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Eur Food Res Technol (2000) 210 : 349–352 Q Springer-Verlag 2000

ORIGINAL PAPER

Patricia Bouchilloux 7 Philippe Darriet


Denis Dubourdieu 7 Robert Henry 7 Sylvia Reichert
Armin Mosandl

Stereodifferentiation of 3-mercapto-2-methylpropanol in wine

Received: 8 June 1999

Abstract 3-Mercapto-2-methylpropanol, an odorous in wines [5–7]. One of these methods is based on low-
compound recently identified in red wines, was ex- temperature vacuum distillation coupled with a specific
tracted from a red wine variety, using a low-tempera- reversible thiol capture on an organomercuric com-
ture vacuum distillation coupled with a specific reversi- pound, sodium p-hydroxymercuribenzoate (p-HMB).
ble thiol capture. The analysis of the diacetylated wine The method applied to red wines has led to the identi-
extract by enantioselective multidimensional gas chro- fication of several thiols, including 3-mercapto-2-me-
matography with mass spectrometry detection revealed thylpropanol (1) [5]. Because its structure contains an
the presence of only one enantiomer, (R)-3-mercapto- asymmetrical carbon, this mercaptoalcohol may have
2-methylpropanol. Although both of the optical iso- two enantiomers which cannot be differentiated by
mers are characterized by the same broth and sweat common gas chromatography coupled to flame pho-
odour, they have very different odour thresholds. The tometric detection or mass spectrometry.
amounts of (R)-3-mercapto-2-methylpropanol in young Some authors have shown that there is a relationship
Cabernet-Sauvignon and Merlot wines can exceed its between the chirality of a molecule and its odour. In
perception threshold, thus suggesting its contribution to particular, various odour descriptors have been attri-
the aroma of these wines. buted to the optical isomers of 3-methylthiohexanol,
whose (R)(–)-enantiomer has a spicy odour and whose
Key words 3-Mercapto-2-methylpropanol 7 Aroma 7 (S)(c)-enantiomer was an exotic fruity one [8].
Enantioselective multidimensional gas Enantioselective analysis makes it possible to deter-
chromatography mine the distribution of certain chiral molecules in var-
ious foods. Weber et al. [9] have shown that the (S)-
enantiomer of 3-methylthiohexanol, which has a fruity
Introduction odour, is 90% present in passion fruit.
In order to define the precise organoleptic role of 1
Recent studies on the characterization of wine aroma in red wines, it is essential to differentiate the two opti-
have revealed the presence of odorous volatile thiols cal isomers and their respective contribution to aroma.
whose odour thresholds are usually very low [1–5]. Enantioselective multidimensional gas chromatography
These reactive compounds are often present at trace with mass spectrometry detection (enantio-MDGC-
levels in wines, so it is difficult to detect them with the MS), using heptakis-(2,3-di-O-methyl-6-O-tert-butyldi-
usual techniques. Specific extraction methods have methylsilyl)-b-cyclodextrin (dimethyl-b-TBDMS) in SE
been developed in order to identify and quantify them 52 as the chiral stationary phase, is a valuable tool for
the enantioselective analysis of volatile compounds
from complex matrices.
P. Bouchilloux 7 P. Darriet (Y) 7 D. Dubourdieu
Faculté d’Œnologie de Bordeaux, 351 Cours de la Libération,
F-33405 Talence, France
Materials and methods
R. Henry
Institut National des Sciences Appliquées de Lyon,
20 Avenue Albert Einstein, F-69621 Villeurbanne, France Enantioselective synthesis of (R)- and (S)-1

S. Reichert 7 A. Mosandl The enantioselective synthesis of (R)- and (S)-1 was carried out
Institut für Lebensmittelchemie, Biozentrum Johann Wolfgang according to Bouchilloux et al. [5]. (R)- and (S)-stereoisomers of
Goethe-Universtät, Marie-Curie-Strasse 9, 1 were produced from (R)- and (S)-3-bromo-2-methylpropanol
D-60493 Frankfurt/Main, Germany (obtained from Aldrich) according to the reaction scheme in
350

Determination of odour thresholds of (R)- and (S)-1

The odour perception thresholds of (R)- and (S)-1 were based on


tasting with a directional test of five increasing concentrations of
each compound in distilled water (or in model solution). The de-
termination of the odour perception threshold corresponded to
the minimum concentration below which 50% of the 35 tasters
statistically failed to detect the difference from the control [10].

Analysis of (R)- and (S)-1 and extract of wine by gas


chromatography-mass spectrometry

Fig. 1 Synthesis of (R)-3-mercapto-2-methylpropanol (1) Gas chromatography-mass spectrometry (GC-MS) was perform-
ed using a Fisons GC 8000 gas chromatograph equipped with a
Fisons MD 800 Quadrupole-MS (electron ionization, 70 eV).
Mass range was set at 40–250 amu. A fused-silica column
(30 m!0.25 mm) coated with SE 52 (film thickness 0.5 mm) was
Fig. 1. During the first step, the substitution of the bromoalcohol used for analysis under the following conditions: column head
by thiourea leads to the formation of (R)-2-methylpropan-3-ol pressure 98 kPa; split injector, temperature 240 7C, split 30 ml/
isothiourea. The alkaline hydrolysis of thiouronium salt and then min; injection volume 1 ml; interface temperature 300 7C; source
consecutive acid treatment give mercaptoalcohol with the same temperature 240 7C; temperature program 40 7C/5 min/2.5 7C
substitution as that of the initial bromoalcohol. The specific rota- min –1/280 7C/20 min.
tions of the (R)- and (S)-1 enantiomers are [a]Dp–15.9 (cp2.73, Retention times: (R)-/(S)-1 19.25 min; wine extract 19.25 min.
THF) and [a]Dpc15.5 (cp2.80, THF) respectively. The MS data of the extract are in agreement with the refer-
ence compound used.

Preparation of wine sample extract


Acetylation of 1 and of extract
Vacuum distillation and thiol capture. A young Cabernet-Sauvig-
non wine (3.5 L; appellation Pauillac, 1997 vintage) was extracted A solution of 1 (20 ml) in dichloromethane was made alkaline
according to Bouchilloux et al. [5]. The total volume of wine was with 4-dimethylaminopyridine. Acetyl chloride (80 ml) was added
fractionated in five samples of 700 ml, each extracted as follows: slowly. After stirring for 3 h, extraction was performed with ether.
700 ml of wine was added to sodium chloride (100 g/l), warmed at The organic layer was washed with 1 N hydrochloric acid and
45 7C and submitted to distillation under vacuum (0.5–1.5 Pa) for aqueous sodium hydrogen carbonate, and dried over sodium
90 min. Simultaneously, the volatile thiols were extracted from sulfate.
wine and placed in a p-HMB solution (36 mg of p-HMB added to The extract was treated under analogous conditions to those
15 ml of 0.1 N sodium hydroxide, q.s. ultrapure water to 100 ml) described for 1.
at 0 7C where they react specifically with the organomercuric com-
pound. After distillation, the solution containing the thiols com-
bined with p-HMB was acidified with 1 N hydrochloric acid to Analysis of diacetylated samples by enantio-MDGC-MS
pH 2.5, and evaporated to dryness. The dry residue was diluted
with ultrapure water (100 ml) and evaporated to dryness, then di- The enantio-MDGC-MS system consisted of a Siemens SiChro-
luted again with ultrapure water (50 ml). mat 2 double oven gas chromatograph (with two independent
temperature controls) with an ITD 800, Finnigan MAT mass se-
Thiol release. The five solutions containing the p-HMB-thiol lective detector, connected to the main column by the ITD trans-
complex were mixed, then adjusted to pH 8 with 10 N sodium hy- fer line with an open split interface. The pre-column was equip-
droxide. Next, the thiols were liberated by addition of an excess ped with a flame ionization detector (FID). Pre- and main column
of L-cysteine (1.9 g L-cysteine-HCl in 50 ml of ultrapure water ad- were connected with a live T-switching device.
justed to pH 8 with 1 N sodium hydroxide). After 30 min of reac- Pre-column: fused-silica capillary (30 m!0.25 mm) coated
tion in the dark, the solution was extracted with dichloromethane with PS 268 (film thickness 0.38 mm); carrier gas hydrogen
(10, 5 and 5 ml successively). The organic phase obtained was 1.4 bar; injector temperature 260 7C, split 30 ml/min; detector
dried with anhydrous sodium sulfate and concentrated under ni- (FID) 280 7C; temperature program 65 7C/5 7C min –1/270 7C; injec-
trogen (100 ml/min) to 20 ml. tion volume 1 ml.

Fig. 2 Analysis of the wine


extract by gas chromatogra-
phy-mass spectrometry
351

Main column: fused-silica capillary (30 m!0.32 mm) coated of a Cabernet-Sauvignon wine were extracted. In order
with a 0.64 mm film of dimethyl-b-TBDMS dissolved in polysilox- to avoid thermodegradation of liable compounds by ex-
an SE 52; carrier gas hydrogen 0.7 bar; detector ITD 800; temper-
ature program 50 7C/1 7C min –1/210 7C. cessively high temperatures [4, 6], vacuum distillation
ITD 800 : transfer line 250 7C; open split interface 250 7C; he- was used. Extraction was coupled with simultaneous
lium sweeping flow 1 ml/min; ion trap manifold 230 7C; electron and reversible thiol trapping by selective reaction of the
ionization 70 eV. thiol group with sodium p-HMB. After liberation, a pu-
Retention times: (R)-enantiomer 70.25 min; (S)-enantiomer
70.59 min; wine extract 70.26 min.
rified extract of volatile thiols was obtained from
MS data of 1-diacetate: 190 (1), 130 (5), 115 (1), 97 (1), 88 wine.
(14), 87 (3), 60 (3), 55 (7), 54 (5), 43 (100). Using enantio-MDGC-MS with dimethyl-b-TBDMS
The reference and compound detected in wine have identical in SE 52 as the chiral stationary phase, it was possible
MS data. to separate the diacetylated enantiomers of 1 in a single
chromatographic run (Fig. 3A). The elution order of
Quantification of (R)-1 in wines the chiral compounds was established by using enantio-
pure standards of defined absolute configurations.
1 was quantified using methods developed for the quantification In the same way, the extract of a Cabernet-Sauvig-
of other volatile thiols in wines [6, 11]. A calibration curve was
obtained by adding five increasing concentrations of (R)-1 to a non wine was derivatized with acetyl chloride. As can
standard red wine containing low quantities of this compound. be seen from Fig. 3B, the enantioselective analysis of
the acetylated extract unequivocally shows that (R)-1 is
the only stereoisomer to be found in wine.
Results and discussion Although described as having the same broth and
sweat odour, (R)-1 has an odour perception threshold
Recent studies on the characterization of wine aroma in water which is 10-fold lower than that of (S)-1 (Ta-
have revealed the presence of a volatile thiol, 1, with a ble 1). Assays in several wines showed that this com-
broth and sweat odour. Because of its stereogenic cen- pound is abundant in young red wines at levels above
ter, two enantiomers, (R)- and (S)-1, are possible. its odour perception threshold (25 to 68 mg/l, Table 2).
In order to reveal the contribution of the aroma-ac- On the other hand, it seems that its level decreases dur-
tive compound 1 to the flavour of wine, it is necessary ing ageing to undetectable values. In Sauvignon blanc
to analyse the genuine enantiomeric ratio of 1 and the wines, (R)-1 is present only at low concentrations
concentrations. For this reason, the volatile substances (~2 mg/l).

Conclusion
Enantio-MDGC-MS was used to demonstrate the ex-
clusive presence of the (R)-enantiomer of 1 in wines.

Table 1 Odour thresholds of enantiopure (R)- and (S)-3-mercap-


to-2-methylpropanol ( 1) in distilled water and model solution

Samples Odour thresholds Odour thresholds


(mg/l of water) (mg/l of model solution a)

(R)-1 3–7 20–27


(S)-1 35–40 120–130
a
The model solution was prepared from an aqueous alcohol solu-
tion (12 v/v %) added to tartaric acid (5 g/l) and adjusted to
pH 3.5 by addition of a 10 N sodium hydroxide solution

Table 2 Quantification of 1 in wine

Samples Vintage Concentration


of (R)-1 (mg/l) a

Pomerol (red wine) 1997 38


Margaux (red wine) 1997 25
Graves Pessac-Léognan (red wine) 1997 68
Sauvignon blanc wine 1 1997 1.2
Sauvignon blanc wine 2 1997 1.7
Fig. 3 A Enantioselective multidimensional gas chromatography St. Julien a (red wine) 1989 2.9
with mass spectrometry detection (enantio-MDGC-MS) analysis St. Julien a (red wine) 1985 1.3
of diacetylated (R)- and (S)-1, including the chromatographic be- St. Julien b (red wine) 1985 4.0
haviour of the two enantiomers. B Enantio-MDGC-MS analysis
a
of diacetylated 1 from wine extract Analysis performed in 1998
352

Indeed, the (R)-enantiomer is much more odorant than 3. Tominaga T, Furrer A, Henry R, Dubourdieu D (1998) Fla-
the (S)-enantiomer. Its concentration in certain young vor Fragrance J 13 : 159–162
4. Bouchilloux P, Darriet P, Dubourdieu D (1998) Vitis
red wines attests its involvement in wine aroma. This 37 : 177–180
compound appears during alcoholic fermentation but 5. Bouchilloux P, Darriet P, Henry R, Lavigne-Cruège V, Du-
its origin is unknown. bourdieu D (1998) J Agric Food Chem 46 : 3095–3099
6. Bouchilloux P, Darriet P, Dubourdieu D (1996) J Int Sci
Acknowledgements We thank Monique Pons for her technical Vigne Vin 30 : 23–29
assistance and the CIVB (Conseil Interprofessionnel des Vins de 7. Tominaga T, Murat ML, Dubourdieu D (1998) J Agric Food
Bordeaux) and the Conseil Régional d’Aquitaine for their finan- Chem 46 : 1044–1048
cial help. 8. Mosandl A, Heusinger G (1984) In: Schreier P (ed) Analysis
of volatiles. De Gruyter, Berlin, pp 343–356
9. Weber B, Dietrich A, Maas B, Marx, A, Olk J, Mosandl A
(1994) Z Lebensm Unters Forsch 199 : 48–50
References 10. Boidron JN, Chatonnet P, Pons M (1988) Connaiss Vigne Vin
22 : 275–294
1. Darriet P, Tominaga T, Lavigne V, Boidron J-N, Dubourdieu 11. Blanchard L (1997) Diplôme d’Etudes Approfondies (Eno-
D (1995) Flavor Fragrance J 10 : 385–392 logie-Ampélologie, Université Bordeaux II, p 2–12
2. Guth H (1997) J Agric Food Chem 45 : 3022–3026

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