The Pennsylvania State University. The Graduate School. Department of Food Science INVESTIGATING THE FORMATION AND FATE OF ETHYL RADICALS IN WINE

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1 The Pennsylvania State University The Graduate School Department of Food Science INVESTIGATING THE FORMATION AND FATE OF ETHYL RADICALS IN WINE A Thesis in Food Science by Gal Y. Kreitman 2013 Gal Y. Kreitman Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science May 2013

2 The thesis of Gal Y. Kreitman was reviewed and approved* by the following: Ryan J. Elias Assistant Professor of Food Science Thesis Advisor Joshua D. Lambert Assistant Professor of Food Science John E. Hayes Assistant Professor of Food Science Robert F. Roberts Professor of Food Science Interim Head of the Department of Food Science *Signatures are on file in the Graduate School ii

3 ABSTRACT The shelf life of wine is greatly affected by its oxidative stability. The nonenzymatic oxidation of wine is thought to proceed by metal-catalyzed reactions capable of oxidizing phenolics and reducing oxygen to quinones and hydroxyl radicals, respectively. Previous studies have focused upon the important reactions that occur between quinones and other wine components, but the fate of oxygen radicals within the context of wine oxidation is less understood. Ethanol is the prime target for hydroxyl radicals in wine due to its abundance (~2 M). I investigated the oxidative stability of model wine solutions with respect to major wine constituents after ethanol (i.e. phenolics, glycerol, and tartaric acid), knowing ethanol forms 1-hydroxyethyl radicals (1-HER) using spin trapping with phenyl-n-tertbutyl nitrone (PBN) paired with electron paramagnetic spectroscopy (EPR). I then focused on the potential reaction between hydroxyl radicals and tartaric acid, and measured other important oxidative markers including hydrogen peroxide consumption and acetaldehyde formation, a known oxidation product of ethanol. As expected, 4- methylcatechol (4-MeC), a model phenolic, accelerated the oxidation of ethanol by hydroxyl radicals, and had resulted in higher 1-HER/PBN adduct formation. Glycerol, despite its presence at relatively high concentrations, did not significantly affect spin adduct formation, suggesting that it was not a major target of hydroxyl radical-mediated oxidation. Interestingly, tartaric acid, seemed to shift from slight antioxidant activity to pro-oxidant activity when oxygen was purged from the model wine system. I investigated the reaction between 1-HER and various wine-related phenolics and thiols, including gallic acid, caffeic acid, ferulic acid, 3-mercaptohexan-1-ol (3MH), cysteine (Cys), and glutathione (GSH) by using competitive spin trapping with detection by EPR and mass spectrometry. In addition, I measured the ability of these compounds iii

4 to prevent the formation of acetaldehyde. The hydroxycinnamic acids, which include caffeic acid and ferulic acid, significantly inhibited 1-HER formation compared to treatment-free model wine by 69% and 55%, respectively. The thiol treatments, Cys, GSH, and 3MH also significantly inhibited 1-HER formation by 88, 87, and 96%, respectively. As expected, I observed similar trends when I measured spin adduct formation with mass spectrometry and when I measured final acetaldehyde yields. This confirms that hydroxycinnamic acids and thiols react with 1-HER under model wine conditions. I also measured the oxidative loss of 3MH, an important varietal thiol, in model wine resulting from either 2-electron quinone trapping or direct reaction with 1- HER. I observed, for the first time, evidence that 1-HER reacts with 3MH; however, the well-known quinone trapping mechanism is likely the dominant mechanism for 3MH loss in wine. Finally, with the knowledge that 1-HER formation in wine is catalyzed by transition metals, I investigated the use of chelators as a means to control 1-HER formation. I focused on the effect of exogenous Fe(II) (bipyridine, Ferrozine) and Fe(III) chelators (EDTA, phytic acid) on 1-HER and acetaldehyde formation using spin trapping with EPR and HPLC-PDA, respectively. I then investigated the ability for these chelator treatments to prevent the oxidative loss of the important aroma-active thiol, 3MH. The Fe(II)-specific chelators were more effective antioxidants than the Fe(III)-specific chelators during the early stages of oxidation, and completely inhibited 1-HER formation compared to the chelator-free control. However, while the addition of Fe(III) chelators was less effective or even pro-oxidative initially, the Fe(III) chelators proved to be more effective antioxidants compared to Fe(II) chelators over the course of the study. In addition, I show for the first time that Fe(II) and Fe(III) chelators can significantly inhibit the oxidative loss of 3MH in model wine. iv

5 TABLE OF CONTENTS LIST OF FIGURES... vii LIST OF SCHEMATICS... ix LIST OF TABLES... x ACKNOWLEDGEMENTS... vii Chapter 1: Literature review Introduction Wine Oxidation Role of Oxygen in Wine Oxidation Role of Transition Metals in Wine Oxidation Catalysis Role of Phenolics in Wine Oxidation Role of Sulfur Dioxide in Wine Oxidation Oxidation of Wine Constituents Reaction with Quinones Hydroxyl radical reaction Hydroxyethyl Radical Reaction Importance of Varietal Thiols in Wine Preventing oxidation Bottle closure and environment Antioxidants Metal Chelation Purpose and Significance Hypothesis and Objectives Chapter 2: Hydroxyl radical scavenging capability of major wine components ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION Chapter 3: Investigation of Ethyl Radical Quenching by Phenolics and Thiols in Model Wine ABSTRACT INTRODUCTION MATERIALS AND METHODS v

6 3.4 RESULTS AND DISCUSSION Chapter 4: The effect of metal chelators on the oxidative stability of model wine ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUDING REMARKS REFERENCES vi

7 LIST OF FIGURES Figure 1: 1-HER/PBN adduct formation under air. All samples contained 12% EtOH, 90μM Fe(II), and 30mM PBN (specific treatments in Table 5) Figure 2: 1-HER/PBN spin adduct formation with varying concentrations of tartaric acid, glycerol, and 4-methylcatechol under Fenton conditions (90μM Fe(II) and 300μM H 2 O 2 ) in aerated model wine (12% EtOH, ph 3.6) and 30mM PBN Figure 3: PBN/1-HER EPR spin intensity after induced Fenton reaction in deoxygenated (black bars) and aerated (grey bars) model wine solution at varying tartaric acid concentration. Note: deoxygenated samples were measured at 3 scans whereas aerated samples were measured at 10 scans Figure 4: Residual H 2 O 2 after induced Fenton reaction in deoxygenated (black bars) and aerated (grey bars) model wine solution at varying tartaric acid concentration Figure 5: Acetaldehyde formation after induced Fenton reaction in deoxygenated (black bars) and aerated (white bars) model wine solution at varying tartaric acid concentration Figure 6: Possible PBN/1-HER adducts formed: (1) oxidized (222 m/z), (2) radical (223 m/z), (3) reduced (224 m/z), and (4 and 5) bi-adducts (268 m/z) Figure 7: Chemical structures of caffeic acid (CA), gallic acid (GA), ferulic acid (FA), 3-mercaptohexan-1-ol (3MH), cysteine (Cys), and glutathione (GSH) Figure 8: Representative experimental EPR spectrum corresponding to the PBN/1-HER spin adduct in model wine conditions Figure 9: EPR spin adduct intensities of PBN/1-HER in the presence of 4-MeC, PBN, and selected treatments in model wine in the absence of oxygen. Spectra were obtained at room temperature after 1 min following the addition of Fe(II) and H 2 O 2. *Statistically significant difference (P < 0.05) from control by one-way ANOVA with Dunnett s post-test Figure 10: 3MH disulfide and 4-MeC-3MH adduct formation from the following treatments: 3MH only (measured immediately); 3MH only; 3MH + 4-MeC; Fenton reaction (Fe(II) + H 2 O 2 ); Fenton reaction (Fe(II) + H 2 O 2 ) + 4-MeC. Reactions were run in model wine (ph 3.6) in the absence of oxygen and measured after 30 min reaction at ambient temperature Figure 11: Total MS spin adduct intensities of all possible PBN/1-HER adducts (oxidized, 222 m/z; radical, 223 m/z; reduced, 224 m/z; bi-adducts, 268 m/z) in the presence of 4-MeC, PBN, and selected treatments in model wine in the absence of oxygen. Spectra were obtained at room temperature after 1 min following the addition of Fe(II) and H 2 O 2. *Statistically significant difference (P < 0.05) from control by one-way ANOVA with Dunnett s post-test vii

8 Figure 12: Acetaldehyde formation in the presence of 4-MeC and phenolic and thiol treatments in model wine in the absence of oxygen. *Statistically significant difference (P < 0.05) from control by one-way ANOVA with Dunnett s post-test Figure 13: 3MH loss resulting from the following treatments: H 2 O 2 only; Fenton reaction (Fe(II) + H 2 O 2 ); Fe(II) + 4MeC; Fenton reaction (Fe(II) + H 2 O 2 ) + 4MeC. Reactions was run in either model wine (ph 3.6) or mannitol solution (ph 3.6) in the absence of oxygen. *Statistically significant difference (P < 0.05) from model wine by Student s t-test Figure 14: Intensity of PBN/1-HER spin adducts (arbitrary units) in the presence of various iron chelator treatments in model wine (12% EtOH, 8 g/l tartaric acid, ph 3.6) and 4-MeC under air at ambient temperature Figure 15: Intensity of PBN/1-HER spin adducts (arbitrary units) in the presence of various iron chelators in pinot gris adjusted to ph 3.6 under air at ambient temperature Figure 16: Acetaldehyde formation over time in the presence of various iron chelator treatments in model wine (12% EtOH, 8 g/l tartaric acid, ph 3.6) and 4- MeC under air at ambient temperature Figure 17: Acetaldehyde formation over time in the presence of various iron chelator treatments in model wine (12% EtOH, 8 g/l tartaric acid, ph 3.6) and 4- MeC under air at 50 C Figure 18: 3MH loss over time in the presence of various iron chelator treatments in model wine (12% EtOH, 8 g/l tartaric acid, ph 3.6) and 4-MeC under air at 50 C viii

9 LIST OF SCHEMATICS Scheme 1: Proposed metal catalyzed reduction of oxygen to hydrogen peroxide and hydroxyl radicals, and subsequent oxidation of ethanol to 1-hydroxyethyl radical... 3 Scheme 2: (A) formation of quinone upon the oxidation of catechol by loss of 2 electrons and 2 protons. (B) metal catalyzed oxidation of catechol and regeneration of Fe(II)... 8 Scheme 3: The competitive reaction of sulfite and iron for hydrogen peroxide Scheme 4: The radical chain reaction of sulfites to yield potent sulfite radicals Scheme 5: The reaction between electrophilic quinone and various nucleophiles found in wine Scheme 6: The reaction of hydroxyl radical with major wine constituents Scheme 7: Proposed reaction of 1-hydroxyethyl radical in wine conditions Scheme 8: 3-mercaptohexan-1-ol production during fermentation Scheme 9: Proposed reaction mechanisms of 1-HER with: (1) ferric ions or oxygen, (2) thiols, (3), and hydroxycinnamic acids Scheme 10: Proposed mechanisms for the loss of 3MH by (1) reaction with 1- HER and subsequent disulfide formation, (2) Michael-type addition reaction to o- quinone, (3) reaction with semiquinone radicals ix

10 LIST OF TABLES Table 1: Sources for introduction of atmospheric oxygen in wine Table 2: Calculated values of relevant reactive oxygen species to wine at ph Table 3: Values obtained by voltammetry in model wine Ph 3.6 and 0.5Mm concentration, with 207 Mv added to give values versus standard hydrogen electrode Table 4: Chemical makeup of wine Table 5: Full factorial design used for investigating the effect of major wine constituents on 1-HER formation x

11 ACKNOWLEDGEMENTS My utmost sincere thanks go to my advisor, Dr. Ryan Elias, for giving me the opportunity to join his lab. I thank him for introducing me to the frustrating yet rewarding field of wine research. Without his patience, support, and dedication this would not have been possible. I would also like to thank my committee members, Dr. Joshua Lambert and Dr. John Hayes, as well as Ms. Denise Gardner for their willingness to talk with me whenever I stopped by. Their insight and guidance have been incredibly useful throughout my time here. I would like to thank the Department of Food Science for providing salary and tuition support. I would also like to thank the Pennsylvania Wine Research and Marketing Board for providing some funding support for this project. I thank all my lab mates and classmates for being supportive of me. They have taught me many valuable skills and helped me develop as a scientist. They have made my experiences here much more enjoyable by being great friends socially and academically. My family and friends at home have always provided love and support, and for that, I am eternally grateful. xi

12 Chapter 1: Literature review 1.1 Introduction Oxygen is ubiquitous in the winemaking process, and can be introduced in virtually every step (Table 1). The impact of oxidation in finished wine can lead to both desirable and deleterious effects depending on the type of wine. For example, oxidation can stabilize the color of red wine by anthocyanin polymerization, resulting in a persistent brick red color which is highly desirable (1). In addition, tannin precipitation and polymerization can result in softening of astringent phenolics in red wine during barrel and bottle aging, yielding a wine with improved mouthfeel (2). Oxidation of white wines, however, can result in browning due to catechin polymerization (3,4) as well as yellowing due to phenolic reactions with oxidation products such as ketones and aldehydes (5,6). Since white wines tend to have a low phenolic content, there are no known improvements in mouthfeel that arise from wine oxidation and, in fact, oxidation may accent the bitterness of some white wines (7,8). Table 1: Sources for introduction of atmospheric oxygen in wine (9,10) Operation Dissolved Oxygen (mg/l) Crushing, Pressing Saturation Racking 2.2 to saturation Pumping Centrifugation 1.0 Filtration 0.7 Bottling 1.6 1

13 Aroma is perhaps the most important attribute affecting the quality of wine but is one of the major attributes that change during oxidation. There are some beneficial effects on aroma due to oxidation, such as the removal of unwanted reductive thiols which contribute to malodorous aromas; however, by the same mechanism, there is a loss of important thiols that are vital in varietal wines as they provide pleasant fruity aromas (11). Oxidation contributes to the generation of various aldehydes from ethanol, organic acids, and sugars in wine (12). These aldehydes are usually associated with wine defects in table and dessert wines, but there are special cases where the presence of these aldehydes is highly desirable, such as in maderized wines (e.g., sherry, Madeira). For the most part, the oxidation of wine post-bottling is rarely beneficial, particularly in white wines, and winemakers are in need of better tools to control these processes. There are relatively few ways to reliably and reproducibly control oxidation reactions in wine. The most commonly employed (and most important) strategy is the careful monitoring of oxygen throughout the winemaking process. Bottle closures, such as metal roll-on tamper evident (ROTE), screwcaps, glass bottle ampoules, among other physical deterrents of oxygen ingress have been proven successful at minimizing the oxygen transfer rate and have been utilized in the industry (13,14). The use of antioxidants has also been employed; the most commonly used antioxidant in wine is sulfur dioxide, which is produced naturally during alcoholic fermentation to a minor extent and is almost always added to the wine at bottling. Glutathione (GSH) and ascorbic acid have also been explored as antioxidants and as potential sulfur dioxide replacements, yet with disappointing results (15 17). The purpose of this review is to provide a concise but comprehensive overview of the oxidative stability of wine in regards to both major and minor wine constituents. In 2

14 addition, beneficial thiols in wine will be reviewed due to their importance in wine. Ways of preventing oxidation, particularly via chelators and metal redox inactivation, will also be reviewed. 1.2 Wine Oxidation The non-enzymatic oxidation of wine yields two important reactive intermediates: quinones and hydroxyl radicals (18). These species are now thought to be produced by the metal-catalyzed oxidation of phenolics during the sequential one-electron reduction of oxygen to hydroperoxyl radical, hydrogen peroxide, hydroxyl radical, and eventually water (19,20) (Scheme 1). Oxygen, phenolics, transition metals, and sulfites all play a major role during this initial oxidation process. OH O H + R OH R OH OH - O 2 HOO HOOH HO Fe 2+ Fe 3+ Fe 2+ Fe 3+ CH 3 CH 2 OH O OH CH 3 CHOH R OH R OH H 2 O Scheme 1: Proposed metal catalyzed reduction of oxygen to hydrogen peroxide and hydroxyl radicals, and subsequent oxidation of ethanol to 1-hydroxyethyl radical. 3

15 1.2.1 Role of Oxygen in Wine Oxidation It is obvious that oxygen is the fuel in the wine oxidation process. Because of this, there is a high interest in controlling for oxygen, and many publications discuss accurate measurement and control of oxygen (21 23). Concentrations of dissolved oxygen are highly variable with temperature and partial pressure, but at room temperature it reaches saturation at approximately 8.4 mg/l (24). As the temperature is decreased, the solubility of oxygen increases, but the rate of oxygen consumption decreases (24). Dissolved oxygen in must and wine reacts with phenolics and sulfites in the presence of transition metals (18,24 26). Oxygen is quite stable in its ground state and is spin forbidden from reacting directly with organic compounds, so it must be activated to its singlet state first. This could potentially occur by photolysis if exposed to light for extended periods of time (18,27), but since this is not often the case, the most common way for oxygen to be activated is by metal catalysis. As shown in Scheme 1, oxygen is eventually reduced to water, but in the process it forms very potent reactive oxygen species, such as the hydroperoxyl radical (HOO ) and hydroxyl radical (HO ), the reduction potential of these reactive oxygen species is reported in Table 2. The metalcatalyzed breakdown of H 2 O 2 to form HO radicals is well known as the Fenton reaction (19). The HO /H 2 O couple has a reduction potential of 2.53 V at ph 3.6, which is high enough to oxidize any given organic molecule in wine at a diffusion-limited rate (18). Table 2: Calculated values of relevant reactive oxygen species to wine at ph 3.6 (28). Reactive oxygen species Redox potential (V) Ground state oxygen (O 2 ) Hydroperoxyl radical (HOO ) 1.22 Hydrogen peroxide (H 2 O 2 ) 0.59 Hydroxyl radical (HO )

16 1.2.2 Role of Transition Metals in Wine Oxidation Catalysis Transition metals are thought to be essential catalysts in the oxidation of wine as they facilitate one electron transfers to oxygen to yield reactive oxygen species. These trace levels of metals can come from a variety of sources including the grapes themselves, dust, fungicide residues, and the winemaking equipment (7). The concentration of iron, copper, and manganese in wine have been reported as mg/L, mg/L, and mg/L respectively (7,29). It has been shown recently that these transition metals, particularly iron and copper, are essential as catalysts for the reduction of oxygen and oxidation of wine components including polyphenols, sulfites, and ethanol (30 33). Ribéreau-Gayon showed that the rate of oxidation could be slowed and eventually stopped in wine by the removal of iron and copper with potassium ferrocyanide (34). This was recently confirmed in another study by Danilewicz and Wallbridge (35). The rate of oxidation is highly dependent on the reduction potential of the Fe(III)/Fe(II) couple (18,28,36). The lower the reduction potential, the greater the reducing power. Therefore, if the reduction potential of the Fe(III)/Fe(II) couple is low, dioxygen will be reduced to its active peroxyl radical form more readily. It also contributes to the reduction of H 2 O 2 to HO radicals via the Fenton reaction (Scheme 1). The reduction potential of the Fe(III)/Fe(II) is dependent on the ligands present in the wine solution. Tartaric acid and phenolics in wine bind to iron and affect its reduction potential and it has been suggested that the reduction potential of Fe(III)/Fe(II) couple in wine ph falls between V (18). Once Fe(II) is oxidized into Fe(III), it quickly reduces back to Fe(II) in the presence of phenolics (36). The iron speciation in wine has been examined, and it has been suggested that the majority of free iron is present as Fe(II) (36,37), though a large fraction of iron is bound to the organic fraction of wine such 5

17 as tartrate (38) and tannins (39). Fe(II) is the major species of iron in wine due to its acidic ph and abundance of phenolics Role of Phenolics in Wine Oxidation Phenolic compounds in wine are mainly extracted from grape skins and seeds, but a minor fraction is also extracted from oak during fermentation (1). The concentration of phenolics depends on the grape variety, the form of oak used, and processing methods. The final concentration of phenolics vary widely across wine varietals and styles; however, the catechin equivalent of white wines are reported to be in the range of 50 mg/l 1500 mg/l (7,40). In general, the concentration of catechin equivalent in white wines is 10-fold less than in red wines. Phenolic compounds in wine can quickly consume oxygen (in the presence of iron) and it has been shown that wines with higher phenolic contents, such as reds, are able to consume more oxygen relative to whites before showing any obvious signs of oxidation (24,41). Wine phenolics are often considered to be antioxidants due to their ability to form a resonance stabilized structure by the delocalization of the free radical (42). However, in wine conditions, the ability to be quickly oxidized propagates the oxidation of wine due to the formation of H 2 O 2 (7) (Scheme 1). There are varying reduction potentials for the different phenolics in wine (Table 3), which in turn have different reaction rates with reactive oxygen species (18,28). In general, the lower the reduction potential, the more readily the phenolic compound oxidizes. It has been shown that polyphenols containing catechol (1,2- diphenol) or gallate (1,2,3-triphenol) moieties are the most easily oxidized due to their low reduction potential (18,43). Wine phenolics that contain a catechol group include catechin, epicatechin, caftaric acid, and condensed tannins. The gallate containing group include gallic acid, gallocatechin, and epigallocatechins (7,12,18). Quercetin has lower reduction potential then most wine phenolics, as it has an extended conjugation 6

18 with the heterocyclic ring providing further stabilization then catechin. This contributes to its quick loss despite its presence in low concentrations (18,44). Table 3: Values obtained by voltammetry in model wine ph 3.6 and 0.5mM concentration (43), with 207 mv added to give values versus standard hydrogen electrode (28). Compound Structure E 3.6 (mv) (+)-Catechin 577 (-)-Epicatechin 568 Caffeic acid 604 Gallic acid 582 Quercetin Methylcatechol 541 1,2-Benzenediol 582 Pyrogallol 468 7

19 Catechols are oxidized to quinones upon the successive loss of two electrons and two protons (18) (Scheme 2A). The two major paths for oxidation of catechols in wine are via metal-catalyzed oxidation and radical-mediated oxidation. The most likely radical to oxidize the catechol in wine is the HOO radical which can react to yield a semiquinone while simultaneously reducing the HOO radical into H 2 O 2 (Scheme 1); however, it is also possible for the phenolics to react with HO radicals and sulfite radicals (30). The semiquinone radical would then react again to be oxidized into a quinone (7,12,18). Phenolic compounds have a pka>9, and at wine ph (range of ), almost all groups are completely protonated to give the neutral form (7,12). In order for metal-catalyzed oxidation reactions to proceed, the catechol needs to be ionized for the metal to bind, which is why oxidation rates are much faster at higher ph as the equilibrium shifts towards the ionic form (7,18). Catechol and gallate groups can reduce an equivalent of two Fe(III) ions to Fe(II) ions (Scheme 2B). Scheme 2 (A) formation of quinone upon the oxidation of catechol by loss of 2 electrons and 2 protons. (B) metal catalyzed oxidation of catechol and regeneration of Fe(II) Quinones are highly electrophilic species and therefore can participate in a variety of reactions leading to detrimental sensory characteristics, by the loss of aromas and browning due to polymerization. In the presence of sulfur dioxide, quinones can be cycled back to their hydroquinone (reduced) form. Due to this ability to regenerate, more 8

20 oxygen can be consumed prior to showing any signs of oxidative characteristics (24). However, this regeneration also allows for the cycling of Fe(III)/Fe(II) and/or further generation of H 2 O Role of Sulfur Dioxide in Wine Oxidation At wine ph, sulfur dioxide is most commonly found in its bisulfite form (HSO 3 - ), which is thought to be the form that is reactive with H 2 O 2. HSO 3 - acts as an important preventive antioxidant in wine as it can react with unstable quinones and remove H 2 O 2 (12,18,31). In addition, HSO - 3 can react with aldehyde and ketone compounds via addition reactions (45) which are formed after the oxidation of major components such as ethanol and tartaric acid. This can reduce the negative aromas associated with these compounds (46) and is considered to be bound sulfur dioxide. However, these reactions are reversible and as oxidation progresses, loss of sulfite will cause a shift in equilibrium and HSO - 3 will become unbound from the aldehydes and ketones. This will remove the masking effect of sulfur dioxide on these oxidized aroma compounds. HSO 3 - reacts with H 2 O 2 by an acid-catalyzed mechanism that does not involve radical intermediates (47) (Scheme 3). By removing H 2 O 2 at a faster rate than the Fenton reaction, HSO 3 - effectively prevents the formation of the HO radical. Scheme 3: The competitive reaction of HSO 3 - and iron for H 2 O 2 HSO 3 - can reduce quinones back to their original hydroquinone form, but it has also been shown that the quinones can react with HSO 3 - to form addition products, both 9

21 of which effectively regenerate the catechol (30,31) (Scheme 5). While HSO - 3 seems to behave mostly as an antioxidant, it has been suggested that its presence is necessary for oxidation to proceed at a fast rate. The reasoning for this is that the presence of HSO - 3 and other nucleophiles can effectively remove the quinone thus shifting the equilibrium and allowing further generation of H 2 O 2 and Fe(II) by oxidation of catechols (32). The balance of both phenolics and sulfites in wine is important for the rate of oxidation in wine. In the presence of oxygen, sulfite autoxidation is catalyzed by metals to promote radical chain propagation. This is prevented by phenolics in wine as they are effective radical scavengers (30). In the absence of phenolics, sulfite autoxidation can yield sulfate radicals, which have comparable reduction potentials to HO radicals (E 3.6 = 2.43 V) (Scheme 4) (30,48). Scheme 4: The radical chain reaction of sulfites to yield potent sulfite radicals Oxidation of Wine Constituents Reaction with Quinones Quinones are oxidation products of catechol and gallate moieties from the primary wine oxidation process (Schemes 1, 2); they are highly unstable and can participate in a variety of reactions. As quinones are highly electrophilic they react with nucleophilic molecules in wine that include amino acids, sulfites, thiols, and flavanols (Scheme 5). 10

22 Scheme 5: The reaction between electrophilic quinone and various nucleophiles found in wine. Quinones can polymerize by reaction with flavanols including catechin and epicatechin (5,49). These polymerization reactions contribute to the browning of white wines, which is undesirable. In the presence of antioxidants including HSO 3 - and ascorbic acid, quinones quickly react and are reduced back to hydroquinones (31,50). The reaction between quinones and amino acids can also occur in wine, which is proceeded by Strecker degradation reactions to yield important aldehydes including methional and phenylacetaldehyde (51) (Scheme 5). 3MH, an important varietal thiol, is significantly depleted by binding of its thiol group with ortho-quinones (11). Upon addition of the thiol to quinone, a catechol adduct is formed (52). The formation of these adducts has been directly shown using electrospray ionization paired with mass spectrometry (53) and nuclear magnetic resonance spectroscopy by both enzymatic and nonenzymatic chemical oxidation (54). 11

23 GSH has been shown to effectively reduce the loss of 3MH in wine (55) due to its reaction with quinones at a more competitive rate compared to 3MH. A recent study by Nikolantonaki and Waterhouse have shown that wine antioxidants including HSO - 3, GSH, and ascorbic acid, as well as hydrogen sulfide, have very high reaction rates with quinones, followed by volatile and varietal thiols, and lastly by nucleophilic amino acids (including methionine, phenylalanine) (56) Hydroxyl radical reaction The HO radical is a very potent oxidant and can abstract a hydrogen atom from practically all organic compounds in wine in a concentration based manner (18,57). Since ethanol is present at the highest concentration in wine at approximately 2 M, it is most likely to react with the HO radical due to its abundance (Scheme 6). The HO radical abstracts the α-hydrogen 85% of the time to form the 1-hydroxyethyl radical (1- HER) and has been confirmed by electron paramagnetic resonance coupled with spin trapping techniques (48). 2-hydroxyethyl radicals are also formed as a minor species. Scheme 6: The reaction of HO radical with major wine constituents. 12

24 However, other minor wine constituents must be taken into consideration when it comes to the HO radical (Table 4). Glycerol and tartaric acid are the most abundant compounds in wine after water and ethanol, present at mm and mm, respectively (46,58). It is expected that approximately 5-10% of the hydroxyl radicals will react with these compounds in a similar fashion to ethanol to produce aldehydes (Scheme 6). It has been reported that tartaric acid and glycerol oxidize to yield glyoxylic acid and glyceraldehyde, respectively (58,59). However, it is worth noting that tartaric acid may not readily react with HO radicals. Tartaric acid binds to Fe(III) and it has been suggested that Fe(III) tartrate complexes are not targets for HO radicals formed from the Fenton reaction (60). In addition, more recent reports suggest that no glyoxylic acid is formed from tartaric acid in the absence of light (61,62). More research needs to be done to fully understand the role of tartaric acid in wine oxidation. Table 4: Chemical makeup of wine (7). Group Typical Range (mm) Group Typical Range (mm) Alcohol Cations 0-64 Polyols Phenolics 0-2 Sugars Esters Acids 7-33 Nitrogenous compounds Anions 1-30 Vitamins Phenolics can be oxidized by the HO radical to form semiquinone radicals and eventually further oxidized to quinones in the presence of other radicals or Fe(III) (Scheme 2). Although phenolics are found at relatively low concentrations in wine relative to ethanol and tartaric acid, it is possible that while phenolics chelate iron during its reduction, the Fenton reaction occurs in close proximity and the HO radical can quickly react with the phenolic (63). 13

25 Hydroxyethyl Radical Reaction 1-HER is highly reducing and can quickly oxidize in the presence of Fe(III) or oxygen to produce acetaldehyde (18,36) (Scheme 7). Upon the reaction of 1-HER with Fe(III), 1-HER will quickly be oxidized and Fe(III) will be simultaneously reduced to Fe(II), which will be able to participate in the Fenton reaction again. In the presence of oxygen, 1-HER forms a 1-hydroxyethyl peroxyl radical intermediate which can then break down to yield acetaldehyde and HOO radical (48). Acetaldehyde can then further react with sulfites and polyphenols. It is also possible for 1-HER to be reduced back to ethanol in the presence of highly reducing compounds, such as phenolics and thiols. Scheme 7: reaction of 1-hydroxyethyl radical in wine conditions It has recently been shown that 1-HER is reactive towards the α,β-unsaturated side chains of hydroxycinammic acids including caffeic and ferulic acids (64). 1-HER attacks the side chain double bond, and then attaches to the α-carbon. This forms a relatively stable benzyl radical which can then be oxidized by Fe(III) to form a 14

26 carbocation which will readily decarboxylate and yield an allylic alcohol (64) (Scheme 7). It has also been shown that in beer isohumulones which contain highly resonant structures can react competitively for 1-HER with POBN spin trap in low levels (65). It has been suggested mechanistically that these isohumulones can either reduce 1-HER to ethanol and/or attach to 1-HER (66). In biological systems, GSH and ascorbic acid can competitively reduce 1-HER radical back to ethanol (67), and there has been evidence for the oxidation of GSH to yield glutathione disulfide (67). This mechanism may also be applicable to sulfites and thiols in wine, as well as phenolics with a low enough reduction potential. We have recently demonstrated that important wine thiols are target of ethyl radicals in model wine conditions (discussed in Chapter 3). 1.3 Importance of Varietal Thiols in Wine Varietal thiols are extremely important and associated with a range of young wines including white, rosé, and red (68). Varietal thiols are usually present in ng/l levels in wine (69) and contribute to pleasant aromas such as grapefruit, passionfruit, and blackcurrant (68). These compounds are produced during fermentation by the cleavage of odorless precursors in grapes by yeast (69 71). The non-varietal thiols are small sulfurous compounds such as hydrogen sulfide and carbon sulfide. They can be produced at high concentrations during fermentation and are associated with wine defects due to their reductive odors such as sweaty and egg-like odors (72), and as such, they are generally harmful to the quality of wine. Some of the most studied varietal thiols in wine include 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA), and 4-mercapto-4-methylpentan-2-one 15

27 (4MMP) (68,73) as they have very low sensory thresholds. They contribute to the beneficial aromas associated with the varietal wines. For the purposes of this review, we will focus on 3MH. 3MH can be generated by 3 known pathways during fermentation (Scheme 8). The major precursor to 3MH is S-3-(hexan-1-ol)-cysteine (Cys3MH). This precursor has been identified in Sauvignon Blanc (74), Merlot, and Cabernet Sauvignon grapes (70), and it has been shown that Cys3MH is cleaved by yeast through its beta lyase activity(75). Another precursor, S-3-(hexan-1-ol)-glutathione (G3MH) has been identified in the grapes of Sauvignon Blanc (76). The two mechanisms by which G3MH can be used to form 3MH could be by acting as a precursor for Cys3MH (77) and as a precursor for 3MH directly (78). 3MH can also be formed from hexanal; E-2-hexanal undergoes a sulfur addition during alcoholic fermentation forming 3MH. (71) Scheme 8: 3-mercaptohexan-1-ol production during fermentation 3MH provides important fruity notes in varietal wines, primarily as passion fruit and grapefruit aromas (69,79,80). 3MH exists as its R- and S- enantiomeric forms, which have sensory thresholds of 50ng/L and 60ng/L, respectively (81). The S- form 16

28 contributes to passion fruit notes (82), whereas the R- form is responsible for grapefruit notes (83). The enatiomeric distribution of 3MH in dry Sauvignon Blanc is approximately 50:50 (84). Despite the presence of 3MH at low levels, it is almost always above the human detection threshold in young white wines such as sauvignon blanc. 3MH is usually present at higher levels compared to the other varietal thiols, ranging from 26-18,000 ng/l in sauvignon blanc, whereas 4MMP is present at levels of 4-40 ng/l (85). It is important to note that 3MH and its acetate (3MHA) both contribute to similar aromas and could potentially have synergistic effects, and both may need to be present to give the key aromas associated with sauvignon blanc and rosé wines (15,43) Since these key aroma compounds are expected to be present in young white wines, their oxidative stability is a major issue. Varietal thiols are labile to oxidation and can be lost within several months depending on storage conditions. This is because thiols are easily oxidized to odorless disulfides under mild oxidative conditions (86,87). They can also be lost quickly to other oxidation products such as quinones (Scheme 5) and radical species (Scheme 7). The use of cork as a bottle closure can also cause partitioning of these volatile compounds into the cork, known as scalping, and could be responsible for loss of fruit aroma in sauvignon blanc (88). It is essential to protect these compounds for a long duration to meet consumer expectations for these fruity wines. 1.4 Preventing oxidation Bottle closure and environment Storage conditions play an important role with respect to the oxidative stability of wine. It is important to store wines in the dark as HO radicals can form via photolysis. UV-photolysis can also generate superoxide and oxidize ethanol to acetaldehyde 17

29 (18,27). In addition, photoactivation of the Fe(III) tartrate complex in wine can cause yellowing in wines by the reaction of glyoxylic acid and phenolics (61). Storage temperature and bottle closure are important as temperature may change the rate of oxygen consumption, and varying bottle closures allow varying oxygen ingress levels (13,14,89). In general, ROTE caps have the lowest oxygen ingress; however, they are usually associated with reductive notes (89,90). Corks that have a higher oxygen ingress rate result in faster oxidation and loss of the fruity aroma, but can also cause problems with loss of varietal thiols as they can partition into the cork (88) Antioxidants Sulfur dioxide is the most commonly used antioxidant in wine, and has been discussed previously. However, in a very small part of the population, sulfites can induce allergic reactions. In addition, some consumers are concerned when they see the contains sulfites warning on wine labels. Because of this, other alternatives have been considered for use. Other antioxidants have been added into wine include ascorbic acid, GSH, and caffeic acid (91). Ascorbic acid has a lower reduction potential than phenolics in wine so it quickly reacts with HOO radicals (18). However, it produces H 2 O 2 and the reactive oxidation product dehydroascorbic acid which can further degrade and react. It also quickly reduces Fe(III) to Fe(II) that can participate in the Fenton reaction (18). HSO - 3 is therefore essential with the addition of ascorbic acid as it is needed to react with H 2 O 2 and dehydroascorbic acid. There is some evidence that ascorbic acid has a beneficial effect on wine color over time by reducing quniones in the presence of sufficient levels of HSO - 3 (15) (Scheme 5). 18

30 GSH is similar to HSO - 3 as it can react with H 2 O 2 and quinones. It can potentially react with 1-HER as seen in physiological conditions, and recent studies suggest the same in model wine (Chapter 3). There have been studies showing its beneficial effects on preventing oxidation and protecting other varietal thiols (55). GSH has a protective effect preventing the loss of fruity notes in wine, and it has been considered to preserve the aroma of young white wine during storage (68). Antioxidants work well by either reacting with the precursors or end products of oxidation, but they are useless when it comes to the HO radicals. Some of these antioxidants actually promote its formation, such as ascorbic acid. Most antioxidants or nucleophiles can in fact promote oxidation by shifting the equilibrium by the removal of quinones Metal Chelation The use of chelators in wine research has been limited. However, in light of recent studies highlighting the essential role that transition metals play in wine oxidation, chelators are of interest due to their ability to disrupt the redox cycling of these metals. As previously discussed, it has been shown that removing trace metals from wine can effectively stop oxidation processes (35). Iron chelators contain ligands that bind to iron, which can effectively sequester iron and disrupt redox cycling, making it unavailable to interact with other ions or molecules in solution. In wine, this could effectively stop the formation of oxygen radicals (Scheme 1). Fe(III) ions have small atomic radii and high charge density compared to Fe(II) and as such, behave as hard Lewis acids. Fe(III) ions have a greater affinity for hard bases such as hydroxide, phenolate, and carboxylate ions (92). Fe(II), on the other hand, is considered a borderline Lewis acid. Fe(II) ions preferentially bind to borderline 19

31 bases containing sulfur, nitrogen, and pyridine ligands (92). Depending on the presence of these specific ligands, the equilibrium can be shifted and result in a different reduction potential of the Fe(III)/Fe(II) couple. Iron is always bound to a ligand in solution and under acidic solutions, Fe(II) hexaaquo-complex is present and reacts slowly with oxygen (18). However, under wine conditions and the presence of the hydroxy acid, tartaric acid, the reduction potential is decreased as Fe(II) is chelated at a 3:1 ratio, and Fe(II) can be quickly oxidized in wine (18). Phenolics also bind to Fe(III), however they must be deprotonated in order for this binding to occur. Catechol and gallate groups are effective metal chelators upon deprotonation. At physiological ph, polyphenols easily deprotonate in the presence of iron and form stable complexes (93). This contributes to the antioxidant activity associated with phenolics at physiological ph but is not applicable for wine. The Fe(III) and catechol or gallate ligand binding is less stable at low ph and the polyphenol quickly reduces the Fe(III) to Fe(II) while simultaneously oxidizing the phenol into a semiquinone radical (Scheme 2B) (94,95). The semiquinone radical is then capable of reducing another Fe(III) by being oxidized into a quinone (93). The regenerated Fe(II) can reduce oxygen and H 2 O 2 which results in ROS generation. This is the main reason phenolics containing catechol and gallate groups in wine behave as pro-oxidants. Synthetic chelators have been used in the food industry as additives to inhibit metal-catalyzed oxidation reactions. One such example is EDTA which has been used to prevent lipid oxidation (96,97). EDTA is a hexadentate ligand and has a strong affinity for Fe(III), and as such, it quickly shifts the equilibrium of iron in wine to form Fe(III). In the presence of oxygen, Fe(II) is quickly oxidized as it reduces oxygen or other ROS such as H 2 O 2. This quickly promotes the production of HOO radicals from oxygen and HO radicals from H 2 O 2. In the presence of EDTA at ph 4, the reduction potential of 20

32 Fe(III)/Fe(II) couple is reduced to 0.12V which allows this quick reaction (98). EDTA has been shown to have pro-oxidant activity in the presence of phenolics and iron (99,100). The reaction is highly dependent on the ph of the system. In a lipid system containing trace metals and EGCG it has been shown that, at neutral ph, EDTA rapidly generated HO radicals, but at ph 3, it inhibited HO radical generation (97). As such, the initial addition of EDTA to wine would result in a prooxidant effect due to the high presence of Fe(II) followed by a quick shift in equilibrium to be oxidized into Fe(III). As redox cycling of iron is not completely inactivated upon binding to EDTA, catechols could potentially reduce Fe(III) while complexed with EDTA by outersphere electron transfer (63,100). Fe(II) chelators such as 2,2 -bipyridine stabilize Fe(II) ions, and can therefore prevent oxygen from binding and being reduced to ROS. In a lipid system, it has been shown that 2,2 -bipyridine increases the concentration of Fe(II) which can generate hydroxyl radicals (97). This is also ph dependent and has been shown to have an antioxidant effect at ph 7 and a prooxidant effect at ph 3. However, the same has not been observed under wine conditions (Chapter 4). Siderophores are small compounds secreted by microorganisms and grasses. They contain sidechains of hydroxamate, catecholate, or a mixture of both, and as such they behave as hard base ligands and have a high affinity for Fe(III) (101). Since they have such a high affinity for iron they can successfully inactivate iron redox cycling. Deferroxamine, for example, is a hexadantate with no free coordination sites that can prevent phenolic oxidation and ROS formation (102). 21

33 1.5 Purpose and Significance Domestic wine consumption has increased steadily over the past few years and is now estimated to be 2.54 gallons per capita as of 2010 (103). The United States is the 4 th largest wine producing country in the world with a reported production of 2,211,300 tons in 2010 and total sales estimated at 32.5 billion dollars (103). Total worldwide production of wine has been reported to be 26,216,967 tonnes in 2010 (104), and as such it is an extremely important commodity. Due to its economic importance, the loss of wine quality due to non-enzymatic chemical oxidation can lead to major financial losses. There are currently few ways to control oxidation in wines. It is particularly important to be able to control oxidation in white wines, as there are no benefits of oxidation after bottling. This is especially important for wines containing varietal thiols such as 3MH, as these compounds contribute to the fruity aromas which are quickly lost during oxidation. Different fermentation processes, bottling, and addition of antioxidants have been considered to reduce oxidation, but they do not prevent it over long periods of time. Currently the only completely effective method for inhibiting oxidation reactions is oxygen exclusion, but this can be deleterious as it results in reductive aromas. Further elucidation of the mechanisms of wine oxidation that lead to both the losses of important aroma compounds as well as the generation of oxidized qualities should be further investigated. Understanding the underlying mechanisms can help further develop tools to prevent wine oxidation in a reproducible manner; tools which winemakers do not currently have. 22

34 1.6 Hypothesis and Objectives Wine quality is important and can be attributed to a variety of factors including color, mouthfeel, flavor, and aroma. There has been an increasing interest in understanding the chemistry behind wine in order to preserve its quality, yet few tools are available to control oxidation. This study will investigate key factors of 1-HER formation via metal catalyzed and radical mediated reactions. In addition, the effect of 1- HER on 3MH and its subsequent loss will be examined. Lastly, the use of metal chelators as a treatment for the prevention of 1-HER formation as well as other oxidation markers will be investigated. I hypothesize that major wine components will affect the formation of 1-HER. In addition, I hypothesize 1-HER can further react with phenolics and thiols in wine and the use of exogenous metal chelators will inhibit the formation of 1-HER and subsequent loss of 3MH in wine. I test this hypothesis by investigating: 1. The mechanism for 1-HER formation as affected by major wine components. 2. The mechanisms of phenolic and thiol loss by reaction with 1-HER. 3. The ability of chelators to inhibit oxidation markers and preventing the oxidative loss of 3MH. 23

35 Chapter 2: Hydroxyl radical scavenging capability of major wine components 2.1 ABSTRACT HO radicals are formed from the metal catalyzed reduction of oxygen, and they react non-selectively. Ethanol is the prime target for HO radicals in wine due to its abundance; however, other major wine constituents may also act as substrates. In this study, the effect of major wine constituents including glycerol, tartaric acid, and phenolics on 1-HER formation was monitored using spin trapping with electron paramagnetic resonance (EPR). Glycerol was not observed to affect 1-HER formation, which is in contrast with previous reports, and 4-methylcatechol (4-MeC) was seen to promote radical formation. Tartaric acid suggested antioxidant effect by inhibition of 1- HER formation. However, upon further investigation, H 2 O 2 consumption and acetaldehyde formation suggest either no activity or prooxidant activity depending on the presence or absence of oxygen. 24

36 2.2 INTRODUCTION The quality of wine is directly linked to its oxidative stability. Wine that is under high oxidative stress is labile to a variety of oxidative reactions that can be deleterious to its quality, particularly in the case of white wine. The most notable quality changes of white wine include color, such as browning or yellowing, and aroma, such as loss of characteristic aroma or the production of aldehydes and ketones. Both changes are clear signs of oxidation. In recent years there has been active research concerning the nonenzymatic chemical oxidation of wine, especially with respect to elucidating the role of transition metal catalysts and identifying free radical intermediates. It appears that HO radicals and quinones are key intermediates to most oxidation related reactions during wine aging (12,18,48). Phenolics, transition metals, and sulfites are all ubiquitous in wine and their concentrations greatly affect the formation of these oxidation intermediates (30,32,35,36,57). The oxidation product of catechols include electrophilic quinones which can react with a variety of nucleophiles by 2-electron reactions in wine, including sulfites, thiols, selected amino acids, and some phenolics (31,49,51,56). HO radicals are formed by the reaction of H 2 O 2 and Fe(II), classically referred to as the Fenton reaction (19,105), and can initiate a variety of radical chain reactions. Due to their reactivity, HO radicals can react with any organic wine component in a diffusion controlled manner (18). It is now known that 1-HER is the major radical species in wine as ethanol ( M) quickly scavenges hydroxyl radicals due to its abundance (48,57) (Scheme 1). Other major wine constituents and potential substrates for HO radicals - include glycerol ( mm) and tartaric acid (13-53 mm) (7,58). The proposed major 25

37 oxidation products of ethanol, tartaric acid, and glycerol are acetaldehyde (24), glyoxylic acid (59), and glyceraldehyde (58), respectively. Scalzo et al. showed that the glycerol can efficiently scavenge HO radicals from wine extracts (106). However, there have been conflicting reports as to whether tartaric acid is a substrate for HO radicals under wine conditions (60). While the oxidation products of these constituents have been identified, the radical scavenging properties of these major wine constituents have not been elucidated in wine solutions. The interaction of flavonoids and the resulting aldehydes has been shown to yield a variety of novel phenolic condensation products which affect the color of wine (6,58, ). While the presence of ethanol, tartaric acid, and glycerol are too high to be significantly depleted by radical reactions, their intermediate oxidation products and final oxidation products may have varying effects on the final wine composition. Since the concentrations of these components vary widely between different wine styles, it is important to know how each of these compounds can affect oxidation. In this study, the fate of HO radicals was measured by following 1-HER formation using a spin trapping EPR spectroscopic technique. The concentration of glycerol, tartaric acid, and phenolics may be sufficiently high to inhibit 1-HER formation. However, the presence of these compounds may also affect the initial formation of the hydroxyl radicals or final acetaldehyde yield, resulting in either prooxidant or antioxidant effects. The aim of this study was to investigate how the presence of tartaric acid, glycerol, and 4-MeC affect the formation of 1-HER, and to confirm their status as Fenton chemistry targets. In addition, the presence of tartaric acid on the consumption of H 2 O 2 and final acetaldehyde yield was investigated as they are important markers of the oxidative progress. 26

38 2.3 MATERIALS AND METHODS Materials. Iron(II) sulfate heptahydrate, 4-methylcatechol, glycerol, xylenol orange tetrasodium salt, and acetaldehyde-dnph analytical standard were obtained from Sigma-Aldrich (St. Louis, MO). 2,4-dinitrophenyl-hydrazine (DNPH) was purchased from MCB laboratory chemicals (Norwood, OH). L-Tartaric acid and D-Sorbitol were obtained from Alfa Aesar (Ward Hill, MA). Hydrogen peroxide (30% w/w; H 2 O 2 ) was obtained from EMD Chemicals (Gibbstown, NJ). The spin trap phenyl-n-tert-butyl nitrone (PBN) was purchased from GeroNova Research Inc. (Garson City, NV). Water was purified through a Millipore Q-Plus system (Milipore Corp., Bedford, MA). All other chemicals and solvents were of analytical or HPLC grade. Model wine preparation. A full factorial experiment (Table 5) was conducted based on the presence or absence of major wine constituents including tartaric acid, glycerol, and 4-MeC. All model solutions contained 12% ethanol and 30mM PBN. Stock solutions of each were made fresh daily in ultrapure water: 20% EtOH containing 50 mm PBN (15% EtOH with or without 37.5 mm PBN for second set of reactions), 10 mm 4-MeC, mm tartaric acid, and 1.3 M glycerol solution. All stock solutions, including pure water were adjusted to ph 3.6 with NaOH and combined immediately prior to the experiment to achieve the final concentration of each treatment as shown in Table 5. For the second set of reactions, tartaric acid was the focus and so the stock solutions were combined to achieve 0 g/l, 4 g/l, and 8 g/l of tartaric acid in 12% EtOH with or without 30 mm PBN. 27

39 Table 5: full factorial design used for investigating the effect of major wine constituents on 1-HER formation. Treatment 4-MeC (mm) Tartaric acid (mm) Glycerol (mm) A B C D E F G H Accelerated model wine oxidation reaction. For accelerated oxidation reactions, 1 ml of each of the given treatments (Table 5) was added to 1.8 ml capacity microcentrifuge tubes and vortexed for 30 sec to achieve air saturation. Fe(II) was added from a 9 mm stock solution (10 μl; 90 μm final concentration) and was allowed to react over 13 hours in the dark. 50 μl aliquots were taken at set time intervals and measured on the EPR. Fenton reaction conditions. Reactions were carried out under either nitrogen gas or air saturation. For the air-saturated treatments, 1 ml of sample was added to 1.8 ml capacity microcentrifuge tube and vortexed for 30 sec to achieve air saturation. For the deoxygenated treatments, 1 ml of samples was added into 5 ml test tube and purged with nitrogen gas for 3 min. following the aeration or deoxygenation, Fenton reagents ([H 2 O 2 ] = 300 μm, [Fe(II)] = 90 μm) added to initiate the oxidation reaction. Stock solutions of Fe(II) (9 mm) and H 2 O 2 (30 mm) were prepared daily in acidified water. Fe(II) (10 μl; 90 μm final concentration) was added added either under nitrogen or air and H 2 O 2 (10 μl, 300 μm final concentration) was then added to initiate the reaction, which was allowed to proceed at ambient temperature for 1 min under nitrogen or air. Sample aliquots were withdrawn and analyzed immediately by EPR or were further treated for analysis for H 2 O 2 by FOX-1 assay and acetaldehyde by DNPH derivatization. 28

40 EPR spin-adduct analysis. 1-HER was measured using spin trapping with PBN and the EPR spectrum observed for the 1-HER/PBN adducts produced a triplet of doublets (hyperfine coupling constants: a N = 15.7 G, a H = 3.3 G) based on previously reported methods (36,48). Sample aliquots (50 μl) were loaded in 50 μl borosilicate micropipets (VWR, Radnor, PA, USA) and the EPR spectra were immediately recorded at room temperature on a Bruker escan R spectrometer (Bruker BioSpin, Rheinstetten, Germany) operating in X-band. The sweep width was set to 50 G and the microwave power was set to mw. The modulation frequency and modulation amplitude were set to khz and 2.45 G, respectively. The receiver gain was set to The conversion time and sweep time were set to msec and sec respectively. The total number of scans per sample was 10. For the deoxygenated samples the total number of scans per sample was 3. Hydrogen Peroxide Analysis. H 2 O 2 was analyzed according to a modified version of the concentrated ferrous oxidation-xylenol orange (FOX) assay (110). FOX reagent contained xylenol orange (1 mm), ferrous sulfate (2.5 mm), and sorbitol (1.0 M) in sulfuric acid solution (0.5 N), and was prepared weekly from stock solutions. The H 2 O 2 analysis was performed by adding the assay solution (20 μl) to diluted model wine (140 μl). The model wine was diluted at a 1:20 ratio with water to remain in the linear portion of the standard curve. Samples were mixed by vortex and allowed to incubate for 30 min at ambient temperature, and were then measured at 560 nm using Thermo Scientific Multiskan spectrophotometer (Thermo Scientific, Waltham, MA) blanked against diluted model wine without any added H 2 O 2. Samples were quantified based on an external 29

41 standard curve prepared with an authentic H 2 O 2 and validated using hydrogen peroxide extinction coefficient ε 240 = 40 M 1 cm 1. No differences between standard curves were observed in the presence or absence of tartaric acid. Acetaldehyde analysis. Acetaldehyde was measured in model wine solutions as its 2,4-dinitrophenylhydrazone (DNPH) derivative by HPLC according to previous methods (33,64). The DNPH solution was prepared by dissolving DNPH (200 mg) in acetonitrile (100 ml), followed by the acidification with 70 wt% perchloric acid (4 ml). Following the Fenton reaction, as described above (in the absence of PBN), 40 μl of sulfuric acid (25 wt%), 240 μl of DNPH, and 100 μl aliquot of model wine in a 1.8 ml capacity microcentrifuge tube. The derivatization reaction was carried out at ambient temperature for 3 h, and then diluted with 480 μl of 60:40 acetonitrile:water. The sample was then filtered over a PTFE syringe tip filter (0.45 μm; 13 mm). Chromatographic separation was achieved isocratically using a ZORBAX Eclipse Plus C18 column (4.6 x 150mm, 5 μm; Agilent Technologies) with a mobile phase consisting of 70:30 methanol:water. The acetaldehyde-dnph derivative was detected using a diode array detector at 365 nm and quantified based on an external standard curve prepared with an authentic acetaldehyde-dnph analytical standard. Statistical analysis. Data were analyzed using either two-way ANOVA and Bonferroni correction or one-way ANOVA and Tukey s HSD to determine differences from control (Minitab 16 Statistical Software, State College, PA, USA). Treatments were significant when p < All experiments were performed in triplicate. 30

42 2.4 RESULTS AND DISCUSSION Effects of major wine constituents on 1-HER formation. Ethanol is the major reactant for oxidation by HO radicals in wine, and is thought to react in a concentration based manner. However, the presence of other major wine constituents can contribute to the reaction with HO radicals. Some may behave as antioxidants due to high concentration and quench the HO radical, whereas others may accelerate HO generation. Some of the major wine constituents after ethanol include glycerol, sugars, tartaric acid, and phenolics. For the factorial experiment, the initial compounds were run under accelerated oxidation conditions under air (Table 5) to attempt to take into accounts most of the major wine components that make up white wines. No oxidation progressed in treatment A which contained only 12% EtOH and 30 mm PBN (Figure 1). Fe(II) can reduce oxygen, however, the oxidation did not progress in the absence of exogenous compounds (i.e. phenolics) that may shift the equilibrium to the product side. In addition, the Fe(II) hexaaquo complex reacts slowly in acidic conditions (111), which further explains why no radical formation was observed over the time frame of the reaction. 31

43 Figure 1: 1-HER/PBN adduct formation under air. All samples contained 12% EtOH, 90μM Fe(II), and 30mM PBN (specific treatments in Table 1). As soon as 4-MeC was added in treatment B, there was a clear spin adduct formation in as a little as 1.2 hours, with a linear increase after 2.3 hours (Figure 1). This is expected as Fe(II) can be oxidized to Fe(III) and in the process reduce oxygen to yield HOO which can then quickly be scavenged by the catechol to form H 2 O 2 (12,18,48) (Scheme 1). HO that is formed by the Fenton reaction quickly reacts with EtOH to yield 1-HER which is trapped by PBN. The removal of HOO by 4-MeC can allow for quick oxidation of Fe(II) due to the equilibrium shift to the product side which will then be oxidized to Fe(III). Due to the reducing nature of the catechol, Fe(III) can then be quickly reduced to Fe(II). It has been suggested that Fe(III) can be reduced to Fe(II) in a matter of minutes (36). 32

44 In treatment C, which contained both 4-MeC and tartaric acid, spin adduct formation was observed within 1.2 hours and there was significantly lower spin adduct formation compared to treatment B after 2.3 hours (Figure 1). This suggests that the presence of tartaric acid inhibits the formation of 1-HER, but does not prevent it. Interestingly, treatment D, which contained only tartaric acid had a similar rate of oxidation and 1-HER formation to treatment C, and by the last time point at 13 hours practically all treatments containing tartaric acid had similar readings (Figure 1). Despite the fact that no phenolics were present to reduce HOO to H 2 O 2 and subsequent HO, the HOO could be reduced to the peroxide in the presence of Fe(II) under these acidic conditions to HO. The reason this was observed in treatments containing tartaric acid and not in treatment A could be due to the fact that the iron tartrate complex is more favorable than the iron hexaaquo complex (18), and the hydroxy group that binds to iron preferentially binds to Fe(III), allowing for the quick oxidation of Fe(II) once it is bound. In addition, it is possible for the HOO (E 3.6 = 1.22 V) to abstract hydrogen from ethanol (E 3.6 = -1.2 V), as it is thermodynamically favorable, although unlikely in the presence of oxygen (28). Treatment E, which contained only 12% EtOH, 30 mm PBN and 130 mm glycerol, also showed no signs of 1-HER formation, similar to treatment A (Figure 1). This suggests that glycerol does not have a significant impact on the rate of oxidation, and this can be further shown by the fact that treatments F, G, and H were practically the same as treatments B, C, and D respectively in terms of spin adduct formation (Figure 1). The presence of glycerol did not seem to change the rate of reaction compared to the comparable treatment without glycerol. This suggests that glycerol, despite its presence at high concentrations in wine, is neither an antioxidant or pro-oxidant. 33

45 In addition to the accelerated oxidation reaction, the same set of treatments was run under a forced Fenton mechanism (Figure 2). The same trends were observed as expected based on Figure 1 and the forced oxidation model. Treatments A, B, C, and D were not significantly different from treatments E, F, G, and H, respectively. These data suggest once again that glycerol, despite its relatively high concentration, does not outcompete ethanol for HO to any meaningful degree. In general, the presence of catechol increased 1-HER spin adduct formation compared to the respective treatment in the absence of catechol. This suggests that 4-MeC is pro-oxidative, which is expected based on previous literature (18,31,57). Despite the short reaction time, it is possible for 4-MeC to very quickly reduce Fe(III), which will drive HO radical formation via the Fenton reaction from H 2 O 2, as Fe(II) is the limiting reactant in this system. 34

46 Spin adduct intensity A B C D E F G H Figure 2: 1-HER/PBN spin adduct formation with varying concentrations of tartaric acid, glycerol, and 4-methylcatechol under Fenton conditions (90μM Fe(II) and 300μM H 2 O 2 ) in aerated model wine (12% EtOH, ph 3.6) and 30mM PBN. Once tartaric acid was added, 1-HER spin adduct formation was significantly inhibited in all treatments (treatment C, D, G, and H) (Figure 2), suggesting either strong inhibition of HO formation or scavenging of HO directly. This was unexpected given that tartrate is capable of complexing Fe(III) to form a relatively stable Fe(III) tartrate complexes, thereby lowering the reduction potential of the Fe(III)/Fe(II) couple; this would be expected to promote the reduction of H 2 O 2 to the reactive HO. In addition, glycerol did not show any inhibition of the spin adduct formation, despite its presence at twice the concentration of tartaric acid. 35

47 Tartaric acid was measured directly in an attempt to quantify any significant loss of the acid over the reaction, which would indicate that tartaric acid is oxidized by scavenging HO directly; however, no differences were observed (data not shown). In fact, it has been stated previously that Fe(III) tartrate complexes are not likely substrates for HO oxidation (60). Glyoxylic acid is suggested to be a major oxidation product of tartaric acid (112,113), yet previous studies have not been able to detect this product under wine conditions (36). In recent studies, it has only been detected in the presence of light, suggesting that glyoxylic acid is generated in wine by photo-oxidative processes (61,62). Effects of tartaric acid on hydrogen peroxide consumption, 1-HER formation, and acetaldehyde formation. In order to get a better understanding of the factors leading to 1-HER/PBN signal inhibition, the effect of tartaric acid concentration was examined more closely on oxidation markers of model wine. There is strong evidence to suggest that tartaric acid behaves as a pro-oxidant in wine by lowering the redox potential of iron and promoting HO formation. In order to understand whether tartaric acid is a prooxidant or antioxidant, varying concentrations of tartaric acid were examined by a series of induced Fenton reactions under both aerated and deoxygenated systems. In the presence of oxygen, 88% and 89% inhibition of 1-HER by 4 g/l and 8 g/l tartaric acid, respectively, compared to the tartrate-free control (Figure 3), which was consistent with the results described above. However, there was no significant difference between 4 g/l and 8 g/l, indicating that this inhibition is not concentration based in the range tested. This effect is likely observed when tartaric acid is in excess to iron, which would always be the case for table wines. When the system was deoxygenated, there 36

48 Spin adduct intensity was no difference between treatments with respect to spin adduct formation, suggesting that tartaric acid is neither inhibiting nor promoting 1-HER formation. The presence of oxygen clearly seems to affect 1-HER radical formation in the presence of Fe-tartrate complexes Deoxygenated Aerated g/l 4 g/l 8 g/l Figure 3: PBN/1-HER EPR spin intensity after induced Fenton reaction in deoxygenated (black bars) and aerated (grey bars) model wine solution at varying tartaric acid concentration. Note: deoxygenated samples were measured at 3 scans whereas aerated samples were measured at 10 scans. 37

49 Due to the discrepancy within the data and contradicting results compared to the literature, both H 2 O 2 consumption and acetaldehyde formation were measured. It is expected that if tartaric acid is, in fact, inhibiting HO formation, less H 2 O 2 would be consumed and vice versa. Acetaldehyde, as the major oxidation product of ethanol, with 1-HER being the intermediate, was also measured to determine whether or not tartaric acid competes for HO under the conditions described. H 2 O 2 consumption was measured after the initial addition of Fe(II) (90 μm), which is expected to swiftly reduce an equimolar concentration H 2 O 2 (i.e., 90 μm) via Fenton reaction. Interestingly, for the aerated solutions, the presence of both 4 g/l and 8 g/l of tartaric acid did not affect the consumption of H 2 O 2 significantly compared to the tartratefree control (Figure 4). This suggests that tartaric acid does not inhibit nor affect H 2 O 2 consumption via Fenton reaction in the presence of oxygen. 38

50 Hydrogen Peroxide (µm) Deoxygenated Aerated g/l 4 g/l 8 g/l Figure 4: Residual H 2 O 2 induced Fenton reaction in deoxygenated (black bars) and aerated (grey bars) model wine solution at varying tartaric acid concentration. When the solution was deoxygenated, there was a significant increase of H 2 O 2 consumption in the absence of tartaric acid in the deoxygenated solution compared to the aerated solution (Figure 4), this was expected as oxygen can react with 1-HER to form hydroperoxyl ethyl radicals which will subsequently degrade to acetaldehyde (48). In the absence of oxygen, 1-HER can directly reduce Fe(III) to Fe(II), thereby regenerating Fe(II) to undergo Fenton reaction (Scheme 7). Once tartaric acid was added, there was a significant increase of H 2 O 2 consumption in both 4 g/l and 8 g/l treatments. This is supported by previous literature as the Fe(III) tartrate solution lowers the reduction potential of Fe(II) thereby making it more reducing (18). 1-HER can then 39

51 quickly regenerate Fe(II) in the absence of oxygen. This suggests that tartaric acid is clearly a pro-oxidant in the absence of oxygen, which is in contrast to the oxygensaturated system. The final ethanol oxidation product, acetaldehyde, was measured to establish if tartaric acid directly competes with ethanol for HO. For the aerated solution, no significant differences in acetaldehyde formation were observed (Figure 5). This supports the observation from the H 2 O 2 consumption, suggesting that the addition of tartaric acid does not affect the rate of radical formation, yet directly contradicts the results obtained from our EPR analysis. In deoxygenated systems, the tartrate-free control had significantly higher acetaldehyde formation compared to the aerated solution (Figure 5), correlating with the H 2 O 2 consumption (Figure 4). Both 4 g/l and 8 g/l tartaric acid significantly increased acetaldehyde formation, however, the difference between these two treatments was not significant. This, once again, directly contradicts the EPR data. 40

52 Acetaldehyde (PPM) 12 Deoxygenated Aerated g/l 4 g/l 8 g/l Figure 5: Acetaldehyde formation after induced Fenton reaction in deoxygenated (black bars) and aerated (white bars) model wine solution at varying tartaric acid concentration. The data suggests that in the absence of oxygen, tartaric acid behaves as a prooxidant by facilitating the Fenton reaction in model wine conditions. In addition, there is no observed difference between the tartaric acid containing (4 g/l and 8 g/l) treatments, thus suggesting that once tartaric is in excess to iron (binds in 3:1 ratio) it shows a similar effect at varying concentrations past saturation (with respect to Fe). In the presence of oxygen, however, slight antioxidant effect by tartaric acid is observable (although not significant), perhaps due to the Fe(III)-tartrate-oxygen complex, which may prevent the reaction between Fe and H 2 O 2 which is present at relatively low levels. However, this needs to be investigated further, particularly at intermediate and low 41

53 concentrations of oxygen that are found in wine conditions. From a practical perspective, care should be taken in conducting EPR studies in wine-like solutions, as tartaric acid affects observed intensities, as discussed above. It is interesting that there was no effect in the absence of oxygen, yet in the presence of oxygen all signals reach similar EPR intensity (Figure 1) further suggesting that tartaric acid may stabilize the signal at a specific level. The effect of tartaric acid needs to be studied further by varying oxygen levels in model wine solutions to further understand the underlying mechanism for oxidation in the presence of both iron and oxygen. 42

54 Chapter 3: Investigation of Ethyl Radical Quenching by Phenolics and Thiols in Published as: Model Wine Kreitman G.Y., Laurie V.F., Elias R.J. Investigation of Ethyl Radical Quenching by Phenolics and Thiols in Model Wine. Journal of Agricultural and Food Chemistry (in press). 3.1 ABSTRACT In the present study, the reaction between 1-HER and various wine-related phenolics and thiols, including gallic acid, caffeic acid, ferulic acid, 3-mercaptohexan-1-ol (3MH), cysteine (Cys), and glutathione (GSH) was studied by using competitive spin trapping with electron paramagnetic resonance (EPR) and mass spectrometry. Previous studies have reported several important reactions occurring between quinones and other wine components, but the fate of 1-HER within the context of wine oxidation is less understood. Furthermore, the ability of these compounds to prevent the formation acetaldehyde, a known non-enzymatic oxidation product of ethanol, was measured. The hydroxycinnamic acids and thiol compounds tested at 5 mm concentrations significantly inhibited spin adduct formation, indicating their reactivity towards 1-HER. In addition, we confirm that the loss of 3MH under model wine conditions is due to quinone trapping as well as 1-HER induced oxidation. 43

55 3.2 INTRODUCTION Nonenzymatic oxidation greatly affects the stability and, thus, economic value of wines. This is particularly true in the case of white wines, in which oxidation results in browning and a loss of important aroma-active compounds that contribute to desirable sensory attributes (114,115). Varietal thiols are produced during fermentation from the enzymatic cleavage of glutathione and cysteine conjugates by yeast activity (69,75,76). These thiols are present at exceedingly low concentrations, but are critically important to the sensory attributes of wine by contributing pleasant aromas (e.g., grapefruit, passionfruit, and blackcurrant) (68). However, these same compounds are labile to oxidation and can be rapidly lost in wines (116), especially if bottled under closures with high oxygen transmission rates or if stored under improper conditions (55,88). In recent studies, it has been shown that these thiols, which are strong nucleophiles, are particularly susceptible to loss by reacting with electrophilic quinones by Michael-type addition reactions resulting from enzymatic (54) and nonenzymatic (31,52,53,116) oxidation reactions. Non-enzymatic wine oxidation is thought to be initiated by the metal-catalyzed reduction of dioxygen by transition metals, particularly iron and copper (12,18,30). Oxygen is reduced to HOO radicals in the presence of reduced transition metals (Scheme 1), and these HOO radicals are thought to react quickly with phenolics bearing a catechol or gallate group to yield H 2 O 2 and semiquionone radicals (18,49). In the presence of Fe(II) or perhaps Cu(I), H 2 O 2 is then further reduced to yield highly reactive HO radicals (E 3.6 = 2.5 V for HO /H 2 O couple) via the Fenton reaction. These HO radicals react at diffusion-limited rates and are thought to react non-selectively with wine components (12,18). As the major non-water component in wine, ethanol is known to be a major target for these radicals. The reaction between HO radicals and ethanol yield ethyl radicals (1-hydroxyethyl radicals and, to a lesser extent, 2-hydroxyethyl 44

56 radicals), which have been shown to be the major radical species in oxidizing wine (48,57). While the fate of 1-HER is not fully understood in wine, it is clear that significant amounts of this radical are subsequently oxidized to acetaldehyde (Scheme 9, Reaction 1). Scheme 9: Proposed reaction mechanisms of 1-HER with: (1) ferric ions or oxygen, (2) thiols, (3), and hydroxycinnamic acids. In recent years, there has been great interest in the stability and fate of thiols in wines, especially 3MH (11,31,54). While thiol loss resulting from Michael-type addition reactions with benzoquinones has been the focus of nearly all of these studies, the present study considers the possibility that thiol loss in wine is also a result of ethyl radical (e.g., 1-HER) oxidation (Scheme 9, Reaction 2). While the HO radical is certainly capable of directly oxidizing thiols (117), this reaction is predicted to be of lesser importance in wine given the non-selectivity of the radical and the relative abundance of ethanol (i.e., a more likely substrate for hydroxyl radical oxidation). 1-HER is reported to have a lower reduction potential (E 3.6 = 1.2 V for the 45

57 CH 3 CH OH/CH 3 CH 2 OH couple (28)) relative to the HO radical (E 3.6 = 2.5 V for HO /H 2 O couple) and, as such, should react more selectively with wine components compared to hydroxyl radicals. Gislason et al recently showed that the α,β-unsaturated side-chains of hydroxycinnamic acids can react directly with 1-HER to form allylic alcohols (Scheme 9, Reaction 3) (64). Similarly, de Almeida et al reported that 1-HER is reactive towards certain isohumulones in beer, resulting in decreased bitterness (65,66). In biological systems, it has been shown that 1-HER is reactive towards GSH, which is oxidized to produce glutathione disulfide (67). In light of recent studies demonstrating the prevalence and relative stability of 1-HER in wine, our objective was to directly assess the reactivity of 1-HER towards several wine-relevant hydroxycinnamic acids and thiols, including Cys, GSH, and 3MH. 46

58 3.3 MATERIALS AND METHODS Materials. Iron(II) sulfate heptahydrate, 4-methylcatechol (4-MeC), ferulic acid (FA), D- mannitol, L-cysteine (Cys), acetaldehyde-dnph analytical standard and catalase from bovine liver were obtained from Sigma-Aldrich (St. Louis, MO). 2,4-dinitrophenylhydrazine (DNPH) was purchased from MCB laboratory chemicals (Norwood, OH). L- Tartaric acid, 3MH, reduced L-glutathione (GSH), 3,4,5-trihydroxybenzoic acid (gallic acid; GA), 3,4-dihydroxycinnamic acid (caffeic acid; CA), and 5,5 -dithiobis(2- nitrobenzoic acid) (99%+) (DTNB) were obtained from Alfa Aesar (Ward Hill, MA). Hydrogen peroxide (30% w/w; H 2 O 2 ) was obtained from EMD Chemicals (Gibbstown, NJ). The spin trap phenyl-n-tert-butyl nitrone (PBN) was purchased from GeroNova Research Inc. (Carson City, NV). Water was purified through a Millipore Q-Plus system (Milipore Corp., Bedford, MA). All other chemicals and solvents were of analytical or HPLC grade. Preparation of model wine. Model wine was prepared according to previous methods (20) by dissolving 8.0 g of tartaric acid in approximately 700 ml of purified water in a 1 L volumetric flask. Absolute ethanol (120 ml) was added and the ph was adjusted to 3.6 using 5 N sodium hydroxide. Sufficient water was added to bring the solution to a final volume of 1 L. Fenton reaction conditions in model wine. The PBN spin trap was dissolved directly into model wine solutions to achieve a final concentration of 5 mm. GA, CA, FA, Cys, GSH, or 3MH were also added directly to the model wine solution to achieve final concentrations of 5 mm; these concentrations were chosen in order to establish competitive kinetic conditions between the spin trap and test compounds. A control consisting of model wine containing only 5 mm PBN was used. All reactions were carried 47

59 out under nitrogen gas. Model wine solutions (1 ml) containing 5 mm of either of the phenolic- or thiol-containing compounds and PBN were transferred to 5 ml test tubes and gently sparged with nitrogen gas via glass dispersion tube at low flow rate for 2 min to achieve deoxygenated conditions. Following deoxygenation, the Fenton reagents ([H 2 O 2 ] = 1 mm, [Fe(II)] = 100 μm) were added to initiate oxidation reactions according to the method described below. Stock solutions of Fe(II) (10mM), 4-methylcatechol (4- MeC) (100 mm), and H 2 O 2 (100 mm) were prepared daily in water acidified with HCl (ph 2) and mixed by vortex. 4-MeC (10 μl; 1mM final concentration), was added to approximate the phenolic fraction of a white wine, and Fe(II) (10 μl; 100 μm final concentration) was added to the model wine under nitrogen. H 2 O 2 (10μL, 1 mm final concentration) was then added to initiate the Fenton reaction, which was allowed to proceed at ambient temperature for 1 min under nitrogen. Sample aliquots were withdrawn and analyzed without delay by EPR and LC/MS (methods described below). For treatments requiring acetaldehyde analysis, experiments were performed as described above except in the absence of PBN. Samples used for acetaldehyde analysis were incubated at ambient temperature for 5 min prior to derivatization (described below). EPR analysis of PBN/1-HER spin adducts. EPR was used to detect PBN/1-HER spin adducts in model wine solution, as described previously (36). Sample aliquots (50 μl) were loaded into borosilicate micropipets (VWR, Radnor, PA, USA) and the EPR spectra were immediately recorded at room temperature on a Bruker escan R spectrometer (Bruker BioSpin, Rheinstetten, Germany) operating in X-band. The sweep width was set to 50 G and the microwave power was set to mw. The modulation frequency and modulation amplitude were set to khz and 2.45 G, respectively. The receiver gain was set to The conversion time and sweep time were set to msec and 48

60 10.49 sec respectively. The total number of scans per sample was 10. The reaction reached a maximum EPR absorbance within one minute and the signal remained constant for 30 minutes before starting to decay (data not shown). The intensity was quantified by adding the maximum and minimum values of the central doublet. HPLC-MS analysis of PBN/1-HER spin adducts. Samples were diluted 1:20 in Millipore water and were filtered over polytetrafluoroethylene (PTFE) syringe tip filters (0.45 μm, 13 mm; AcrodiscTM, Ann Arbor, MI, USA). The HPLC system consisted of a binary pumping system (Shimadzu LC-10ADvp) with high pressure mixing and sample introduction by means of a Shimadzu SIL 10ADvp autosampler. PBN/1-HER spin adducts were separated on a ZORBAX Eclipse Plus C18 column (4.6 x 150 mm, 5 μm; Agilent Technologies, Santa Clara, CA, USA). The mobile phase consisted of 0.1% v/v formic acid (A) and 0.1% v/v formic acid in methanol (B). The PBN/1-HER adducts were eluted by gradient according to the following program: 0 min, 0% B; 0-5 min, 80% B; 5-18 min, 90% B; min, 90% B; min, 0% B; min, 0% B. Detection and quantification of spin adducts were achieved using a Waters Quattro micro triple quadrupole mass spectrometer (Waters Laboratory Informatics, Milford, MA, USA) coupled to the HPLC. Mass spectra were collected in negative ion mode using electrospray ionization (ESI). The ESI capillary spray was held at 0.50 kv. The cone source voltage was set to 60 V and the source temperature was set to 120 C. The desolvation gas flow was 250 L/h. Selected ion monitoring mode was set to monitor ions with m/z of 178, 222, 223, 224, and 268, which correspond to unreacted PBN, oxidized PBN-1-HER adduct, radical PBN-1-HER adduct, reduced PBN-1-HER adduct, and the PBN-HER bi-adduct, respectively (Figure 6). Due to the complexity of having pure standards for all of these adducts, their quantification was performed based on raw areas. 49

61 Figure 6: Possible PBN/1-HER adducts formed: (1) oxidized (222 m/z), (2) radical (223 m/z), (3) reduced (224 m/z), and (4 and 5) bi-adducts (268 m/z). Acetaldehyde analysis. Acetaldehyde was measured in model wine solutions as its DNPH derivative by HPLC according to previous methods (33,64). Following the Fenton reaction, as described above (in the absence of PBN), 40 μl of sulfuric acid (25%) and 240 μl of DNPH reagent were added to a 100 μl aliquot of model wine in a 1.8 ml capacity microcentrifuge tube. The derivatization reaction was carried out at ambient temperature for 3 h, after which 480 μl of 60:40 acetonitrile:water was added to the sample. The sample was then filtered over a PTFE syringe tip filter (0.45 μm; 13 mm). Chromatographic separation was achieved isocratically using a ZORBAX Eclipse Plus C18 column (4.6 x 150mm, 5 μm; Agilent Technologies) with a mobile phase consisting of 70:30 methanol:water. The acetaldehyde-dnph derivative was detected using a diode array detector at 365 nm and quantified based on an external standard curve prepared with an authentic acetaldehyde-dnph analytical standard. Quantification of 3MH loss. In order to study the loss of 3MH by 1-HER, an alternative model wine solution, in which the ethanol was replaced by mannitol, was also prepared 50

62 alongside a standard model wine (i.e., with ethanol). Mannitol, unlike ethanol, does not produce stable radicals that could react with 3MH. To prepare this solution, mannitol (22 g) was added to a 100 ml solution containing 8 g/l tartaric acid at ph 3.6 with a final mannitol concentration of 1.2 M, despite the fact that this is approximately half the concentration of ethanol in model wine, it still achieves an overwhelming molar excess to 3MH. To either deoxygenated mannitol or deoxygenated ethanol model wine solutions, a final concentration of 100 μm 3MH was established. Fenton reagents and 4-MeC solutions were prepared as described above. The reagents were added to achieve Fenton conditions with or without 4-MeC; in addition, controls which included H 2 O 2 only or 4-MeC and Fe(II) were added to verify that the reaction is attributed to radical and quinone addition type reactions. Reaction vessels were capped and held at ambient temperature for ten minutes. 3MH was measured in model wine solutions using the Ellman s assay (118). Following the Fenton reaction, 25 μl of catalase (100 IU) was added directly to model wine solutions followed by 100 μl of phosphate/tris buffer (1 M; ph 8.1) to achieve a final ph of 7.0. Catalase was added to prevent further reaction by H 2 O 2 formed at this ph, as previously documented (33). DTNB (375 μl; 2 mm) in phosphate buffer (100 mm; ph 7.0) was then added and the reaction was allowed to proceed for 10 min. The DTNB derivative was then quantified using a Genesys 10S UV- Vis spectrophotometer (ThermoScientific, Waltham, MA, USA) at 412 nm. Quantification of 3MH was based on an external standard curve. Statistical analysis. Data were analyzed using one-way ANOVA and Dunnett s posttest or Student s t-test to determine differences from control (Minitab 16 Statistical Software, State College, PA, USA). Treatments were significant when p < All experiments were performed in triplicate. 51

63 Figure 7: Chemical structures of caffeic acid (CA), gallic acid (GA), ferulic acid (FA), 3- mercaptohexan-1-ol (3MH), cysteine (Cys), and glutathione (GSH). 52

64 3.4 RESULTS AND DISCUSSION Analysis of PBN/1-HER adducts by EPR. GA, CA, FA, Cys, GSH, and 3MH (Figure 7) were investigated for their ability to quench 1-HER in model wine solutions in which oxidation was initiated by exogenous Fenton reagents (i.e., Fe(II) and H 2 O 2 ). The HO radicals formed under these conditions are extremely reactive and are thought to react with the organic fraction of wine in a concentration-dependent manner. Ethanol (~2 M in wine) is present in molar excess compared to other wine components, and is thus predicted to be the major target of HO radicals, resulting in the generation of hydroxyethyl radicals (36,57). It has been previously shown that the accelerated oxidation conditions (i.e., Fenton reaction) employed in the present study yield the same radicals that result from the unforced oxidation of wine (36,57). Treatment (GA, CA, FA, Cys, GSH, and 3MH) concentrations (5 mm) used in this study are higher than what is typically present in wine, but were chosen to establish equimolar concentrations with PBN in order to create competitive kinetic conditions. 53

65 Figure 8: Representative experimental EPR spectrum corresponding to the PBN/1-HER spin adduct in model wine conditions. A triplet of doublets was observed in the EPR spectrum of oxidized model wine samples (Figure 8), with hyperfine coupling constants (a N = 15.5 G, a H = 3.3 G) indicative of PBN/1-HER adducts (36). As expected, the intensity of PBN/1-HER spin adduct formation for GA was not significantly different from the control (Figure 9). GA was, in essence, used as a negative control as it plays a similar role to 4-MeC which was present in all other treatments. GA was used at 5 mm to verify that the gallate group does not react with either 1-HER or HO radicals to a significant extent at the concentrations used. As these reactions were run in the absence of oxygen, Fe(II) was not considered to be limiting in the Fenton reaction, as 1-HER quickly reduce Fe(III) under low oxygen conditions (Scheme 9, Reaction 1) (36). If the experiment was 54

66 PBN Spin Adducts (EPR intensity) conducted in the presence of oxygen, 1-HER would be more likely to react with oxygen to form peroxyl radicals rather than reduce Fe(III) to Fe(II) ions. In the absence of a reducing agent capable of cycling Fe(III) back to its Fe(II) state, it would be expected that the PBN/1-HER spin adduct intensity observed in the GA treatment would be higher than the control, as gallate groups facilitate Fe(III)/Fe(II) redox cycling (18,31) * * 1 * * * 0 Control GA CA FA Cys GSH 3SH Figure 9: EPR spin adduct intensities of PBN/1-HER in the presence of 4-MeC, PBN, and selected treatments in model wine in the absence of oxygen. Spectra were obtained at room temperature after 1 min following the addition of Fe(II) and H 2 O 2. *Statistically significant difference (P < 0.05) from control by one-way ANOVA with Dunnett s posttest. Significantly lower PBN/1-HER spin adducts were observed in solutions containing hydroxycinnamic acids (FA and CA) and thiol-containing compounds (Cys, GSH, 3MH) compared to the control model wine (Figure 9). Based on the results seen 55

67 from the GA treatment, it is unlikely that the concentrations used were sufficiently high to compete with ethanol for HO radicals, as ethanol is present at ca. 400 molar excess. Therefore, these results suggest evidence of direct competition between the treatments and PBN for 1-HER. CA inhibited the production of PBN/1-HER spin adducts by ca. 69% compared to the control, despite the presence of a catechol group and a similar reduction potential to that of GA (18). A decrease in observed spin adducts can be attributed to the high reactivity of CA s α,β-unsaturated side-chain group, which has recently been shown by Gislason et al to efficiently scavenge 1-HER at the α-position (64). The resulting complex is a stable benzyl radical and in the presence of Fe(III), rearranges to its carbocation form and, eventually, into an allylic alcohol (64). The ability of FA to compete with PBN for 1-HER was also investigated, as it contains an α,β-unsaturated side-chain yet, unlike CA, does not contain a catechol group and therefore does not form an o-benzoquinone upon oxidation. As was the case with CA, FA was observed to scavenge 1-HER radicals, thereby suppressing PBN/1-HER adduct formation by ca. 55% compared to the control. The thiol-containing treatments inhibited spin adduct formation to the greatest extent, indicating their reactivity towards 1-HER under wine conditions. Cys, GSH, and 3MH supressed the formation of PBN/1-HER spin adducts by 88, 87, and 96%, respectively (Figure 9). It is possible that these compounds showed high reduction in spin adduct formation due to the fact that thiol groups have a relatively low reduction potential and can effectively reduce 1-HER. Reduction potentials do not necessarily predict the reaction rates. For the 2-electron reduction potential, RSH/RSSR couple is a stronger reducing agent than polypenols (119), however, a sulfhydryl group has a reduction potential of approximately V for the RSH/RS couple at wine ph (31), this is lower than the catechol system (E 3.6 = -1.0 V for catechol/semiquinone couple). This 56

68 Area (AU) indicates that upon the formation of a thiyl radical, it is likely quickly scavenged by a catechol which is present in excess to the thiol in wine. We observed that the dimerized form of 3MH was formed when undergoing a Fenton reaction in the absence of 4-MeC, but we did not observe significance in the presence of 4-MeC (Figure 10). Although it is possible for the disulfide to form, as it has been observed in botrytized and aged wines (120) by reducing 1-HER back to ethanol while simultaneously forming a thiyl radical (Scheme 2, Reaction 2) MH disulfide 4-MeC-3MH adduct t = 0 t = 30 4-MeC Fenton Fenton+4-MeC Figure 10: 3MH disulfide and 4-MeC-3MH adduct formation from the following treatments: 3MH only (measured immediately); 3MH only; 3MH + 4-MeC; Fenton reaction (Fe(II) + H 2 O 2 ); Fenton reaction (Fe(II) + H 2 O 2 ) + 4-MeC. Reactions were run in model wine (ph 3.6) in the absence of oxygen and measured after 30 min reaction at ambient temperature. 57

69 Analysis of PBN/1-HER adducts by LC-MS. In order to confirm the EPR spin trapping results reported above, PBN/1-HER adducts were also measured using LC-MS. This was important due to the potential instability of the EPR-active form of the adduct under the conditions employed in the study. Both hydroxylamine and nitrone forms of the spin adducts (i.e., reduced and oxidized radical adducts, respectively) are EPR-silent species due to the loss of their unpaired electrons. Previous studies have demonstrated that under some conditions (e.g., biological systems), reducing agents such as Cys and GSH can result in the reduction of paramagnetic radical spin adducts to their EPR-silent hydroxylamine forms (121). It has also been shown that under beer conditions, α-(4- pyridyl-1-oxide)-n-tert-butylnitrone (POBN)/1-HER adducts, which are structurally analogous to PBN/1-HER, can be oxidized to their nitrone forms or react with a second 1-HER to yield a bi-adduct, both of which are undetectable by EPR (65). Therefore, the nitrone, nitroxide radical, hydroxylamine, and bi-adduct forms of the PBN/1-HER adducts were measured by LC-MS (222 m/z, 223 m/z, 224 m/z, and 268 m/z, respectively) to account for any losses of the EPR-active nitroxide adducts. 58

70 Figure 11: Total MS spin adduct intensities of all possible PBN/1-HER adducts (oxidized, 222 m/z; radical, 223 m/z; reduced, 224 m/z; bi-adducts, 268 m/z) in the presence of 4-MeC, PBN, and selected treatments in model wine in the absence of oxygen. Spectra were obtained at room temperature after 1 min following the addition of Fe(II) and H 2 O 2. *Statistically significant difference (P < 0.05) from control by one-way ANOVA with Dunnett s post-test. No significant differences between the control and treatments with respect to formation of the nitrone (oxidized form of the adduct, 222 m/z) were observed (Figure 11). This was expected as samples were analyzed immediately following the induction of the Fenton reaction and there was limited opportunity for oxidation of the nitroxide radical adduct. Samples that were allowed to stand for extended periods prior to LC-MS analysis were found to contain higher peak area of bi-adducts and nitrone forms, and lower peak area of hydroxylamine and nitroxide radical forms (data not shown). The peak area for the EPR-active PBN/1-HER adduct (nitroxide radical; 223 m/z) detected in 59

71 model wine solutions containing the GA treatment was not significantly different from the control; however, the total nitroxide radical peak area was significantly lower for the hydroxycinnamic acid and thiol treatments compared to the control, thus corroborating our EPR spin trapping results. Interestingly, the total peak area value for the reduced (hydroxylamine) PBN/1-HER adduct (224 m/z) for 3MH was significantly lower than those observed in the control. Previous studies have suggested that the addition of a thiol (e.g., GSH) to a pre-formed PBN/1-HER adduct does not, in fact, affect EPR signal intensity, but would effectively inhibit spin adduct formation if the thiol were present prior to the reaction (67), thereby suggesting competition with PBN for 1-HER. With respect to bi-adduct (268 m/z) formation, the peak area for the GA treatment was significantly higher than the control, yet for the hydroxycinnamic acids (CA, FA), there was no difference from control. Bi-adduct formation was significantly lower in the thiol-containing treatment compared to control. Acetaldehyde analysis. With respect to acetaldehyde formation (i.e. an alternative way to asses ethanol oxidation), the results were consistent with the above reported EPR and LC-MS analysis of 1-HER. GA did not significantly prevent acetaldehyde formation compared to the control (Figure 12). Significantly less acetaldehyde was observed in samples containing the hydroxycinnamic acid and thiol treatments compared to the control. The overall trend for acetaldehyde formation was similar to 1-HER formation, as measured both by EPR and LC-MS, thus supporting our proposed mechanism. 60

72 Acetaldehyde (μm) * * * * * 0 Control GA CA FA Cys GSH 3SH Figure 12: Acetaldehyde formation in the presence of 4-MeC and phenolic and thiol treatments in model wine in the absence of oxygen. *Statistically significant difference (P < 0.05) from control by one-way ANOVA with Dunnett s post-test. Contribution of 1-HER to 3MH loss. In order to directly assess the extent to which a test thiol (3MH in this case) is oxidized by 1-HER, wine samples were oxizided in either the presence or absence of 4-MeC. This was done to determine the relative contribution of quinone reactions (via 1,4-Michael addition reactions) versus 1-HER induced oxidation reactions to the overall loss of the model thiol under wine conditions. Mannitol, a sugar alcohol that is a known substrate for hydroxyl radicals (122), was substituted for ethanol in some experiments. Unlike ethanol, mannitol has been shown to be rapidly degraded following its reaction with hydroxyl radicals and, thus, would not be expected to promote thiol (e.g., 3MH) oxidation as 1-HER would. 61