were lower than 1:3 O2:RSH.

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

Under normal conditions, the concentration of H2S in wine (0.3 – 1 µM) would generally

be lower than that of other thiols, such as the combined pool of GSH (up to 40 µM) and Cys, homo-

Cys and Cys analogues (20 µM).92–94,185,198 Therefore, to better model a real wine situation, the

oxidation of H2S in the presence of an excess of thiols (Cys, 6SH, and 3SH) was examined in model

wine with the combination of Fe/Cu described above (Figures 3.7A-D). The final concentration of

added H2S was targeted to be double that of the Cu(II) concentration that was established in the

model wine, based on the initial 2:1 H2S:Cu(II) molar ratio. In these experiments, a haze was

formed initially, presumably due to insoluble Cu(I)-thiol complexes.55 However, no black-green

CuS precipitate was observed at the end of the reaction, indicating that the Cu(I)-complex did not

aggregate to the point of precipitation under conditions that were designed to closely mimic real

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wine conditions. This observation may explain why precipitates are not observed when Cu(II) is

added to wine containing H2S. The reduction of Cu(II) also explains the absence of the highly

insoluble Cu(II)S, which may have been expected to form.91 Compared to H2S, the three thiols were

present in large molar excess, but H2S was still quickly oxidized, with at least 60% of free H2S

removed within 5 min in all treatments (Figures 3.7A-D). By 24 h, there was virtually no H2S

remaining in the four experiments, and even after all free H2S was depleted, the remaining free

thiol continued to oxidize without precipitation of a copper-complex.

Figure 3.7. Total thiol and H2S loss on addition of Fe(III) (200 µM) and Cu(II) (50 µM) to (A)

6SH (300 µM) + H2S (100 µM); (B) 3SH (300 µM) + H2S (100 µM); (C) Cys (300 µM) + H2S

(100 µM); (D) Fe(III) (100 µM) and Cu(II) (25 µM) to Cys (300 µM) + H2S (50 µM) to air saturated

model wine. Error bars indicate standard deviation of triplicate treatments.

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

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

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

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reaction ratios (Figured 3.8A and 3.8B). Under both conditions (i.e., high and low metal

concentrations), virtually all Fe(III) was reduced to Fe(II) within the first few minutes of the

experiment; however, in the high metal treatment, Fe(II) quickly reoxidized to Fe(III). The high

metal concentration treatment caused all H2S and Cys to be oxidized within 2 h whereas the low

metal treatment required 24 h. The total combined Cys+H2S consumption was 302 and 326 µM for

the high and low treatments, respectively, with corresponding total O2 consumption of 132 and 138

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

~1:2.3 O2:Cys+H2S, irrespective of metal concentration, and was intermediate between the

expected 1:3 ratio for Cys and 1:1.5 ratio for H2S. However, the total concentration of AC generated

was quite different between the two systems. The high metal concentration treatment resulted in

150 µM of generated AC, whereas the low metal treatment resulted in 81 µM of AC. Figures 3.8A

and 3.8B correspond to approximately 1:1 AC:O2 ratio in the high metal system and a 1:2 AC:O2

ratio in the low metal system. This could be explained by the fact that a higher concentration of

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

(Figure 3.3).

Figure 3.8. Total concentrations of Fe(III), Fe(II), O2 (consumed), thiol, and AC in Cys+H2S

treatment containing low and high metal concentration. (A) Low Fe (100 µM) and Cu (25 µM), (B) High Fe (200 µM) and Cu (50 µM). Error bars indicate standard deviation of triplicate treatments.

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3.4.7 Formation of mixed organic polysulfanes

When H2S and 6SH were oxidized together in the presence of Cu(II) and Fe(III), the

formation of 6SH-polysulfane was evident; these were present with up to five linking S atoms

(n=5), as determined by HPLC-MS (Figures B.1 and B.2). These were not detected when 6SH was

oxidized in the absence of H2S. Similar results were obtained with H2S and 3SH (data not shown),

revealing that in a mixed thiol system, as is typical of wines, the formation of mixed disulfides and

polysulfanes would be expected in the initial Cu(II) fining process. This is consistent with the

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

H2S, methanethiol, and ethanethiol.100 When H2S was oxidized alone, MBB derivatization followed

by HPLC-MS analysis indicated the presence of up to S5-bimane, with sequential fragmentation

losses of m/z 32 (Figure B.3). These species would likely remain bound to Cu(I)72 or potentially to

Fe(II),112 but importantly, mixed-thiol disulfides and organic polysulfanes could contribute to the

recurrence of H2S post-bottling. The release of thiols from disulfides via sulfitolysis is a likely

scenario invoked by the presence of sulfite, which was recently found to react with disulfides

resulting in the release of a free thiol and the formation S-sulfonated products in wine.44 Further

research is underway to investigate the importance of these compounds on the evolution of sulfidic

off-odors in wine.

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

H2S and thiols. Accordingly, the presence of H2S and thiols was shown to rapidly reduce Cu(II),

with the resulting Cu(I) then able to rapidly reduce Fe(III). This process occurs more quickly than

when H2S and thiols react directly with Fe(III). The iron redox cycle is then completed as Fe(II) is

re-oxidized to Fe(III) by oxygen. Oxygen reacts in the Fenton reaction to produce acetaldehyde so

it is unlikely that it adds to sulfur to form sulfur oxyanions to any significant extent.

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Though these studies were conducted at initial air saturation in order better to follow the

oxidative processes, it was argued in Part 1 of this investigation that aspects of the proposed

mechanisms would apply to Cu fining conducted under anaerobic conditions. Under such

conditions, all the Cu(II) would be quickly reduced to Cu(I) by H2S and thiols, and the Cu(I) would

be oxidized by any Fe(III) that might remain. The reaction would then be expected to stop until O2

was introduced as a result of racking, filtration, or bottling.

Copper fining quickly oxidizes H2S, but the subsequent interaction with other transition

metals and wine constituents needs to be better understood. The interaction of other metals in wine

including Zn, Al, and Mn, which are present at an average of 0.54, 0.41, and 0.97 mg/L,

respectively, should also be considered in future studies, as they are present in significant quantities

and have been shown to influence the evolution of volatile sulfur compounds in wine over time.70

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Chapter 4


Wine. Part 3: Manganese Catalyzed Oxidation and Interaction with Iron and

Copper.

4.1 ABSTRACT

Recent work suggests that manganese has a modest activity in catalyzing polyphenol and

sulfite oxidation in wine. Furthermore, manganese is known to mediate thiol and H2S oxidation in

aquatic systems. It was therefore of interest to investigate the interaction of manganese with iron

and copper toward catalyzing thiol and H2S oxidation under wine-like conditions. Sulfhydryl

compounds (cysteine, 6-sulfanylhexan-1-ol, and H2S) were reacted with Mn(II) alone or in

combination of Fe(III) and Cu(II) in model wine, and the concentrations of sulfhydryl, oxygen, and

acetaldehyde were monitored over time. The reaction of thiols with manganese resulted in radical

chain reaction paired with large oxygen uptake and generation of sulfur oxyanions. H2S did not

generate free thiyl radicals, and had minimal interaction with Mn(II). When Cu(II) was introduced,

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

4.2 INTRODUCTION

Iron and copper catalyze non-enzymatic wine oxidation by reducing oxygen, which is

paired with oxidation of ethanol, polyphenolics, and sulfhydryls.52,54–56,59 However, few studies

have examined the mechanistic involvement of other transition metals on the oxidation in wine.

Manganese has been proposed to have an effect at mediating wine oxidation, and is present at

concentrations similar to Fe (~1 mg/L average around the world114,199). Mn has been reported to

catalyze browning in sherry wine in combination with iron,200 increase acetaldehyde production in

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red wines,115 and decrease volatile sulfur compounds concentrations during storage in both red and

white wines.70,117 Furthermore, recent work demonstrated modest catalytic activity of Mn in model

wine and Sauvignon Blanc in the presence of Fe and Cu.116

Mn(III) is a strongly oxidizing species which can be readily reduced to Mn(II) by wine

constituents. Recent work demonstrated that when Mn(III) is added to model wine, it forms a

Mn(III)-tartrate complex with a UV-absorbance maximum at ~240 nm and a shoulder at ~300

nm.116 Under wine pH conditions the Mn(III)-tartrate complex is unstable, with Mn(III) being

reduced, presumably by the tartaric acid ligand.116 It is therefore expected that essentially all Mn

should exist as Mn(II) under wine conditions, and likely remains bound to organic acids (i.e. tartaric

and malic acid).

The reduction potential of the Mn(III)/Mn(II) redox couple is considerably higher than that

of the Fe(III)/Fe(II) system and Mn cannot readily redox cycle in wine conditions.116 The reaction

of O2, H2O2, or Fe(III) with Mn(II) to generate Mn(III) is thermodynamically disfavored and is

found to proceed very slowly if at all in model wine.116 However, Mn(II) is a very effective catalyst

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

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

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

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

to regenerate Fe(III) to continue the process.58

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

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Fe(II) reacts with O2 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 H2O2.58

It was found that Mn(II) may play a role in reacting with Fe(III)-superoxo intermediate and driving

the reaction forward (Figure 4.2).116 The reduction of this complex regenerates Mn(III) which can

further oxidize wine constituents. It was found that added Mn(II) does not affect the Fenton reaction

under wine conditions, but it may play a role in directly oxidizing tartaric acid.116

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

Under aquatic environments, the reaction of organic thiols and H2S with Mn(III) has been

shown to be faster than that of organic acids.74,202 It is therefore possible that these substrates may

be preferentially oxidized even in the presence of excess tartaric and malic acids. Based on recent

work on the interaction of Fe, Cu, and Mn in wine oxidation, it would be of interest to investigate

the possible catalytic action of Mn in mediating the oxidation of thiols and H2S and its interaction

with Fe and Cu in wine conditions.


4.3.1 Chemicals

4-methylcatechol (4-MeC), L-Cysteine (Cys), 6-sulfanylhexan-1-ol (6SH), and

manganese(II) sulfate monohydrate, and iron(II) sulfate heptahydrate were obtained from Sigma-

88

Aldrich (St. Louis, MO). 2,4-Dinitrophenylhydrazine (DNPH) was purchased from MCB

laboratory chemicals (Norwood, OH), and L-tartaric acid and 5,5’-dithiobis(2-nitrobenzoic acid)

(DTNB) were obtained from Alfa Aesar (Ward Hill, MA). Copper(II) sulfate pentahydrate was

purchased from EMD Chemicals (Gibbstown, NJ), and sodium hydrosulfide hydrate (as a source

of H2S) was purchased from Acros Organics (Geel, Belgium). Iron(III) chloride hexahydrate was

purchased from Mallinckrodt Chemicals (St. Louis, MO). Water was purified through a Millipore

Q-Plus system (Milipore Corp., Bedford, MA). All other chemicals and solvents were of analytical

or HPLC grade and solutions were prepared volumetrically, with the balance made up with Milli-

Q water unless specified otherwise.

4.3.2 Model Wine Experiments




For H2S and Cys, an aqueous stock solution of each (approximately 0.4 M) were freshly

prepared, whereas 6SH was added directly by syringe during experimentation. Aqueous stock

solutions of Cu(II) sulfate (~50 mM), Fe(II) sulfate (~50 mM), Fe(III) chloride(~200 mM), and

Mn(II) sulfate (~200 mM) were freshly prepared. For Mn experiments, Mn(II) (100 μM) was added

to air saturated model wine containing H2S, 6SH, or Cys treatments (1 L, 150 μM each) and

thoroughly mixed. An additional treatment was prepared with Cys containing 4-MeC (1 mM) prior

to the addition of Mn(II). For Mn and Fe combination experiments, Mn(II) (100 μM) and Fe(III)

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

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

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

89

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


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.


Glass B.O.D. bottles were fitted with PSt3 oxidots and oxygen readings were taken per

time point using a NomaSense O2 P6000 meter (Nomacorc LLC, Zublon, NC). Initial O2

concentrations ranged from 6.6 – 7.0 mg/L. Further details were reported in Chapter 2.

4.3.4 Spectrophotometric measurements

UV-vis spectra of the treatments were recorded at each time point using 10 mm quartz

cuvettes (model wine blank) and measured using Agilent 8453 UV-Vis spectrophotometer

(Agilent, Santa Clara, CA). Determination of Fe(III) concentration was achieved by measurement

of absorbance at 336 nm associated with the Fe(III)-tartrate complex.197

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

Further details were reported in Chapter 2.

90

4.3.5 HPLC Analyses


dinitrophenylhydrazone (DNPH) derivative with an external standard curve (10 – 220 μM) by

HPLC as described in Chapter 2.

Oxidized species formed by the reaction of 6SH were monitored using LC-MS/MS. Mass

spectra were obtained using ESI- and ESI+ and full scan between m/z 100-1000. The compounds

were also monitored by UV absorbance at 210 nm. Mobile phases consisted of 0.1% v/v formic

acid (A) and 0.1% v/v formic acid in acetonitrile (B) with a linear gradient according to the

following program: 0 min, 5% B; 8 min, 95% B; 10 min, 95% B; 10.1 min, 5% B; 12 min, 5% B.

The ESI capillary spray voltage was set to 4 kV, the sample cone voltage was 25 V, the source

temperature was 120 °C, and the desolvation gas flow was 650 L/h. ESI- with multiple reaction

monitoring was utilized for detection of the 6SH-sulfonate using the same parameters described

above and collision energy of 20 eV. The 6SH-sulfonate was monitored at m/z 181→81.


4.4.1 Reaction of Cys with Mn

When Cys (150 μM) was oxidized in the presence of Mn(II) (100 μM) in air-saturated

model wine, it was found that the consumption of Cys (118 µM) was accompanied with a large O2

(208 µM) uptake (Figure 4.3A and 4.3B). As with sulfite autoxidation (Figure 4.1), Mn(II) would

have to be oxidized for the process to proceed. It seems likely the oxidation of Cys is also initiated

by traces of iron contaminating the model wine used in this study. However, Fe(III)-mediated

oxidation of Cys does not appear to generate free thiyl radicals in model wine (Chapter 3). It is

91

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) O2 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 MnO2 mediated thiol oxidation and dissolution of

the polymeric complex suggest that oxygen in MnO2 is displaced by thiols, resulting in

Mn(IV)SR.74 Subsequent intra-molecular electron transfer generates Mn(III)OH and a thiyl

radical.74 The resulting Mn(III)OH complex, which may be analogous to Mn(III)-tartrate in wine,

92

readily co-ordinates with thiols and the resulting Mn(III)SR quickly dissociates releasing Mn(II)

and a thiyl radical.74 Mn(III) could directly oxidize tartaric acid, but the reaction rate between

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

During the oxidation of Cys, there was an initial induction period (approximately 8 hr) in

which minimal Cys and O2 were consumed (Figure 4.3A and 4.3B). Presumably, during this time

build-up of reactive intermediates could have occurred, similar to sulfite autoxidation.201 Oxygen

was quickly consumed until the system became anoxic, containing less than 50 μg/L (~1.5 µM) O2.

After approximately 120 h, 118 µM of Cys were consumed along with 208 µM O2, giving a O2:Cys

molar ratio of ~1.8:1. This overall molar ratio suggests that a large amount of O2 adds to Cys,

resulting ultimately in the formation of cysteine sulfonic acid, but other oxidation products could

include disulfides and sulfinic acids.203

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

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

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

generate the sulfonyl peroxyl radical (RSO2OO•), which is a very strong oxidant,203,204 and could

oxidize Mn(II). The sulfonyl peroxide (RSO2OOH) would be generated, which could undergo

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

demonstrated that H2O2 is not a sufficiently strong oxidant to oxidize Mn(II),116 however, RSO2OO•

could be capable of oxidizing Mn(II) to Mn(III). HO• radicals would in turn abstract hydrogen from

ethanol to yield a hydroxyethyl radical (1-HER), finally producing acetaldehyde (AC).

93

Figure 4.4. Proposed mechanism of Mn(III)-catalyzed radical chain reactions of thiols in air

saturated model wine resulting in thiyl radical intermediates which subsequently oxygen and

ethanol.

There are difficulties associated with measuring initial thiyl radical generation by Mn-

mediated oxidation, but there has been some indirect evidence through sulfur addition products to

double bonds.205 The thiyl radicals may dimerize to yield a disulfide, and this would be the case if

the Mn(II)-thiol radical complex polymerized and disulfide formation occurred in situ as is the case

in Cu(II)- and Fe(III)-mediated thiol oxidation (Chapters 2 and 3). However, this was not the

outcome in the case of Mn(III). It would appear that the free thiyl radical is released, which quickly

reacts with O2 as discussed above. The possibility that Cys coordinates with Mn(II) to catalyze the

reduction of O2 was considered, but it is not expected that the thiolate group will bind to Mn(II) at

wine pH,206 similar to Fe(II).109,110

The reaction observed for Mn is unlike that which was observed in the previous studies

focusing on Fe and Cu mediated oxidation, which appeared to result in a concerted oxidation of

sulfhydryls and the generation of the corresponding disulfide (Chapters 2 and 3). No evidence was

found for the formation of thiyl radicals or subsequent formation of sulfonic acids in these latter

systems described. However, Mn(III)-mediated oxidation shows convincing, yet indirect evidence

for the formation of thiyl radical which may subsequently react with O2 and eventually yield a

sulfonic acid (see results for 6SH).

Along with the O2 consumption, AC was generated (Figure 4.1C), and with 184 µM

generated it gives an AC:O2 ratio of 1:1.1. The above mechanism (Figure 4.4) would indicate a 1:2

94

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

the sulfonyl peroxyl radical (RSO2OO•), 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 O2 consumption (Figure

4.3B). As a radical scavenger, the catechol intercepts intermediate radicals and, as in sulfite

autoxidation, prevents radical chain propagation. Consequently, Mn alone should not catalyze thiol

oxidation in wine, where polyphenols are present. A more detailed examination of the Mn-

catalyzed reaction products was therefore not undertaken.

4.4.2 Reaction of Cys with Mn+Fe

When Mn(II) and Fe(III) (100 µM each) were combined there was a longer induction

period compared to Mn(II) alone, which could be explained by the presence of a large excess of

Fe(III), which would delay Fe(II) oxidation.197 Despite the longer induction period, it appears that

overall molar ratios in the presence of Fe(III) remained similar (Figures 4.3A-C): 201 µM of O2

was consumed along with 118 µM Cys, giving a total of 1.7:1 O2:Cys molar reaction ratio. This

again would suggest that O2 is incorporated into the Cys molecule to form cysteine oxyanions,

presumably with cysteine sulfonic acid being a major product.

The Fe(III)-tartrate absorbance was also measured (Figure 4.3D), and approximately 15%

of Fe(III) had been reduced shortly after initiating the reaction. The concentration started to

decrease at the last time point (168 h) and approximately 18% of Fe(III) was reduced to Fe(II),

presumably due to the absence of O2 at that point with residual Cys reducing Fe(III). The measured

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

95

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

AC directly, especially when O2 concentrations were low.

Although the presence of traces of iron was thought necessary to initiate Mn(II) oxidation

to Mn(III) (Figure 4.2), an addition of a small (10 µM) amount of Fe(II) to the solution along with

Mn(II) was investigated to see its effect on the induction period. However, the results were similar

to that of Mn(II) alone (Figure 4.3B), which suggests that the reduction of trace amounts of Fe(III)

to Fe(II) by Cys is not the rate limiting step for the initial reactive intermediate buildup.

4.4.3 Reaction of Cys with Mn+Fe+Cu

When Cu(II) (25 µM) was added along with Fe(III) and Mn(II) (100 µM each), there was

a rapid consumption of Cys with small amount of O2 uptake (Figures 4.3A and 4.3B). Based on

previous work, it would be expected that Cu(II) would be rapidly reduced by Cys to Cu(I), which

would subsequently reduce Fe(III), cycling Cu(II) until all Fe and Cu are reduced (Chapter 3). The

concentration of Cys was increased from 150 µM to 200 µM to account for the initial rapid uptake

of 150 µM Cys, and to allow subsequent oxidation to be monitored.

After initiating the reaction, the majority of Fe(III) (80%) was reduced to Fe(II) within 5

min (Figure 4.3D), which was paired with the reaction of 133 µM Cys and minimal O2

consumption (Figures 4.3A and 4.3B). The initial and subsequent reaction appear to be dominated

by the presence of Cu, preventing Mn-mediated thiol oxidation and subsequent radical formation.

After 48 h, 184 μM Cys was consumed along with the 74 μM O2 consumed to give ~1:2.5 O2:Cys

molar ratio. This ratio was slightly lower but consistent with that of Fe+Cu system, which resulted

in a ~1:2.7 O2:Cys molar ratio (Chapter 3). A total of 46 μM of AC was generated (Figure 4.3C),

giving a AC:O2 molar ratio of ~1.6:1, which was lower than the ~2:1 ratio observed in the Fe+Cu

system alone.

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It appears that with Mn(II) alone or Fe(III)+Mn(II), Mn promotes the generation of

cysteinyl radicals which quickly react with oxygen and result in large O2 uptake (Figure 4.4).

However, when Cu is present it appears to dominate and oxidation reverts to the Cu catalyzed

mechanism that would yield disulfide. Nonetheless, it does appear that the presence of Mn(II)

catalyzed the reoxidation of Fe(II) in the presence of O2, as observed by the fast reoxidation of

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

4.4.4 Reaction of 6SH

Previous work on Fe(III)-mediated oxidation of 6SH showed that the reaction proceeded

extremely slowly, which would affect Fe(II) generation (Chapter 3). Consequently, the oxidation

of 6SH was found to proceed relatively slowly with Mn(II) (Figure 4.5A-C). This may indicate

the importance of Fe(III) reduction to Fe(II) and subsequent formation of the Fe(III)-superoxo

complex to generate Mn(III) and drive the reaction forward (Figure 4.2). The Mn(II)-catalyzed

oxidation of Cys is much faster than that of 6SH, and may be explained by the greater ability of

Cys to reduce Fe(III).

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Figure 4.5: Reaction of 6SH (150 or 200 μM) with Mn(II) (100 μM), Fe(III) (100 μM), and Cu(II)

(25 μM) in air saturated model wine. (A) 6SH consumption, (B) O2 consumption, (C) acetaldehyde

generation, and (D) %Fe(III)-tartrate based on absorbance at 336 nm. Error bars indicate standard deviation of triplicate treatments.

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

(Figures 4.5A and 4.5B). In the case of Mn(II)-mediated oxidation, which is expected to be

initiated by trace iron contamination, approximately 14 μM O2 and 23 μM of 6SH were consumed

over a 192 h period. This resulted in a ~1:1.6 O2:6SH ratio, which is lower than that observed with

Cys (Figure 4.3). This perhaps indicates that not as much O2 is incorporated into the thiol. A small

amount of AC (7 μM) was generated, which would correspond AC:O2 molar ratio of 1:2.

When Fe(III) (100 μM) was added along with Mn(II) (100 μM), the reaction proceeded

more quickly (Figures 4.5A and 4.5B), indicating a synergistic effect between the metals, unlike

the case of Cys (Figure 4.3A). There was a total consumption of 46 μM O2 and 55 μM 6SH. This

resulted in a ~1:1.2 O2:6SH ratio, which is higher than that with Mn(II) alone. AC (49 μM) was

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

Cys. Monitoring the Fe(III)-tartrate concentration over time indicated that virtually all Fe remained

as Fe(III) throughout the experiment (Figure 4.5D). Evidently any Fe(II) generated was rapidly re-

oxidized.

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

(100 µM) and Cu(II) (25 µM) compared to the other two metal combinations (Mn or Mn+Fe). With

the Fe+Mn+Cu combination, 65 µM of O2 was consumed with 189 of 6SH within 72 h, giving a

1:2.9 O2:6SH molar reaction ratio (Figure 4.5A and 4.5B). AC (58 µM) was also produced giving

a ~1.1:1 AC:O2 molar reaction ratio (Figure 4.5D). These ratios, which are similar to those obtained

with the Fe+Cu system (Chapter 3) indicate that Cu catalysis dominated in the presence of Mn,

which was similarly observed for the Cys system. The low O2 uptake relative to thiol oxidation

points to the disulfide being the main product and that the O2 is reduced to H2O2 to produce an

equivalent of AC.

Mn(II) (100 µM) alone produced a slow oxidation of 6SH (150 µM), with a 1:1.6 O2:6SH

molar reaction ratio. The reaction is accelerated by Fe(III) (100 µM) with a 1:1.2 O2:6SH molar

reaction ratio. The higher O2 uptake relative to that of the Cu containing system (1:2.9 O2:6SH

molar reaction ratio) points to the formation of oxyanions as with Cys. Clearly, Fe and Mn interact

as Fe(III) (200 µM) alone does not catalyze the oxidation of 6SH (Chapter 3).

The oxyanion products of 6SH were analyzed by LC-MS. Using MS/MS, the 6SH-sulfonic

acid was observed near the column void volume in the Mn+Fe system (Figure C.1), whereas it was

not present in the initial 6SH stock or Mn+Fe+Cu mediated oxidation. Furthermore, it was observed

that several oxidized disulfide species were formed including thiol-sulfinate, thiol-sulfonate,

sulfinyl-sulfone, and α-disulfone (Figure C.2) in the Fe+Mn system. The same species were

observed in the Mn-only system except for the α-disulfone, presumably due to the relatively slow

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reaction and insufficient concentration for detection. In the Mn+Fe+Cu system, the thiol-sulfinate

was observed, but this was a smaller response than the other two systems, despite the higher

consumption of 6SH. This may indicate that Mn(III)-mediated oxidation did occur to some extent,

but the disulfide due to Cu- mediated oxidation was still deemed to be the major product in the

system as discussed above.

Several mechanisms could be proposed to explain how these oxidation products arise; it is

possible that the 6SH-sulfinate was one of the predominant intermediates that can then

disproportionate to the various observed oxidation products.144 However, as with Cys, the

conditions in which O2 is present in large excess to form sulfur oxyanion species is unlikely under

real wine conditions. Nonetheless, the thiyl radical is the likely precursor for the formation of these

products. Furthermore, the formation of a glutathione-hydroxycinnamic acid product has been

observed and proposed to be initiated by the glutathione thiyl radical.135

4.4.5 Reaction of H2S

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

O2 consumption or appreciable amount of H2S consumed (Figures 4.6A and 4.6B). It would be

expected that trace contamination by Fe would be present in this system as well, resulting in

generation of trace amounts of Mn(III). However, unlike Cys and 6SH, H2S can be considered a

sulfhydryl compound capable of donating two electrons. Furthermore, the generation of a

hydrosulfide radical would be thermodynamically unfavorable and it would quickly react with

metals to either reform H2S or lose an electron to form elemental sulfur.207 The reduction of Mn(III)

by H2S likely proceeds through an inner-sphere mechanism. In this process, two equivalents of

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

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

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trace amounts of Fe, which would be capable of oxidizing Mn(II), and the fact that H2S would not

result in buildup of reactive intermediates, Mn-mediated oxidation of H2S does not occur under the

conditions described. Similarly, no AC was generated (Figure 4.6C).

Figure 4.6. Reaction of H2S (150 or 200 μM) with Mn(II) (100 μM), Fe(III) (100 μM), and Cu(II)

(25 μM) in air saturated model wine. (A) H2S consumption, (B) O2 consumption, (C) acetaldehyde

generation, and (D) %Fe(III)-tartrate based on absorbance at 336 nm. Error bars indicate standard

deviation of triplicate treatments.

When Fe(III) (100 µM) was added in combination to Mn(II), H2S was slowly consumed

over time, along with O2 (Figures 4.6A and 4.6B). Presumably, the interaction occurs in the same

manner as described previously with Fe(III) whereby H2S reduces two equivalents of Fe(III) to

Fe(II) with its oxidation to S0 (Chapter 3). Over time, Fe(II)-tartrate reduces O2, resulting in

generation of H2O2, and subsequent Fenton reaction to generate hydroxyl radicals. Overall, 54 µM

of O2 were consumed in conjunction with 84 µm of H2S, giving an O2:H2S molar ratio of ~1:1.6,

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which is consistent with the results for Fe alone (Chapter 3). This result, along with Mn-only

oxidation, suggests that the reaction is driven primarily by Fe in this system. However, Mn does

play a role in reoxidizing Fe(II), and the relative amount of Fe(II) in the system was much lower

than with Fe-alone (Figure 4.6D and Chapter 3).

When Cu(II) (25 µM) was added along with Mn(II) and Fe(III) to H2S (200 µM), the results

were similar to those obtained with the Fe+Cu combination (Chapter 3). During the process, there

was a fast initial uptake of H2S, with approximately 58 µM consumed within 5 min. At the end of

the reaction at around 120 h, there was 115 µM O2 consumed along with 180 µM of H2S (Figures

4.6A and 4.6B), again resulting in a ~1:1.6 O2:H2S molar reaction ratio. Therefore, it would appear

that the addition of Mn to the H2S system does not alter the course of the reaction. H2S is likely

oxidized to S0 and the reduced metals are re-oxidized by O2.207 However, if other thiols were also

present, it would be expected that polysulfanes would be formed (Chapters 3 and 5). Mn(II) seems

to play an important role in oxidizing Fe(II), as virtually all Fe was re-oxidized at the end of the

reaction, whereas in the Fe+Cu system approximately 40% of Fe(II) remained reduced at the end

of the reaction (Figure 4.6D, Chapter 3). Approximately 53 µM of acetaldehyde was generated

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

4.5 CONCLUSIONS

Mn(II) was found to catalyze Cys and 6SH oxidation with high O2 consumption relative to

that of the thiol. It is proposed, therefore, that thiyl radicals are released and subsequently add O2

to produce sulfur oxyanions. It may be concluded that Mn(II)-catalyzed oxidation is a radical chain

reaction initiated by traces of Fe, in a similar manner to sulfite autoxidation. Consequently, 4-MeC

was found to inhibit the Mn(II) catalyzed reaction, presumably by intercepting intermediate radicals

so preventing radical chain propagation.

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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 H2S and occurs without O2 consumption and likely generates S0. When present, O2 is

reduced by Cu(I) or Fe(II) to produce H2O2, which undergoes the Fenton reaction to generate AC.

When Fe, Mn and Cu are combined, the catalytic activity of Cu dominates so that thiol oxidation

by Cu(II) occurs with minimal radical formation. Therefore, Mn(II) alone should not catalyze thiol

oxidation in wine. Nonetheless, Mn(II) appears to promote reoxidation of Fe(II); whether Mn is

capable of specifically catalyzing thiol oxidation needs to be investigated further using a more

complete model wine system and in real wines.

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Chapter 5

Investigating Volatile Sulfur Compound Precursors and Practical

Applications

5.1 ABSTRACT

The addition of Cu(II) to model systems containing H2S and thiols demonstrated the

generation of polysulfanes, rather than simply forming insoluble Cu(II)S as previously assumed. It

was therefore of interest to investigate the formation of mixed disulfides and polysulfanes in model

and white wine samples. It was found that at relatively low concentrations of H2S and methanethiol

(MeSH) (100 µg/L of each), Cu(II)-fining resulted in the generation of MeSH-glutathione disulfide

and trisulfane in white wine as determined by qTOF LC/MS. The reduction of the resulting non-

volatile disulfides may then play a role in the recurrence of undesirable sulfidic odors. Therefore,

the ability of Cu(II) and bisulfite (SO2), ascorbic acid, and cysteine to promote the catalytic scission

of diethyl disulfide (DEDS) was investigated. It was found that the combination of SO2 along with

Fe and Cu depleted more DEDS than the other treatments. Furthermore, a method for releasing

volatile sulfur compounds from their precursors as a diagnostic test was investigated using tris(2-

carboxyethyl)phosphine (a reducing agent) and bathocuproine disulfonic acid (a chelator). The

addition of the reagents successfully released H2S and MeSH from red and white wines that were

free of reductive faults at the time of addition.

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5.2 INTRODUCTION

Sulfidic off-odors in wine have been a serious quality issue for decades and, when detected

in the course of winemaking, are generally controlled by sparging, aerative pump overs, splash

racking, and/or the use of copper fining.2,41,90 Chapters 2 – 4 of this dissertation focused heavily on

elucidating the initial mechanisms of oxidation responsible for removal of these undesirable

compounds using H2S and model thiols. It was found that the addition of Cu(II) oxidized thiols to

disulfides and the presence of H2S together with thiols resulted in polysulfanes as a result of

oxidation. Furthermore, the complete loss of aroma – but not necessarily redox activity – occurs

when thiols and H2S are bound to a metal complex as Cu(I)-SR. It is therefore apparent that the

volatile sulfur compounds (VSCs) are not readily removed from wine in an insoluble complex that

can be filtered, but rather generate redox active compounds that remain in the wine as soluble

components.

In the post-bottling period, in which a wine is assumed to be free of faults, it has been well

established that wine may accumulate undesirable sulfidic odors during the aging period, especially

when O2 ingress is limited.47,48,186 There have been numerous studies suggesting that the most

common VSCs responsible for post-bottling reductive aroma are H2S, MeSH, and dimethyl sulfide

(DMS).50,57,70 There have been several hypotheses proposed to explain the mechanism(s) that

underlie the generation of these sulfidic off-odors; these include bisulfite reduction,209 thioacetate

and thioether hydrolysis,41 and sulfidic off-aroma generation from strecker degradation of sulfur-

containing amino acids.71,178 Another well accepted hypothesis is the reduction of symmetrical

disulfides of MeSH and ethanethiol (EtSH), which typically have 10-50-fold higher sensory

detection thresholds than their respective free thiols.1,185 However, the rates that influence these

reactions, and their relevance under wine conditions remain unknown.

105

Recent work has suggested that upwards of 99% H2S and ~70% MeSH may be effectively

bound with transition metals (e.g., Cu, Fe, Zn) in wine, and that accelerated anaerobic aging results

in the release of these complexes.80,81 From the work described in chapters 2 – 4 of this thesis, it is

apparent that Cu(I)-SR complex generation is fast. Although this mechanism has not been studied

under anaerobic conditions, Cu(I)-SR is unlikely to easily react or oxidize in the absence of O2.

Nonetheless, disulfides and polysulfanes are generated in the presence of H2S and thiols in the

initial Cu(II) fining process with no O2 uptake. Subsequent oxidation of Cu(I)-SR upon O2 ingress

likely results in further generation of disulfides and polysulfanes.

It was therefore of interest to further investigate the generation of disulfides and

polysulfanes under real wine conditions, and to examine how they may contribute to reduced off-

odors in wine. Given that thiols typically have lower detection thresholds compared to their

corresponding disulfides, and that mixed disulfides may have no perceptible odor, the release of

free thiols via disulfide reduction or scission reactions could result in reductive odors becoming

apparent in a wine that had previously been free of faults. This was examined for diethyl disulfide

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

Furthermore, working under the assumption that metal complexes and

disulfides/polysulfanes play a crucial role as potential precursors for these sulfidic odors, a method

for their quick release has been developed and validated with the ultimate goal of informing

winemakers if their product is susceptible to reductive off-aromas in the post-bottling period. This

would afford them the opportunity to take steps to control this – for example, through proper bottle

closure selection.

106


5.3.1 Materials

L-Cysteine (Cys), L-cystine, ethanethiol (EtSH), diethyl disulfide (DEDS), sodium

thiomethoxide (as a source of MeSH), ferrous sulfate hexhydrate, tris(2-carboxyethyl)phosphine

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

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

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

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

(as a source of H2S) was purchased from Acros Organics (Geel, Belgium). Iron(III) chloride

hexahydrate was purchased from Mallinckrodt Chemicals (St. Louis, MO). Water was purified

through a Millipore Q-Plus system (Milipore Corp., Bedford, MA). All other chemicals and

solvents were of analytical or HPLC grade, and solutions were prepared volumetrically, with the

balance made up with Milli-Q water unless specified otherwise.

5.3.2 Preparation of model wine and real wine samples

5.3.2.1 Disulfide and polysulfane generation




Either glutathione (GSH, 500 µM) or cysteine (Cys, 500 µM) were added to model wine

and mixed thoroughly. H2S (250 µM) and/or MeSH (250 µM) were subsequently added to the

107

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

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

Fe(III) (100 µM) and Cu(II) (50 µM) were subsequently added and thoroughly mixed. The

solutions (25 mL) were stored in the dark in capped 50 mL capacity polypropylene tubes under air.

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

Commercial white wine blend was purchased locally to which GSH was added to achieve

a final concentration of 50 µM. H2S and MeSH were subsequently added to achieve the following

three treatment concentrations: 100 µg/L, 500 µg/L, and 5000 µg/L. Following the addition of the

sulfhydryl-containing compounds, Fe(III) (5 mg/L) and Cu(II) (1 mg/L) were added and the

resulting solutions were mixed thoroughly. The samples (100 mL) were stored in the dark in

stoppered 100 mL volumetric flasks and analyzed the following day by HPLC-QTOF.

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

Model wine was prepared as described above; however, cystine (400 µM) was added prior

to pH adjustment for this experiment and mixed until it dissolved. Afterwards, sample aliquots (~30

mL) were adjusted to pH 2, 3, 4, 5, or 11 using hydrochloric acid (5 M) or sodium hydroxide (10

M). Following pH adjustment, BCDA (1 mM) was added followed by Cu(II) (100 µM). A control

sample was prepared which contained only BCDA (1 mM) and Cu(II) (100 µM) over the pH range

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

(1mM), and Cu(II) (100 µM) over the pH range described above. The samples were analyzed over

time for BCDA-Cu(I) generation as described below. Experiments were conducted in triplicate.

108

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

Model wine (pH 3.6) was prepared as described above and deoxygenated with argon until

the dissolved oxygen concentration fell below 50 µg/L as measured by a NomaSense O2 P6000

meter (Nomacorc LLC, Zublon, NC) equipped with a dipping probe. Following deoxygenation,

model wine solutions were transferred to an anaerobic chamber to equilibrate overnight. The

following day, diethyl disulfide (DEDS, 50 µg/L) was added from a stock solution by syringe to

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

(AA), Cu(II) sulfate, and Fe(II) sulfate were added from freshly made stock solution to yield final

concentrations outlined in Table 5.1.

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

Treatment Cys SO2 AA Cu(II) Fe(II)

T1 - - - - -

T2 - - 50 mg/L - - T3 - 50 mg/L - - -

T4 - - - 1 mg/L 5 mg/L

T5 12 mg/L - - 1 mg/L 5 mg/L T6 - - 50 mg/L 1 mg/L 5 mg/L

T7 - 50 mg/L - 1 mg/L 5 mg/L

T8 12 mg/L 50 mg/L 50 mg/L 1 mg/L 5 mg/L

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

Biological Oxygen Demand (B.O.D.) bottles (Wheaton, Millville, NJ), allowing the solution to

overflow, at which point the bottles were immediately capped with ground glass stoppers in order

to completely eliminate headspace. The glass reservoir of the B.O.D. bottles was topped off with

water and covered with 2 layers of parafilm and aluminum foil to prevent evaporation. The bottles

were covered in aluminum foil and stored at 40 °C. One B.O.D. bottle was sacrificed per time point

and used for further GC analysis as described below. Samples were prepared by transferring 1 mL

of sample aliquot into a 20 mL amber GC vial containing 9 mL of saturated brine (350 g/L NaCl)

109

and capped immediately based on previously described methodology in order to release metal-thiol

complexes.80 Experiments were conducted in duplicate.

5.3.2.4 Release and reduction of bound VSCs

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

8 mg/L) or in an anaerobic chamber (dissolved [O2]: <100 µg/L). The model wine was spiked with

a combination of H2S (100 µg/L), MeSH (100 µg/L), and EtSH (100 µg/L). One sample aliquot

was transferred to a 60 mL B.O.D. bottle and capped without headspace using the procedure

described above. The remaining sample fraction was spiked with Cu(II) sulfate (1 mg/L) and the

resulting solution was transferred to a B.O.D. bottle and stored overnight. The following day, 10

mL sample aliquot of the control was transferred to a 20 mL amber GC vial and capped

immediately. Sample aliquots (10 mL) of the Cu(II) sulfate-containing sample were transferred to

five 20 mL amber GC vials. One sample was used as a positive control (i.e. no reagents added) and

capped. The other four treatments included: TCEP (tris(2-carboxyethyl)phosphine, 1 mM), BCDA

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

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

The experiments were conducted in triplicate.

Six commercial Pennsylvania wines were obtained locally. The bottles were opened, and ca. 50

mL of wine were carefully transferred to beakers using a serological pipette while taking care to

avoid agitation, and these aliquots were immediately transferred to an anaerobic chamber. One 10

mL sample aliquot was used as a control for determination of free VSCs in the original wine

samples. The other four treatments (TCEP, BCDA, TCEP+BCDA, TCEP+BCDA+Cys) were

prepared as described above.

110

5.3.3 Methods of analysis

5.3.3.1 HPLC

Samples (5 µL) were separated by reversed-phase HPLC using a Prominence 20 UFLCXR

system (Shimadzu, Columbia MD) with a Waters (Milford, MA) BEH C18 column (100 mm × 2.1

mm, 1.7 µm particle size) maintained at 55 °C and a 20 minute aqueous acetonitrile gradient, at a

flow rate of 250 µL/min. Solvent A was HPLC grade water with 0.1% formic acid and Solvent B

was HPLC grade acetonitrile with 0.1% formic acid. The initial mobile phase conditions were 97%

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

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

(QTOF) MS with Duospray™ ion source (AB Sciex, Framingham, MA) using electrospray

ionization (ESI) conditions. The ESI capillary voltage was set at 5.5 kV in positive ion mode or 4.5

kV in negative ion mode, with a declustering potential of 80 V. The mass spectrometer was operated

in Information-Dependent Acquisition (IDA ) mode with a 100 ms survey scan from 100 to 1200

m/z, and up to 20 MS/MS product ion scans (100 ms) per duty cycle using a collision energy of 50

eV with a 20 eV spread.

5.3.3.2 GC

Samples were analyzed using an Aglient 5890 gas chromatograph (Santa Clara, CA)

equipped with a Gerstel MPS2 autosampler (Linthicum, MD) and coupled to a pulsed flame

photometric detector (PFPD). Instrument control and data analysis were performed with Agilent

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

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

111

was 35 °C, which was held for 3 min, and then ramped to 100 °C at a rate of 10 °C/min, and finally

ramped to to 220 °C at a rate of 20 °C/min. The programmable temperature vaporizer (PTV) inlet

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

TX, USA) detector was maintained at 250 °C using the default flow rates suggested by the

manufacturer. Emission was monitored from 6 to 24.9 msec.

The samples were stored in a cooled sample tray at 4 °C. The vial was incubated at 60 °C

for 10 min with agitation at 500 rpm. Using a Gerstel 1.0 mL headspace (HS) syringe kept at 60

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

1:2 split ratio.

5.3.3.3 UV-Vis

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

Standard solution consisted of excess Cys (5 mM), which was added in order to ensure that Cu(I)

remained in its reduced state. An external standard curve of the Cu(I)-BCDA complex was prepared

in model wine, and absorbance values were recorded at 484 nm using a 10 mm quartz cuvette

against a model wine blank. The baseline measurements of the control samples were subtracted

from the treatment samples for each pH value.


5.4.1 Disulfide and polysulfane generation

We showed that Cu(II) fining results in near immediate Cu(II) reduction along with

oxidation of H2S and thiols in Chapter 2 of this thesis. We subsequently showed that the oxidation

112

of 6-sulfanylhexan-1-ol (6SH) and H2S resulted in the formation of 6SH polysulfane with up to 5

linking sulfur atoms between the 6SH molecules in Chapter 3. With this knowledge, we

investigated whether mixed disulfides and polysulfanes could be formed with wine relevant thiols.

Two non-volatile thiols, Cys and GSH, were used in these experiments as they represent the major

fraction of free sulfhydryl functionality in wine and are typically present at concentrations that far

exceed those of VSCs. MeSH and H2S, which are two of the primary sulfhydryl-containing

compounds associated with sulfidic off odors in wine were also added. Fe(III) and Cu(II) were then

added to mimic copper fining and wine oxidation. Although these experiments were conducted

under air, it is expected that the initial oxidation reaction of the sulfhydryls paired with Cu(II)

reduction will occur in the same manner as would be expected in the absence of O2. The

concentrations of sulfhydryls used in this model system far exceed those found in wine, but were

used to readily assess and detect oxidation products.

Test solutions were allowed to oxidize overnight, after which point they were analyzed

using LC-Q-TOF. Cys polysulfanes were observed up to n=6 (Table 5.2) for the treatment

containing the combination of Cys+H2S. Similarly, the oxidation of the GSH+H2S combination

treatment resulted in GSH polysulfanes up to n=7 (Table 5.3). When MeSH was added along with

H2S, the symmetrical polysulfanes for Cys and GSH (Tables 5.2 and 5.3) were formed, and the

presence of the mixed disulfide and polysulfanes was also readily observed. In the case of Cys,

Cys-MeSH disulfide and polysulfanes were observed up to n=6 (Table 5.4), and GSH-MeSH was

observed up to n=8 (Table 5.5). The corresponding spectrum can be found in the appendix (Figures

D.1 – D.4). The Cu(II)-mediated oxidation process results in disulfides, but it is clear that it does

not result strictly in the generation of symmetrical disulfides. Furthermore, it appears that when

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

polysulfanes.

113

Table 5.2. Cys-polysulfanes identified by LC-QTOF after reacting Cys (500 µM) and H2S (250

µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model wine.

S (n) Molecular formula

M+H monoisotopic mass

Retention time (min)

S/N ratio

Intensity (ion count)

1 C3H7NO2S 122.027 ± 0.005 0.99 1027.4 52270

2 C6H12N2O4S2 241.031 ± 0.005 0.99 6820.7 685100

3 C6H12N2O4S3 273.003 ± 0.005 0.99 3737.2 319400

4 C6H12N2O4S4 304.975 ± 0.005 1.22 39805.8 190900

5 C6H12N2O4S5 336.947 ± 0.005 2.38 203.6 9045

6 C6H12N2O4S6 368.919 ± 0.005 3.41 47.4 612.2

Table 5.3. GSH-polysulfanes identified by LC-QTOF after reacting GSH (500 µM) and H2S (250

µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model wine.




S/N ratio Intensity (ion count)

1 C10H17N3O6S 308.091 ± 0.005 1.28,1.42 4650, 1855 2308000, 1180000

2 C20H32N6O12S2 613.159 ± 0.005 1.29, 1.49,

1.66

6070.9, 3741.3,

6289.6

2166000, 1019000,

1143000

3 C20H32N6O12S3 645.131 ± 0.005 2.29, 2.51 6033.1,

13107.5

1382000, 1413000

4 C20H32N6O12S4 677.103 ± 0.005 3.46 8178.4 634300

5 C20H32N6O12S5 709.075 ± 0.005 4.25 1150.6 28550

6 C20H32N6O12S6 741.043 ± 0.005 5.1 161.4 1513

7 C20H32N6O12S7 773.020 ± 0.005 5.87 27.2 67.57

Table 5.4. Mixed Cys-MeSH disulfide and polysulfanes identified by LC-QTOF after reacting Cys (500 µM), H2S (250 µM), and MeSH (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air

saturated model wine.




S/N ratio Intensity (ion count)

2 C4H9NO2S2 168.015 ± 0.005 1.48 2683.8 201400

3 C4H9NO2S3 199.987 ± 0.005 3.1 3843.1 134200

4 C4H9NO2S4 231.959 ± 0.005 4.68 1154.5 31140

5 C4H9NO2S5 263.931 ± 0.005 6.27 805.5 6398

6 C4H9NO2S6 295.903 ± 0.005 7.75 146.7 915

114

Table 5.5. Mixed GSH-MeSH disulfide and polysulfanes identified by LC-QTOF after reacting

GSH (500 µM), H2S (250 µM), and MeSH (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in

air saturated model wine.

S (n) Molecular formula M+H monoisotopic mass


S/N ratio Intensity (ion

count) 2 C11H19N3O6S2 354.079 ± 0.005 3.32 68539.5 3601000

3 C11H19N3O6S3 386.051 ± 0.005 4.69 36202.7 2277000

4 C11H19N3O6S4 418.023 ± 0.005 6.03 19465.6 703200

5 C11H19N3O6S5 449.995 ± 0.005 7.33 5645.9 120200

6 C11H19N3O6S6 481.967 ± 0.005 8.48 13701.3 17660

7 C11H19N3O6S7 513.939 ± 0.005 9.5 1293.9 2361

8 C11H19N3O6S8 545.911 ± 0.005 10.47 40 337

The masses associated with the higher oxidation states of sulfur, including sulfenic,

sulfinic, and sulfonic acids, as well as oxidized disulfides, could not be observed. This may indicate

that during the process of Fe(III) and Cu(II) oxidation, free sulfur radicals are not generated to an

appreciable degree that would result in a detectable amount of sulfur oxyanions. As discussed in

Chapter 2 and 3, the sulfhydryl likely remains anchored onto the metal center during the electron

transfer oxidation process, giving disulfides and polysulfanes as the exclusive products. This also

indicates that while O2 plays an important role in the re-oxidation of the metals and in accepting

electrons via metal catalysis, O2 does not play a “direct” role in sulfhydryl-mediated oxidation in

the case of Fe(III) and Cu(II), which is unlike that of Mn(III) which results in free radical generation

and subsequent O2 uptake (Chapter 4).

The recognition that both symmetrical and asymmetrical disulfides and polysulfanes are

generated under the conditions described above is important, as winemakers generally assume the

that symmetrical disulfides are exclusively generated during wine oxidation.210 Research that has

focused on reduction of symmetrical disulfides (DMDS and DEDS) to explain the generation of

MeSH and EtSH has not found good correlation between the two.49,126,127 It is possible that large

amounts of MeSH and EtSH are, in fact, bound as non-volatile disulfides in combination with Cys

115

and/or GSH, which would not be detectable by the standard analytical practices (e.g., GC analysis)

that are typically used for VSCs.1

It was important to verify that the reactions described above are also relevant and possible

under real wine conditions. In order to test this, GSH (50 µM) was added to a commercial white

wine blend (i.e, the average GSH concentration in young Sauvignon blanc wines).211 Along with

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

100, 500, and 5000 µg/L. The wines were subsequently oxidized by the addition of Cu(II) (1 mg/L)

and Fe(III) (5 mg/L). At the highest treatment level (5000 µg/L each of H2S and MeSH), the mixed

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

(Table 5.6). At 500 µg/L, the formation of polysulfanes up to n=5 was detected. At the lowest

concentration, the peak corresponding to the mixed MeSH-GSH was apparent and the trisulfane

was detected (Table 5.6).

Table 5.6. Identified mixed GSH-MeSH disulfide and polysulfanes in white wine spiked at various

concentrations of H2S and MeSH by LC-QTOF.

H2S and MeSH added S(n)

Retention time

(min) 100 µg/L 500 µg/L 5000 µg/L

S/N ratio intensity S/N ratio intensity S/N ratio intensity

2 4.5 74.3 3732 259.6 9736 105.6 5623

3 5.9 33.2 885 280.1 8833 207.3 10800

4 7.3 - - 146 2903 245.4 7373

5 8.6 - - 36 251.5 222 2273

6 9.7 - - - - 25.5 588.8

7 10.67 - - - - 26.1 400.1

8 11.57 - - - - 11.9 126

Winemakers are advised to avoid and minimize O2 throughout the Cu(II) fining process to

prevent disulfide generation. However, we have demonstrated that the initial Cu(II) reduction will

result in inevitable formation of disulfides and mixed disulfides in a manner that is independent of

116

O2. The presence of O2 will reoxidize the metals and cause further oxidation of sulfhydryl

compounds. More realistically, MeSH will typically be present in wine at ~1 – 5 µg/L; however,

the concentration of GSH and the transition metals used here are in molar excess to MeSH, and so

the reaction is expected to be similar under wine conditions. The generation of mixed disulfides at

trace concentrations that are nonvolatile, as described here, could potentially act as precursors for

reductive odor generation post-bottling, and needs to be further investigated.

5.4.2 Disulfide scission

The mechanisms for disulfide reduction in wine, as well as the conditions and parameters

that favor this reduction, remain ambiguous. The involvement of transition metals, bisulfite, and

ascorbic acid all appear to be capable of playing a role in the redox status of sulfur compounds

(Chapter 1). It has been hypothesized that disulfide reduction in wine generates volatile thiols with

low detection thresholds; however, recent work has failed to show depletion of symmetrical

disulfides and corresponding thiol generation.49,127 As described above, mixed disulfides are

expected to form, and may play a role in thiol generation.

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

VSC in wine during the post-bottling period. It is possible that Cu and other transition metals may

be involved in disulfide bond scission via concomitant electrophilic and nucleophilic attack (Figure

1.9 – pg 39).144 In this mechanism, an electrophilic species (E+), such as Cu(II), may bind to the

disulfide bond making the overall complex more electrophilic and causing the disulfide bond to

become more susceptible towards nucleophilic attack. The nucleophilic species (Nu-) could be

water, but under wine conditions, sulfite, other thiols, and ascorbic acid may play a more important

role as nucleophiles. This reaction could potentially result in the release of potent VSCs. If Cu(II)

and a thiol behave as the electrophilic and nucleophilic species, respectively, the reaction with the

117

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).

118

0 5 0 1 0 0 1 5 0

0

5 0

1 0 0

1 5 0

T im e (h o u rs )

Cu

(I)

(M

)

p H 2

p H 3

p H 4

p H 5

p H 1 1

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 H2S and MeSH.71

The possibility that disulfides are cleaved at higher pH to generate H2S and MeSH is, therefore,

consistent with the results shown here.

One confounding factor that needs to be taken into account is that BCDA makes Cu(II) a

much stronger oxidant, driving the reaction forward in a matter of days. It may be expected that

this reaction could also occur under wine conditions in the absence of BCDA, but it would be a

much slower process over several weeks to months. The ability of other nucleophilic species (e.g.,

thiols, sulfite, ascorbic acid) to accelerate the reaction could not be tested using this protocol as

they are capable of directly reducing Cu(II) to Cu(I).

119

5.4.3 Reactivity of diethyl disulfide

To determine the practical relevance of the above described mechanism (i.e., the proposed

electrophile-assisted nucleophilic cleavage of disulfides), the reaction was further investigated

under model wine conditions. In this experiment, 50 µg/L of DEDS was used as a model disulfide.

The treatments added were common nucleophilic species in wine that included cysteine, bisulfite,

and ascorbic acid in the presence or absence Fe(II) and Cu(II) (refer to Table 5.1). The samples

were stored anaerobically at 40 °C to mimic accelerated reductive aging and were monitored over

time by GC-PFPD (Table 5.7). The samples were diluted with a strong brine prior to analysis to

release thiols from their metal complex as described previously.80 It is expected that Cu(I)SR would

be formed upon the cleavage of the disulfide.

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

Diethyl disulfide (µg/L)

Treatment Day 4 Day 8 Day 18

T1 47.9 ± 3.0 Aa 42.1 ± 2.7 Aa 45.4 ± 0.0 Aa

T2 45.9 ± 4.1 Aa 39.6 ± 2.4 Bab 41.2 ± 4.2 ABab

T3 44.4 ± 2.9 Aa 36.4 ± 3.7 Bab 36.4 ± 0.6 Bbc

T4 44.6 ± 2.9 Aa 36.4 ± 0.6 Bab 37.4 ± 0.0 Bbc

T5 40.7 ± 1.1 Aa 35.7 ± 1.0 ABab 34.7 ± 1.5 Bbc

T6 44.6 ± 0.8 Aa 35.1 ± 0.2 Bab 32.9 ± 0.7 Bc

T7 40.4 ± 1.6 Aa 32.6 ± 1.0 Bb 24.4 ± 5.0 Cd

T8 42.4 ± 2.0 Aa 36.0 ± 1.5 Bab 34.5 ± 2.8 Bbc

* Results are shown ± standard deviation of the means. Rows with different capital letters indicate

significant differences over time (p < 0.05), whereas columns with different lower case letters case

indicate significant differences between treatments (p < 0.05).

The concentration of DEDS was observed to fluctuate in the control treatment (T1) during

this experiment; however, there was no significant difference in its concentration over the 18 day

period. T2 was not significantly different than the control, but all other treatments had significantly

120

(p<0.05) lower DEDS concentration compared to control at day 18, which was particularly evident

for T7. There was no detectable concentration of EtSH generated in any of the samples.

The decrease of DEDS over time in the sample treatments could indicate disulfide scission,

however, the fact that EtSH was not detected was surprising. A possible explanation is that the

brine dilution could have brought the concentration of EtSH to below the detection limit of the

instrument. It is also possible that the generated EtSH reacted further to form the corresponding

nonvolatile mixed disulfides with Cys and organic thiosulfate with sulfite. In a previous study

where aging trials were performed with EtSH and DEDS using stable isotope dilution, it was found

that even without aeration both EtSH and DEDS concentrations were decreased.127

Sulfite was observed to play a role in decreasing DEDS concentration, with a significantly

lower value for T3 measured compared to control at day 18 (Table 5.7). Furthermore, the

combination of sulfite and transition metals (T7) were significantly lower than the control (T1) and

sulfite without metals (T3), suggesting a synergistic effect in the reaction with DEDS.

The interconversion of DEDS in the presence of sulfite (sulfitolysis) to form free EtSH and

the corresponding organic thiosulfate (Bunte salt) has been previously investigated in model wine,

and it has been claimed that ca. 700 days would be necessary to generate EtSH to a level that

exceeds the odor detection threshold.43 Simiar to thiol-disulfide interchange, sulfitolysis is a base-

catalyzed reaction and is not expected to occur to a significant degree under wine conditions.

Sulfitolysis proceeds as shown in Figure 1.8 (pg 38), with sulfite cleaving the disulfide to generate

a free thiol and corresponding Bunte salt. The Bunte salt may then undergo acid-catalyzed cleavage

to generate the corresponding free thiol and sulfate. Recent reports have shown sulfitolysis occurs

under wine conditions causing the cleavage of glutathione disulfide and cystine to generate the

corresponding Bunte salt, which appeared to be relatively stable,44 although previous work with

DEDS assumes that the rate limiting step is the formation of the Bunte salt and not its hydrolysis.43

121

Together with transition metals, the reaction could be accelerated based on the reaction depicted in

Figure 1.9 (pg 39).

Ascorbic acid alone (T2) did not result in a decrease in DEDS concentration over the 18

day period, although the combination of ascorbic acid and transition metals (T6) did cause a

significant decrease compared to the control and T2 within that same period. However, while the

value for T6 was lower than T4, this was not statistically significant and so the effect between

transition metals with or without ascorbic acid could not be differentiated. Ascorbic acid is

frequently used during bench trials to assess and compare aroma of wines in order to determine if

disulfides are present in the wine. In the trial, ascorbic acid is added in excess to release disulfides

with an incubation time of a few minutes, followed by the addition of Cu(II) sulfate to remove the

generated thiols.124 If the resulting odor disappears after the addition of Cu, the type of reductive

compound is attributed to disulfides in wine. Surprisingly, much like the copper fining practice, the

aforementioned practice has been commonplace in the wine industry for several decades, yet the

mechanism that causes the reduction under wine conditions, and the degree to which it proceeds,

remains unknown. Recent work suggests that Cu(I)SR is an important nonvolatile precursor for

releasing H2S and thiols, and that ascorbic acid may have an effect at reducing or displacing these

complexes. Based on the results described here, ascorbic acid in a simple model system is not

capable of reducing disulfides, and may require the involvement of transition metals.

Ascorbic acid may be capable of reducing disulfide bonds, and like sulfitolysis and thiol-

disulfide interchange, it appears to proceed faster under high pH conditions. The reaction likely

occurs via the involvement of the mono- and di-anion of ascorbic acid, whereas the undissociated

acid has negligible reactivity in cleaving RSSR as well as RSNO, which may have a similar reaction

pathway to the disulfide.159–161 The mechanism for ascorbate-mediated cleavage of the disulfide is

122

unknown, but it has been suggested that the presence of transition metal ions, such as copper and

iron, facilitate disulfide cleavage.159

The treatment containing Cys and transition metals (T5) was significantly lower than

control at day 18, and while it was lower than T4, this was not statistically significant (Table 5.7).

Interestingly, similar results were obtained for T8, which contained sulfite, AA, and Cys. It was

expected that the combination would play a role at further decreasing DEDS concentrations;

however, this was not the case and the decrease was inhibited compared to T7.

These results demonstrate that transition metals and sulfite play an important role in loss

of disulfides over time under wine conditions. However, the results relating to the generation of the

corresponding thiols remain inconclusive and need to be further investigated. As a simple disulfide,

DEDS may not be as reactive as mixed disulfides containing GSH or Cys with VSCs (e.g., MeSH

or EtSH), as their tridentate ability may bind to the metal more effectively and drive the reaction

forward. We have shown that the generation of these mixed disulfides is possible, and their reaction

should be investigated further. Furthermore, sulfitolysis of disulfides of either symmetrical or

assymetrical disulfides containing MeSH and EtSH may generate the corresponding Bunte salt with

MeSH and EtSH, and these compounds may be susceptible to acid-catalyzed cleavage and

subsequent release of VSCs.

Although it is not expected that polysulfanes would be generated at sufficiently high

concentrations to contribute to the generation of sulfidic off odors in wine, these species are likely

to be more reactive due to their ability to simultaneously coordinate with several sulfur atoms and,

therefore, act as a multi-dentate ligands to metal ions. The ability of metals to bind directly to the

sulfur chain may therefore promote subsequent reductive or oxidative cleavage.154

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5.4.4 Predicting a wine’s ability to exhibit reductive off-odors

At present, winemakers have limited options for controlling, or even predicting, the

development of reductive off-odors in the post-bottling period. Cu(II) additions are common for

the control of free thiols prior to bottling, but little can be done once the wine is bottled. There are

methods for quantifying various reductive aroma precursors in wine (e.g., disulfides and thioesters),

however, this practice is both time consuming and expensive, and is thus not practical for most

winemakers. Providing winemakers with the tools for predicting the evolution of VSCs in a specific

lot of wine would be extremely useful and would inform further remedial actions.

We have demonstrated that the Cu-fining process may generate non-volatile mixed

disulfides and metal complexes. A wine may, therefore, lack a reduced aroma profile despite the

presence of significant amounts of disulfides and metal complexes; however, once these molecules

are cleaved, as described above, the resulting thiol compounds are capable of causing wine

spoilage. The objective of this project was to develop a simple, fast, inexpensive, and reliable

method kit for testing a wine’s ability to exhibit reductive odors during the post-bottling period by

the dissociation of VSCs. Our goal was to demonstrate a practical application of the fundamental,

mechanistic work described in previous chapters of this thesis.

Commonly encountered VSCs (H2S, MeSH, and EtSH) were added to model wine at a

final concentration of 100 µg/L, at which point Cu(II) sulfate was added at 1 mg/L to simulate

copper fining process. As described previously, this would result in the formation of the

corresponding disulfides, polysulfanes, and Cu(I)SR complexes (Chapter 2). Afterwards,

treatments for their reduction were added and then analyzed using GC-PFPD (Table 5.8). As

expected, Cu(II) addition, which was in molar excess to the VSCs, resulted in a complete loss of

all sulfhydryl compounds.

124

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

model wine.

H2S MeSH EtSH Treatment Average

peak area

Recovery

(%)

Average

peak area

Recovery

(%)

Average peak

area

Recovery

(%)

control 6977.2 ± 151 100 485.2 ± 47.9 100 4857.3 ± 92.2 100

Cu(II) 0 ± 0 0 0 ± 0 0 0 ± 0 0

Cu+TCEP 155.6 ± 60.8 2.23 337.0 ± 22.2 69.47 3457.4 ± 182.1 71.18

Cu+BCDA 0 ± 0 0 0 ± 0 0 0 ± 0 0

Cu+TCEP+BCDA 254.8 ± 54.3 3.65 328.7 ± 30.0 67.74 3454.1 ± 235.2 71.11

Cu+TCEP+BCDA +Cys

242.1 ± 21.1 3.47 353.7 ± 34.1 72.90 3682.1 ± 276.5 75.81

The addition of TCEP resulted in the release of ~70% of MeSH and EtSH, but was

relatively ineffective in releasing H2S (~2% release). TCEP is a reagent capable of reducing a

disulfide (S-S) into two free thiols (-SH), and the strength of the resulting phosphorus-oxygen bond

makes the reaction irreversible (Figure 5.2).212,213 The reagent is practically odorless and will not

interfere with the aroma associated with free thiol compounds, and can quickly react at acidic wine

conditions and reduce disulfides and polysulfanes.

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

Surprisingly, BCDA alone failed to result in the release of any of the tested sulfhydryl

compounds, which would have been expected to be bound as Cu(I)SR complexes to some extent.

We had previously shown that BCDA is capable of displacing the insoluble Cu(I)-6SH aggregate

(Chapter 2). Recent work has shown that the metal complex-bound forms of H2S and MeSH could

be responsible for VSC generation.80,81 It appears that anaerobic aging results in a decrease of bound

forms and the release of the volatile fraction. As the experimental conditions were conducted under

air, the lack of release of the corresponding sulfhydryls could therefore be attributed to their

125

oxidation. In a separate experiment, large amounts of H2S and Cu(II) sulfate were combined in

model wine to form the non-volatile CuS nanoparticles. The addition of TCEP resulted in the

release of H2S as noted by smell, however, BCDA addition did not result in H2S release. Addition

of barium hydroxide to the solution after BCDA addition resulted in a fine white precipitate due to

BaSO4, suggesting that H2S had been oxidized to sulfate.

The use of BCDA and TCEP in combination yielded results similar to that of TCEP alone.

The addition of Cys in combination of BCDA and TCEP resulted in a slight increase in the recovery

of the thiols (Table 5.8). Cys was added in excess to act as a reducing agent for Cu(II) and to serve

as a sacrificial thiol. If excess Cu(II) remains, BCDA may oxidize the released volatile thiol fraction

to subsequently reduce Cu(II) to Cu(I).

Results obtained under aerobic conditions showed reasonable recovery of MeSH and EtSH,

but were insufficient in the case of H2S. Even in the presence of excess reducing agents, it appears

that O2 interferes with the recovery of labile H2S, and so the experiment was repeated under

anaerobic conditions for H2S (Table 5.9).

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

Treatment Average peak area Recovery (%) control 6355.8 ± 740.1 100

Cu(II) 0 ± 0 0

Cu+TCEP 4348 ± 121.7 68.41

Cu+BCDA 16.7 ± 14.6 0.26

Cu+TCEP+BCDA 4798.5 ± 392.8 75.50

Cu+TCEP+BCDA+Cys 5935.6 ± 23.5 93.39

A markedly higher recovery was observed with TCEP in the absence of O2, resulting in 68%

recovery compared to ~2% in the presence of oxygen. For BCDA, virtually no H2S was recovered,

presumably due to the presence of excess Cu(II) in solution. The combination of BCDA and TCEP

resulted in a 75% recovery of H2S, giving a slight increase compared to TCEP alone. When Cys

126

was present along with TCEP and BCDA, the recovery was further increased to 93%. This is

apparently due to the presence of excess Cys that was capable of reducing Cu(II) to Cu(I), thereby

preventing the oxidation of released H2S by Cu(II).

Having established the conditions for optimal sulfhydryl compound release and recovery, these

conditions and reagents were then used to analyze six commercial Pennsylvania red and white

wines in order to determine their ability to release VSCs (Table 5.10).

Table 5.10: Concentrations of H2S and MeSH in three PA white wines and three PA red wines

before and after addition of treatment reagents. None of the wines released detectable amounts of EtSH before or after the kit was used.

WW1 WW2 WW3 H2S (µg/L) MeSH (µg/L) H2S (µg/L) MeSH

(µg/L)

H2S (µg/L) MeSH

(µg/L)

control 2.50 ± 0.11 2.00 ± 0.07 2.25 ± 0.07 2.57 ± 0.01 ND ND

Cu+TCEP+BCDA

50.34 ± 2.16 2.93 ± 0.01 79.61 ± 5.72 4.25 ± 0.43 43.28 ± 5.70 2.43 ± 0.05

Cu+TCEP+BCDA+Cys

51.94 ± 4.14 2.69 ± 0.09 79.61 ± 1.84 4.25 ± 0.46 46.02 ± 4.12 2.33 ± 0.03

RW1 RW2 RW3

H2S (µg/L) MeSH (µg/L) H2S (µg/L) MeSH

(µg/L)

H2S (µg/L) MeSH

(µg/L)

control ND ND ND ND 2.22 ± 0.01 ND

Cu+TCEP+BCDA

26.94 ± 0.88 87.93 ± 0.54 45.74 ± 2.80 3.89 ± 0.08 4.47 ± 0.24 8.04 ± 0

Cu+TCEP+BCDA+Cys

32.83 ± 1.76 88.94 ± 0.75 46.35 ± 2.86 3.56 ± 0.04 5.19 ± 0.49 8.37 ± 0.09

EtSH was not detected in any of the samples before or after the addition of the reagents,

however free H2S and MeSH ranged from undetectable concentrations to 2.50 µg/L and 2.57 µg/L,

respectively. In all cases, H2S and MeSH were released in the wines above their reported threshold

upon the addition of the test reagents. H2S release ranged from 5.19 to 79.61 µg/L, and MeSH

ranged from 2.33 to 8.37 µg/L, with an outlier at 88.94 µg/L. These concentrations were consistent

with the study reported by Franco-Luesma and Ferreira, and were consistent with the fact that over

50% of MeSH and 90% of H2S are bound.80 It appears that the addition of Cys improved recovery

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slightly in some of the wines, however, it was mostly ineffective. This could be explained by the

fact that wines would likely already contain thiols such as Cys and GSH in excess, and that copper

will likely be present in its reduced Cu(I) form under reductive conditions.

TCEP may have some activity with respect to reducing copper and dissociating its thiol

complex, and can also reduce sulfoxides (e.g. DMSO to DMS), although these were not quantified.

The precise mechanism governing the release of VSCs cannot be elucidated from the results

outlined here; however, recent work suggests that 60 – 90% of H2S release and 24 – 48% of MeSH

release is attributed to metal complex dissociation.81 The remaining portion is due to de novo

formation, which could be attributed to disulfide reduction, although there are also other pathways

proposed for generation of VSCs.

While the anaerobic preparation of the samples is not practical from a winery perspective,

these results can easily be adapted to work as a kit in a winery setting. The samples can be prepared

by transferring ~20 – 30 mL of wine to a 50 mL polypropylene tube with a screw cap. The sample

can be deoxygenated with nitrogen, argon, or sodium bicarbonate. Alternatively a sample of the

wine can be taken from the bottom of the tank and carefully transferred to avoid oxygen ingress.

The reagents can be made into a kit with a packet containing 5 mg each of TCEP, BCDA, and Cys.

The reagents are added to the wine, followed by capping the tube and mixing. After 5 – 10 min, the

wine sample is evaluated by informal sensory analysis; if VSCs are present above their odor

detection thresholds, they will be readily apparent.

The use of the reagents described above is an effective way of quickly releasing VSCs,

which are indicative of a wine’s ability and potential to exhibit reductive odors after bottling. Such

a kit needs to be tested compared to natural reductive bottle aging processes to verify that any of

the results obtained correlate with VSC generation. The dissociation of the metal complexes as well

as reduction disulfide and polysulfanes is done at a very high efficiency by the reagents, and it is

128

unlikely that the generation will proceed to such extent under typical wine aging. Nevertheless,

such a semi-quantitative kit may be able to predict potential for a wine to exhibit reductive odors

post-bottling. If the wine exhibits reductive off odors, the winemaker can take preventative

measures including consideration for copper additions, sparging, bottle closures, and wine aging.

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Chapter 6

Conclusions and Recommendations for Future Work

6.1 Summary

In this dissertation, I examined the interaction of transition metals with H2S and thiols in

model wine conditions. I found that copper plays a central role at mediating redox reactions of

sulfhydryl compounds, and is capable of oxidizing thiols and H2S to disulfides and polysulfanes

and form Cu(I)-SR metal complexes. The formation of disulfides, polysulfanes, and Cu(I)-SR

complexes occurs without oxygen uptake, and will therefore similarly occur in wineries when

Cu(II) fining is employed. I observed that the presence of thiols also inhibits the precipitation of

CuS, presumably by interfering with bulk crystal formation. Furthermore, Cu(I)-SR is not inert,

and can react in the presence of oxygen and catalyze Fenton-like reaction and subsequent ethanol

oxidation.

I found that when Fe(III) is added in combination of H2S and thiols, the oxidation of H2S

and thiols and reduction of Fe(III) to Fe(II) occurs with the generation of disulfides. However, the

reaction is drastically slower compare to that of Cu(II), furthermore, Fe(II) did not appear to play

a major role in binding to H2S and thiols. When Fe(III) and Cu(II) used in combination, the reaction

was much faster than either of the metal alone, suggesting a synergistic reaction. It was found that

Cu(I)-SR is generated within seconds, and is subsequently oxidized by Fe(III). Cu(II) is reduced

again to Cu(I)-SR in the presence of excess H2S and thiols, resulting in fast reduction of both Cu(II)

and Fe(III). Fe(II) appeared to react faster with oxygen than Cu(I)-SR, driving the overall reaction

faster in the presence of oxygen. When H2S and 6SH were oxidized in the presence of Fe and Cu,

I was able to detect polysulfanes up to 5 linking sulfur atoms.

130

I had also investigated the effect of manganese at catalyzing thiol and H2S oxidation. I

found that unlike the reaction with Fe and Cu, Mn resulted in the generation of free thiyl radicals

and subsequent radical chain reaction. This resulted in the generation of sulfonic acids and various

oxidized disulfide species. However, in the presence of polyphenolics, which are abundant in wine,

the thiyl radicals are quickly scavenged. Furthermore, when Cu(II) is added, it appears that the Cu-

driven reaction dominates and limits thiyl radical formation. Nonetheless, it appears that Mn may

accelerate the reaction and also generate transient thiyl radicals during wine oxidation.

Lastly, I had demonstrated that applying Cu-fining in white wine which had added H2S and

MeSH resulted in the generation of mixed GSH-MeSH disulfide and trisulfane. This compound is

nonvolatile and may release MeSH under post-bottling conditions. I have demonstrated that Fe and

Cu in combination of reducing agents (SO2, Cys, and ascorbic acid) play a key role in disulfide

scission under anaerobic conditions. Given that disulfides, polysulfanes, and metal sulfide

complexes may play a crucial role in the generation of sulfidic odors post-bottling, I developed a

method kit to force their reduction and dissociation. I have successfully released H2S and MeSH

from wines previously free of sulfidic faults. This protocol may aid winemakers in predicting their

wine’s ability to exhibit sulfidic odors and therefore take action.

6.2 Future Work

6.2.1 Interaction of H2S and Thiols with Zinc

Zn(II) is known to have similar binding properties with sulfide as Cu(II), but it does not

redox cycle. The reaction displayed by Zn(II) is a simple substitution reaction generating Zn(II)S.

There is evidence showing the binding of Zn with H2S in wine and beer, but whether it effects

overall redox reactions in wine need to be further investigated.

131

6.2.2 Interaction of reducing agents and disulfides

Under physiological conditions, ascorbic acid and glutathione have an intricate

relationship, with glutathione reducing dehydroascorbic acid to ascorbic acid. However, it has also

been suggested that ascorbic acid could reduce disulfide bridges with release of free thiols. In my

work investigating DEDS reduction, ascorbic acid alone was ineffective at reducing DEDS without

the addition of Fe and Cu. Further work should investigate the interaction of transition metals and

ascorbic acid at reducing and/or dissociating VSC precursors.

6.2.3 Using alternative treatments to Cu(II) fining

Cu(II) salts are extremely effective at removing free sulfhydryl functionalities, but they

may result in accumulation of copper and oxidation products that release post-bottling. The use of

physically bound copper could prove effective at providing the beneficial effects of copper while

minimizing its downsides. Preliminary work reported in Appendix E showed promising results but

this needs to be investigated further. The work has shown that the use of a bound Cu-iminodiacetic

acid complex encapsulated in a PDMS material was effective at removing free H2S and EtSH while

limiting the accumulation of metal sulfides and disulfides. There are numerous types of support

materials and methods for synthesizing copper particles, and it is worthwhile to explore further to

avoid the use of the free Cu(II) salt.

6.3 Concluding Remarks

Cu(II) fining is a commonly utilized process for the control of sulfidic odors in wine in

both small and largescale wineries. This work demonstrates how Cu(II) interacts with both H2S and

132

thiols and which major products are formed. It was found that disulfides, polysulfanes, and Cu(I)-

SR complexes are readily formed regardless of oxygen concentration. Fe and Mn play a role at

catalyzing the redox reactions, but do not change the resulting oxidation products. Because the

oxidation products remain redox active, they may reduce and/or dissociate under reductive wine

conditions, resulting in the release of H2S and MeSH. Fe and Cu in combination of reducing agents

in wine play a key role at mediating the reduction of these compounds. This work provides a

foundation and basis for future work in effectively controlling sulfidic odors in wine post-bottling.

133

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157

Appendix A. Supplementary information for Chapter 2

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

mBBr + cys + h2s

m/z100 125 150 175 200 225 250 275 300 325 350 375 400 425

%

0

100310

309

223

191113 149117 153 211

225

292238

288247

312

378313367

345 394 397 425

158

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

mBBr + cys + h2s

m/z100 150 200 250 300 350 400 450 500 550 600 650 700

%

0

100413

412

412

191113 190

149 192 267221 379300 310 357

415

450481 627511 605

531549 673

694

159

Figure A.3. Chromatographic profile of combined MRM spectra. Rt 7.97 min – Cys-bimane (m/z

310→223); 12.59 min – sulfide-dibimane (m/z 413→191); 13.63 min – 6SH-bimane (m/z 323→222).

t=0m R2 D

Time5.00 10.00 15.00 20.00 25.00

%

0

10012.59

7.97

13.63

160

Appendix B: Supplementary information for Chapter 3.

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

µM) and Cu(II) (50 µM).

6MH+H2S Ox

Time20.00 21.00 22.00 23.00 24.00 25.00

%

0

100n = 2 n = 3

n = 5

n = 4

161

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

6MH+H2S Ox

m/z125 150 175 200 225 250 275 300 325 350 375 400

%

0

100

%

0

100

%

0

100

%

0

100214

214

158124

141 199197171

215

353

345216

329309282249236301

363

365

313

214

158116124 159 179 199 215

279237249

331

315

317

331

333352

334

281

147116

124214158

299

282301

249

116267

n = 2

n = 3

n = 4

n = 5

162

Figure B.3. ESI- mass spectrum of S5-bimane obtained from reaction of H2S (300 µM) with Fe(III)

(200 µM) and Cu(II) (50 µM) followed by MBB derivatization.

163

Appendix C. Supplementary information for Chapter 4

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).

181>81 sulfoante fragmentation in 6SH-Fe/Cu(+Mn)

Time2.00 4.00 6.00 8.00 10.00

%

0

100

GYK160408_5 MRM of 1 Channel ES- TIC452

9.568.15

7.847.233.661.490.73 2.832.64

5.454.896.69 8.52

9.68

181>81 sulfoante fragmentation

Time2.00 4.00 6.00 8.00 10.00

%

0

100

GYK160408_4 MRM of 1 Channel ES- TIC

1.62e3

1.871.76

0.26

1.360.40

1.972.02

2.17

2.329.709.498.847.68 9.96

164

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.

6SH+MN+FE

Time7.50 8.00 8.50 9.00 9.50 10.00

%

0

100

GYK160506_4 Scan ES- 329

5.72e5

8.90

8.85

8.328.167.967.75 8.54 8.58

8.97 10.079.04

9.789.559.479.11 9.94

6SH+MN+FE

Time7.50 8.00 8.50 9.00 9.50 10.00

%

0

100

7.50 8.00 8.50 9.00 9.50 10.00

%

0

100

7.50 8.00 8.50 9.00 9.50 10.00

%

0

100

7.50 8.00 8.50 9.00 9.50 10.00

%

0

100

GYK160506_6 Scan ES+ 315

6.98e6

9.02

8.898.528.087.877.70 8.25

10.039.299.89

9.489.60 10.08

GYK160506_6 Scan ES+ 299

5.29e6

8.85

7.69 8.167.918.75

8.548.39

8.89

8.9110.089.989.00

9.779.619.379.33

GYK160506_6 Scan ES+ 283

5.54e7

8.56

9.60

GYK160506_6 Scan ES+ 267

4.42e7

9.70

165

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

6SH+MN+FE+CU

Time7.50 8.00 8.50 9.00 9.50 10.00

%

0

100

7.50 8.00 8.50 9.00 9.50 10.00

%

0

100

7.50 8.00 8.50 9.00 9.50 10.00

%

0

100

7.50 8.00 8.50 9.00 9.50 10.00

%

0

100

GYK160506_9 Scan ES+ 315

1.61e6

10.0710.019.869.599.549.359.20

8.888.337.69 8.007.83

8.12 8.808.63

GYK160506_9 Scan ES+ 299

1.49e6

10.0810.03

9.759.719.278.898.748.327.847.75 8.09

8.678.96 9.44

9.94

GYK160506_9 Scan ES+ 283

1.13e7

8.55

7.877.66 8.428.267.97

10.078.66

9.999.489.428.778.91

9.22 9.51

GYK160506_9 Scan ES+ 267

1.13e8

9.699.72

166

Appendix D. Supplementary information for Chapter 5

Figure D.1. Identified Cys-polysulfanes by LC-QTOF after reacting Cys (500 µM) and H2S (250

µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model wine. The insert shows the maximum abundance based on percent of each given mass.




S/N ratio

Intensity (AU)

1 C3H7NO2S 122.027 ± 0.005 0.99 1027.4 52270

2 C6H12N2O4S2 241.031 ± 0.005 0.99 6820.7 685100

3 C6H12N2O4S3 273.003 ± 0.005 0.99 3737.2 319400

4 C6H12N2O4S4 304.975 ± 0.005 1.22 39805.8 190900

5 C6H12N2O4S5 336.947 ± 0.005 2.38 203.6 9045

6 C6H12N2O4S6 368.919 ± 0.005 3.41 47.4 612.2

167

Figure D.2. Identified GSH-polysulfanes by LC-QTOF after reacting GSH (500 µM) and H2S (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model wine. The insert shows the

maximum abundance based on percent of each given mass.




S/N ratio Intensity (AU)

1 C10H17N3O6S 308.091 ± 0.005 1.28,1.42 4650, 1855 2308000, 1180000

2 C20H32N6O12S2 613.159 ± 0.005 1.29, 1.49,

1.66

6070.9, 3741.3,

6289.6

2166000, 1019000,

1143000

3 C20H32N6O12S3 645.131 ± 0.005 2.29, 2.51 6033.1,

13107.5

1382000, 1413000

4 C20H32N6O12S4 677.103 ± 0.005 3.46 8178.4 634300

5 C20H32N6O12S5 709.075 ± 0.005 4.25 1150.6 28550

6 C20H32N6O12S6 741.043 ± 0.005 5.1 161.4 1513

168

Figure D.3. Identified mixed Cys-MeSH disulfide and polysulfanes by LC-QTOF after reacting

Cys (500 µM), H2S (250 µM), and MeSH (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model wine. The insert shows the maximum abundance based on percent of each given

mass.





2 C4H9NO2S2 168.015 ± 0.005 1.48 2683.8 201400

3 C4H9NO2S3 199.987 ± 0.005 3.1 3843.1 134200

4 C4H9NO2S4 231.959 ± 0.005 4.68 1154.5 31140

5 C4H9NO2S5 263.931 ± 0.005 6.27 805.5 6398

6 C4H9NO2S6 295.903 ± 0.005 7.75 146.7 915

169

Figure D.4. Identified mixed GSH-MeSH disulfide and polysulfanes by LC-QTOF after reacting

GSH (500 µM), H2S (250 µM), and MeSH (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model wine. The insert shows the maximum abundance based on percent of each given

mass.

S (n) Molecular formula M+H monoisotopic mass



2 C11H19N3O6S2 354.079 ± 0.005 3.32 68539.5 3601000

3 C11H19N3O6S3 386.051 ± 0.005 4.69 36202.7 2277000

4 C11H19N3O6S4 418.023 ± 0.005 6.03 19465.6 703200

5 C11H19N3O6S5 449.995 ± 0.005 7.33 5645.9 120200

6 C11H19N3O6S6 481.967 ± 0.005 8.48 13701.3 17660

7 C11H19N3O6S7 513.939 ± 0.005 9.5 1293.9 2361

8 C11H19N3O6S8 545.911 ± 0.005 10.47 40 337

170

Appendix E. Preliminary studies using Cu(II) sulfate alternatives for the

control sulfidic odors in wine

The use of PDMS-encapsulated copper particles as an alternative to copper fining was

investigated. Given the importance that disulfides, polysulfanes and, and metal-thiol complexes

play with respect to wine quality, an alternative to copper fining has been investigated using a

variety of bound copper particles. If the copper fining process could be conducted without the risk

of leaving residual copper and sulfidic odor precursors was available, the potential for post-bottling

generation of sulfidic off-odors could be dramatically decreased. It was found that the use of certain

encapsulated materials was more effective at removing H2S and EtSH and their oxidized precursor

compounds compared to traditional Cu(II) sulfate additions.

Methodology. Various copper and silver particles were encapsulated in a thin PDMS film

and kindly donated by Martin Schmitt. The PDMS film treatments were placed inside a 300 mL

B.O.D. bottle prior to the experiment and allowed to equilibrate in the anaerobic chamber

overnight. Model wine was spiked with H2S (50 µg/L) and EtSH (50 µg/L), and then immediately

transferred to B.O.D. bottles containing the PDMS film treatment. For every experiment, a control

sample was included which was transferred to a B.O.D. bottle containing no PDMS film treatments.

After 24 hours, one 10 mL sample aliquot was transferred to a 20 mL amber GC vials and capped

immediately to determine free H2S and EtSH in solution. A separate 10 mL aliquot was spiked with

TCEP (1 mM), BCDA (1 mM), and Cys (1 mM) for determination of residual bound forms of H2S

and EtSH. The experiments were conducted in duplicate in an anaerobic chamber.

Bound Cu-particles. The use of copper fining may result in the generation of sulfhydryl

metal complexes and disulfides which can be subsequently reduced post-bottling and cause

chemical spoilage of the wine. There are numerous downsides to the use of Cu(II) salts, but they

171

are extremely effective in the removal of free thiol functionality. For these reasons, the use of Cu(II)

as physically or chemically bound forms could be an effective way of removing thiols without the

introduction of unwanted precursors for VSCs.

A variety of PDMS encapsulated copper and silver particles were explored in the

experiments described in this section. PDMS was used here to provide a barrier that both prevents

the migration of metal particles into the wine while also allowing for the migration of VSCs into

the capsule. Once the sulfhydryl-containing compound reacts with the metal (i.e., Cu or Ag), it

should become physically immobilized as the metal complex forms. The release of the resulting

disulfide from the PDMS film is less likely due to the higher molecular weight of the compound

needing to transport out of the PDMS film, furthermore, the compounds may potentially scalp into

the PDMS material. The results for removal and regeneration of H2S and EtSH are described below

(Tables E.1 and E.2).

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

Treatment % removal* % removal after "reduction"* Relative % regenerated

PDMS

(negative control)

8.8 3.2 8.2 -1.0 6.7 0.0

Cu sulfate

(positive control)

100.0 100.0 50.0 61.2 50.0 38.8

Cu powder 91.1 67.4 62.2 56.3 31.7 16.6

CuIDA 76.5 100.0 46.6 78.1 39.0 21.9

immobilized CuIDA

99.4 100.0 72.3 75.5 27.2 24.5

Cu foil 42.9 34.3 40.8 31.1 5.1 9.2

Cu oxide 100.0 92.3 64.9 67.7 35.1 26.7 Cu stearate 84.3 99.4 55.2 67.3 34.5 32.3

Ag powder 38.9 29.0 37.1 25.8 4.7 11.0

Ag acetate 100.0 98.3 40.1 42.1 59.9 57.2 Ag

encapsulated

98.6 93.5 72.9 79.3 26.1 15.1

Ag stearate 22.4 36.1 19.4 29.2 13.7 19.3

172

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

Treatment % removal* % removal after "reduction"* Relative % regenerated

PDMS

(negative

control)

17.5 8.8 13.1 6.3 25.0 27.7

Cu sulfate (positive

control)

100.0 100.0 20.3 15.0 79.7 85.0

Cu powder 84.0 68.1 61.0 55.7 27.5 18.1 CuIDA 74.9 100.0 40.6 60.8 45.8 39.2

immobilized

CuIDA

90.4 100.0 66.7 62.1 26.2 37.9

Cu foil 67.3 62.9 64.0 56.4 4.9 10.2

Cu oxide 100.0 86.2 69.8 49.9 30.2 42.1

Cu stearate 84.6 97.1 63.3 67.2 25.2 30.7

Ag powder 70.3 57.7 68.4 53.5 2.6 7.4 Ag acetate 100.0 93.9 68.9 64.7 31.1 31.0

Ag

encapsulated

100.0 93.7 87.0 79.0 13.0 15.8

Ag stearate 47.7 61.1 44.4 56.3 6.9 7.8

All treatments were more effective with respect to removing H2S and EtSH compared to

the PDMS film negative control, although some scalping by the PDMS material was observed.

None of the treatments except Cu(II) sulfate resulted in consistent 100% removal of H2S and EtSH,

but the immobilized CuIDA, Cu oxide, Ag acetate, and encapsulated Ag were very effective.

However, after forcing the reduction of the model wine (see section 5.4.4), Cu sulfate had the most

H2S and EtSH regenerated compared to all treatments (except for Ag acetate, for unknown reasons).

Some of the treatments varied widely between the two experimental replicates, which may be due

to holes in some of the PDMS sachets.

Some compromises will have to be made such that a complete removal of VSCs can occur

within a reasonable time frame in a winery, but the treatment must also result in the least disulfides

and metal-thiols after use. A few of the treatments were particularly effective at preventing

accumulation of either disulfides and/or metal-bound VSCs (Cu foil, Ag powder, Ag stearate) but

173

also reacted slowly, resulting in incomplete removal of the VSCs within 24 hours. The immobilized

CuIDA and encapsulated Ag cation exchange (and perhaps the Cu oxide) resulted in almost

complete removal of VSCs with less generation after 'reduction' compared to copper sulfate.

Although these results are preliminary in nature, they may provide a useful alternative for

copper fining to limit the negative aspects associated with it. Further analysis is needed to measure

residual free metal ions in solution.

Vita

Gal Y. Kreitman

Education

Ph.D. Food Science, The Pennsylvania State University, University Park, PA, 2016 M.S. Food Science, The Pennsylvania State University, University Park, PA, 2013

B.S. Food Science, The Pennsylvania State University, University Park, PA, 2011

Publications


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

Food Chem. 2016, 64, 4095-4104. Kreitman, G.Y.; Danilewicz, J.C.; Jeffery, D.W.; Elias, R.J. Reaction Mechanisms of Metals with

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

J. Agric. Food Chem. 2016, 64, 4105-4113. Kreitman, G.Y., Cantu, A., Waterhouse, A.L., Elias, R.J. Effect of Metal Chelators on the Oxidative

Stability of Model Wine. J. Agric. Food Chem. 2013, 61, 9480–9487.

Kreitman, G.Y., Laurie, V.F., Elias, R.J. Investigation of ethyl radical quenching by phenolics and thiols in model wine. J. Agric. Food Chem. 2013, 61, 685–92.

Presentations

Kreitman G.Y., Elias R.J. What’s that smell?! Predicting Reductive Aroma in Wine (invited talk). PA Wine Marketing and Research Board Symposium, State College, PA, 2016. Oral

Presentation

Kreitman G.Y., Danilewicz J.C., Elias R.J. A Mechanistic Investigation of Copper-Mediated Oxidation of Thiols in Model Wine. 66th Annual Meeting of the American Society for

Enology and Viticulture, Portland, OR. 2015. Poster Presentation.

Kreitman G.Y. and Elias R.J. The Role of Copper in the Evolution of Sulfur Compounds in Wine (invited talk). PA Wine Marketing and Research Board Symposium, State College, PA, 2015.

Oral Presentation

Kreitman G.Y., Cantu A., Waterhouse A.L., Elias R.J. Controlling oxidation of model wine using

metal chelators. 65th Annual Meeting of the American Society for Enology and Viticulture, Austin, TX. 2014. Oral Presentation.

Kreitman G.Y., Elias R.J. Oxidative loss of thiols in model wine solution by 1-hydroxyethyl

radicals (invited talk). 244th National Meeting & Exposition of the American Chemical Society, Philadelphia, PA. 2012. Oral Presentation.

Awards

PA Wine Marketing and Research Program Grant Recipient (2015, 2016)

American Wine Society Educational Foundation Scholarship (2015)

American Society for Enology and Viticulture (2014, 2015)

Penn State College of Agricultural Sciences Competitive Grants Winner (2014)

reaction mechanisms of transition metals with …

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