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Copper(II) mediated hydrogen sulfide and thiol oxidation to disulfides and organic polysulfanes, and their reductive cleavage in wine: Mechanistic elucidation and potential applications Gal Y Kreitman, John C Danilewicz, David William Jeffery, and Ryan J. Elias J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05418 • Publication Date (Web): 05 Mar 2017 Downloaded from http://pubs.acs.org on March 5, 2017

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Journal of Agricultural and Food Chemistry

Copper(II) Mediated Hydrogen Sulfide and Thiol Oxidation to Disulfides and Organic Polysulfanes, and their Reductive Cleavage in Wine: Mechanistic Elucidation and Potential Applications Gal Y. Kreitman†, , John C. Danilewicz‡, David. W. Jeffery§, Ryan J. Elias*,† ⊥

†

Department of Food Science, College of Agricultural Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802, United States ‡

44 Sandwich Road, Ash, Canterbury, Kent CT3 2AF, United Kingdom

§

School of Agriculture, Food and Wine, Waite Research Institute, The University of Adelaide, PMB 1, Glen Osmond, South Australia 5064, Australia

Present address: Mondelēz International, Inc., 100 Deforest Avenue, East Hanover, NJ 07936, United States ⊥

* To whom correspondence should be addressed. Tel: +1 (814) 865-5371 Fax: +1 (814) 863-6132 E-mail: [email protected]

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ABSTRACT

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Fermentation-derived volatile sulfur compounds (VSCs) are undesirable in wine and are

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often remediated in a process known as copper fining. In the present study, the addition of Cu(II)

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to model and real wine systems containing hydrogen sulfide (H2S) and thiols provided evidence

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for the generation of disulfides (disulfanes) and organic polysulfanes. Cu(II) fining of a white wine

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spiked with glutathione, H2S, and methanethiol (MeSH) resulted in the generation of MeSH-

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glutathione disulfide and trisulfane. In the present study, the mechanisms underlying the

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interaction of H2S and thiols with Cu(II) is discussed, and a prospective diagnostic test for releasing

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volatile sulfur compounds from their non-volatile forms in wine is investigated. This test utilized

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a combination of reducing agents, metal chelators, and low oxygen conditions to promote the

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release of H2S and MeSH, at levels above their reported sensory thresholds, from red and white

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wines that were otherwise free of sulfidic off-odors at the time of addition.

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KEYWORDS: Hydrogen sulfide, thiols, copper, polysulfanes, wine aroma

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INTRODUCTION

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Sulfidic off-odors in wine present a serious quality issue due to their adverse effects to

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overall wine aroma.1,2 When detected in the course of winemaking, such off-odors are routinely

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treated through the use of copper fining prior to bottling.3–5 During the post-bottling period, even

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in wine deemed to be free of faults at the time of bottling, VSCs that are responsible for the

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undesirable odors may accumulate during storage, especially when oxygen ingress through the

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closure is limited.6–9 The most common VSCs responsible for so-called “reductive” sulfidic off-

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odors that can arise post-bottling are H2S, MeSH, and dimethyl sulfide (DMS).1,10 The generation

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of DMS, whose aroma is often described as “canned corn”, is linked to non-enzymatic hydrolysis

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of S-methylmethionine over a wine’s aging period;11,12 however, the pathways for the generation

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of H2S and MeSH, described as “rotten egg” and “rotten cabbage”, respectively, have yet to be

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established. Several hypotheses have been proposed to explain the non-enzymatic mechanism(s)

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responsible for the generation of H2S and MeSH in finished wine (i.e., post-fermentation). These

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mechanisms have been suggested to include bisulfite reduction,13,14 thioacetate and thioether

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hydrolysis,3,15 and Strecker degradation of sulfur-containing amino acids.16,17 The reduction of

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symmetrical disulfides has also been proposed leading to MeSH and EtSH, with these thiols having

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markedly lower (ca. 10-50 times) sensory detection thresholds than their respective disulfides.1,18

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While the oxidation of thiols plausibly explains disulfide formation, the underlying mechanisms

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by which H2S and thiols are subsequently released remain to be elucidated.

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Recent work suggests that the interaction of transition metals, particularly copper, with

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sulfur-containing compounds under anaerobic (reductive) conditions is correlated with the release

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of H2S and MeSH.6,9,19,20 In order to understand, in mechanistic terms, the role of copper in the

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generation of potential latent sulfur forms, our group has recently focused on elucidating the initial



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reactions involving the metal-catalyzed oxidation of sulfhydryls that are responsible for the

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“removal” of undesirable VSCs (Figure 1).21 In that work, we presented evidence that Cu(II)

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initially coordinates with two sulfhydryl moieties to produce complex (1), followed by an electron

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transfer reaction from sulfur to give Cu(I). The generation of the disulfide and polysulfanes appears

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to occur in a concerted manner in close proximity to the metal and without the release of free thiyl

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radicals, such as in dimeric complex (2).21 The resulting copper-thiolate (Cu(I)-SR) species (3)

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subsequently aggregates to further stabilize Cu(I). Initially, in the absence of oxygen, both thiol

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and H2S oxidation products are produced as well as Cu(I) complexes. In a similar manner

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formation of a mixed complex involving H2S (4) would result in formation of a hydrodisulfide (5).

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Incorporation of this product into a Cu-complex (6) would in turn result in trisulfane (7). The

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resultant very fine (Cu(I)-SR)n aggregates remain reactive, behaving as soluble species wherein

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Cu(I) is highly reductive and is readily oxidized by Fe(III) and O2 upon exposure to air. It is

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apparent, therefore, that the VSCs responsible for sulfidic off-odors cannot be readily removed by

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filtration from wine as an insoluble complex after copper fining, but rather generate redox-active

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compounds that remain in the wine effectively as soluble components.

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The dissociation of metal-sulfide complexes could be partially responsible for the release

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of H2S and MeSH under reductive wine conditions; however, in some wines it has been shown

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that up to 42% and 76% of H2S and MeSH, respectively, are generated through other pathways.20

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However, it is also possible that the cleavage of disulfides via sulfitolysis could serve as a pathway

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for VSC release during storage.14,22 With respect to disulfide formation, recent work seemed to

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show that Cu(II) had a remarkable effect (more so than oxygen) on the generation of disulfides

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that arose from oxidation of varietal thiols 3-sulfanylhexan-1-ol and 3-sulfanylhexyl acetate in

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wines, leading to the hypothesis that disulfides play a key role as aroma reservoirs in wine.23 In


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another study, Cu(II)-catalyzed oxidation of model compound 6-sulfanylhexan-1-ol (6SH) in the

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presence H2S not only resulted in the formation of 6SH-disulfide, but also polysulfanes consisting

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of up to five linking sulfur atoms between the 6SH constituents.24

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Due to the relative abundance of thiol-containing compounds such as glutathione (GSH)

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and cysteine (Cys) in wine, and in light of the preceding results, it is likely that the use of Cu(II)

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to treat off-odors due to MeSH and H2S would generate a variety of asymmetrical (i.e., mixed)

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disulfides and organic polysulfanes (Figure 1). The generation of polysulfanes in hydroalcoholic

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media has been hypothesized to occur in the presence of H2S and thiols via a perthiol

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intermediate.25 Recent work demonstrated the presence of transient species of hydropolysulfides

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which, some have argued, contribute to the “minerality” of wine and can further react to generate

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perthiols.26 Furthermore, polysulfide species are thiophilic species that may further react to

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generate additional mixed polysulfanes.27,28 The presence of polysulfanes and polysulfides in wine

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is therefore indicative of there being forms of latent VSCs beyond simple disulfides that have yet

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to be fully considered.

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The observations outlined above lead to the following aims for the present study: (1) to

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confirm metal-catalyzed generation of disulfides and organic polysulfanes in model and real

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wines; and (2) to adapt a method to force the the dissociation of metal sulfides and reduction of

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disulfides and organic polysulfanes to release H2S and thiols. The ultimate goal being to provide

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a tool for winemakers to determine whether their product is susceptible to the appearance of

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sulfidic off-aromas in the post-bottling period.

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MATERIALS AND METHODS

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Materials. L-Cysteine (Cys), L-cystine, ethanethiol (EtSH), diethyl disulfide (DEDS), sodium

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thiomethoxide (as a source of MeSH), ferrous sulfate hexahydrate, tris(2-carboxyethyl)phosphine

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

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

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

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

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(as a source of H2S) was from Acros Organics (Geel, Belgium) and ferric chloride hexahydrate

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was from Mallinckrodt Chemicals (St. Louis, MO). Water was purified through a Millipore Q-

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Plus system (Milipore Corp., Bedford, MA). All other chemicals and solvents were of analytical

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or HPLC grade, and solutions were prepared volumetrically, with the balance made up with Milli-

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Q water unless specified otherwise. Six commercial Pennsylvania wines (three white and three red

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wines) from the Lake Erie American Viticultural Area were purchased locally.

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Preparation of model wine and real wine samples

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Disulfide and polysulfane generation in model wine. Model wine was prepared by

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dissolving tartaric acid (5 g/L) in water, followed by the addition of ethanol to yield a final

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concentration of 12% v/v. The solution was adjusted to pH 3.6 with sodium hydroxide (10 M) and

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brought to volume with water.

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Either glutathione (GSH, 500 µM) or cysteine (Cys, 500 µM) were added to model wine

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and mixed thoroughly. H2S (250 µM) and/or MeSH (250 µM) were subsequently added to the

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

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


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Fe(III) (100 µM) and Cu(II) (50 µM) were added and the solutions were thoroughly mixed. The

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solutions (25 mL) were stored in the dark at room temperature in capped 50 mL capacity

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polypropylene tubes under air. The samples were analyzed after 24 hours by ultra fast liquid

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chromatography (UFLC) tandem QTOF-MS, as described below.

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Disulfide and polysulfane generation in white wine. GSH was added to commercial

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white wine blends to achieve a final concentration of 50 µM. H2S and MeSH were subsequently

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added to achieve the following three treatment concentrations of each sulfhydryl: 100 µg/L, 500

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µg/L, and 5000 µg/L. Following the addition of the sulfhydryl-containing compounds, Fe(III) (5

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mg/L) and Cu(II) (1 mg/L) were added and the resulting solutions were mixed thoroughly. The

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samples (100 mL) were stored in the dark in stoppered 100 mL volumetric flasks and analyzed

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after 24 hours by UFLC-QTOF-MS.

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Release and reduction of bound VSCs. Initial experiments were conducted in either air

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saturated model wine (dissolved [O2]: 7–8 mg/L) or in an anaerobic chamber (dissolved [O2]:

Copper(II)-Mediated Hydrogen Sulfide and Thiol ... - ACS Publications

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