New insights on thiol precursors catabolism by yeast during wine fermentation: identification of the N-Acetyl-L-Cysteine conjugate

Abstract

Understanding the catabolism of thiol precursors is essential for understanding the revelation of varietal thiols in wine. For many years, knowledge of these precursors has been limited to the S-conjugates of glutathione, cysteine (Cys3SH) and the dipeptides g-GluCys and CysGly, without being able to explain the full origin of 3-sulfanylhexan-1-ol (3SH) in wines. Cysteine was the one considered as the final intermediate before the revelation of 3SH by yeast. Inspired by the glutathione detoxification pathway of xenobiotics, we identified the 3-S-(N-Acetyl-Cysteinyl)hexan-1-ol (NAC3SH) as a new metabolite, by developing (a) a dedicated organic synthesis strategy, (b) a targeted LC-MS/MS analysis method and (c) filiation studies under oenological conditions.In practice, we synthesized NAC3SH by Michael addition of N-Acetyl-L-Cysteine onto trans-2-hexenal in 50/50 water/acetonitrile followed by reduction with NaBH4 in situ. After purification by preparative HPLC, the two diastereomers of NAC3SH were successfully isolated. Characterization was done by both 1D and 2D homonuclear 1H and heteronuclear 1H /13C NMR spectroscopy and quantification by 1D 1H NMR (qNMR). An analytical method for NAC3SH was developed by LC-MS/MS using Cys3SH-d2 as internal standard. The resolution of the two diastereomers could not be achieved under our conditions and they were therefore analyzed in 50/50 equimolar mixture. The method has been validated and showed very satisfactory analytical performances (accuracy = 102%, linearity: R2 = 0.976, LOQ = 6.9 µg/L, LOD = 23 µg/L, repeatability: CV).