Article pubs.acs.org/JAFC
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H NMR: A Novel Approach To Determining the Thermodynamic Properties of Acetaldehyde Condensation Reactions with Glycerol, (+)-Catechin, and Glutathione in Model Wine
Ana L. Peterson and Andrew L. Waterhouse* Department of Viticulture and Enology, University of California, One Shields Avenue, Davis, California 95616, United States S Supporting Information *
ABSTRACT: As wine oxidizes, ethanol is converted to acetaldehyde, but its accumulation is not predictable, due to poorly characterized reactions with alcohols, SO2, thiols, flavanols, and others. Measurement of these components has been thwarted by equilibria into the other forms during sample preparation. NMR spectra can be taken on intact samples and is thus ideal for this situation. Equilibria of acetaldehyde with glycerol, (+)-catechin, and glutathione were studied separately in model wine solutions at pH 3−4 by 1H NMR and 2D (1H−1H) COSY spectra. Glycerol acetals had equilibrium constants between 1.14 ± 0.056 and 2.53 ± 0.043 M−1, whereas ethylidene-bridged (+)-catechin dimers and glutathione thiohemiacetals had more favorable equilibria: from (3.92 ± 0.13) × 103 to (6.13 ± 0.32) × 103 M−2 and from 10.18 ± 0.22 to 11.17 ± 0.47 M−1, respectively. These data can be used to create accurate measures of acetaldehyde in its various forms and, consequently, offer insight into wine oxidation. KEYWORDS: acetaldehyde, (+)-catechin, glutathione, glycerol, equilibrium constants
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wines undergoing biological aging.10 Four heterocyclic acetal isomers are produced from glycerol, cis- and trans-dioxolane (1a,b) and cis- and trans-dioxane (2a,b), and have been identified as oxidation markers in fortified wines, the amount correlating with barrel age in Madeira and Port wines in which slow oxidation is ongoing.6,11 Acetaldehyde reacts with flavan-3-ols and anthocyanins, altering the overall color and mouthfeel (astringency)12 with condensation and polymerization reactions.13 Bridging reactions between flavanoids (i.e., 5) have been studied in real and model wine solutions.14−16 These reactions are reversible, and one method has been described to recover the acetaldehyde from these products.7 Acetaldehyde can also act as a nucleophile and attack the electrophilic portion of the flavylium cation (anthocyanin), forming a pyran ring and yielding vitisin B, (6).17 Addition of SO2 to wine for the prevention of oxidative and microbial spoilage results in an equilibrium between various S(IV) species, bisulfite (HSO3−) being the predominant form at wine pH. Bisulfite can also undergo nucleophilic addition with carbonyl compounds in wine, yielding hydroxyalkylsulfonic acids (7). The dissociation constant for bisulfite-bound acetaldehyde under wine-relevant conditions is well documented and indicates that the sulfonate product is highly favored.18 The sensitivity of wine consumers to SO2 has led to investigations into the use of glutathione as an alternative antioxidant, specifically its ability to scavenge quinones under wine-like conditions.19 The addition of this thiol nucleophile to
INTRODUCTION Aging is crucial to the development of a red wine and occurs partly through its exposure to oxygen as first demonstrated by Pasteur via comparing wine sealed in an ampule with oxygen versus no headspace.1 Wine oxidation is dependent on the method of aging, wine composition, and other factors. The exposure of wine to oxygen induces a cascade of radical reactions and subsequent generation of new compounds, many of which can alter the aroma, color, and even mouthfeel of wine. The interaction between oxygen and various wine components has been studied in numerous model wine experiments to determine the mechanisms by which ethanol and other substrates are oxidized.2,3 The aroma complexity and pigmentation of a red wine can increase with modest oxygen exposure.4 Micro-oxygenation accelerates the aging process through controlled additions of oxygen; however, metrics to monitor the process are lacking, but necessary to gain better control over the changes and thus wine quality. Currently, aged and micro-oxygenated wines are monitored in production by sensory analysis or, in some research situations, by measuring acetaldehyde.5−7 The electrophilicity of acetaldehyde leads to its participation in reversible reactions with various nucleophiles in wine, including alcohols, thiols, sulfur dioxide SO2, and flavonoids. Consequently, its measurement is complicated by the formation of these adducts, and measurement of free acetaldehyde does not reflect the total amount present or the amount of oxidation that has occurred (Figure 1). In an aqueous solution, acetaldehyde rapidly (acid is a catalyst)8 equilibrates with water, forming the hydrate (4),9 and a parallel reaction with ethanol yields the hemiacetal (3a) and acetal (1,1-diethoxyethane, 3b). Diethyl acetal has been reported to have a fruity character in commercial sherry © XXXX American Chemical Society
Received: May 6, 2016 Revised: August 9, 2016 Accepted: August 13, 2016
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DOI: 10.1021/acs.jafc.6b02077 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. Molecular depiction of some of the equilibria formed between acetaldehyde and its various derivatives: trans- and cis-dioxolane (1a and 1b), trans- and cis-dioxane (2a and 2b), ethyl hemiacetal (3a) and diethoxyethane (3b), acetaldehyde hydrate (4), ethylidene-bridged catechin dimer (5), pyranoanthocyanins (vitisin B, 6), hydroxyethylsulfonic acid (7), and the glutathione adduct (8).
acetaldehyde yields a thiohemiacetal (8).20 Thus, the glutathione thiohemiacetal is another possible pathway for acetaldehyde consumption. Gas chromatographic methods for acetaldehyde involve solid-phase microextraction,21 followed by derivatization with PFBHA.22 Liquid chromatography methods use DNPH23 or cysteamine.24 The addition of heat and/or changed pH for derivatization methods will catalyze the hydrolysis of acetals25 and cleavage of ethylidene bridges7 as well as hydrolyze hydroxysulfonates,23 altering free acetaldehyde. These procedures will therefore create interferences for measurements of free acetaldehyde. Although many addition products of acetaldehyde have been studied in wine, the concentration of free acetaldehyde is not usually reported.26,27 To determine the equilibrium point of these reactions, the concentration of all components must be known. Because the available methods likely measured some of the addition products, but at best were tested against only one of the addition reactions, it was necessary to find a method for the analysis of reaction solutions that did not disrupt or shift the equilibria and could measure all components. Although less sensitive than chromatographic methods (5.1 μg/L28), NMR spectroscopy (1.1 mg/L29) is capable of the simultaneous detection of many components and has minimal sample preparation and, thus, would not affect reaction equilibria. NMR has advanced its use for quantitation of components and interactions in real and model wine solutions.30,31 The development of new pulse sequences for the suppression of water and ethanol32 and the optimization of 2D experiments such as diffusion-ordered NMR spectroscopy (DOSY) to investigate changes in wine through aging33 have improved the selectivity, sensitivity, and resolution of wine analysis by NMR. NMR studies of acetaldehyde in aqueous solutions have revealed the equilibrium that exists between acetaldehyde and its hydrated form.34,35 NMR has also been used to measure the acetaldehyde equilibria with hemiacetals (acetal intermediates)25 and, more recently, the formation of acetaldehyde
hydrate as well as poly(oxymethylmethylene) glycols (hydrate polymerization products).36 Nikolantonaki et al. successfully applied NMR spectroscopy to the analysis of acetaldehyde and other carbonyls in the free and SO2-bound forms in wine to overcome measurement difficulties caused by their instability and chemical reactivity.29 This prompted our investigation and optimization of 1H NMR for the measurement of acetaldehyde interactions with other nucleophiles in model wine solutions. Under wine-relevant conditions, 1H NMR was used to investigate the equilibria between acetaldehyde and its derivatives in interactions with sulfites, flavonols, and thiols.
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MATERIALS AND METHODS
Chemicals and Reagents. Acetaldehyde (99.5%) and glutathione (98%, reduced) were purchased from Acros Organics (Morris Plains, NJ, USA). Acetaldehyde diethyl acetal (99%), 3-trimethylsilylpropionic-2,2,3,3-d4 acid sodium salt (98 atom % D), deuterium oxide (99.9 atom % D), glycerol (99.5%, ACS reagent), (+)-catechin hydrate (98%), and (+)-tartaric acid (≥99.5%) were all obtained from SigmaAldrich (St. Louis, MO, USA). Sodium hydroxide solution (10.05− 9.95 N certified) was purchased from Fisher Scientific (Fair Lawn, NJ, USA). Deuterium-labeled ethanol (ethanol-d6, 99% D,
