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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Straightforward Syntheses of Deuterated Precursors to be Usedas Powerful Tracers under Fermentative ConditionsFlorine Cavelier 1 , Aurélie Roland 2 , Alain Razungles 2 , andRémi Schneider 31 IBMM UMR CNRS 4247, Montpellier, 34095, France; 2 UMR 1083 Sciences Pourl’œnologie INRA-SupAgro-Université Montpellier I, Montpellier, 34060, France;3 Institut Français de la Vigne et du Vin, Montpellier, 34060, FranceIntroductionThe S-3-(hexan-1-ol)-glutathione (G3MH, 1) and S-3-(4-methylpentan-2-one)-glutathione(G4MMP, 2), initially identified in Sauvignon Blanc grapes [1,2], were presumed to beprecursors of powerful odorant thiols found in wines: the 3-mercaptohexan-1-ol (3MH) [3]and the 4-mercapto-4-methylpentan-2-one (4MMP) [4] respectively. These thiols, alsocalled varietal thiols, were reminiscent of grape fruit and blackcurrant bud and contributepositively to the fruity notes of young wines. The biogenesis of such thiols in wine is verycomplicated and not entirely understood. To bring new insights in the 3MH and 4MMPbiogenesis, labeled analogues of G3MH and G4MMP were synthesized and used as tracersin fermentation experiments to investigate potential relationship with these thiols underenological conditions.Results and DiscussionThis study intended to provide few amounts of deuterated analogues of glutathioneconjugates occurring in grapes. First synthesis of 1 in deuterated form required lengthy(7 steps) and tedious efforts in order to obtain the desired product with a very moderateyield [5]. Other work reported the direct synthesis of 1 and 2 using a conjugate addition ofglutathione on (E)-2-hexenal with pyridine giving pure products in satisfactory yields [6].Unfortunately, we did not succeed in reproducing this reported synthesis. Therefore, wedeveloped a new strategy for the 1-d 2 synthesis in racemic form as well as for the 2-d 10 .Conjugate addition of glutathione on the (E)-2-hexenal led to 1 after a reduction stepwith sodium borohydride (Figure 1). The conjugate addition was the only limiting step(incomplete, generation of by-products), whereas the reduction facilitated the production ofthe expected compound in good yield and purity as already reported [5,6]. To improve theglobal reaction yield and the purity of the desired product, investigations were necessary inorder to optimize the conjugate addition [7]. The optimal conditions were thus defined:room temperature, slow addition of (E)-2-hexenal in 3 portions, pH=8 (phosphate buffer),stirring for 10 h. Then, the same conditions, including an additional reduction step withsodium borohydride, were applied to the formation of 1-d 2 with the (E)-2,3-[²H 2 ]-2-hexenalas starting material [6].HOOCNH 2NHOHSONH1. (E)-2-hexenal or (E/Z)-2-hexenal d 2phosphate bufferCOOH pH=8RT, 10h2. NaBH 4, 2h, RT11 %HOOCNH 2C H 3OONHNHS1H or DOHCOOHH or DHOOCNH 2NHOHSONH1. Mesityl oxide or mesityl oxide d 10pyridineCOOH RT, 36hHOOC80 %NH 2ONHSOONH2CH 3 or CD 3CH 3 or CD 3COOHCH 3 or CD 3H or DFig. 1. Synthesis of natural and labeled G3MH (1) and G4MMP(2).48