Quantitative determination of sulfur containing wine odorants at sub-ppb levels. 1. Synthesis of the deuterated analogues

Article abstract of DOI:10.1021/jf0004715

[²H₁₀]-4-Sulfanyl-4-methylpentan-2-one (d₁₀-SMP), [²H₂]-3-sulfanylhexan-1-ol (d₂-3SH), and [²H₅]-3-sulfanylhexyl acetate (d₅-3SHAc), the labeled analogues of impact odorants of wines and other foods, were synthesized to be used for the quantitative determination of the natural compounds in white and red wines by stable isotope dilution assay. The sulfidation was achieved by Michael addition, on mesityl oxide or ethyl hex-2-enoate, respectively, of the sulfhydryl anion generated in situ from triphenylsilanethiol and potassium fluoride under phase transfer conditions. The labeling of4-sulfanyl-4-methylpentan-2-one(SMP) was obtained from the commercial starting material, [²H₆]-acetone, so that this method could be used to synthesize ¹³C-labeled SMP from ¹³C-labeled acetone. The labeling of 3-sulfanylhexan-1-ol (3SH) and 3-sulfanylhexyl acetate (3SHAc) was obtained from reduction with lithium aluminum deuteride of the Michael adduct ethyl 3-sulfanylhexanoate and [²H₃]-acetylation. During the synthesis, 3SH and 3SHAc were partially oxidized to their disulfide, which were reduced back to the thiols by an additional reduction step; the tertiary thiol SMP was less sensitive to this oxidation.

Full text of DOI:10.1021/jf0004715

J. Agric. Food Chem. 2000, 48, 5819−5823
5819
Qu a n tita tive Deter m in a tion of Su lfu r Con ta in in g Win e Od or a n ts a t
Su b-p p b Levels. 1. Syn th esis of th e Deu ter a ted An a logu es
Yorgos Kotseridis,* J ean-Lo¨ıc Ray, Christian Augier, and Raymond Baumes
Unite´ Biopolyme`res Aroˆmes, INRA-IPV, 2 Place Viala, 34060 Montpellier, Cedex 01, France
[2H10]-4-Sulfanyl-4-methylpentan-2-one (d10-SMP), [2H2]-3-sulfanylhexan-1-ol (d2-3SH), and [2H5]-
3-sulfanylhexyl acetate (d5-3SHAc), the labeled analogues of impact odorants of wines and other
foods, were synthesized to be used for the quantitative determination of the natural compounds in
white and red wines by stable isotope dilution assay. The sulfidation was achieved by Michael
addition, on mesityl oxide or ethyl hex-2-enoate, respectively, of the sulfhydryl anion generated in
situ from triphenylsilanethiol and potassium fluoride under phase transfer conditions. The labeling
of 4-sulfanyl-4-methylpentan-2-one (SMP) was obtained from the commercial starting material, [2H6]-
acetone, so that this method could be used to synthesize 13C-labeled SMP from 13C-labeled acetone.
The labeling of 3-sulfanylhexan-1-ol (3SH) and 3-sulfanylhexyl acetate (3SHAc) was obtained from
reduction with lithium aluminum deuteride of the Michael adduct ethyl 3-sulfanylhexanoate and
[2H3]-acetylation. During the synthesis, 3SH and 3SHAc were partially oxidized to their disulfide,
which were reduced back to the thiols by an additional reduction step; the tertiary thiol SMP was
less sensitive to this oxidation.
Keyw or d s: Wine aroma; 4-sulfanyl-4-methylpentan-2-one; 3-sulfanylhexan-1-ol; 3-sulfanylhexyl
acetate; labeling; deuterium; disulfide
INTRODUCTION
1998a; Tominaga et al., 1998b; Bouchilloux et al., 1998),
and probably of wines of other varieties.
Sulfur containing compounds exhibit, in general,
intense smelling properties due to their extremely low
odor thresholds. Depending on their levels in beverages
and foods they contribute either favorably to the aroma
or to off-flavor. Thus 4-sulfanyl-4-methylpentan-2-one
(SMP) was described to have a pleasant box tree and
blackcurrant bud odor at trace levels in Sauvignon
wines but an unpleasant catty urine odor at higher
levels (Darriet, 1993). Its occurrence in Chenin blanc
and Colombar wines was hypothesized in 1981 (Du
Plessis and Augustyn, 1981), but it was positively
identified in Sauvignon and Scheurebe wines only
recently (Darriet, 1993; Darriet et al., 1995; Guth, 1997).
3-Sulfanylhexan-1-ol (3SH), first identified in yellow
passion fruits (Engel and Tressl, 1991; Weber et al.,
1994; Werkhoff et al., 1998) has an odor reminiscent of
grape fruit and passion fruit. It was recently reported
in Sauvignon blanc wines (Tominaga et al., 1998a) and
in red Bordeaux wines (Merlot, Cabernet Sauvignon
cultivars) (Bouchilloux et al., 1998; Kotseridis and
Baumes, 2000). Its acetate, 3-sulfanylhexyl acetate
(3SHAc), reminiscent of exotic fruits, was also first
identified in yellow passion fruits (Engel and Tressl,
1991; Werkhoff et al., 1998; Weber et al., 1995) and later
in wines (Tominaga et al., 1996; Bouchilloux et al.,
1998). These compounds have extremely low odor
thresholds (Tominaga et al., 1996; Tominaga et al.,
1998a; Tominaga et al., 1998b; Bouchilloux et al., 1998)
and are impact odorants of Sauvignon, Merlot, Cabernet
Sauvignon (Tominaga et al., 1996; Tominaga et al.,
The levels of these powerful odorant compounds were
reported in the sub-ppb range in white and red wines
(Bouchilloux et al., 1996; Bouchilloux et al., 1998;
Tominaga et al., 1998b). As discussed previously the use
of an internal standard whose chemical structure is
different from that of the analyte does not allow an
accurate quantification (Guichard 1988), especially in
the case of thiols which are very reactive species
(Hofmann et al., 1996). Thus a stable isotope dilution
assay (SIDA) was the most accurate method for the
quantitative determination of these trace odorants.
Hence, we reported in this paper the synthesis of [2H10]-
4-sulfanyl-4-methylpentan-2-one (d10-SMP), of [2H2]-3-
sulfanylhexan-1-ol (d2-3SH), and of [2H5]-3-sulfanylhexyl
acetate (d5-3SHAc) which will be used as labeled
standards in the quantitative determination of their
natural analogues in wines, as reported in the following
paper (Kotseridis et al., submitted to J . Agric. Food
Chem.).
EXPERIMENTAL PROCEDURES
Ch em ica ls. Mesityl oxide (98 wt %), tetrahydrofuran,
anhydrous (99.9 wt %), deuterium oxide (D2O, 99.8 atom %
D), deuterated sulfuric acid (D2SO4, 98 wt %, solution in D2O),
[2H6]acetone (d6-acetone, 99.8 atom % D), [2H3]acetic acid (99.9
+ atom % D), [2H3]acetyl chloride (99 + atom % D), lithium
aluminum deuteride (98 atom % D), 18-crown-6 ether (99.5 +
wt %), triphenylsilanethiol (98 wt %), potassium fluoride
anhydrous powder (99.99 + wt %), magnesium oxide (98 wt
%), sodium hydrosulfide hydrate (NaSH × H2O), cyclohexane
(99+ %), (E)-hex-2-enal (98 wt %), carbon tetrachloride anhy-
drous (99.9 + wt %) and lithium diisopropylamide mono-
(tetrahydrofuran) complex solution (LDA, 1.5 M in cyclohex-
ane) were all purchased from Aldrich Chemical Co. Inc.
Concentrated sulfuric acid (95-97%), chlorhydric acid (32%),
acetone (GR for analysis), acetic acid (glacial), diethyl ether,
* Author to whom correspondence should be addressed
(telephone 30 31 936 616; fax 0030 31 902 132; e-mail
yorgos@erevnon.com). Permanent address: Erevnon - Food
Technology, Poliviou str. 81-83, GR-54351 Thessaloniki, Greece.
10.1021/jf0004715 CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/21/2000

5820 J. Agric. Food Chem., Vol. 48, No. 12, 2000
Kotseridis et al.
F igu r e 1. Mass spectrum of 4-mercapto-4-methylpentan-2-
one.
F igu r e 2. Mass spectrum of [2H10]-4-mercapto-4-methylpen-
tan-2-one.
pentane, dichloromethane (ultrapure grade), molecular sieve
(4 Å), and silica gel 60 (230-400 mesh ASTM) were all
obtained from Merck, 64271 Darmstadt Germany. 2,6-Di-tert-
butyl-p-cresol (>99 wt %) and zinc (purum powder) were
purchased from Fluka Chemie AG, CH-9470 Buchs.
3-Sulfanylhexan-1-ol (97 wt %), 3-sulfanylhexyl acetate (97
wt %), and ethyl (E)-hex-2-enoate (96 wt %) were purchased
from Interchim, Montlucon (France).
cyclohexane which were boiled gently for 72 h. The reaction
mixture was then cooled to room temperature, and the solvent
was concentrated under vacuum at 35 °C: 2.35 g (21.8 mmol;
40%) of deuterated mesityl oxide were obtained; EI-MS (70
eV), m/z (%) 90(100)-62(82)-89(65)-61(51)-107(35)-108(33)-46-
(32)-42(26).
[2H10]-4-Sulfanyl-4-methylpentan-2-one. In a flame dried
conical flask 108 mg of [2H10]mesityl oxide (1 mmol), 584 mg
of triphenylsilanethiol (2 mmol), 264 mg of 18-crown-6 ether
(1 mmol), 116 mg of potassium fluoride (2 mmol), and 10 mg
of 2,6-di-tert-butyl-p-cresol (0.045 mmol) were added to 5 mL
of dry tetrahydrofuran (THF) and stirred at room temperature
under nitrogen atmosphere for 2 h. The dark green reaction
mixture was acidified, filtered on glasswool, and concentrated
under vacuum at 35 °C, and the residue was taken up in 2
mL of pentane. This solution was further purified by flash
chromatography on silica gel as described above. d10-SMP (67
mg, 47%) was obtained as a colorless oil. Its chemical purity
was 85% as determined by GC-EIMS. As the byproducts,
triphenylsilane and the crown ether had no impact in its use
in SIDA; it was not further purified. CI-MS (methane): m/z
140-143 (M + H+; 1-1.5%), 46 (CD3CO+, 100%); EI-MS, see
Figure 2.
Syn th esis of [2H2]-3-Su lfa n ylh exa n -1-ol. Ethyl 3-Sulfa-
nylhexanoate: In a conical flask containing 10 mL of anhy-
drous tetrahydrofuran (THF), 573 mg of ethyl (E)-hex-2-enoate
(4 mmol), 2.41 g of triphenylsilanethiol (8 mmol), 1.057 g of
18-crown-6 ether (4 mmol), 465 mg of potassium fluoride (8
mmol), and 20 mg of 2, 6-di-tert-butyl-p-cresol (0.09 mmol)
were added and stirred at room temperature, under nitrogen
atmosphere for 2 h. Then the dark green reaction mixture was
acidified, filtered on glasswool, and concentrated at 35 °C
under vacuum. The residue was taken up in 5 mL of glacial
acetic acid, added with 1.2 g of powdered zinc (18.2 mmol) and
refluxed under nitrogen atmosphere for 4 h. Then the reaction
mixture was concentrated at 50 °C under vacuum. The residue
was taken up in 2 mL of pentane, and the solution was further
purified by flash chromatography on silica gel with pure
pentane (200 mL) and then with pentane/diethyl ether (95/5,
v/v, 200 mL) as eluent. Ethyl 3-sulfanylhexanoate (450 mg,
2.56 mmol, 64%) was obtained as a colorless oil; EI-MS, see
Figure 3a.
[2H2]-3-Sulfanylhexan-1-ol. In a flame dried three-necked
flask, fitted with a dropping funnel and a nitrogen inlet, 152
mg of lithium aluminum deuteride (LiAlD4, 3.6 mmol) was
carefully added to 10 mL of dry diethyl ether (freshly distilled
from CaCl2), and the mixture was stirred for further 10 min.
After the formation of a milky suspension, a solution of ethyl
3-sulfanylhexanoate (528 mg, 3 mmol) in 3 mL of dry diethyl
ether was added carefully via the dropping funnel. Stirring
was continued for further 3 h, and then 2 mL of diethyl ether
saturated with water was added carefully to the reaction
mixture. The ether layer was separated, and the aqueous
phase was acidified to pH 1 with sulfuric acid (2 mol/L), then
extracted with pentane, and concentrated at 30 °C under
vacuum. The residue was taken up in acetic acid and treated
with powdered zinc as above for ethyl 3-sulfanylhexanoate to
yield 202 mg (1.48 mmol, 49%) of crude [2H2]-3-sulfanylhexan-
1-ol. As its chemical purity determined by GC-EIMS was 95%,
All glassware were cleaned by washing with acetone,
alcohol, and dichloromethane, followed by oven baking at 100
°C prior to use.
In str u m en ta l An a lysis. GC/EIMS analysis was carried out
using a Hewlett-Packard HP gas chromatograph 5890 series
II fitted with a 30 m fused-silica column (0.32 mm i.d. and 0.5
µm film thickness), coated with DB WAX (J &W Scientific). The
injection of the extracts (3 µL) was on-column at 35 °C; then
the temperature of the injector was increased 180 °C/min to
250 °C, till the end of the run. The carrier gas was Helium 6.0
(Linde gaz, Marseille), with a flow-rate of 1.35 mL/min. The
oven temperature program was 35 °C (for 3 min), then
increased at 3 °C/min to 245 °C, and held at this temperature
for a further 20 min. The GC instrument was coupled to a
5989A mass selective detector and a MS chemstation (HP-UX).
The electron impact (EI) energy was 70 eV, and the quadrupole
temperature was set at 250 °C.
GC-CIMS analysis was carried out using a Varian CP 3800
gas chromatograph fitted with the same column as above and
coupled to a Varian Saturn 2000. The GC conditions were the
same as above except that the initial temperatures of the
injector and of the oven were respectively 20 °C and 60 °C.
The temperatures of the transfer line, the manifold, and the
trap were respectively 250, 40, and 170 °C. The reactant gas
was methane, the emission current was 20 µA, and the
maximum ionization and reaction times were respectively 2
ms and 60 ms.
A Varian 500 MHz NMR spectrometer was used to record
the 1H- and 13C NMR spectra. Sample spectra were from
deuterated chloroform solutions; TMS (0 ppm δ) and CDCl3
(77.0 ppm δ) were used as reference.
Syn th esis of 4-Su lfa n yl-4-m eth ylp en ta n -2-on e. In a
conical flask 98 mg of mesityl oxide (1 mmol), 584 mg of
triphenylsilanethiol (2 mmol), 264 mg of 18-crown-6 ether (1
mmol), 116 mg of potassium fluoride (2 mmol), and 10 mg of
2, 6-di-tert-butyl-p-cresol (0.045 mmol) were added to 5 mL of
dry tetrahydrofuran (THF) and stirred at room temperature
under nitrogen atmosphere for 2 h. Then the dark green
reaction mixture was acidified, filtered on glasswool, and
concentrated under vacuum. The residue was taken up in 2
mL of pentane, and the solution was further purified by flash
chromatography on silica gel using pentane (200 mL) and then
pentane/diethyl ether (5/95, 200 mL) as eluent. SMP (92.4 mg,
70%) was obtained as a colorless oil; 13C NMR (CDCl3) δ 31.8
(C-1), 32.5 (C-5, C-5′), 41.4 (C-4), 57.7 (C-3), 206.7 (C-2). 1H
NMR (CDCl3) δ 1.40 (s, C-4-(CH3)2), 2.09 (s, (H-1)3), 2.35 (s,
SH), 2.70 (s, (H-3)2). EI-MS, see Figure 1.
Syn th esis of [2H10]-4-Su lfa n yl-4-m eth ylp en ta n -2-on e.
[2H10]Mesityl Oxide. The porous thimble of a Soxhlet extraction
apparatus was filled with a mixture of magnesium oxide and
of molecular sieve (4 Å). The apparatus was fitted to a flask
containing 4 mL (54.4 mmol) of [2H6]acetone and 16 mL of

Synthesis of Deuterated Sulfur Containing Wine Odorants
J. Agric. Food Chem., Vol. 48, No. 12, 2000 5821
F igu r e 4. Mass spectrum of [2H5]-3-sulfanylhexyl acetate.
F igu r e 5. 4-Sulfanyl-4-methylpentan-2-one synthesis.
methane, and stirring was continued for 15 min. The organic
phase was separated in a separatory funnel, centrifuged for 5
min at 9000g (4 °C), dried over sodium sulfate, then concen-
trated to about 1 mL under vacuum at 30 °C, and analyzed
by GC/MS (full scan mode) to check if any exchange had
occurred.
F igu r e 3. Mass spectra of (a) ethyl 3-sulfanylhexanoate and
(b) [2H2]-3-sulfanylhexan-1-ol.
it was used in SIDA without further purification; 1H NMR
(CDCL3) 0.85 (t, J ) 7 Hz, (H-6)3), 1.32 (d, J ) 7.9 Hz, SH),
1.34-1.50 (m, (H-4)2, (H-5)2), 1.56 (br dd, J ) 14.3 Hz, J ) 9.5
Hz, H-2), 1.88 ((br dd, J ) 14 Hz, J ) 3.7 Hz, H-2′), 2.88 (m,
H-3); the integration of the signal for the residual H-1 relative
to that of the signal for H-3 showed an isotopic purity of 98%,
consistent with that of the deuteride used. CI-MS (methane):
m/z 119 (M + H+ - H2O; 40%), 85 (M + H+ - H2O - H2S;
100%); EI-MS, see Figure 3b.
RESULTS AND DISCUSSION
The key step of the synthesis of SMP, 3SH, and
3SHAc was the Michael addition of the sulfhydryl anion
generated in situ from triphenylsilanethiol and potas-
sium fluoride under phase transfer conditions. Triphen-
ylsilanethiol was reported previously as a solid hydrogen
sulfide equivalent in the ring opening of epoxides (Brit-
tain and Gareau, 1993). The labeling was obtained
either in the beginning of the synthesis for SMP
(deuterated starting material, [2H6]acetone) or in the
end for 3SH and 3SHAc (reduction with lithium alumi-
num deuteride and [2H3]-acetylation.
4-Su lfa n yl-4-m eth ylp en ta n -2-on e Syn th esis. Syn-
thesis of SMP was attempted following the synthetic
route proposed by Guth (1997). In our hands, the yield
of the first step of this synthesis, the acid catalyzed
condensation of acetone, determined using GC/MS, was
very low (mesityl oxide yield 4%), and the final yield of
SMP was lower than 1%. Modifications were attempted
to improve the mesityl oxide yield. Ten times more
sulfuric acid and use of cyclohexane as a solvent at 50
°C yielded up to 40% mesityl oxide. However, the same
conditions, using D2SO4 and acetone-d6, yielded only
13% of deuterated mesityl oxide. Basic conditions by
preforming the enolate at low temperature and then
adding acetone (Kleschick et al., 1977) yielded up to 33%
of diacetone alcohol and about the same yield for mesityl
oxide, but the same conditions using acetone-d6, yielded
only 12% of deuterated mesityl oxide.
Syn th esis of [2H5]-3-Su lfa n ylh exyl Aceta te. [2H5]-3-
Sulfanylhexyl acetate was prepared using the synthetic route
reported previously for the unlabeled compound (Heusinger
and Mosandl, 1984). Under strictly anhydrous conditions and
under nitrogen, [2H2]-3-sulfanylhexan-1-ol (80 mg, 0.6 mmol)
was added to 3 mL of anhydrous carbon tetrachloride (CCl4),
and the solution was stirred at room temperature for 10 min.
[2H3]Acetyl chloride (128 µL, 1.8 mmol) was added, and the
reaction mixture (monitored by GC/MS) was further stirred
for 5 h at room temperature, under nitrogen, and then
concentrated at 35 °C under vacuum. The residue was taken
up in [2H3]acetic acid and treated with powdered zinc as above
for ethyl 3-sulfanylhexanoate, to yield 56 mg (0.3 mmol, 50%)
of crude [2H5]-3-sulfanylhexyl acetate used in SIDA without
further purification (chemical purity 95% as determined by
GC/EIMS). 1H NMR (CDCL3) δ 0.86 (t, J ) 7 Hz, (H-6)3), 1.31
(d, J ) 7.3 Hz, SH), 1.33-1.60 (m, (H-4)2, (H-5)2), 1.65 (br dd,
J ) 14.1 Hz, J ) 9.2 Hz, H-2), 1.94 (br dd, J ) 14 Hz, J ) 4.3
Hz, H-2′), 2.81 (m, H-3); the integration of the signal for the
residual H-1 relative to that of the signal for H-3 showed an
isotopic purity of 98% for those positions, while the signal for
the acetate group was too small to be accuretely integrated,
consistent with the high isotopic purity of the [2H3]acetyl
chloride used. CI-MS (methane): m/z 164 (M + H+ - H2O;
60%), 119 (M + H+ - d3AcOH; 65%), 46 (CD3CO+, 100%); EI-
MS, see in Figure 4.
Sta bility of d 10-SMP a t Win e p H. In a 0.5 L Erlenmeyer
0.328 mg of d10-SMP (in 1 mL pentane) and 0.4 g of tartaric
acid were added to 100 mL of an ethanol/water solution (12/
88 v/v), and the pH of the mixture was adjusted to 2.8 with
potassium bicarbonate and stirred at room temperature for
24 h. Then the flask was placed in an ice bath under
nitrogen: 30 mL of pentane/dichloromethane (2/1 v/v) was
added, and the mixture was stirred during 15 min at 700 rpm.
The mixture was supplemented with 20 mL of dichloro-
The best yield of deuterated mesityl oxide was ob-
tained in the one-pot basic condensation of acetone with
magnesium oxide and dehydration of the intermediate
deuterated 4-hydroxy-4-methylpentan-2-one (diacetone
alcohol) (Figure 5). These reactions were carried out in
a Soxhlet extraction apparatus using cyclohexane as a
solvent, which azeotropically distilled with water and
acetone, and mixing in the porous thimble the base

5822 J. Agric. Food Chem., Vol. 48, No. 12, 2000
Kotseridis et al.
F igu r e 6. Synthesis of [2H2]-3-sulfanylhexan-1-ol and of [2H5]-
3-sulfanylhexyl acetate.
necessary for the condensation and a molecular sieve
to trap the water formed from the dehydration. Thus
the reaction equilibrium was displaced toward the
deuterated mesityl oxide, which was then drained back
to the flask by the solvent where it remained due to its
higher boiling point. The solvent allowed the use of
small amounts of the labeled acetone to prevent further
condensation of mesityl oxide. The mass spectrum of the
deuterated mesityl oxide showed a molecular ion at m/z
108 and fragment ions at m/z 62 (C4D7+) and 46
(COCD3+), which was consistent with the incorporation
of 10 deuterium atoms in the molecule.
The second step was the addition of the sulfhydryl
anion to mesityl oxide using triphenylsilanethiol and
potassium fluoride under phase transfer catalysis condi-
tions (Figure 5). The conditions of this reaction, deter-
mined for the unlabeled SMP, allowed for the obtaining
of SMP in 70% yield. Its structure was confirmed by 1H-
and 13C NMR (Experimental Procedures) and MS
(Figure 1). When the starting material was the deuter-
ated mesityl oxide synthesized above, the yield in
deuterated SMP was 47%. As triphenylsilanethiol con-
tained a SH group and as the workup was made with
water, two protons were incorporated in the interchange-
able positions of the molecule (Figure 5). That was
consistent with the molecular ion at m/z 142 and the
fragment ion at m/z 109 (M+ - SH) in its mass
spectrum, showing the occurrence of 10 deuterium
atoms in the molecule (Figure 2). In fact, the clusters
of molecular ions in the EI- and CI-MS of the product
obtained showed that it was a mixture of isotopomers
containing on average 10 deuterium atoms, each of them
retaining the deuterated gem-dimethyl moiety.
F igu r e 7. Mass spectra of (a) bis-(1’-carbethoxypent-2’-yl)-
disulfide, (b) [2H4]bis-(1’-hydroxyhex-3’-yl)disulfide, and (c)
[2H10]bis-(1’-acetoxyhex-3’-yl) disulfide.
that was further purified by flash chromatography on
silica gel. Although the following step should have
reduced the disulfide, this additional step made easier
the flash chromatography purification.
The second step was the reduction with lithium
aluminum deuteride of the ethyl ester compound to the
corresponding alcohol, [2H2]-3-sulfanylhexan-1-ol, la-
beled on the first carbon of the aliphatic chain. During
the workup of the basic reaction mixture, the product
was partially oxidized to the corresponding disulfide
(mass spectrum see in Figure 7b); therefore a reduction
step under acidic conditions was used again to obtain a
crude product containing only [2H2]-3-sulfanylhexan-1-
As the labeling of the synthesized SMP was obtained
from the starting material, [2H6]acetone, this synthesis
could be used to obtain the [13C6]-labeled SMP from the
much more expensive [13C3]acetone.
[2H2]-3-Su lfa n ylh exa n -1-ol syn th esis. The synthe-
sis of 3-sulfanylhexan-1-ol starting from (E)-hex-2-enal
and hydrogen sulfide was reported previously (Winter
et al., 1976; Engel and Tressl, 1991). As the use of
hydrogen sulfide was an unpleasant operation, its anion
was generated as above from triphenylsilanethiol under
phase transfer conditions, but the yield obtained was
not satisfactory (25%), probably due to the instability
of the unsaturated aldehyde in these conditions.
However, these conditions allowed for obtaining in
good yield ethyl 3-sulfanylhexanoate starting from ethyl
(E)-hex-2-enoate (Figure 6). Despite the care being
taken to prevent oxidation of the sulfanyl group, the
corresponding disulfide (Figure 7a) was obtained as a
secondary product (about 30% as assessed by GC/MS).
Thus a reduction step was carried out to obtain a crude
product containing mainly ethyl 3-sulfanylhexanoate,
1
ol with an estimated total yield of 31%. The H NMR
showed the disappearance of the signal for H-1 and H-1′,
reported at 3.74 ppm in the unlabeled compound (Kle-
schick et al., 1977) and the collapse of the multiplets
for H-2 and H-2′ to two doublets of doublet with broad
lines due to the residual couplings with D-1 and D-1′.
The mass spectrum was consistent with this labeling
(Figure 3).
[2H5]-3-Su lfa n ylh exyl Aceta te Syn th esis. The syn-
thesis of [2H5]-3-sulfanylhexyl acetate was achieved
using the synthetic route proposed by Heusinger and
Mosandl (1984), slightly modified. Thus the acetylation
of the synthesized [2H2]-3-sulfanylhexan-1-ol was car-
ried out with [2H3]acetyl chloride in carbon tetrachlo-
ride, affording [2H5]-3-sulfanylhexyl acetate with an
estimated yield of 50% (Figure 6). The reaction was

Synthesis of Deuterated Sulfur Containing Wine Odorants
J. Agric. Food Chem., Vol. 48, No. 12, 2000 5823
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Kotseridis, Y.; Baumes, R. Identification of impact odorants
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monitored by GC/MS and stopped when the hydroxyl
function was acetylated in order to avoid the slower
acetylation of the sulfanyl group. As a small amount of
the corresponding disulfide was detected by GC/MS
(mass spectrum see in Figure 7c) an additional reduc-
tion step, using [2H3]acetic acid and powdered zinc, was
used. The 1H NMR was similar to that of 3SH; its
comparison with the spectrum reported for the unla-
beled compound (Engel and Tressl, 1991) confirmed the
labeling of the acetate moiety and of the methylene in
position 1. The mass spectrum was consistent with this
labeling (Figure 4).
Sta bility of d 10-SMP a t Win e p H. Contrary to d2-
3SH and d5-3SHAc, d10-SMP contained enolizable deu-
terium atoms that could be back exchanged during the
analysis process (Kotseridis et al., submitted to J . Agric.
Food Chem.). Thus d10-SMP was treated in a model wine
(pH 2.8) for 24 h at room temperature. Using GC/MS
in the full scan mode, no proton-deuterium exchange
in d10-SMP was observed. This experiment allowed its
use as labeled standard in the conditions used to
determine quantitatively SMP in wines process (Kot-
seridis et al., submitted to J . Agric. Food Chem.).
However, should a labeled SMP be used in conditions
favoring any D/H exchange, 13C-labeled SMP could be
synthesized by the method described above.
CONCLUSIONS
Kotseridis, Y.; Ray, J . L.; Lepoutre, J . P. L.; Baumes, R.
Quantitative determination of Sulfur containing wine odor-
ants at sub-ppb levels. Part II: Development and application
of a Stable Isotope Dilution Assay. Submitted to J . Agric.
Food Chem.
Pearce, T. J . P.; Peacock, J . M.; Aylward, F.; Haisman, D. R.
Catty odours in food: reactions between Hydrogen Sulphide
and unsaturated ketones. Chem. Ind. (London) 1967, 1562-
1563.
Tominaga, T.; Darriet, P.; Dubourdieu, D. Identification de
l’ace´tate de 3-mercaptohexanol, compose´ a forte odeur de
buis, intervenant dans l′aroˆme des vins de Sauvignon. Vitis
1996, 35, 207-210.
Tominaga, T.; Furrer, A.; Henry, R.; Dubourdieu, D. Identi-
fication of new volatile thiols in the aroma of Vitis Vinifera
L.var. Sauvignon. Flavour Fragnance J . 1998, 13, 159-
162.
Tominaga, T.; Murat, M. L.; Dubourdieu, D. Development of
a method for analyzing the volatile thiols involved in the
characteristic aroma of wine made from Vitis Vinifera L.cv.
Sauvignon. J . Agric. Food Chem. 1998, 46, 1044-1048.
Weber, B.; Maas, B.; Marx, A.; Olk, J .; Mosandl, A. Enantio-
meric distribution of the chiral sulphur-containing alcohols
in yellow and purple passion fruits. Z. Lebensm. Unters.
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Weber, B.; Maas, B.; Mosandl, A. Stereoisomeric distribution
of some chiral sulphur-containing trace components of
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2441.
The methods reported in this paper to synthesize d10-
SMP, d2-3SH, and d5-3SHAc were relatively easy and
presented fairly good yields. The use of a solid hydrogen
sulfide equivalent (Brittain and Gareau, 1993) rendered
the preparation of these thiols a not unduly unpleasant
operation. Furthermore these methods seemed of quite
general application to be used in the preparation of some
other deuterated thiols and of 13C-labeled SMP.
We also observed that these sulfanyl compounds were
easily oxidized to their disulfides, especially when basic
conditions were used, so that additional reduction steps
were necessary. This observation was in agreement with
the oxidative instability of some thiols occurring in food
flavor (Hofmann et al., 1996), but SMP, which was a
tertiary thiol, was found to be less sensitive to this
oxidation. Moreover, no formation of its disulfide was
observed during the experiment to test its stability
under acidic conditions.
However, this oxidative instability of thiols showed
the necessity of SIDA for their quantification, particu-
larly in wines in which they occurred at trace levels.
Development and application of such a method will be
reported in the following paper (Kotseridis et al.,
submitted to J . Agric. Food Chem.).
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Received for review April 12, 2000. Revised manuscript
received August 29, 2000. Accepted September 11, 2000.
Darriet, P.; Tominaga, T.; Lavigne, V.; Boidron, J . N.; Du-
bourdieu, D. Identification of a powerful aromatic compo-
J F0004715