Odorous impact of volatile thiols on the aroma of young botrytized sweet wines: Identification and quantification of new sulfanyl alcohols

Article abstract of DOI:10.1021/jf062582v

Specific extraction of volatile thiols using sodium p- hydroxymercuribenzoate revealed the presence of three new sulfanylalcohols in wines made from Botrytis-infected grapes: 3-sulfanylpentan-1-ol (II), 3-sulfanylheptan-1-ol (III), and 2-methyl-3-sulfanylbutan-1-ol (IV). The first two have citrus aromas, whereas the third is reminiscent of raw onion. In addition, 2-methyl-3-sulfanylpentan-1-ol, which has a raw onion odor, was tentatively identified. Like 3-sulfanylhexan-1-ol (I), already reported in Sauternes wines, compounds II, III, and IV were absent from must. They were found in wine after alcoholic fermentation, and their concentrations were drastically higher when Botrytis cinerea had developed on the grapes. In the commercial botrytized wines analyzed, the mean levels of II, III, and IV were 209, 51, and 103 ng/L, respectively. Despite their low odor activity values, sensory tests showed additive effects among I, II, and III, thus confirming their olfactory impact on the overall aroma of botrytized wines.

Full text of DOI:10.1021/jf062582v

J. Agric. Food Chem. 2007, 55, 1437−1444 1437
Odorous Impact of Volatile Thiols on the Aroma of Young
Botrytized Sweet Wines: Identification and Quantification of
New Sulfanyl Alcohols
ELISE SARRAZIN,† SVITLANA SHINKARUK,‡,§ TAKATOSHI TOMINAGA,*,†
†
BERNARD BENNETAU,‡ ERIC FREÄ ROT,# AND DENIS DUBOURDIEU
Faculte´ d’Œnologie, Institut des Sciences de la Vigne et du Vin, UMR 1219, INRA, Universite´ Victor
Segalen Bordeaux 2, 351 cours de la Libe´ration, 33405 Talence Cedex, France; Laboratoire de Chimie
Organique et Organome´tallique, UMR 5802, CNRS, Universite´ Bordeaux 1, 351 cours de la
Libe´ration, 33405 Talence Cedex, France; and Ecole Nationale d’Inge´nieurs des Travaux Agricoles de
Bordeaux, 1 cours du Ge´ne´ral de Gaulle, CS 40201, 33175 Gradignan Cedex, France; and Corporate
R&D Division, Analysis and Perception, Firmenich SA, Route des Jeunes 1,
P.O. Box 239, CH-1211 Geneva 8, Switzerland
Specific extraction of volatile thiols using sodium p-hydroxymercuribenzoate revealed the presence
of three new sulfanylalcohols in wines made from Botrytis-infected grapes: 3-sulfanylpentan-1-ol
(II), 3-sulfanylheptan-1-ol (III), and 2-methyl-3-sulfanylbutan-1-ol (IV). The first two have citrus aromas,
whereas the third is reminiscent of raw onion. In addition, 2-methyl-3-sulfanylpentan-1-ol, which has
a raw onion odor, was tentatively identified. Like 3-sulfanylhexan-1-ol (I), already reported in Sauternes
wines, compounds II, III, and IV were absent from must. They were found in wine after alcoholic
fermentation, and their concentrations were drastically higher when Botrytis cinerea had developed
on the grapes. In the commercial botrytized wines analyzed, the mean levels of II, III, and IV were
209, 51, and 103 ng/L, respectively. Despite their low odor activity values, sensory tests showed
additive effects among I, II, and III, thus confirming their olfactory impact on the overall aroma of
botrytized wines.
KEYWORDS: Botrytized wines; 3-sulfanylpentan-1-ol; 3-sulfanylheptan-1-ol; 2-methyl-3-sulfanylbutan-
1-ol; 3-sulfanylhexan-1-ol; 2-methyl-3-sulfanylpentan-1-ol; additive effects
INTRODUCTION
ing to citrus nuances. 3-Sulfanylhexan-1-ol and 4-methyl-4-
sulfanylpentan-2-one belong to the chemical family of volatile
thiols, among the most powerful odorants of all. In botrytized
wines, Tominaga et al. evidenced surprisingly high concentra-
tions of 3-sulfanylhexan-1-ol (8, 9). The contribution of volatile
thiols in botrytized wine aroma was recently confirmed by Bailly
et al. (10), who reported on new polyfunctional thiols, such as
3-methyl-3-sulfanylbutanal. In addition, Sarrazin et al. (7)
reported the presence of other, not yet identified, volatile thiols
that may be involved in the citrus nuances of botrytized wines.
In this paper, we report the identification of three new volatile
thiols in wines made from Botrytis-infected grapes, as well as
their quantification in wines made from grapes with different
degrees of botrytization. The contribution of these new sulfa-
nylalcohols to the characteristic aroma of botrytized wines was
then examined by sensory analysis.
Sweet wines made from Botrytis-infected grapes are very
famous throughout the world. They are produced from overripe
grapes affected by the Botrytis cinerea fungus under specific
climatic conditions, alternating foggy mornings with sunny
afternoons (1). Due to the unusual composition of the grapes,
these specialty wines are characterized by an exceptional range
of aromas, evoking not only citrus but also crystallized and dried
fruits, as well as honey. Aroma research aimed at explaining
these typical nuances has focused mainly on heterocyclic
odoriferous compounds, such as sotolon [4,5-dimethyl-3-hy-
droxy-2(5H)-furanone] (2, 3), γ- and δ-lactones (4-6), and
3(2H)-furanones (7). However, these compounds cannot explain
the distinctive citrus nuances of young botrytized wines.
Among the volatile compounds found in botrytized wine, two
odoriferous molecules have already been identified as contribut-
MATERIALS AND METHODS
* Corresponding author (telephone +33-5 40 00 64 89; fax +33-5 40
00 64 68; e-mail takatoshi.tominaga@oenologie.u-bordeaux2.fr).
† Universite´ Victor Segalen Bordeaux 2.
Wines Analyzed. The botrytized wines were all made from Semillon
and Sauvignon blanc grapes and were from the following appellations
of the Bordeaux region: Sauternes (2001, 2002, and 2003), Loupiac
(2002 and 2003), and Barsac (2001).
‡ Universite´ Bordeaux 1.
§ Ecole Nationale d’Inge´nieurs en Travaux Agricoles de Bordeaux.
# Firmenich SA.
10.1021/jf062582v CCC: $37.00
© 2007 American Chemical Society
Published on Web 01/24/2007

1438 J. Agric. Food Chem., Vol. 55, No. 4, 2007
Sarrazin et al.
Figure 1. Structures of 3-sulfanylhexan-1-ol (I), 3-sulfanylpentan-1-ol (II), 3-sulfanylheptan-1-ol (III), and 2-methyl-3-sulfanylbutan-1-ol (IV).
Figure 2. Synthetic pathway from
R,â-unsaturated aldehydes to 3-sulfanyl-1-alcohols.
Standard Products. Sodium p-hydroxymercuribenzoate, 5,5′-dithio-
bis(2-nitrobenzoic acid), trans-2-pentenal (95%) trans-2-methyl-2-
butenal (98%), thioacetic acid (96%), and lithium aluminum hydride
were purchased from Sigma-Aldrich Chemicals (L’Isle d’Abeau,
France). 4-Methoxy-2-methylbutane-2-thiol was purchased from Oxford
Chemicals (Grasse, France). 3-Sulfanylhexan-1-ol (I) (>95%) was
obtained from Lancaster (Bischheim, France). 3-Sulfanylheptan-1-ol
(III) (>95%) was kindly donated by Firmenich SA (Gene`ve, Switzer-
land).
column-head pressure of 22 psi and a flow rate of 1 mL/min. Each
GC-O analysis was performed by three experienced judges.
Identification and Quantification by Gas Chromatography-
Mass Spectrometry (GC-MS). A 3 µL sample of each concentrated
extract was analyzed on a 6890N gas chromatograph (Agilent Tech-
nologies) under the conditions described above. The detector was a
mass spectrometer (MS 5973, Agilent Technologies), functioning in
EI mode (70 eV), and was connected to the GC with a transfer line
heated to 250 °C. Mass spectra were taken over the m/z 40-300 range.
Volatile thiols were identified on the basis of their linear retention
indexes and a comparison of MS fragmentation patterns obtained in
SCAN mode on two capillaries (BP20 and BPX5) with those of
reference compounds previously reported.
The three new volatile thiols (II-IV, Figure 1), as well as
3-sulfanylhexan-1-ol (I), were quantified in SIM mode, selecting the
following ions: m/z 134, 116, and 100 for I, m/z 120, 102, and 86 for
II and IV, and m/z 148, 114, and 96 for III. They were quantified
using m/z 134 for I, m/z 120 for II and IV, and m/z 148 for III. The
internal standard, 4-methoxy-2-methylbutane-2-thiol, was detected with
the m/z 134 and 100 ions. Calibration curves were determined using a
dry white wine supplemented with dilute alcohol solutions containing
I, II, III, and IV, at final concentrations ranging from 0 to 9000 ng/L
for I, from 0 to 400 ng/L for II, from 0 to 200 ng/L for III, and from
0 to 200 ng/L for IV. The concentrations of volatile thiol standards
were previously determined according to Ellman’s method (12) using
5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). For each concentration, the
volatile thiols were extracted from the wine according to the process
described above. Repeatability of the measuring system was assessed
over a series of five extractions. The quantification limit was calculated
as the minimum concentration that generated a peak signal 10 times
higher than the signal from background noise.
Purification of Volatile Thiols. The volatile thiols were specifically
extracted by reversible combination of the thiols with sodium p-
hydroxymercuribenzoate (p-HMB) as described by Tominaga et al. (11).
A 500 mL sample of wine, containing 2.5 nmol of 4-methoxy-2-
methylbutane-2-thiol as internal standard, was extracted with two
successive additions of 100 mL of dichloromethane in a 1 L flask with
magnetic stirring for 10 min each time. The combined organic phases
were centrifuged at 4000g for 15 min to break the emulsion and
separated in a separating funnel. The organic phase obtained was then
extracted with two successive additions of 20 mL of p-HMB solution
[1 mM in Trizma base [2-amino-2-(hydroxymethyl)-1,3-propanediol]
at 0.2 M] for 10 min each time. The two aqueous phases were combined
and then loaded on a strongly basic anion-exchange column (1.5 × 3
cm) (Dowex 1-1 × 2-100). The column was then washed with 50 mL
of sodium acetate buffer (0.1 M, pH 7). The volatile thiols were released
from the thiol-p-HMB complex fixed on the column by percolating
with 60 mL of cysteine solution (10 g/L) adjusted to pH 7 with NaOH
(10 M). The eluate containing the volatile thiols was collected in a
100 mL flask, and 0.5 mL of ethyl acetate was added. The eluate was
extracted twice with dichloromethane (4 and 3 mL, respectively) for
10 min each time, under magnetic stirring. The two organic phases
were combined, dried over anhydrous sodium sulfate, and then
concentrated under nitrogen flow in a 10 mL graduated tube to
approximately 200 µL. The concentrate was then transferred to a 1
mL vial and concentrated to 25 µL.
Gas Chromatography Coupled Simultaneously with Olfactom-
etry (GC-O) and Flame Photometric Detection (GC-FPD). Olfactory
analyses were carried out using a Hewlett-Packard 5890 gas chromato-
graph (Agilent Technologies, Palo Alto, CA) equipped with a flame
photometric detector (from Agilent Technologies) and a sniffing-port
(ODO-1 from SGE, Ringbow, Australia). Three microliters of each
concentrated extract was injected by a splitless injector (230 °C; purge
time ) 1 min, purge flow ) 50 mL/min) at oven temperature (45 °C)
onto a type BP20 capillary column [SGE, 50 m, 0.22 mm internal
diameter (i.d.), 0.25 µm film thickness] or a type BPX5 fused silica
capillary column (SGE, 50 m, 0.25 mm i.d., 1.0 µm film thickness).
For all analyses, the temperature program was as follows: 45 °C for
10 min, raised at 3 °C/min to 230 °C (BP20 column) and 250 °C (BPX5
column), followed by a 20 min isotherm. The flame photometric
detector was held at 230 °C. The carrier gas was hydrogen with a
Synthesis. 3-Sulfanylpentan-1-ol (II) and 2-methyl-3-sulfanylbutan-
1-ol (IV) were synthesized in two steps by the Michael-type addition
of thioacetic acid to corresponding R,â-unsaturated aldehydes, followed
by reducing the carbonyl group to alcohol (Figure 2). Our approach
was similar to combinatorial synthesis previously described by Ver-
meulen et al. (13).
1
Experimental Equipment. H and 13C NMR spectra were recorded
with a Bruker AC-300 FT (1H, 300 MHz; 13C, 75 MHz), using TMS
as an internal standard. Chemical shifts (δ) and coupling constants (J)
are expressed in parts per million and hertz, respectively. IR spectra
were recorded with a Perkin-Elmer Paragon 1000 FT-IR spectropho-
tometer. Thin-layer chromatography (TLC) was performed on SDS TLC
plates: thickness ) 0.25 mm, particle size ) 15 µm, pore size ) 60
Å. Merck silica gel 60 (70-230 mesh and 0.063-0.200 mm) was used
for flash chromatography. Spots were revealed with UV as well as with
KMnO4 (0.05% in water). Diethyl ether was dried by refluxing a
solution containing sodium wires and benzophenone under nitrogen
and distilled immediately before use. The thioacetic acid was distilled

New Volatile Thiols in Young Botrytized Wines
J. Agric. Food Chem., Vol. 55, No. 4, 2007 1439
immediately before use. All moisture-sensitive reactions were carried
out in an argon atmosphere in oven- or flame-dried glassware.
3-Sulfanylpentan-1-ol. Thioacetic Acid S-(1-Ethyl-3-oxopropyl)
Ester (1). trans-2-Pentenal (3.35 g, 3.9 mL, 40 mmol) was added
dropwise to ice-cooled freshly distilled thioacetic acid (4.27 g, 4 mL,
56 mmol) under an argon atmosphere. The ice bath was then removed
and the reaction mixture stirred at room temperature for 16 h. Thioacetic
acid excess was removed under reduced pressure. The crude product
was purified by silica gel chromatography (petroleum ether/diethyl ether
70:30, Rf ) 0.44), resulting in a colorless oil (4.9 g, 77%).
COCH3), 2.60 [m, 1H, -CH(CH3)-CH(CH3)-S-], 3.96 [qd, J ) 7.53
Hz, J ) 4.89 Hz, 1H, -CH(CH3)-CH(CH3)-S-], 9.59 (s, 1H, CHO);
13C NMR (CDCl3) δ 10.4 [-CH(CH3)-CH(CH3)-S-], 17.8 [-CH-
(CH3)-CH(CH3)-S-], 30.6 (COCH3), 38.6 [-CH(CH3)-CH(CH3)-
S-], 50.9 [-CH(CH3)-CH(CH3)-S-], 195.0 (CH3CO), 202.3 (-CHO).
(l,u)-2-Methyl-3-sulfanylbutan-1-ol (IV). The purified compound
(l,u)-2 enriched in like enantiomers (l:u ) 2:1) (1.2 g, 7.5 mmol) was
treated with lithium aluminum hydride (0.57 g, 15 mmol) under the
same conditions as described for II. The silica gel chromatography
(petroleum ether/diethyl ether 70:30) of the crude product gave the
title compound as a colorless oil (0.76 g, 85%) with purity >95% (GC-
MS and 1H NMR). The two diastereoisomers [like (RR+SS) and unlike
(SR+RS)] cannot be separated by standard silica gel chromatography,
TLC (petroleum ether/diethyl ether 80:20), Rf ) 0.28; MS (EI, 70
eV), m/z (%) 160 (M+, 4), 132 (2), 117 (35), 100 (9), 89 (13), 85 (10),
1
75 (9), 56 (15), 55 (19), 43 (100); H NMR (CDCl3) δ 0.93 (dd, J )
1
but do have different H and 13C NMR spectra. The chemical shift
7.9 Hz, J ) 6.8 Hz, 3H, -CH3), 1.62 (m, 1H, 4-Ha, -CH-CH2-
CH3), 1.69 (m, 1H, 4-Hb, CH-CH2-CH3), 2.28 (s, 3H, COCH3), 2.69
(m, 2H, -CH-CH2-CHO), 3.84 (tt, J2-3 ) J3-4 ) 6.8 Hz, 1H, -CH2-
CH-CH2-), 9.64 (dd, 1H, J ) 3.0 Hz, J ) 1.9 Hz, CHO); 13C NMR
(CDCl3) δ 11.3 (-CH3), 27.5 (-CH-CH2-CH3), 30.6 (COCH3), 39.9
(-CH2-CH-CH2-), 48.3 (-CH-CH2-CHO), 195.1 (CH3CO),
200.0 (-CHO); IR, ν 952, 1114, 1354, 1425, 1690, 1724, 2922, 2966
assignment as well as the l:u ratio of diastereoisomer pairs was
determined by NMR (1H, 13C, confirmed by 2D NMR analysis) and
compared with literature data (14). The l:u ratio was the same as in
the starting material, 2:1.
TLC (petroleum ether/diethyl ether 60/40), Rf ) 0.40; MS (EI, 70
eV), m/z (%) 120 (M+, 34), 102 (6), 89 (22), 86 (100), 71 (76), 69
(39), 61 (76), 60 (81), 55 (55), 45 (46); IR, 1036, 1379, 1449, 2559,
cm-1
.
2876, 2926, 2964, 3358 cm-1
.
3-Sulfanylpentan-1-ol (II). The purified compound 1 (1.83 g, 11.4
mmol) in 10 mL of dry diethyl ether was slowly added to an ice-cold
suspension of lithium aluminum hydride (0.87 g, 22.8 mmol) in 30
mL of dry diethyl ether under an argon atmosphere. The reaction
mixture was allowed to warm to room temperature for 3 h and then
cooled again (ice bath). The suspension was carefully treated with cold
saturated aqueous ammonium chloride solution (100 mL), and the pH
was adjusted to pH 2-3 with 1 M hydrochloric acid. The organic phase
was separated, and the aqueous layer was extracted with diethyl ether
(2 × 50 mL). The combined organic phases were washed with saturated
sodium chloride solution (2 × 50 mL), dried over magnesium sulfate,
filtered, and concentrated under reduced pressure to give the title
compound as a colorless oil (1.15 g, 90%). The purity of the compound
obtained, determined by GC-MS and 1H NMR, was >95%. This
compound was thus used without further purification.
1
(l)-2-Methyl-3-sulfanylbutan-1-ol. H NMR (CDCl3) δ 0.86 [d, J
) 6.8 Hz, 3H, -CH(CH3)-CH(CH3)SH], 1.25 (d, J ) 7.9 Hz, 1H,
SH), 1.32 [d, J ) 7.2 Hz, 3H, -CH(CH3)-CH(CH3)SH], 1.80 [m,
1H, -CH(CH3)-CH(CH3)SH], 2.63 (br s, OH), 3.23 [apparent quintet
d, JCH-CH3 ) 7.9 Hz, JCH-SH ) 7.2 Hz, JCH-CH ) 3.8 Hz, 1H, -CH-
2
3
(CH3)-CH(CH3)SH], 3.48 (dd, JCHH ) 10.6 Hz, JCH2-CH ) 6.0 Hz,
2
3
1H, 1Ha, -CH2OH), 3.57 (dd, JCHH ) 10.6 Hz, JCH2-CH ) 7.9 Hz,
1H, 1Hb, -CH2OH); 13C NMR (CDCl3) δ 10.9 [-CH(CH3)-CH(CH3)-
SH], 23.6 [-CH(CH3)-CH(CH3)SH], 36.9 [-CH(CH3)-CH(CH3)-
SH], 41.6 [-CH(CH3)-CH(CH3)SH], 65.8 (-CH2OH).
1
(u)-2-Methyl-3-sulfanylbutan-1-ol. H NMR (CDCl3) δ 0.96 [d, J
) 7.1 Hz, 3H, -CH(CH3)-CH(CH3)SH], 1.32 [d, J ) 7.1 Hz, 3H,
-CH(CH3)-CH(CH3)SH], 1.49 (d, J ) 7.1 Hz, 1H, SH), 1.75 [m,
1H, -CH(CH3)-CH(CH3)SH], 2.63 (br s, OH), 3.10 [apparent
sextuplets, JCH-CH3 ≈ JCH-SH ≈ JCH-CH ≈ 7.1 Hz, 1H, -CH(CH3)-
CH(CH3)SH], 3.58 (d, J ) 10.5 Hz, 2H, -CH2OH); 13C NMR (CDCl3)
δ 13.5 [-CH(CH3)-CH(CH3)SH], 21.7 [-CH(CH3)-CH(CH3)SH],
37.3 [-CH(CH3)-CH(CH3)SH], 43.1 [-CH(CH3)-CH(CH3)SH], 65.6
(-CH2OH).
Microvinification. Must Preparation. Eight kilograms of Semillon
and Sauvignon blanc grapes were picked in the same vineyard (Chaˆteau
d’Yquem, Sauternes, France, 2005) at four stages in B. cinerea
development: healthy (grapes not infected by B. cinerea), pourri plein
(grapes entirely botrytized but not desiccated, picked 2 weeks after
healthy grapes), pourri roˆti (grapes botrytized and desiccated, picked
2 weeks after full-rotten grapes), and late pourri roˆti (shriveled grapes
left for a further 10 days before picking). Grapes were crushed in a
pneumatic press under a CO2 atmosphere and left to settle with 50
mg/L SO2 for 24 h at 12 °C. The mean grape volume was determined
by measuring the must volume from 1000 grapes. Sugar concentrations
varied from 217 to 400 g/L, depending on the stage of B. cinerea
development. The assimilable nitrogen content was estimated by using
the So¨rensen method (15) and corrected to 190 mg/L in all must samples
by adding Thiazote (Laffort Œnologie, Bordeaux, France) before
alcoholic fermentation (16).
Fermentation. Must was inoculated with Saccharomyces cereVisiae
(strain Zymaflore ST-Laffort Œnologie, Bordeaux, France) precultured
for 24 h (200 mg/L) (17) and fermented in 750 mL sterile bottles (650
mL must per bottle). Yeast strain establishment was assessed by
comparing the initial industrial yeast karyotype with the biomass, using
pulsed field electrophoresis (18). Fermentation took place in a tem-
perature-controlled environment at 23 °C and was monitored by CO2
release (16, 17). Every experiment was carried out in triplicate. When
the required alcoholic concentration, that is, 13% vol, was reached,
fermentation was stopped by adding sulfur dioxide solution (300 mg/
L).
TLC (petroleum ether/diethyl ether 60:40), Rf ) 0.35; MS (EI, 70
eV), m/z (%) 120 (M+, 34), 102 (6), 86 (100), 75 (25), 73 (47), 69
(84), 61 (53), 57 (96), 47 (24), 45 (25); 1H NMR (CDCl3) δ 0.97 (t, J
) 7.3 Hz, 3H, -CH3), 1.36 (d, J ) 7.5 Hz, 1H, SH), 1.50 (m, 1H,
4-Ha, CH-CH2-CH3), 1.65 (m, 1H, 4-Hb, -CH-CH2-CH3), 1.70
(m, 1H, 2-Ha, -CH-CH2-), 1.90 (m, 1H, 2-Hb, -CH-CH2-CH2-
OH), 2.83 (m, 1H, -CH2-CH-CH2-), 3.02 (br s, 1H, OH), 3.74 (m,
2H, -CH2OH); 13C NMR (CDCl3) δ 11.4 (-CH3), 32.1 (-CH-CH2-
CH3), 39.5 (-CH2-CH-CH2-), 40.9 (-CH-CH2-CH2OH), 60.3
(-CH2OH); IR, ν 1053, 1461, 2590, 3340 cm-1
.
2-Methyl-3-sulfanylbutan-1-ol. (l, u)-Thioacetic Acid S-(1,2-Di-
methyl-3-oxopropyl) Ester) (2). trans-2-Methyl-2-butenal (3.35 g, 3.9
mL, 40 mmol) was treated with thioacetic acid under the conditions
described for thioacetic acid S-(1-ethyl-3-oxopropyl) ester to produce
a colorless oil (4.3 g, 67%). The two diastereoisomers [like (RR+SS)
and unlike (SR+RS)] cannot be separated by standard silica gel
chromatography, but have different 1H and 13C NMR spectra. Chemical
shift assignment and the ratio of l:u diastereoisomer pairs was
determined by NMR (1H, 13C, confirmed by 2D NMR analysis) and
compared with literature data (14). The l:u ratio was 2:1.
TLC (petroleum ether/diethyl ether 70:30), Rf ) 0.51; MS (EI, 70
eV), m/z (%) 160 (M+, 1), 132 (8), 117 (46), 100 (9), 89 (29), 82 (10),
77 (9), 56 (44), 55 (27), 43 (100); IR, ν 955, 1114, 1354, 1380, 1452,
1691, 1727, 2920, 2972 cm-1
.
1
(l)-Thioacetic Acid S-(1,2-Dimethyl-3-oxopropyl) Ester. H NMR
(CDCl3) δ 1.11 [d, J ) 7.17 Hz, 3H, -CH(CH3)-CH(CH3)-S-], 1.34
[d, J ) 7.53 Hz, 3H, -CH(CH3)-CH(CH3)-S-], 2.29 (s, 3H,
COCH3), 2.60 [m, 1H, -CH(CH3)-CH(CH3)-S-], 4.00 [qd, J ) 7.53
Hz, J ) 4.89 Hz, 1H, -CH(CH3)-CH(CH3)-S-], 9.60 (s, 1H, CHO);
13C NMR (CDCl3) δ 10.4 [-CH(CH3)-CH(CH3)-S-], 19.2 [-CH-
(CH3)-CH(CH3)-S-], 30.7 (COCH3), 39.2 [-CH(CH3)-CH(CH3)-
S-], 51.0 [-CH(CH3)-CH(CH3)-S-], 194.7 (CH3CO), 202.6 (-CHO).
1
(u)-Thioacetic Acid S-(1,2-Dimethyl-3-oxopropyl) Ester. H NMR
Sensory Tests. Determining Odor Thresholds. Perception thresholds
of synthesized II and III were assessed by directional triangular tests
of five increasing concentrations in ultrapure water (Milli-Q, Millipore,
(CDCl3) δ 1.12 [d, J ) 7.17 Hz, 3H, -CH(CH3)-CH(CH3)-S-], 1.25
[d, J ) 7.53 Hz, 3H, -CH(CH3)-CH(CH3)-S-], 2.30 (s, 3H,

1440 J. Agric. Food Chem., Vol. 55, No. 4, 2007
Sarrazin et al.
Table 1. Odorous Zones (OZ) Corresponding to Volatile Thiols Detected in Specific Extracts of Botrytized Wines
a LRI, linear retention index calculated on both BP20 and BPX5 capillarie. b Coincidence of LRI and odors on two capillary columns (BP20 and BPX5). c Mass spectrum
in agreement with spectra of synthesized substances on two columns (BP20 and BPX5). d The two pairs of diastereoisomers (like and unlike) have the same LRI.
Bedford, MA) or in model dilute alcohol solution (5 g/L tartaric acid,
12%v/v ethanol, pH 3.5). The solutions were presented in glasses
corresponding to AFNOR (Association Franc¸aise des Normes) stan-
dards. The odor perception threshold was the minimum concentration
below which at least 50% of 45 tasters statistically failed to distinguish
the sample from the control (19).
4-methyl-4-sulfanylpentan-2-ol (23). OZ 4 and 5, reminiscent
of cooked leeks, correspond to 3-sulfanylbutan-1-ol (24) and
3-methyl-3-sulfanylbutan-1-ol (23), respectively. OZ 6, smelling
of broth, corresponds to 2-methyl-3-sulfanylpropan-1-ol (25).
Finally, OZ 10, with a grapefruit aroma, corresponds to
3-sulfanylhexan-1-ol (I) (23). However, under our conditions
we did not detect the two sulfanylaldehydes (3-methyl-3-
sulfanylbutanal and 3-sulfanylheptanal) recently reported by
Bailly et al. (10).
Study of AdditiVe Effects among Volatile Thiols. To assess additive
effects among the volatile thiols identified, triangular tests were carried
out in model solution, as follows. A control solution containing 5000
ng/L of I was compared with three test solutions containing 5000 ng/L
of I, together with 200 ng/L of II (test A) or 50 ng/L of III (test B),
or both at the same time (test C). The solutions were presented in glasses
corresponding to AFNOR standards and coded with three-digit numbers
(20). The panel consisted of 49 tasters, and the significance of the results
was determined according to the binomial law.
Among the four unknown odoriferous zones (OZ 7, 8, 9, and
11), two were reminiscent of citrus zest and grapefruit (OZ 8
and 11), whereas the other two had onion and sweat odors (OZ
5 and 7). GC-MS on two capillary columns with different
polarities was used to obtain three mass spectra corresponding
to OZ 7, 8, and 11. The three fragmentation patterns strongly
indicated the presence of a thiol and a hydroxyl group, due to
the loss of m/z 34 and m/z 18, respectively (Figure 3). Therefore,
the volatile compounds associated with OZ 7, 8, and 11 were
assumed to be sulfanyl alcohols. Moreover, the compound
corresponding to OZ 7 was characterized by a loss of the m/z
15 ion, due to a methyl substitution. On the basis of their mass
spectra and linear retention indices compared with those of
synthetic substances (13, 26), these three compounds were
identified as 2-methyl-3-sulfanylbutan-1-ol (OZ 7, IV), 3-sul-
fanylpentan-1-ol (OZ 8, II), and 3-sulfanylheptan-1-ol (OZ 11,
III). Like 3-sulfanylhexan-1-ol (I), 4-methyl-4-sulfanylpentan-
2-one, and 4-methyl-4-sulfanylpentan-2-ol, these three new
volatile thiols are strong odorants, with the O and S functions
in the 1,3-position. To our knowledge, none of these three
RESULTS AND DISCUSSION
Identification of Three New 3-Sulfanylalcohols in Botry-
tized Wines. Volatile thiols were extracted selectively from a
commercial botrytized wine, using p-HMB. The extract was
analyzed using GC-O, GC-FPD, and GC-MS. As shown in
Table 1, 11 odoriferous zones (OZ), corresponding to sulfur-
containing compounds, were detected. Seven of them (OZ 1,
2, 3, 4, 5, 6, and 10) had already been identified in wines,
including five (OZ 1, 2, 3, 5, and 10) previously reported in
botrytized wines (7-10). OZ 1, smelling of cooked meat,
corresponds to 2-methyl-3-furanthiol (21). OZ 2, with a strong
box-tree odor, corresponds to 4-methyl-4-sulfanylpentan-2-one
(22). OZ 3, reminiscent of citrus and grapefruit, corresponds to

New Volatile Thiols in Young Botrytized Wines
J. Agric. Food Chem., Vol. 55, No. 4, 2007 1441
Figure 3. Mass spectra of 3-sulfanyl-2-methylbutan-1-ol (OZ 4, IV) (a), 3-sulfanylpentan-1-ol (OZ 5, II) (b), and 3-sulfanylheptan-1-ol (c) (OZ 8, III)
(isolated from wine).
volatile thiols had ever been described in wine before. Com-
pounds II and IV were previously reported as odorous com-
pounds in human axillary sweat (27, 28), whereas III was
recently identified in Ruta chalepensis (29). In addition, a
position isomer of IV, 3-methyl-2-sulfanylbutan-1-ol, was
recently reported in lager beer aroma (30).
Concerning the last odorous zone, reminiscent of raw onion
and sweat (OZ 9), no signal (peak) or spectrum was obtained
by GC-FPD or GC-MS. Nevertheless, retention indices on two
capillary columns and olfactory descriptors correlated well with
those of 2-methyl-3-sulfanylpentan-1-ol (13), already identified
in raw onion (31). A comparison of retention indices and sensory
descriptors of this odorous zone with those of synthesized
2-methyl-3-sulfanylpentan-1-ol confirmed the presence of this
volatile thiol.
According to our previous olfactometric study (7), using GC-
AEDA with three experienced judges, II, III, and IV were
among the most potent odorants in young botrytized wines.
Therefore, they were thought to contribute to botrytized wine
aroma and had to be examined. Compound I, already known
to make a significant contribution to botrytized wine aroma (7-
10), was also studied.

1442 J. Agric. Food Chem., Vol. 55, No. 4, 2007
Table 2. Calibration Line Parameters
Sarrazin et al.
showed that compounds II, III, and IV were absent before
alcoholic fermentation, like I (data not shown). Therefore, this
demonstrated that these three new volatile thiols were formed
during fermentation. Previous studies showed that I, present in
a cysteinylated conjugate precursor form in must, was released
by yeast during alcoholic fermentation (33). As compounds II,
III, and IV have chemical structures similar to that of I, they
are likely to be formed by the same pathway. Furthermore, II
and IV have been recently shown to be released through the
cleavage of cysteine precursors by a C-S lyase produced by
axillary bacteria (27, 34, 35). However, Schneider et al. (36)
described an alternative pathway to explain the genesis of
3-sulfanylhexan-1-ol and 4-methyl-4-sulfanylpentan-2-one in
wine, starting from the corresponding conjugated carbonyl
compounds. Further research is now needed to understand the
origin of these new volatile compounds in wines made from
Botrytis-infected grapes. Moreover, the role of the B. cinerea
metabolism in their formation requires further study.
repeat-
ability
quantifi-
cation
limit
linear
range
(ng/L)
validation
range
(ng/L)
(n
)
5)
calibration
line
compd
(%)
R 2
(ng/L)
I
II
III
IV
3
6
6
6
y
y
y
y
)
)
)
)
68.737x
113.73x
145.69x
203.88x
0.9939
0.9996
0.9941
0.9982
0−9000
0−400
0−200
0−200
3.1
2.8
3.7
4.8
3.1−
2.8−
3.7−
4.8−
9000
400
200
200
Standard Curves, Sensitivity, and Repeatability. Table 2
shows the calibration lines, R2, repeatability, and quantification
limits obtained for each of the four compounds. The recovery
rate for the volatile thiols was calculated according to the method
described by Tominaga et al. (32) and was >70%, irrespective
of the quantity added.
Impact of B. cinerea Development on Grapes on Concen-
trations of I-IV in Wine. B. cinerea development on grapes
is well-known to deeply modify grape composition and, thus,
the volatile compounds present in the corresponding wines.
Semillon and Sauvignon blanc grapes were picked on the same
plots at different stages in botrytization (healthy, pourri plein,
pourri roˆti, and late pourri roˆti). They were crushed and
microfermented. Specific extraction of volatile thiols was carried
out on must and wine from each grape sample to assay the three
new compounds (II-IV), as well as 3-sulfanylhexan-1-ol (I).
As shown in Table 3, wines made from healthy grapes contained
only trace levels of II, III, and IV and a few hundred nanograms
per liter of I. On the contrary, the three new volatile thiols were
found in every wine made from both varieties of rotten grapes,
whereas concentrations of I were considerably higher. Further-
more, the levels of the four volatile thiols increased as B. cinerea
developed on the grapes. Considering the thiol to grape volume
ratio between the healthy and pourri plein stages, the increase
in the four volatile thiols could not be explained simply by the
decrease in grape volume. Then, between the pourri plein and
the pourri roˆti stages, grapes were severely desiccated and
decreased significantly in volume. The increase in volatile thiols
in the wine was, therefore, mainly explained by desiccation.
Finally, due to rainy weather between the pourri roˆti and the
late pourri roˆti stages, the grapes absorbed water and increased
slightly in volume. This led to a dilution of the grape content
that partially masked the increase in volatile thiol levels.
These results demonstrated the predominant role of the B.
cinerea metabolism on the sulfanyl alcohol contents in wine.
Specific extraction of volatile thiols from botrytized must
Sensory Impact of Compounds II and III on Botrytized
Wine Aroma. The olfactory impact of these new sulfanyl
alcohols on the overall aroma of botrytized wines was examined
by determining their odor thresholds, in water and model dilute
alcohol solution, and their mean levels in commercial wines.
Table 4 lists olfactory descriptions of these three new com-
pounds and their perception thresholds. The aroma of compound
II is somewhat reminiscent of grapefruit, but its perception
threshold is much higher than that of compound I (620 ng/L in
water, 950 ng/L in model dilute alcohol solution). Compound
III has a citrus odor and a relatively low odor threshold (10
ng/L in water, 35 ng/L in model dilute alcohol solution). No
sensory tests were possible for compound IV. Due to the
presence of two asymmetric centers, significant olfactory
differences have already been described between its like and
unlike pairs: the odor perception threshold of the like pair is
100-fold higher than that of the unlike pair (14). The same
drastic sensory differences were found for 3-mercapto-2-
methylpentan-1-ol (37), illustrating the predominant role of
chirality in the olfactory perception of molecules with two
asymmetric centers. As no information concerning the config-
uration of IV in botrytized wines was obtained under our GC
conditions, no sensory tests could be performed.
In a second stage, seven commercial botrytized wines
(vintages from 2001-2003) from the Bordeaux region were
analyzed to assay the mean levels of these new sulfanyl alcohols.
As shown in Table 5, the three new volatile thiols were
consistently found in botrytized wines, and the mean levels
obtained for II, III, and IV were 209, 51, and 103 ng/L,
Table 3. Quantitative Assays (Nanograms per Liter) of the Three New Volatile Thiols (II−IV) and 3-Sulfanylhexan-1-ol (I), at Different Stages in
Botrytization [Comparison with Decrease in Mean Grape Volume (Milliliters per Grape)]
mean
grape
volumea
variation of
mean grape
volume (%)
variety
Botrytis stage
I
II
tre
III
tr
IV
tr
Semillon
healthyb
pourri plein
pourri roˆti
0.85
0.68
0.37
0.38
100
80
44
195c 58d
±
2326
3678
6334
±
±
±
419
1765
1267
93
124
291
±
±
±
14
54
128
34
50
±
±
±
5
26
13
67
118
134
±
±
±
9
61
55
late pourri roˆti
45
118
Sauvignon
healthy
0.78
0.52
0.21
0.29
100
67
27
161
3003
9648
9319
±
±
±
±
27
tr
141
348
375
tr
95
263
258
tr
50
185
185
pourri plein
pourri roˆti
late pourri roˆti
300
1544
2050
±
±
±
8
42
71
±
±
±
39
92
44
±
±
±
5
20
18
37
a Based on the volume obtained from crushing 1000 grapes (mL/grape). b healthy, grapes not infected by B. cinerea; pourri plein, grapes entirely botrytized but not
desiccated, picked 2 weeks after healthy grapes; pourri roˆti, grapes botrytized and desiccated, picked 2 weeks after pourri plein grapes; late pourri roˆti, shriveled grapes
left a further 10 days before picking. c Mean value (n
) )
3). d Standard deviation s (n 3). e Traces.

New Volatile Thiols in Young Botrytized Wines
J. Agric. Food Chem., Vol. 55, No. 4, 2007 1443
masked by that of I. On the contrary, there was a significant
difference with a risk of 0.1% between the control solution and
the solution containing both II and III (test C, 29/49). According
to Guadagni et al. (39), as I, II, and III belong to the same
class of compounds and have the same odors, these results
demonstrated additive effects. This kind of molecular interaction
among volatile thiols has also recently been described between
3-methyl-3-sulfanylbutanal and 2-methylfuran-3-thiol, giving a
strong bacon-petroleum odor (10).
Therefore, this demonstrated that compounds II and III had
a considerable impact on the overall aroma in the presence of
other volatile thiols, such as I, although they had low individual
OAVs. As previously described (39-40), the aromatic profile
of complex matrices, such as wine, cannot solely be explained
by the odorants with the highest OAVs. Due to molecular
interactions, volatile compounds with lower OAVs may also
contribute to the overall aroma through additive effects.
Although these compounds have odors different from that of
wine, they may play a predominant role. Further work is now
required, particularly on compound IV, to improve our under-
standing of the character and complexity of botrytized wine
aromas.
Table 4. Olfactory Descriptions and Perception Thresholds of the New
Volatile Thiols (I−IV) Identified in Botrytized Wines (Nanograms per
Liter) in Water and Model Solution (5 g/L Tartaric Acid, 12% v/v
Ethanol, pH 3.5)
olfactory perception threshold
ref compd
olfactory description
water
model solution
I
II
III
IV
grapefruit
citrus, sulfur
grapefruit
17a
620
10
60a
950
35
raw onion, sweat
ndb
nd
a Tominaga et al. (23). b Not determined.
Table 5. Quantitative Assays (Nanograms per Liter) and Odor Activity
Values of Some Volatile Thiols (I IV) in Young Botrytized Wines from
Various Appellations of the Bordeaux Region
−
appellation vintage
I
II
III
IV
149
87
120
83
Doisy Dae¨ne Sauternes
Tour Blanche Sauternes
Cantegrille Barsac
Doisy Dae¨ne Sauternes
Dauphine´ Loupiac
Rondillon
Doisy Dae¨ne Sauternes
Dauphine´ Loupiac
Rondillon
2001
2001
2001
2002
2002
7033
299
217
223
203
235
63
59
44
48
72
5606
5034
4765
4749
105
ABBREVIATIONS USED
2003
2003
5386
2450
199
91
44
26
96
84
I, 3-sulfanylhexan-1-ol; II, 3-sulfanylpentan-1-ol; III, 3-sul-
fanylheptan-1-ol; IV, 2-methyl-3-sulfanylbutan-1-ol; GC-O, gas
chromatography-olfactometry; GC-FPD, gas chromatography-
flame photometry detection; GC-MS, gas chromatography-
mass spectrometry; OAV, odor activity value; OZ, odorous
zone; p-HMB, sodium p-hydroxymercuribenzoate.
mean level
5003 (83)a 209 (0.2) 51 (1.5) 103 (ndb)
a (number): odor activity value. b Not determined.
Table 6. Triangular Tests Involving 49 Tasters
ACKNOWLEDGMENT
control
solution
correct answers/
total answers
We thank Dr. Philippe Darriet in the Faculte´ d’Oenologie
(Talence, France) and Dr. Sandrine Garbaye in the Chaˆteau
d’Yquem (Sauternes, France) for their help.
test
test solution
p
A
B
C
5000 ng/L I
5000 ng/L I
5000 ng/L I
+
+
+
200 ng/L II
50 ng/L III
200 ng/L II
5000 ng/L I
5000 ng/L I
5000 ng/L I
17/49
13/49
29/49
NSa
NS
0.001
+
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50 ng/L III
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JF062582V