Synthesis of the Individual Diastereomers of the Cysteine Conjugate of 3-Mercaptohexanol (3-MH) KEVIN H. PARDON, †,# SEAN D. GRANEY, § DIMITRA L. CAPONE, †,# JAN H. SWIEGERS, †,# MARK A. SEFTON, †,# AND GORDON M. ELSEY* ,†,§,# The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, South Australia 5064, Australia; School of Chemistry, Physics and Earth Sciences, Flinders University, P.O. Box 2100, Adelaide, South Australia 5001, Australia; and Cooperative Research Centre for Viticulture, P.O. Box 154, Glen Osmond, South Australia 5064, Australia The individual diastereoisomers of the cysteine conjugate of 3-mercaptohexanol (4) were synthesized with high isomeric purity (>98%). On treatment with Apotryptophanase enzyme, the 3R diastereoi- somer of 4 gave an 82% yield of the R enantiomer of 1, with no trace of the 3S enantiomer present. Conversely, the 3S diastereoisomer of 4 gave the 3S enantiomer of 1 (43%) accompanied by a trace of the 3R form (S/R ) 98.5:1.5), reflecting the diastereomeric purity of the cysteine conjugate. The same stereochemical outcome was observed when the individual diastereoisomers of 4 were added to fermentations with the Saccharomyces cerevisiae AWRI 1655 yeast strain, which gave 1 in 1% yield. A d 10 -analogue of 1 was synthesized and used as an internal standard to determine, by gas chromatography-mass spectrometry (GC-MS), the amounts of 1 formed in these transformations. KEYWORDS: Wine; aroma, flavor; 3-MH; cysteine conjugates; thiols INTRODUCTION In recent years, the contribution of thiol-containing com- pounds to the aroma and flavor of wines has been an important focus for research (1). Many such compounds are extremely potent, with aroma detection thresholds typically in the nano- grams per liter range, and are characterized by distinctive aroma qualities. Three thiols (Figure 1) that have been found to be important to the aroma of wine are 3-mercaptohexanol (3-MH, 1), the O-acetate of this compound (3-MHA, 2), and 4-mercapto- 4-methylpentan-2-one (4-MMP, 3). 1 is characterized by a tropical fruit/passion fruit aroma and was first identified in yellow passion fruit (2–4). It has subsequently been identified in several wine varieties (5–7). 2 has also been described as having a tropical fruit aroma and was found to occur, in passion fruit, predominantly as the S enantiomer (8). 3 has an aroma reminiscent of box tree and black currant but, at higher concentrations, has been found to have a rather unpleasant cat- urine odor (9–11). The importance of thiols to the overall aroma of a wine is exemplified by the fact that 3 was found to be the most potent aroma compound (of 42 measured) in one study (9). These thiols themselves are not present in grape juice in measurable concentration prior to fermentation. Rather, 1 and 3 are believed to accumulate as L-cysteine and glutathione conjugates (12–15), with the amino acid moiety of the former being cleaved by the action of the yeast during fermentation (16–18). 2 is presumed to be formed from 1 during fermentation as a result of yeast alcohol acetyltransferase activity (19). More recently, Schneider et al. have shown that volatile thiols can be formed by the addition of hydrogen sulfide, produced by yeast under fermentation conditions, to olefinic precursors of 1 and 3 (20). However, under the conditions reported, this alternative mode of generation accounted for approximately 10% only of the total thiols generated. One factor that must be taken into consideration with 1 and 2 (but not 3) is the stereochemistry at the carbon to which the sulfur is attached, as, for both compounds, the two enantiomers have differing sensory properties (21). The cysteine conjugate of 1 has two diastereomeric forms, namely, 3S-4 and 3R-4. Thus, the stereochemical outcome of the transformation of the conjugate 4 by the yeast may be dependent on the starting configuration of 4. Wakabayashi et al. (18) prepared a 1:1 diastereomeric mixture of 4, and when this mixture was subjected to enzymatic cleavage, using two different sources of -lyase, the two enantiomers of 1 were found in similar proportions. As an expansion of our earlier work concerning the liberation of 3 from its cysteine conjugate (16, 17), we have now synthesized the two cysteine conjugate forms of 4, in diaste- reomerically pure fashion, to investigate the stereoselectivity of thiol formation more thoroughly and to obtain authentic samples for characterizing grape composition. * Corresponding author (telephone +61 8 8201 3071; fax +61 8 8201 2905; e-mail Gordon.Elsey@flinders.edu.au). † The Australian Wine Research Institute. # Cooperative Research Centre for Viticulture. § Flinders University. 3758 J. Agric. Food Chem. 2008, 56, 3758–3763 10.1021/jf8000444 CCC: $40.75  2008 American Chemical Society Published on Web 05/08/2008