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 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
ii
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
iii
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
iv
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
v
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.
vi
TABLE OF CONTENTS
LIST OF FIGURES .................................................................................................................... x
LIST OF TABLES .................................................................................................................... xv
ACKNOWLEDGEMENTS .................................................................................................... xvii
Chapter 1 Literature Review....................................................................................................... 1
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
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
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
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
Figure 3.1. Reduction of oxygen by Fe(II) to yield hydrogen peroxide without the release of hydroperoxyl radicals. ............................................................................................. 64
xii
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)
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 4.3. 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
xiii
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 –
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
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
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
xv
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
H2S (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air saturated model
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
xvi
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
xvii
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.
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,
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
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
Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model
Wine. Part 2: Iron and Copper Catalyzed Oxidation.
Published as:
Kreitman, G.Y.; Danilewicz, J.C.; Jeffery, D.W.; Elias, R.J. Reaction Mechanisms of Metals with
Hydrogen Sulfide and Thiols in Model Wine. Part 2: Iron- and Copper- Catalyzed Oxidation. J.
Agric. Food Chem. 2016, 64, 4105-4113.
3.1 ABSTRACT
Sulfidic off-odors arising during wine production are frequently removed by Cu(II) fining.
In Part 1 of this study, the reaction of H2S and thiols with Cu(II) was examined; however, the
interaction of iron and copper is also known to play an important synergistic role in mediating non-
enzymatic wine oxidation. The interaction of these two metals in the oxidation of H2S and thiols
(cysteine, 3-sulfanylhexan-1-ol, and 6-sulfanylhexan-1-ol) was therefore examined under wine-like
conditions. H2S and thiols (300 μM) were reacted with Fe(III) (100 or 200 μM) alone and in
combination with Cu(II) (25 or 50 μM), and concentrations of H2S and thiols, oxygen, and
acetaldehyde were monitored over time. H2S and thiols were shown to be slowly oxidized in the
presence of Fe(III) alone, and were not bound to Fe(III) under model wine conditions. However,
Cu(II) added to model wine containing Fe(III) was quickly reduced by H2S and thiols to form Cu(I)-
complexes, which then rapidly reduced Fe(III) to Fe(II). Oxidation of Fe(II) in the presence of
oxygen regenerated Fe(III) and completed the iron redox cycle. In addition, sulfur-derived
oxidation products were observed, and the formation of organic polysulfanes was demonstrated.
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 MATERIALS AND METHODS
3.3.1 Chemicals
L-Cysteine (Cys), monobromobimane (MBB), 6-sulfanylhexan-1-ol (6SH), and
diethylenetriaminepentaacetic acid (DTPA) were obtained from Sigma-Aldrich (St. Louis,
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
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 (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
Oxygen Demand (B.O.D.) bottles (Wheaton, Millville, NJ), allowing the solution to overflow, and
bottles were capped immediately with ground glass stoppers, eliminating headspace. The glass
reservoir of the B.O.D. bottles was topped off with water daily. The bottles were stored in the dark
at ambient temperature. One B.O.D. bottle was sacrificed per time point per replicate and used for
further analyses. All experiments were conducted in triplicate and contained their own series of
sacrificial bottles.
3.3.3 Determination of oxygen consumption
Glass B.O.D. bottles were fitted with PSt3 oxidots and oxygen readings were taken per
time point using a NomaSense O2 P6000 meter (Nomacorc LLC, Zublon, NC). Further details were
reported in Part 1.55
3.3.4 Spectrophotometric measurements
UV-vis spectra of the treatments were recorded at each time point using 10 mm quartz
cuvettes (model wine blank) and measured using Agilent 8453 UV-Vis spectrophotometer
(Agilent, Santa Clara, CA). Determination of Fe(III) concentration was achieved by measurement
of absorbance at 336 nm associated with the Fe(III)-tartrate complex.58
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.
Acetaldehyde was measured in model wine treatment solutions as its 2,4-
dinitrophenylhydrazone (DNPH) derivative with an external standard curve (10 – 220 μM) by
HPLC as described in Part 1.55
Polysulfides were formed by the reaction of H2S (300 μM) with Cu(II) (50 μM) and
Fe(III) (200 μM). A sample was derivatized using MBB as described above with the same
HPLC separation parameters. Mass spectra were obtained using ESI- and full scan between
m/z 100-1000. 6SH and 3SH polysulfanes were obtained by adding H2S (100 μM), Fe(III)
(200 μM), and Cu(II) (50 μM) to 6SH or 3SH (300 μM). The organic polysulfanes were
detected by UV absorbance at 210 nm and verified using MS detection with ESI+ and full
scan between m/z 100-1000. Mobile phases consisted of 0.1% v/v formic acid (A) and 0.1%
v/v formic acid in acetonitrile (B) with a linear gradient according to the following
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
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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
77
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)
(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.
83
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
Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model
Wine. Part 3: Manganese Catalyzed Oxidation and Interaction with Iron and
Copper.
4.1 ABSTRACT
Recent work suggests that manganese has a modest activity in catalyzing polyphenol and
sulfite oxidation in wine. Furthermore, manganese is known to mediate thiol and H2S oxidation in
aquatic systems. It was therefore of interest to investigate the interaction of manganese with iron
and copper toward catalyzing thiol and H2S oxidation under wine-like conditions. Sulfhydryl
compounds (cysteine, 6-sulfanylhexan-1-ol, and H2S) were reacted with Mn(II) alone or in
combination of Fe(III) and Cu(II) in model wine, and the concentrations of sulfhydryl, oxygen, and
acetaldehyde were monitored over time. The reaction of thiols with manganese resulted in radical
chain reaction paired with large oxygen uptake and generation of sulfur oxyanions. H2S did not
generate free thiyl radicals, and had minimal interaction with Mn(II). When Cu(II) was introduced,
Cu-mediated oxidation dominated in all treatments and Mn-mediated radical reaction was limited.
4.2 INTRODUCTION
Iron and copper catalyze non-enzymatic wine oxidation by reducing oxygen, which is
paired with oxidation of ethanol, polyphenolics, and sulfhydryls.52,54–56,59 However, few studies
have examined the mechanistic involvement of other transition metals on the oxidation in wine.
Manganese has been proposed to have an effect at mediating wine oxidation, and is present at
concentrations similar to Fe (~1 mg/L average around the world114,199). Mn has been reported to
catalyze browning in sherry wine in combination with iron,200 increase acetaldehyde production in
86
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.
87
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 MATERIALS AND METHODS
4.3.1 Chemicals
4-methylcatechol (4-MeC), L-Cysteine (Cys), 6-sulfanylhexan-1-ol (6SH), and
manganese(II) sulfate monohydrate, and iron(II) sulfate heptahydrate were obtained from Sigma-
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
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 (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
Oxygen Demand (B.O.D.) bottles (Wheaton, Millville, NJ), allowing the solution to overflow, and
bottles were capped immediately with ground glass stoppers, eliminating headspace. The glass
reservoir of the B.O.D. bottles was topped off with water daily. The bottles were stored in the dark
at ambient temperature. One B.O.D. bottle was sacrificed per time point per replicate and sample
aliquots were stored at -80 °C until further analyses. All experiments were conducted in triplicate
and contained their own series of sacrificial bottles.
4.3.3 Determination of oxygen consumption
Glass B.O.D. bottles were fitted with PSt3 oxidots and oxygen readings were taken per
time point using a NomaSense O2 P6000 meter (Nomacorc LLC, Zublon, NC). Initial O2
concentrations ranged from 6.6 – 7.0 mg/L. Further details were reported in Chapter 2.
4.3.4 Spectrophotometric measurements
UV-vis spectra of the treatments were recorded at each time point using 10 mm quartz
cuvettes (model wine blank) and measured using Agilent 8453 UV-Vis spectrophotometer
(Agilent, Santa Clara, CA). Determination of Fe(III) concentration was achieved by measurement
of absorbance at 336 nm associated with the Fe(III)-tartrate complex.197
For H2S, Cys, 6SH, and 3SH, total concentration was analyzed using Ellman’s assay.
Further details were reported in Chapter 2.
90
4.3.5 HPLC Analyses
Acetaldehyde was measured in model wine treatment solutions as its 2,4-
dinitrophenylhydrazone (DNPH) derivative with an external standard curve (10 – 220 μM) by
HPLC as described in Chapter 2.
Oxidized species formed by the reaction of 6SH were monitored using LC-MS/MS. Mass
spectra were obtained using ESI- and ESI+ and full scan between m/z 100-1000. The compounds
were also monitored by UV absorbance at 210 nm. Mobile phases consisted of 0.1% v/v formic
acid (A) and 0.1% v/v formic acid in acetonitrile (B) with a linear gradient according to the
Table 5.4. Mixed Cys-MeSH disulfide and polysulfanes identified by LC-QTOF after reacting Cys (500 µM), H2S (250 µM), and MeSH (250 µM) with Fe(III) (100 µM) and Cu(II) (50 µM) in air
saturated model wine.
S (n) Molecular formula
M+H monoisotopic mass
Retention time (min)
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
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.
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) Molecular formula
M+H monoisotopic mass
Retention time (min)
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.
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.
S (n) Molecular formula
M+H monoisotopic mass
Retention time (min)
S/N ratio Intensity (AU)
2 C4H9NO2S2 168.015 ± 0.005 1.48 2683.8 201400
3 C4H9NO2S3 199.987 ± 0.005 3.1 3843.1 134200
4 C4H9NO2S4 231.959 ± 0.005 4.68 1154.5 31140
5 C4H9NO2S5 263.931 ± 0.005 6.27 805.5 6398
6 C4H9NO2S6 295.903 ± 0.005 7.75 146.7 915
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
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
Kreitman, G.Y.; Danilewicz, J.C.; Jeffery, D.W.; Elias, R.J. Reaction Mechanisms of Metals with
Hydrogen Sulfide and Thiols in Model Wine. Part 1: Copper Catalyzed Oxidation. J. Agric.
Food Chem. 2016, 64, 4095-4104. Kreitman, G.Y.; Danilewicz, J.C.; Jeffery, D.W.; Elias, R.J. Reaction Mechanisms of Metals with
Hydrogen Sulfide and Thiols in Model Wine. Part 2: Iron- and Copper- Catalyzed Oxidation.
J. Agric. Food Chem. 2016, 64, 4105-4113. Kreitman, G.Y., Cantu, A., Waterhouse, A.L., Elias, R.J. Effect of Metal Chelators on the Oxidative
Stability of Model Wine. J. Agric. Food Chem. 2013, 61, 9480–9487.
Kreitman, G.Y., Laurie, V.F., Elias, R.J. Investigation of ethyl radical quenching by phenolics and thiols in model wine. J. Agric. Food Chem. 2013, 61, 685–92.
Presentations
Kreitman G.Y., Elias R.J. What’s that smell?! Predicting Reductive Aroma in Wine (invited talk). PA Wine Marketing and Research Board Symposium, State College, PA, 2016. Oral
Presentation
Kreitman G.Y., Danilewicz J.C., Elias R.J. A Mechanistic Investigation of Copper-Mediated Oxidation of Thiols in Model Wine. 66th Annual Meeting of the American Society for
Enology and Viticulture, Portland, OR. 2015. Poster Presentation.
Kreitman G.Y. and Elias R.J. The Role of Copper in the Evolution of Sulfur Compounds in Wine (invited talk). PA Wine Marketing and Research Board Symposium, State College, PA, 2015.
Oral Presentation
Kreitman G.Y., Cantu A., Waterhouse A.L., Elias R.J. Controlling oxidation of model wine using
metal chelators. 65th Annual Meeting of the American Society for Enology and Viticulture, Austin, TX. 2014. Oral Presentation.
Kreitman G.Y., Elias R.J. Oxidative loss of thiols in model wine solution by 1-hydroxyethyl
radicals (invited talk). 244th National Meeting & Exposition of the American Chemical Society, Philadelphia, PA. 2012. Oral Presentation.
Awards
PA Wine Marketing and Research Program Grant Recipient (2015, 2016)
American Wine Society Educational Foundation Scholarship (2015)
American Society for Enology and Viticulture (2014, 2015)
Penn State College of Agricultural Sciences Competitive Grants Winner (2014)