Mechanism of Interaction of Polyphenols, Oxygen, and Sulfur Dioxide in Model Wine and Wine

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1 Mechanism of Interaction of Polyphenols, Oxygen, and Sulfur Dioxide in Model Wine and Wine John C. Danilewicz, 1 * John T. Seccombe, 2 and Jonathan Whelan 2 Abstract: The interaction of oxygen, sulfur dioxide, and 4-methylcatechol (4-MeC) was studied in a model wine containing catalytic concentrations of iron and copper in order to provide further evidence that when a catechol and oxygen interact, hydrogen peroxide and a quinone are formed, both of which react with. The aerial oxidation of the catechol in the presence of benzenesulfinic acid (BSA) slowly produced the BSA-quinone adduct in high yield. It was also quickly prepared by adding ferric chloride, demonstrating that the quinone is cleanly produced in this model wine and that the catechol is rapidly oxidized by Fe(III) ions. This reaction is important in the catalytic function of the metal. The oxygen and molar reaction ratio was 1:2, which is consistent with one mole equivalent of reacting with hydrogen peroxide and a second with the quinone. When BSA was added to the system to trap the quinone the ratio was reduced to 1:1. The rate of reaction of oxygen and increased with catechol concentration. However, the rate of reaction of oxygen was also markedly accelerated by and by BSA, and it is proposed that substances that react with quinones accelerate catechol autoxidation. When 4-MeC was oxidized in the presence of, ~38% of the quinone that was formed reacted with bisulfite to produce the sulfonic acid adduct and most of the remainder was reduced back to the catechol. The O 2 molar reaction ratio in two red wines was 1:~1.7, suggesting that some nucleophilic substances may be competing with bisulfite for quinones. The rate of reaction of oxygen was also accelerated by in red wine. Key words: sulfur dioxide, oxygen, catechols, iron The mechanism of interaction of sulfur dioxide and oxygen has been comprehensively studied over the last hundred years, not least because of its great importance in producing acid rain. In the atmosphere as in wine, is hydrated and exists mainly as the bisulfite ion (HSO 3- ). Two molecules react with one of oxygen to produce two sulfate ions, and it has long been proposed that the same reaction occurs in wine. Consequently, it was envisaged that, by reacting with oxygen, protected vulnerable wine constituents from oxidation (Ribéreau-Gayon et al. 2000, Clarke and Bakker 2004). However, the reaction rate of oxygen with is quite slow relative to that which can occur in wine, and it has been argued that the main antioxidant action of is to react with hydrogen peroxide produced as a result of polyphenol oxidation (Boulton et al. 1996). Fe(II) catalyzes the reduction of hydrogen peroxide by way of the Fenton reaction to produce hydroxyl radicals, which are highly reactive and will oxidize ethanol to acetaldehyde (Danilewicz 2003, Waterhouse and Laurie 2006). The interaction of with oxygen is in fact quite complex (Scheme 1). It involves a metal-catalyzed radi Sandwich Road, Ash, Canterbury, Kent CT3 2AF, UK, and 2 Department of Wine, Plumpton College (University of Brighton), Ditchling Road, Plumpton, Nr Lewes, East Sussex BN7 3AE, UK. *Corresponding author (tel: ; jdanilewicz@btconnect. com) Manuscript submitted August 2007; revised December 2007 Copyright 2008 by the American Society for Enology and Viticulture. All rights reserved. 128 Scheme 1 Radical chain reaction involved in bisulfite oxidation. cal chain reaction in which oxidized forms of transition metals such as Fe(III) initiate the process by oxidizing bisulfite to the sulfite radical (SO 3 ). This radical reacts rapidly with oxygen, producing the highly oxidizing per- oxomonosulfate radicals (SO 5 ), which by reacting with bisulfite via two pathways (A and B in Scheme 1), produce sulfate and regenerate sulfite radicals to continue the chain process (Brandt et al. 1994, Brandt and van Eldik 1995, Connick et al. 1995). Catechols are known to block this reaction, presumably by scavenging intermediate peroxomonosulfate radicals, and so prevent radical chain propagation. The direct interaction of oxygen and bisulfite is therefore very unlikely to occur to a significant degree in wine because of the radical scavenging activity of polyphenols (Danilewicz 2007). The overall initial oxidative process that occurs in wine is shown in Scheme 2. It is proposed that oxygen and the catechol do not interact directly, but do so by redox cycling iron between them, possibly with the assistance of copper, the metals performing an essential catalytic function (Danilewicz 2007). The process is initiated by

2 Interaction of Polyphenols, Oxygen, and 129 the coordination of the catechol with Fe(III). The complex is unstable, electron transfer within the complex resulting in the formation of Fe(II) and the semiquinone radical, which dissociate. The semiquinone may then be oxidized by Fe(III) or disproportionate to produce the quinone, while the Fe(II) is reoxidized to the ferric state by oxygen to continue its catalytic function. As a result of the latter interaction, oxygen is reduced first to the hydroperoxyl radical and then to hydrogen peroxide, probably by the catechol (Danilewicz 2003, Waterhouse and Laurie 2006, du Toit et al. 2006). Bisulfite is known to react rapidly with hydrogen peroxide but it should also be capable of reacting with the quinone. Studies of the interaction of bisulfite with 1,4-benzoquinone have shown that at ph 3.5 approximately half was reduced back to 1,4-benzenediol and half underwent Michael-type 1,4-addition to give the sulfonic acid (LuValle 1952, Youngblood 1986). These transformations can be represented as shown in Scheme 3 for a catechol. The reduction mechanism has been proposed to explain some observations (Saucier and Waterhouse 1999, Cheynier et al. 1989a). However, the addition route is an alternative possibility, with the irreversible formation of the stable sulfonic acid. This is supported by the observation that 3,4-dihydroxybenzenesulfonic acid is produced when catechol is oxidized by tyrosinase in the presence of at ph 6.5 (Wedzicha et al. 1987). The aim of this work was to examine the relative molar reaction ratio of oxygen, a catechol, and in a model wine to see if it is consistent with the proposed mechanism. When a simple catechol is oxidized, two mole equivalents of should be taken up for each mole of oxygen consumed (Scheme 3); the same molar ratio as if had interacted directly with oxygen. However, it has been demonstrated that the rate of oxidation is dependent on catechol concentration (Danilewicz 2007), and a further aim was to demonstrate that the quinone was Scheme 2 Proposed catalytic action of iron and copper in the oxidation of catechols to produce quinones and hydrogen peroxide. Scheme 3 The interaction of bisulfi te with hydrogen peroxide and qui- The interaction of bisulfite with hydrogen peroxide and quinones following catechol oxidation. indeed produced in the presence of and to determine how the two interact. If all the quinone were reduced back to the catechol, then the catechol concentration would appear to remain constant, so that it would seem that it had not been involved in the oxidative process. Some work was also undertaken to show that the mechanism also applied to real wine. Materials and Methods Materials. Water (ISO 3696:1987 grade 3) and copper(ii) sulfate pentahydrate (BDH AnalaR grade) were obtained from VWR International (Lutherworth, UK). Water (HPLC gradient grade) and sodium hydroxide (AR grade) were from Fluka (Pool, UK). Other materials were ethanol (absolute for microbiology), iron(ii) sulfate heptahydrate, 4-methylcatechol (4-MeC), benzenesulfinic acid (BSA) sodium salt, (R)-cysteine, 6-mercaptohexan-1- ol (6-MHex), and acetonitrile (Chromasolv, HPLC gradient grade) (Sigma-Aldrich, Dorset, UK); potassium metabisulfite (Kadifit, Geisenheim AG, Germany); tartaric acid (AR grade) (Fischer Scientific, Loughborough, UK); and glacial acetic acid (Scientific and Chemical Supplies, Bilston, UK). Kieselgel 60 F 254 plates (Merck, Darmstadt, Germany) were used for thin-layer chromatography and compounds were visualized by UV light and by spraying with aqueous potassium permanganate. 1 H NMR spectra were recorded using a Unity INVA 600 MHz NMR spectrometer (Varian, Palo Alto, CA). Electrospray-mass spectrometry (ES-MS) was performed on a ThermoFinnigan LCQ Classic instrument (Thermo Fisher Scientific, Waltham, MA). Preparation of model wine solutions. (+)-Tartaric acid (8.0 g) was dissolved in water (~800 ml) in a 1-L volumetric flask. Ethanol was added to give a 12% (v/v) final concentration for the dissolved oxygen studies and 12.5% for HPLC experiments. 10 ml of a freshly made up solution of Fe(II) sulfate heptahydrate (248.9 mg) in water (100 ml) and 1 ml of a solution and Cu(II) sulfate pentahydrate (58.9 mg) in water (100 ml) were added to give a final concentration of 5 mg/l Fe and 0.15 mg/l Cu. This was followed by solid potassium metabisulfite to give the required free concentration. The ph was increased to 3.60 with 2.5 N sodium hydroxide, also adding water progressively to the mark as the required ph was approached. The concentration of tartaric acid was selected so that it would simulate the action of all wine acids and produce a final titratable acidity of ~4 g/l. Establishment of O 2 molar reaction ratio in a closed system. A small amount of the above model wine solution was withdrawn to wash in the required amount of 4-MeC and BSA, when the latter was included. The resulting solution was saturated with oxygen by shaking in air and then sealed in 12 ~60-mL glass bottles with rubber-lined metal screwcaps, ensuring the exclusion of any air bubbles and then stored in the dark. Maximum and minimum temperatures were noted during the course of each experiment. Initial free concentration was

3 130 Danilewicz et al. determined in duplicate and O 2 concentration in triplicate as described below. Three bottles were taken at each time point so as to measure free and O 2 concentrations in triplicate. Mean values (±SD) were calculated and figures drawn using Excel software (Microsoft, Redmond, WA). Where error bars denoting ±SD are not shown they were smaller than the data point symbol dimensions. Effect of (R)-cysteine and 6-MHex on rate of O 2 consumption in a closed system. (R)-Cysteine, 6-MHex, and 4-MeC were dissolved as required in the model wine solution, to which was not added. Initial O 2 concentration was measured and then followed over time in triplicate as described above. Unlike the odorant wine thiols, 6-MHex has an unpleasant odor. Oxygen and measurement. An HI-9146 dissolved oxygen meter fitted with a HI Clarke type electrode (Hanna Instruments, Leighton Buzzard, UK) was used to measure oxygen. The manufacturer specifies a resolution and limit of detection (LOD) of the meter as 0.01 mg/l O 2. For measurements the bottle cap was quickly removed and a small stirrer bar inserted followed by the electrode, which displaced ~13 ml liquid. The electrode tip was lowered to ~5 mm of the briskly stirred magnetic bar. Readings stabilized within 30 sec and then remained stable for more than 5 min, showing that, although the system was not sealed during measurement, no measurable amount of external oxygen reached the measurement area during that time. This finding applied to oxygen concentrations down to ~1 mg/l. To test the air tightness of the sealed bottles, water was degassed by boiling to zero oxygen and the bottles sealed. Zero oxygen readings were obtained each day for 4 days. For, free and bound concentration was measured by the aeration-oxidation method (Iland et al. 2004, Ough and Amerine 1988). Changes in 4-MeC and its reaction products in a continuously aerated system. Model wine solutions (150 ml), in which required amounts of 4-MeC and BSA were dissolved were placed in 750-mL clear glass bottles, sealed with a plastic cap, and maintained in the dark at ambient temperature, with maximum and minimum temperatures recorded during each series of experiments. The large air headspace ensured that solutions remained at or near aerial oxygen saturation as <2% of the available oxygen reacted. The bottles were only brief ly exposed to light when analyzed, at which time the solutions were shaken and stoppers briefly removed. The concentration of 4-MeC and its reaction products were determined by HPLC. Free concentration was also measured as described above. Experiments were performed in triplicate. Mean values (±SD) were obtained and treated as described above. HPLC analysis. Analyses were carried out using two Shimadzu LC-6A pumps and a SPD-6A UV-vis detector (Shimadzu, Milton Keynes, UK). A Hypersil ODS C 18 RPC column (Thermo Electron Corp., Waltham, MA; 250 mm x 4.6 mm; particle size 5 µm) was used, fitted with a 5-µm column guard (Hypersil ODS 3.0 mm). Injection of samples was performed using a Rheodyne 7725i injection port (IDEX Corp., Northbrook, IL) fitted with a 20-µL injection loop. Isocratic elution was performed using a 6:4 mixture of acetonitrile and 3% aqueous acetic acid, at a f low rate of 1.0 ml/min. Solutions of compounds to be analyzed were injected in triplicate at concentrations of 50, 25, 12.5, and 6.25 mg/l in 0.8% aqueous tartaric acid to obtain a calibration curve, using a detection wavelength of 295 nm, which was close to the absorption maximum of 4-MeC (298 nm). This wavelength provided the best sensitivity to construct its calibration curve, which of the compounds studied demonstrated the greatest variability. The simultaneous quantification of other compounds studied, such as BSA, the BSA-quinone adduct, and 4-MeC sulfonic acid, was also conveniently conducted at that wavelength. Model wine solutions were diluted 1:10 prior to injection. Data was collected and analyzed using Clarity software version (DataApex, Prague, Czech Rep.) on a Windows XP system. Typical elution times were sulfonic acid 1.97 min, BSA 2.47 min, 4-MeC 3.63 min, and BSA-quinone adduct 3.87 min. Wines. Two red wines were examined: AOC Beaujolais, Domaine des Communes, 2005 (Dupond, Villefranche, France) and Willowglen, Shiraz 2005 (De-Bortoli, Bilbul, South East Australia). Measurement of O 2 molar reaction ratio and rate of consumption of O 2 in a closed system. Free concentration was adjusted by adding solid potassium metabisulfite. The wine was then saturated with oxygen by shaking in air and sealed in 12 ~60-mL bottles. Dissolved oxygen and concentration were determined as for model wine. However, both free and bound were determined as effects on total concentration had to be taken into account. Model wine solutions contained only very small amounts of bound due to contaminating acetaldehyde in the ethanol. In the experiment where was removed, a molar equivalent of hydrogen peroxide was added to react both with free and bound. Total removal was confirmed by measurement. Excess hydrogen peroxide was not used. 4,5-Dihydroxy-2-methylbenzenesulfonic acid potassium salt. 4-Methylcatechol (2.0 g) was dissolved in warm concentrated sulfuric acid (2 ml, sp gr 1.84) giving a red oil, which solidified on standing at room temperature for 24 hr. The mixture was cautiously dissolved in water (20 ml), diluted with an equal volume of methanol, and the resulting solution basified to ph 6 with solid K 2 CO 3, added cautiously in small portions. On cooling, the K 2 SO 4 that was produced was filtered off and the filtrate evaporated to dryness under reduced pressure. The resulting solid was extracted three times with hot methanol, which on evaporation gave a yellow foam. The crude product was dissolved in hot ethanol, decanting off from a small amount of insoluble brown tar. On cooling, an initial crop (720 mg) of a light buff solid crystallized out. Concentration of the filtrate gave a second crop (777 mg) of the required compound as a white solid. R f 0.45

4 Interaction of Polyphenols, Oxygen, and 131 (butan-1-ol:hoac:h 2 O, 25:6:25 top layer). 1 H NMR (D 2 O): δ 7.36 (s, 1H), 6.84 (s, 1H), 2.44 (s, 3H). ES-MS m/z (M-H + ) -. 4-Benzenesulfonyl-5-methylbenzene-1,2-diol. 4-Methylcatechol (434 mg, 3.5 mmol) and BSA sodium salt (574 mg, 3.5 mmol) were dissolved in model wine (250 ml), which was shaken in air and placed in a 500-mL flask in the dark. The product began to crystallize out after 5 days, and when left with periodic air saturation over 2.5 months the suspension was extracted with EtOAc. The extract was washed with water, dried over MgSO 4, and on evaporation gave an oil which crystallized to a white solid (841 mg, 91%). Recrystallization from aqueous ethanol gave white crystals (760 mg, 82%). R f 0.31 (EtOAc: hexane, 1:1). 1 H NMR (DMSO-d 6 ): δ 7.86, 7.85 (2t, 2H), 7.77, 7.76, 7.75 (3t, 1H), 7.70, 7.69, 7.68 (3t, 2H), 7.61 (s, 1H), 6.78 (s, 1H), 2.26 (s, 3H). ES- MS m/z (M-H + ) -. For rapid synthesis, 4-methylcatechol (496 mg, 4 mmol) and BSA sodium salt (656 mg, 4 mmol) were dissolved in model wine (200 ml). Solid FeCl 3.6H 2 O (2.16 g, 8 mmol) was added in small portions over 10 min to the stirred solution. Product began to crystallize out within ~5 min and was left to stir for a further 3 hr; ph had dropped to ~2. The mixture was extracted with EtOAc, the extract washed with dilute aqueous tartaric acid, followed by water and dried over MgSO 4. Evaporation gave an oil which crystallized to a white solid (914 mg, 86%). Recrystallization as above gave the required product (754 mg, 72%). R f 0.31 (as above). Results and Discussion BSA has been used in mechanistic studies to trap quinones with which it reacts rapidly in a 1,4-Michael fashion (Scheme 4) (Cheynier et al. 1989a, 1989b). In an initial experiment, therefore, model wine containing 4-MeC was exposed to air in the dark in the presence of an equimolar amount of the sulfinic acid. The quinone- BSA adduct began to crystallize out as glistening crystals after a few days, and when the solution was left for 2.5 months with periodic aerial oxygen saturation, it was isolated in 82% recrystallized yield. This reaction mixture remained clear over that period, but the same reaction mixture to which BSA had not been added became orange-brown, suggesting that the pigment was derived from the quinone. The BSA-adduct was also prepared quickly by adding two equivalents of ferric chloride to the above mixture, when the adduct crystallized out almost immediately and was isolated in 72% yield. If the mechanism for formation of the BSA- adduct is accepted, these results show that the quinone is indeed formed quite cleanly in these model wine conditions on exposure to oxygen, when catalytic amounts of iron and copper are included, and that the catechol does also react rapidly with Fe(III) to produce it. It is proposed that this latter interaction with iron is a key initial process in the autoxidation of catechols (Scheme 2). The O 2 molar reaction ratio was examined first. Model wine containing and increasing concentrations of 4-MeC was saturated with aerial oxygen and sealed in 60-mL screwcapped bottles as described previously. The reduction in oxygen and concentration was measured over time, and the rate of reaction of both increased with 4-MeC concentration (Figure 1). Clearly, such an increase in rate of reaction would not occur if simply reacted with oxygen without catechol involvement. The O 2 molar reaction ratio was found to be close to 1:2 as required by the above mechanism. BSA (1.64 g/l; 10 mm) was then added to this model wine system, containing 4-MeC (300 mg/l; 2.4 mm), and (44.8 mg/l; 70 µm), to see if it could compete with bisulfite for the quinone. The O 2 molar reaction ratio was reduced to 1:1 (Figure 2; curves b)o 2 and b) ), indicating that now only reacted with hydrogen peroxide. For comparison, curves d)o 2 and d) were obtained in the same system when BSA was omitted, and in which a 1:1.93 molar reaction ratio is evident. It was then observed that the rate of oxygen reaction was markedly accelerated by itself (Figure 2; curves a)o 2 and d)o 2 ), and also by BSA in the absence of (curves a)o 2 and c)o 2 ). The marked acceleration in rate of oxygen reaction by was observed at 4-MeC concentrations ranging from 80 mg/l to 1.2 g/l (Figure 3). As discussed above, is unlikely to react directly with oxygen because of the inhibitory action of the catechol. Neither is BSA oxidized in these model wine conditions; it acts simply as a nucleophile. Otherwise, the adduct would not be formed in such high yield in the above Scheme 4 1,4 Michael addition of a benzenesulfinate anion to a quinone to give a BSA adduct. Figure 1 Rate of reaction of O 2 and in the presence of different concentrations of 4-MeC in a model wine. Starting values in mg/l: (a) 50.4; O , (b) 49.6; O , (c) 44.0; O Molar reaction ratios were calculated from the last data points.

5 132 Danilewicz et al. experiments in which equimolar amounts of 4-MeC and BSA were oxidized. As will be discussed below, bisulfite also reacts with the quinone, and it was suspected that this property, which it shares with BSA, may explain the accelerating effect. The interaction of the Fe(III)-Fe(II) and 1,4-benzoquinone 1,4-hydroquinone redox systems is an equilibrium process (Baxendale et al. 1951), but whether this is so in wine for catechols is uncertain due to the greater instability of 1,2-benzoquinones. However, the respective quinones of caffeic acid, (+)-catechin, and 4-MeC can be observed by HPLC, when these compounds are rapidly oxidized with polyphenoloxidase, but these quinones are unstable, being vulnerable to attack by nucleophiles (Richard-Forget et al. 1992, Cheynier et al. 1989b). Attempts to speculate on the redox state of a wine, that is the position of polyphenol-quinone equilibria, from reduction potentials is not possible as it is now understood that the observed potential is due principally to the oxidation of ethanol at the anode. This is balanced by the reduction of oxygen, the rate of which, and hence the magnitude of the recorded potential, is dependent on the oxygen concentration at the instant of measurement (Vivas et al. 1993). In the absence of oxygen, when low potentials are recorded, protons take the place of oxygen and are reduced to hydrogen. At the concentrations found in wine, polyphenols are not sufficiently electroactive to make a significant contribution, as for example red and white wines behave similarly with respect to changes in reduction potential (Kilmartin and Zou 2001). The two-electron reduction potential for the catechol quinone and the oxygen hydrogen peroxide couples is 0.58 V and 0.57 V, respectively, at wine ph (Danilewicz 2003), and so the net difference in potential (ΔE) is close to zero for the interaction of the catechol with oxygen to produce the quinone and hydrogen peroxide (Scheme 5). From the relationship -ΔG = 2FΔE for a two-electron process and where F is the Faraday constant, it is evident that the change in free energy for the process is close to zero. It is proposed that the resulting slow reaction only proceeds forward because of the reaction of the quinone. This displaces the reversible process, which, however, would be unlikely to reach equilibrium, allowing the net production of Fe(II) to reduce oxygen (Scheme 5). Consequently, it is further proposed that the acceleration of the oxidative process by bisulfite and BSA is due to their ability to intercept the quinone as it is formed. The same mechanism has been proposed elsewhere for the marked acceleration produced by glutathione on the rate of transcaftaric acid oxidation by polyphenoloxidase (Cheynier and Van Hulst 1988). The authors provide evidence that the oxidation is reversible, the quinone being simultaneously reduced as the catechol is oxidized. Consequently by trapping the quinone as it is formed, glutathione accelerates net oxidation. In view of the results obtained by Cheynier and Van Hulst (1988), the effect of thiols on rate of oxygen consumption induced by 4-MeC was also briefly investigated, initially using (R)-cysteine as a model thiol. Glutathione was not oxidized in their polyphenoloxidase experiment, but in our model wine conditions (Figure 4), (R)-cysteine is oxidized much more rapidly than the catechol, so obscuring any accelerating effect. In contrast, Cilliers and Singleton (1990) found that cysteine did not cause the consumption of oxygen and slowed that produced by catechols at ph 7 and above, when no metals were deliberately added. As with catechols and, the oxidation of thiols is a free radical process catalyzed by metals such as Fe and Cu (Lamfrom and Nielsen 1957, Smith et al. 1994). It is proposed that thiols form an unstable complex with Fe(III) or Cu(II), electron transfer resulting in Figure 2 Effect of and BSA on rate of reaction of O 2 in model wine containing 300 mg/l 4-MeC, and effect of BSA on the O 2 molar reaction ratio. Figure 3 Increase in rate of reaction of oxygen in the presence at increasing 4-MeC concentrations. Scheme 5 The coupled oxidation reduction of a catechol and oxygen by redox cycling of iron. The effect of nucleophiles is to drive the process forward by reacting with the quinone.

6 Interaction of Polyphenols, Oxygen, and 133 the formation of a thiyl radical (RS ) and reduction of the metals to Fe(II) and Cu(I), respectively (Scheme 6). The thiyl radical may either react with oxygen to produce the oxidizing sulfinic acid-derived radical (R ) or by reacting with a thiolate ion produce the reducing disulfide radical anion (RSSR ) (Schäfer et al. 1978). In this latter route oxygen is consumed by returning the reduced metals to their oxidized state, so producing hydroperoxyl radicals (HO 2 ) and hydrogen peroxide. The relative rate of the two processes will be ph-dependent, as under acid conditions thiolate ion concentration will be low, the pk a of simple thiols being ~10. The rapid oxidation of cysteine is probably due to its ability to form a strong bis-bidentated complex with Cu(I), which readily reacts with oxygen (Bagiyan et al. 2004, Pecci et al. 1997). Simpler thiols, which do not contain a second metal-binding functionality, should form weaker complexes, which may react more slowly. That appears to be so with 6-MHex (Figure 4), which was chosen as a model for the odorant wine thiols (Tominaga et al. 1998, Bouchilloux et al. 1998). After a small initial consumption of oxygen, which was probably due to the oxidation of Fe(II), 6-MHex caused no further significant consumption of oxygen over 5 days at the low concentrations used in this study. However, there was an increase in rate of oxygen consumption when 4-MeC was added compared with 4-MeC alone (Figure 4; curves b and c). In fact the observed rate was faster than that produced by 4-MeC alone at twice the concentration. The ability of bisulfite to compete effectively with thiols for quinones explains the protecting effect of with respect to loss of odorant thiols on wine oxidation (Blanchard et al. 2004). A further point is that the reduction potential of the RS / RSH couple is calculated as 1.16 V at wine ph from the value found at ph 7 (Surdhar and Armstrong 1987). That is higher than the semiquinone/catechol couple of 0.85 V (Danilewicz 2003). Consequently, catechols should protect thiols from oxidation by reducing any thiyl radicals that might be produced back to the thiol. The acceleration in rate of 4-MeC reaction by BSA was also observed in model wine when maintained at or near aerial oxygen saturation, using HPLC to determine the rate of reaction of 4-MeC and the rate of formation of the BSA-adduct (Figure 5). At hr, 89.3 mg/l of the catechol had reacted in the presence of BSA, while mg/l of the adduct had formed; a 94% yield. The adduct began to crystallize out beyond that time. Here again the catechol was cleanly oxidized to the quinone and BSA was acting simply as a nucleophile. By comparison, the rate of reaction of 4-MeC in the absence of or BSA was very slow (Figure 5). The interaction of with the quinone derived from 4-MeC was studied in model wine, which was maintained at or near aerial oxygen saturation. Free concentration was determined over time together with the concentration of 4-MeC and its sulfonic acid derivative by HPLC. A reference sample of the sulfonic acid was prepared by sulfonation of 4-MeC as described in the experimental section. The initial 4-MeC concentration was 250 mg/l and that of free was 76.5 mg/l (Figure 6). On extrapolation of the regression line for concentration, it fell by 48.9 mg/l (7.64 x10-4 mol/l) at hr. At that time the amount of sulfonic acid derivative that was produced was 30.0 ± 1.5 mg/l (1.46 ± 0.07 x 10-4 mol/l), which translates to a ~38% yield, on the assumption that half the consumed had been available to the quinone. The concentration of 4-MeC was depleted by 21.6 ± 5.3 mg/l (1.74 ± 0.4 x 10-4 mol/l). Since the O 2 molar Figure 4 Effect of (R)-cysteine and 6-MHex on oxygen consumption by 4-MeC in model wine: (a) 6-MHex (81.4 mg/l, 6.06 x 10-4 mol/l); (b) 4-MeC (600 mg/l, 4.83 x 10-3 mol/l); (c) 4-MeC (600 mg/l) + 6-MHex (81.4 mg/l); (d) (R)-cysteine (107 mg/l, 6.09 x 10-4 mol/l); (e) (R)-cysteine (107 mg/l) + 4-MeC (600 mg/l); (f) (R)-cysteine (764 mg/l, 4.35 x 10-3 mol/l). Scheme 6 Proposed mechanism for the metal catalyzed interaction of thiols with oxygen. Figure 5 Effect of BSA (1.64 g/l) on rate of 4-MeC (250 mg/l) oxidation in model wine maintained at or near aerial oxygen saturation without.

7 134 Danilewicz et al. reaction ratio is 1:2, the amount of oxygen and hence the amount of catechol that reacted should have been (3.82 x 10-4 mol/l), that is 47.4 mg/l of the catechol, indicating that between 43 and 56% of the quinone had been reduced back to the catechol. Although the 4-MeC assay gave somewhat variable results in this study, it is apparent that much less 4-MeC was actually consumed than the amount that should have reacted. In this experiment, between ~6 and ~19% of the catechol that was initially oxidized was unaccounted for. It is possible, therefore, that up to half that amount could have condensed with the quinone. It could be argued that in the above experiment no quinone reduction had actually occurred and that the catechol simply had not been oxidized in the first place. To explore this possibility, BSA was added in an attempt to trap the quinone before it could be reduced (Figure 7). At hr, and 4-MeC concentration fell by 52.8 mg/l (8.24 x 10-4 mol/l) and by 48.2 mg/l (3.88 x 10-4 mol/l), respectively, and during that time 98.8 mg/l (3.74 x 10-4 mol/l) of the quinone-bsa adduct and 12.6 mg/l (6.17 x 10-5 mol/l) of the sulfonic acid had formed. Bisulfite will react with hydrogen peroxide, add to the quinone, and also reduce it back to the catechol (Scheme 7). However, some of the quinone will also be intercepted by BSA. By simple algebraic substitution using the above values, it was calculated that 27% of the quinone was reduced, 10% added bisulfite to produce the sulfonic acid, and 62% formed the BSA adduct. In all, 72% of the quinone was trapped as addition compounds. The proportion of products is likely to be dependent on the relative concentrations of the nucleophiles. The competition study between and BSA previously discussed was performed at a lower relative concentration, and all the quinone appears to have reacted with the BSA (Figure 2). The mechanism by which bisulfite in effect transfers two electrons and two protons to the quinone to reduce it back to the catechol is intriguing. Kinetic studies investigating the addition of bisulfite to 1,4-benzoquinone indicate the rapid reversible formation of the 1,2-adduct before bisulfite addition (Youngblood 1986). These transformations for a 1,2-benzoquinone system are shown in Scheme 8. It is proposed that nucleophilic attack by water on sulfur of the 1,2-adduct might achieve the necessary transformation to produce the catechol. The catechol 4-substituent may exert an inhibitory steric effect on addition to the neighboring ortho-position. Consequently, 1,2-addition and hence reduction may be relatively more favored in flavanols, which have larger catechol 4-substituents than 4-MeC. The rate of oxygen and consumption in a red 2005 Beaujolais wine under the conditions of the above experiments is shown (Figure 8). Some bound dissociated as free concentration was reduced, and so it was necessary to take total consumption into account. Although the rate of reaction varied, a smooth reduction in oxygen concentration was observed. The initial O 2 Figure 6 Change in concentration of 4-MeC, sulfonic acid, and on oxidation of model wine maintained at or near aerial oxygen saturation. Starting concentrations: 250 mg/l 4-MeC and 75.6 mg/l free. Figure 7 Formation of sulfonic acid and BSA-adduct and reduction in and 4-MeC concentration on oxidation of model wine maintained at or near aerial oxygen saturation. Starting concentrations: 4-MeC 251 mg/l, free 81.6 mg/l, BSA 1.64 g/l. Scheme 7 Reaction of with the quinone produced when 250 mg/l 4-MeC was oxidized under aerial oxygen saturation in model wine containing 81.6 mg/l free and 1.64 g/l BSA. Scheme 8 Proposed mechanisms for the reaction of bisulfite with quinones: 1,4 and 1,2 addition of bisulfite to a quinone resulting in the formation of the sulfonic acid and reduction to the catechol.

8 Interaction of Polyphenols, Oxygen, and 135 Figure 8 Reaction of oxygen and in a Beaujolais 2005 wine. Initial concentration: free 36.7 mg/l, bound 41.4 mg/l; O mg/l. Figure 9 Effect of on rate of reaction of oxygen in red wine, Beaujolais, Dupond 2005 (starting concentrations shown). molar reaction ratio at 21 hr was 1:2 as in model wine but the ratio dropped to 1:1.7 as the reaction continued. It is possible that in wine nucleophilic polyphenols are better able to compete with bisulfite for quinones as concentration falls (Tao et al. 2007). The same overall pattern was also observed in a 2005 Australian Shiraz wine (data not shown). More data is required, but it could demonstrate that the concentration of may control the ability of quinones to condense with polyphenols in red wine when exposed to oxygen, such as in microoxygenation and barrel aging. The accelerating effect on oxygen consumption was also observed in red wine. The free concentration was increased to 51.2 mg/l in one portion of the Beaujolais wine and removed entirely with hydrogen peroxide in a second. Both were saturated with aerial oxygen, and the consumption of oxygen was markedly slowed by the removal of (Figure 9). Conclusions These studies have provided further evidence that the rate of reaction of oxygen and in model wine is dependent on the concentration of the catechol. Clearly, therefore, does not simply react with oxygen to pro- tect vulnerable polyphenols from oxidation as has long been assumed. It is well established that the autoxidation of is a radical chain reaction, which is blocked by radical scavenging polyphenols and hence the direct interaction of oxygen and should not occur in wine conditions. Trapping experiments using BSA have shown that the quinone is cleanly produced on oxidation of a catechol in these model wine conditions. Furthermore, the O 2 molar reaction ratio of 1:2 indicates that reacts not only with hydrogen peroxide but also with this quinone. That has been confirmed by the identification of the sulfonic acid, which is formed in significant amounts under these experimental conditions. Results are consistent with the remainder of the quinone being reduced back to the original catechol, as has been shown for 1,4-benzoquinone. The addition of BSA as a competing nucleophile prevents the reaction of bisulfite with the quinone. In real wine it is therefore possible that nucleophiles such as polyphenols and odorant thiols could compete with bisulfite for quinones, depending on concentration. This has implications in the deliberate exposure of wine to oxygen such as in microoxygenation and barrel aging when concentration could affect the results. The quinones derived from caffeic acid, (+)-catechin, and especially (-)-epicatechin are considerably less stable than the one derived from 4-MeC; therefore, the behavior of these substances in these model wine conditions needs to be investigated to establish if the above findings are more broadly applicable to real wine catechols. A fuller study is also necessary to better understand the interaction of thiols with the polyphenol-oxygen- system, which would help in managing the removal of undesirable thiol odorants and preserving desirable ones of grape origin. An important finding is that accelerates the reaction of oxygen in model wine conditions and in red wine, which we propose is due to the reaction of bisulfite with quinones. If so, it would suggest that the broad conclusions drawn in these studies regarding the interaction of bisulfite and other nucleophiles with quinones also apply to wine. Literature Cited Baxendale, J.H., H.R. Hardy, and L.H. Sutcliffe Kinetics and equilibria in the system ferrous ion + ferric ion + hydroquinone + quinone. Trans. Faraday Soc. 47: Bagiyan, G.A., I.K. Koroleva, N.V. Soroka, and A.V. Ufimtsev Kinetics of the catalytic oxidation reactions of thiol compounds in aqueous solutions in the presence of copper ions. Kin. Cataly. 45: Blanchard, L., P. Darriet, and D. Dubourdieu Reactivity of 3-mercaptoheptanol in red wine: Impact of oxygen, phenolic fractions, and sulfur dioxide. Am. J. Enol. Vitic. 55: Bouchilloux, P., P. Darriet, R. Henry, V. Lavigne-Cruège, and D. Dubourdieu Identification of volatile and powerful odorous thiols in Bordeaux red wine varieties. J. Agric. Food Chem. 46:

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