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Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model Wine. Part 1: Copper Catalyzed Oxidation. 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.6b00641 • Publication Date (Web): 30 Apr 2016 Downloaded from http://pubs.acs.org on May 9, 2016

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Journal of Agricultural and Food Chemistry

Reaction Mechanisms of Metals with Hydrogen Sulfide and Thiols in Model Wine. Part 1: Copper Catalyzed Oxidation. Gal Y. Kreitman†, John C. Danilewicz§, David. W. Jeffery‡, Ryan J. Elias*,†

†

Department of Food Science, 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

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

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commonly encountered in wine production. These odors are usually removed by the process of

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Cu(II) fining – a process that remains poorly understood. The present study aims to elucidate the

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underlying mechanisms by which Cu(II) interacts with H2S and thiol compounds (RSH) under

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wine-like conditions. Copper complex formation was monitored along with H2S, thiol, oxygen,

7

and acetaldehyde concentrations after addition of Cu(II) (50 or 100 μM) to air saturated model

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wine solutions containing H2S, cysteine, 6-sulfanylhexan-1-ol, or 3-sulfanylhexan-1-ol (300 μM

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

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

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of Cu(II) to Cu(I) which reacted with oxygen. H2S was observed to initially oxidize rather than

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form insoluble copper sulfide. The proposed reaction mechanisms provide an insight into the

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extent to which H2S can be selectively removed in the presence of thiols in wine.

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KEYWORDS: H2S, thiols, copper, oxidation, wine aroma, reaction mechanism


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INTRODUCTION

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Volatile sulfur containing compounds (VSCs) have a major impact on the sensory quality of

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wine.1–3 Typically, VSCs have exceedingly low aroma detection thresholds (i.e., μg/L to ng/L) and,

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depending on their structure, can have beneficial or deleterious effects with respect to consumer

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acceptance. Grape-derived varietal thiols, such as 3-sulfanylhexan-1-ol (3SH), 3-sulfanylhexyl

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acetate (3SHA), and 4-methyl-4-sulfanypentan-2-one (4MSP), contribute pleasant aromas (e.g.,

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grapefruit, passionfruit, and blackcurrant).4–6 On the other hand, the production of fermentation-

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related VSCs, such as H2S, methanethiol (MeSH), and ethanethiol (EtSH), can result in the

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development of undesirable odors, often described as rotten egg, putrefaction, sewage and burnt

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rubber, that are obviously detrimental to wine quality.1,7,8 These odors are generally most evident at

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low oxygen concentrations and are described to be sulfidic off-odors. Wines that display such odors

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are described as having reductive character.

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The accumulation of sulfidic off-odors is a common problem for winemakers and is usually

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remedied by splash racking in order to volatilize and/or oxidize VSCs or, classically, by the use of

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copper fining.2,7,9 In this latter practice, Cu(II) is added as its sulfate or citrate salt whereby it is

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assumed to remove H2S by forming a highly insoluble colloidal CuS precipitate (Figure 1),9,10 which

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can be subsequently removed from the wine by racking and/or filtration. The mechanism for copper

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fining remains poorly understood and there are known disadvantages to the process. In the case of

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disulfides, thioacetates, and cyclic sulfur compounds, which can also contribute unpleasant sulfidic

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off-odors, copper fining is ineffective due to the absence of a free thiol group.2,7 Copper fining can

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also cause significant losses of beneficial thiol compounds (e.g. 3SH, 3SHA, 4MSP) that are

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important to the varietal character of a wine.11 Furthermore, other thiols could interfere with the fining

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process by competing for Cu(II) given that the average combined concentration of cysteine (Cys), N-

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acetylcysteine and homocysteine is reported to be ca. 20 µM in a number of white wines, while the 3 ACS Paragon Plus Environment


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average concentration of glutathione (GSH) is reported to be ca. 40 µM in wines made from

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Sauvignon blanc.12–15 These nonvolatile thiols would be in large molar excess to the exogenous

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copper (3–6 µM) used in a fining operation, and would far exceed the concentration of H2S (ca. 300

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nM)16 when copper fining is considered. Furthermore, a recent study by Clark et al.17 demonstrated

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the practical difficulty of removing CuS from wine, even with filtration, as the precipitate may not be

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observed.10 This lack of precipitate formation would leave residual copper in wine that can contribute

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to a series of redox-mediated reactions in the post-bottling period, as elaborated below.

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After bottling, the concentration of sulfidic off-odors can increase, especially under reductive

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conditions when oxygen exposure is limited such as when screw cap closures are used.11,18,19

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Although the causative mechanism remains unclear, wine appears to contain precursors that are able

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to produce H2S and MeSH.20,21 The formation of H2S from the Strecker degradation of Cys has been

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previously reported,22 while some have suggested that H2S may be formed by the direct reduction of

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sulfate or sulfite.19 It has also been shown that thiols can be reversibly bound by iron and copper,23,24

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and that wines containing higher copper concentrations can accumulate sulfidic off-odors during

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bottle aging.11,25 While transition metals are known to be essential for catalyzing oxidation reactions

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in wine,26 Cu, Fe, Mn, Zn, and Al have more recently been shown to synergistically affect the

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evolution of VSCs under anaerobic storage conditions.25

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In order to understand how wines develop sulfidic off-odors during storage, it is essential to

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understand how H2S and thiols react in the presence of oxygen and transition metals prior to bottling.

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The identification of reaction products may then allow potentially troublesome precursors to be

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targeted. Recent studies in this area have advanced our general mechanistic understanding of iron-

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catalyzed wine oxidation; however, the role of copper remains poorly understood. The goal of this

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present study is to determine the underlying mechanism of Cu-catalyzed H2S and thiol oxidation

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under wine conditions. 4 ACS Paragon Plus Environment

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MATERIALS AND METHODS

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Chemicals. 4-Methylcatechol (4-MeC), L-cysteine (Cys), monobromobimane (MBB), 5,5-

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dimethyl-1-pyrroline N-oxide (DMPO), bathocuproinedisulfonic acid (BCDA) disodium salt, 6-

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sulfanylhexan-1-ol (6SH), and diethylenetriaminepentaacetic acid (DTPA) were obtained from

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Sigma-Aldrich (St. Louis, MO). 2,4-Dinitrophenylhydrazine (DNPH) was purchased from MCB

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laboratory chemicals (Norwood, OH) and L-tartaric acid, 3SH, and 5,5’-dithiobis(2-nitrobenzoic

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acid) (DTNB) were obtained from Alfa Aesar (Ward Hill, MA). Cupric sulfate pentahydrate was

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purchased from EMD Chemicals (Gibbstown, NJ), TRIS hydrochloride from J.T. Baker (Center

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Valley, PA), and sodium hydrosulfide hydrate (as a source of H2S) was purchased from Acros

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Organics (Geel, Belgium). Water was purified through a Millipore Q-Plus system (Milipore Corp.,

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Bedford, MA). All other chemicals and solvents were of analytical or HPLC grade, and solutions

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were prepared volumetrically, with the balance made up with Milli-Q water unless specified

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otherwise.

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Model wine experiments. Model wine was prepared by dissolving tartaric acid (5 g/L) in

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water, followed by the addition of ethanol to yield a final concentration of 12% v/v. The solution was

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adjusted to pH 3.6 with sodium hydroxide (10 M) and brought to volume with water. For H2S and

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Cys, an aqueous stock solution of each (0.5 M) was freshly prepared, whereas 6SH and 3SH were

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added directly by syringe during experimentation (Figure 2). An aqueous stock solution of Cu(II)

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sulfate (0.1 M) was prepared freshly. In certain experiments, 4-MeC (1 mM) was added prior to the

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addition of H2S and thiol compounds, and Cu(II). H2S, Cys, 6SH, or 3SH were added to air saturated

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model wine (1 L, 300 μM) followed by thorough mixing. Cu(II) was added to H2S, Cys, and 6SH (50

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μM) or 3SH (100 μM) and thoroughly mixed. For mixed H2S and Cys system, H2S (100 µM) and Cys

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

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and thorough mixing. The solution was immediately transferred to 60 mL glass Biological Oxygen 5 ACS Paragon Plus Environment


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Demand (B.O.D.) bottles (Wheaton, Millville, NJ), allowing the solution to overflow, and bottles

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were capped immediately with ground glass stoppers, thereby eliminating headspace. The glass

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reservoir of the B.O.D. bottles was topped off with water daily. The bottles were stored in the dark at

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ambient temperature. One B.O.D. bottle was sacrificed per time point per replicate and used for

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further analyses. All experiments were conducted in triplicate and had their own series of sacrificial

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bottles.

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For experiments focusing on 6SH-disulfide formation, one experiment was prepared as

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described above and followed over time. For additional experiments for deciphering immediate

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disulfide generation, model wine (3 mL) containing 6SH (600 μM) in a glass test tube was

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deoxygenated for 2 min under argon with stirring. After sparging, Cu(II) was added at varying

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concentrations (50, 100, or 200 µM) under argon and reacted with stirring for 5 minutes. The solution

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was then immediately analyzed to determine 6SH and 6SH-disulfide concentrations (described

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below). In experiments involving 4-MeC or DMPO, these compounds were dissolved directly into

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model wine to achieve a final concentration of 1 mM prior to addition of Cu(II) (100 µM).

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Determination of oxygen consumption. Prior to the experiment, 60 mL glass B.O.D. bottles

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containing PSt3 oxidots (Nomacorc LLC, Zublon, NC) were filled with air saturated model wine for

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a minimum of 2 hours to allow the oxidots to equilibrate. One B.O.D. bottle was used as a model

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wine control (i.e., did not contain a treatment) and two other bottles were used as technical duplicates

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to determine oxygen concentration for each treatment replicate (3 treatment replicates total). Thus,

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immediately after the addition of Cu(II) solution, the model wine used for equilibration was discarded

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and the respective treatment solution was instantly transferred into the bottles. Oxygen readings were

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taken per time point using NomaSense O2 P6000 meter (Nomacorc LLC, Zublon, NC), and data were

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normalized to the model wine reference sample. Starting oxygen concentrations were approximately

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7 mg/L (~220 µM) in all solutions. 6 ACS Paragon Plus Environment

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Cu-complex formation and dissolution. 6SH-Cu(I) complex was prepared by adding Cu(II)

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(100 µM) to model wine (1 L) containing 6SH (400 µM). The immediately formed precipitate was

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vacuum filtered with a 0.45 µm nylon membrane (Wheaton, Millville, NJ), washed with water

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followed by ethyl acetate in order to remove residual disulfide, and dried under vacuum. In an

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anaerobic chamber (95% Ar, 5% H2), ~1 mg of the solid was added to water containing approximately

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5× molar excess of BCDA. This mixture was stirred for approximately 30 min until all of the solid

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dissolved. 6SH, 6SH-disulfide, and Cu(I) concentrations were measured as described below.

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Spectrophotometric measurements of thiols. UV-vis spectra were recorded on an Agilent

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8453 UV-Vis spectrophotometer (Agilent, Santa Clara, CA). Determination of Cu binding to H2S and

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thiols was determined by measurement over 200-700 nm. The concentration of H2S, Cys, 6SH, and

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3SH was determined using Ellman’s reagent (DTNB).27 An aliquot of sample (100 μL) diluted with

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model wine (900 μL) was treated with a solution of DTNB (400 μL, 2 mM) in phosphate buffer (10

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mM, pH 7.0) followed by addition of TRIS-phosphate buffer (100 μL, 1 M, pH 8.1). The mixture was

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left at ambient temperature for 30 min before the absorbance was measured at 412 nm against a blank

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consisting of model wine, DTNB solution, and TRIS-phosphate buffer in the proportions specified

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above.

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Spectrophotometric measurement of Cu(I)-BCDA. Cu(I) concentration was analyzed

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using the BCDA assay.28 Treatment and standard solutions consisted of excess Cys (5 mM) to ensure

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Cu(I) remained in its reduced state. An external standard curve of the Cu(I)-BCDA complex was

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prepared in model wine, and absorbance values were recorded at 484 nm against a model wine blank.

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HPLC analyses of thiols. MBB derivatization was used to determine each H2S and Cys

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concentrations in the mixed system based on a modification of a previous method.29 MBB reagent

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(40 mM) was prepared anaerobically by dissolving the solid in acetonitrile. Aliquots of the reagent 7 ACS Paragon Plus Environment


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were stored at -80 °C. Briefly, a sample aliquot (70 μL) was mixed with an equal volume of TRIS-

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HCl buffer (100 mM) containing DTPA (0.1 mM) at pH 9.5, followed by the immediate addition of

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MBB (10 μL; 40 mM). The reaction was allowed to proceed aerobically at room temperature in the

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dark for 30 min before the addition of sulfuric acid (50 μL, 200 mM) and 6SH-bimane internal

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standard (50 μL). 6SH-bimane was prepared following a sulfide-dibimane synthesis described

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previously.29 Samples were filtered through PTFE syringe tip filters (0.45 μm, 13 mm filter diameter;

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

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Quantitative analysis was performed with a Shimadzu LC-VP series HPLC (Columbia, MD)

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interfaced to a Waters Quattro micro triple quadrupole mass spectrometer (Milford, MA) that was

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operated with MassLynx software. Bimane adducts were separated on a ZORBAX Eclipse Plus C18

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column (2.1 x 150 mm, 5 μm) with a guard column of the same material at a flow rate of 0.2 mL/min

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with mobile phases consisting of 0.1% v/v formic acid (A) and 0.1% v/v formic acid in acetonitrile

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(B) and a linear gradient according to the following program: 0 min, 2% B; 9 min, 50% B; 14 min,

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100% B; 18 min, 100% B; 19 min, 2% B; 26 min, 2% B.

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Detection of bimane adducts was performed using negative ion electrospray ionization (ESI-

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) with multiple reaction monitoring (MRM) (Figures S1-S3). The ESI capillary spray voltage was set

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to 4 kV, the sample cone voltage was set to 25 V, and the source temperature was 120 °C. The

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desolvation gas flow was 450 L/h and collision energy was set to 20 eV. The mass transition of

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sulfide-dibimane was monitored at m/z 413→191, cysteine-bimane was monitored at m/z 310→223,

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and the internal standard 6SH-bimane was monitored at m/z 323.2→222.2. An external standard curve

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was prepared for sulfide-dibimane and Cys-bimane and data were normalized to the 6SH-bimane

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internal standard.


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For experiments involving 6SH and its disulfide, quantitative analysis was performed using

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the HPLC system described above and UV detection at 210 nm with external standard calibration

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curves. Separation was achieved at a flow rate of 0.2 mL/min with mobile phases consisting of 0.1%

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v/v formic acid (A) and 0.1% v/v formic acid in acetonitrile (B) and a linear gradient according to the

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

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For experiments involving dissolution of 6SH-Cu complex with BCDA, the same

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chromatographic conditions described for 6SH and its disulfide were followed. However, the BCDA

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peak could not be resolved from that of 6SH at 210 nm, therefore detection of 6SH was performed

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using ESI+ with selective ion monitoring (SIM) at m/z 135 with an external calibration curve. The

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ESI capillary spray voltage was set at 4 kV, the sample cone voltage was set to 25 V and the source

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temperature was 120 °C. The desolvation gas flow was 650 L/h.

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HPLC analysis of catechols. For experiments containing 4-MeC, quantitative analysis was

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performed with the HPLC system described above and UV detection at 280 nm with an external

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standard calibration curve. 4-MeC was separated on an Ultra Aromax column (2.1 x 150 mm, 5 μm)

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with a guard column of the same material at a flow rate of 0.2 mL/min with mobile phases consisting

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of 0.1% v/v formic acid (A) and 0.1% v/v formic acid in acetonitrile (B) and a linear gradient

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according to the following program: 0 min, 30% B; 3 min, 30% B; 12 min, 100% B; 20 min, 100%

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B; 20.1 min, 30% B; 25 min, 30% B. The putative formation of oxidation products including catechol-

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thiol adducts and condensed units was monitored both at 280 nm and with negative ion ESI-MS (total

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ion chromatogram m/z 100-1000).

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HPLC analysis of acetaldehyde. Acetaldehyde was measured in model wine treatment

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solutions as its 2,4-dinitrophenylhydrazone (DNPH) derivative by HPLC as described previously30

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with the following modification: the sample was centrifuged at 15000 × g at 4 °C for 10 min. The

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supernatant was then transferred to an HPLC vial for further analysis. 9 ACS Paragon Plus Environment


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Copper determination. For each given time point, samples were mixed in B.O.D. bottles and

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then filtered through a 0.45 um PTFE syringe filter. The resulting filtrate (5 mL) was digested by the

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addition of 30% hydrogen peroxide (3 mL) and sulfuric acid (100 μL) based on modification of

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previous reported methodology.31 The samples were heated in a convection oven at 110 °C overnight

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before being reconstituted to 5 mL with 0.1 M nitric acid. Samples were analyzed by inductively

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coupled plasma optical emission spectroscopy (Agilent 700 Series, Santa Clara, CA) using a

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vertically aligned torch and with monitoring at 324.7 nm.

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EPR analysis. Loss of the electron paramagnetic resonance (EPR) signal for active Cu(II)

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(0.5 mM) in model wine was monitored after the metal solution was mixed with the respective H2S

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and thiol treatments (1.5 mM). Samples were transferred to a cuvette and snap frozen in liquid

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nitrogen. Continuous wave EPR spectra were acquired on a Bruker ESP300 X-band spectrometer

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

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

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Instruments, Abingdon, UK). Data acquisition and control of experimental parameters were

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performed using the EWWIN 2012 software package. Instrument settings were as follows:

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temperature, 100 K; microwave power, 2 mW; modulation frequency, 9480 MHz; modulation

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amplitude, 20 dB; scan range, 2000 G.

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RESULTS

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The reactivity of Cu(II) with H2S, which is the primary target of Cu fining, and the following

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three thiols was investigated under wine conditions (Figure 2): (1) Cys, which also represented homo-

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Cys and Cys derivatives, (2) 6SH to represent primary thiols, and (3) 3SH to represent secondary

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thiols. With H2S Cu(II) addition resulted in an immediate uptake of ~1.4 (72 µM) mole equivalents 10 ACS Paragon Plus Environment

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of H2S, the remainder was then fully consumed within 72 h. However, with the thiols, the immediate

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uptake increased to approximately two equivalents (Figure 3), with initial consumption of 101 and

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121 µM for Cys and 6SH, respectively, the remainder then being fully consumed within 48 h. The

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varietal thiol 3SH reacted in the same manner but more slowly, with 2 mole equivalent of 3SH (210

208

µM) consumed relative to Cu(II) added after 2 hours, and was not fully reacted after 168 h (Figure

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3).

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EPR analysis showed that Cu(II) was immediately reduced to Cu(I) due to loss of

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paramagnetic Cu(II) signal by Cys, 6SH and H2S; again, 3SH reacted more slowly (Figure 4A), with

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Cu(II) reduction being complete after 2 h (data not shown). The apparent formation of a Cu(I)

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complex was observed by UV spectroscopy (Figure 4B). Absorbance increased markedly from 200-

214

400 nm by the addition of H2S and Cys to model wine containing Cu(II), but did not produce a distinct

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absorbance maximum above 220 nm. In contrast, 6SH showed a maximum at 353 nm, and 3SH had

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absorbance maxima at 282 and 311 nm (Figure 4B).

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The addition of Cu(II) to H2S in model wine resulted in a clear golden colored solution that

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yielded a green/black precipitate over time, whereas a haze that developed with the three thiol

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treatments (Cys, 6SH, 3SH) aggregated to form a fine white/yellow precipitate. This was particularly

220

evident for 6SH, as essentially all the Cu(I) complex was removed by filtration (0.45 µm) from 5 to

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45 min after mixing (Figure 5A). Filtration at earlier time points and measurement of residual copper

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remaining in solution confirmed that the 6SH aggregate formed rapidly and could be removed from

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solution by filtration after 5 min (Figure 5B). However, at the last time point, copper had been released

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from the insoluble Cu(I) complex in a copper form that could not be removed by a 0.45 µm filter.

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3SH reacted in the same manner, but more slowly. For the H2S treatment, ca. 60% of the copper was

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removed by filtration within 5 min and up to 24 h. After 72 h, there was a green-black precipitate.

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Approximately 90% of copper was then removed from solution (Figure 5B). 11 ACS Paragon Plus Environment


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The aggregate initially formed from the reaction between Cu(II) and 6SH on drying gave a

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fine powder, which was solubilized in water containing BCDA (a Cu(I) selective chelator28). The

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insoluble Cu(I)-complex dissolved as BCDA displaced the thiolate ligand, yielding 1.17 ± 0.02 mM

231

Cu(I), as determined by UV spectrophotometry, and 1.17 ± 0.13 mM 6SH was released, as determined

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by HPLC-MS, giving a ~1:1 Cu(I):6SH molar ratio with minimal disulfide formation (data not

233

shown).

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When H2S (75 µM) and Cys (468 µM) were added together to model wine in the presence of

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Cu(II), ca. 53 and 135 µM of H2S and Cys, respectively, were consumed within 5 min (Figure 6).

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Together this gives 189 µM of sulfhydryl compounds consumed with added 100 µM Cu(II) which

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translates to a ~2:1 binding ratio of H2S + Cys:Cu(II). Subsequent reaction resulted in complete loss

238

of H2S within 40 min and Cys after 48 h. While a visible precipitate was observed at the end of the

239

reaction (74 h), it was not observed to the same extent as was the case with H2S alone.

240

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

241

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

242

3.6 µM, respectively (data not shown). In addition, the oxidation of 6SH (240 μM), in the presence

243

of 50 µM Cu(II) was monitored over time in air saturated model wine (Figure 7). After 262 h, 231 ±

244

2.5 µM of the thiol reacted and 116 ± 2.7 µM disulfide was produced. Approximately 69 ± 8.0 µM

245

O2 was consumed in this reaction (Figure 7), giving an O2:thiol molar reaction ratio of ~1:3.3.

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To further examine the mechanism of disulfide formation using 6SH as a model, an attempt

247

was made to intercept potential intermediate thiyl radicals with the o-quinone-producing 4-MeC, and

248

the radical trap DMPO. However, no change in disulfide formation was observed by HPLC upon

249

addition of Cu(II) (100 µM) to model wine containing 6SH (600 µM) and 4-MeC or DMPO (1.0 mM)

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under anaerobic conditions (data not shown).


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Oxygen consumption was also measured in model wines containing the H2S and thiol

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treatments, as well as a combination treatment consisting of Cys+H2S (Figure 8). Minimal O2 uptake

253

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