Synthesis of Wine Thiol Conjugates and Labeled Analogues: Fermentation of the Glutathione Conjugate of 3-Mercaptohexan-1-ol Yields the Corresponding Cysteine Conjugate and Free Thiol

Abstract

Synthesis of the putative wine thiol precursor 3-S-glutathionylhexan-1-ol (Glut-3-MH) has been undertaken to provide pure reference materials for the development of HPLC-MS/MS methods for precursor quantitation in grape juice and wine, and for use in fermentation experiments. Labeled thiol conjugates were also prepared for use as internal standards. Purification and fermentation of a single diastereomer of Glut-3-MH with VIN13 (CSL1) yielded not only the (R)-enantiomer of the wine impact odorant 3-mercaptohexan-1-ol (3-MH) but also the cysteine conjugate intermediate as a single (R)-diastereomer, as determined by HPLC-MS/MS. Chiral GC-MS was used to quantify the total amount of (R)-3-MH released from the ferments, resulting in a molar conversion yield of the glutathione conjugate of about 3%. Enzymatic degradation of the single (R)-Glut-3-MH diastereomer with a gamma-glutamyltranspeptidase confirmed the stereochemical relationship to the related cysteine conjugate. This is the first demonstration that Glut-3-MH can liberate 3-MH under model fermentation conditions, where the cysteine conjugate is also formed in the process. This furthers our understanding of the nature of wine thiol precursors and opens avenues for additional studies into formation and interchange of wine thiols and their precursors.