Synthesis of the individual diastereomers of the cysteine conjugate of 3-mercaptohexanol (3-MH)

Article abstract of DOI:10.1021/jf8000444

The individual diastereoisomers of the cysteine conjugate of 3-mercaptohexanol (4) were synthesized with high isomeric purity (>98%). On treatment with Apotryptophanase enzyme, the 3R diastereoisomer of 4 gave an 82% yield of the R enantiomer of 1, with no trace of the 3S enantiomer present. Conversely, the 3S diastereoisomer of 4 gave the 3S enantiomer of 1 (43%) accompanied by a trace of the 3R form (S/R = 98.5:1.5), reflecting the diastereomeric purity of the cysteine conjugate. The same stereochemical outcome was observed when the individual diastereoisomers of 4 were added to fermentations with the Saccharomyces cerevisiae AWRI 1655 yeast strain, which gave 1 in 1% yield. A d₁₀-analogue of 1 was synthesized and used as an internal standard to determine, by gas chromatography-mass spectrometry (GC-MS), the amounts of 1 formed in these transformations.

Full text of DOI:10.1021/jf8000444

3758 J. Agric. Food Chem. 2008, 56, 3758–3763
Synthesis of the Individual Diastereomers of the
Cysteine Conjugate of 3-Mercaptohexanol (3-MH)
KEVIN H. PARDON,†,# SEAN D. GRANEY,§ DIMITRA L. CAPONE,†,#
JAN H. SWIEGERS,†,# MARK A. SEFTON,†,# AND GORDON M. ELSEY*,†,§,#
The Australian Wine Research Institute, P.O. Box 197, Glen Osmond,
South Australia 5064, Australia; School of Chemistry, Physics and Earth Sciences,
Flinders University, P.O. Box 2100, Adelaide, South Australia 5001, Australia; and Cooperative
Research Centre for Viticulture, P.O. Box 154, Glen Osmond, South Australia 5064, Australia
The individual diastereoisomers of the cysteine conjugate of 3-mercaptohexanol (4) were synthesized
with high isomeric purity (>98%). On treatment with Apotryptophanase enzyme, the 3R diastereoi-
somer of 4 gave an 82% yield of the R enantiomer of 1, with no trace of the 3S enantiomer present.
Conversely, the 3S diastereoisomer of 4 gave the 3S enantiomer of 1 (43%) accompanied by a trace
of the 3R form (S/R ) 98.5:1.5), reflecting the diastereomeric purity of the cysteine conjugate. The
same stereochemical outcome was observed when the individual diastereoisomers of 4 were added
to fermentations with the Saccharomyces cerevisiae AWRI 1655 yeast strain, which gave 1 in 1%
yield. A d10-analogue of 1 was synthesized and used as an internal standard to determine, by gas
chromatography-mass spectrometry (GC-MS), the amounts of 1 formed in these transformations.
KEYWORDS: Wine; aroma, flavor; 3-MH; cysteine conjugates; thiols
INTRODUCTION
conjugates (12–15), with the amino acid moiety of the former
beingcleavedbytheactionoftheyeastduringfermentation(16–18).
2 is presumed to be formed from 1 during fermentation as a
result of yeast alcohol acetyltransferase activity (19). More
recently, Schneider et al. have shown that volatile thiols can be
formed by the addition of hydrogen sulfide, produced by yeast
under fermentation conditions, to olefinic precursors of 1 and
3 (20). However, under the conditions reported, this alternative
mode of generation accounted for approximately 10% only of
the total thiols generated.
In recent years, the contribution of thiol-containing com-
pounds to the aroma and flavor of wines has been an important
focus for research (1). Many such compounds are extremely
potent, with aroma detection thresholds typically in the nano-
grams per liter range, and are characterized by distinctive aroma
qualities. Three thiols (Figure 1) that have been found to be
important to the aroma of wine are 3-mercaptohexanol (3-MH,
1), the O-acetate of this compound (3-MHA, 2), and 4-mercapto-
4-methylpentan-2-one (4-MMP, 3). 1 is characterized by a
tropical fruit/passion fruit aroma and was first identified in
yellow passion fruit (2–4). It has subsequently been identified
in several wine varieties (5–7). 2 has also been described as
having a tropical fruit aroma and was found to occur, in passion
fruit, predominantly as the S enantiomer (8). 3 has an aroma
reminiscent of box tree and black currant but, at higher
concentrations, has been found to have a rather unpleasant cat-
urine odor (9–11). The importance of thiols to the overall aroma
of a wine is exemplified by the fact that 3 was found to be the
most potent aroma compound (of 42 measured) in one study
(9).
One factor that must be taken into consideration with 1 and
2 (but not 3) is the stereochemistry at the carbon to which the
sulfur is attached, as, for both compounds, the two enantiomers
have differing sensory properties (21). The cysteine conjugate
of 1 has two diastereomeric forms, namely, 3S-4 and 3R-4. Thus,
the stereochemical outcome of the transformation of the
conjugate 4 by the yeast may be dependent on the starting
configuration of 4. Wakabayashi et al. (18) prepared a 1:1
diastereomeric mixture of 4, and when this mixture was
subjected to enzymatic cleavage, using two different sources
of ꢀ-lyase, the two enantiomers of 1 were found in similar
proportions.
These thiols themselves are not present in grape juice in
measurable concentration prior to fermentation. Rather, 1 and
3 are believed to accumulate as L-cysteine and glutathione
As an expansion of our earlier work concerning the liberation
of 3 from its cysteine conjugate (16, 17), we have now
synthesized the two cysteine conjugate forms of 4, in diaste-
reomerically pure fashion, to investigate the stereoselectivity
of thiol formation more thoroughly and to obtain authentic
samples for characterizing grape composition.
* Corresponding author (telephone +61 8 8201 3071; fax +61 8
8201 2905; e-mail Gordon.Elsey@flinders.edu.au).
† The Australian Wine Research Institute.
# Cooperative Research Centre for Viticulture.
§ Flinders University.
10.1021/jf8000444 CCC: $40.75  2008 American Chemical Society
Published on Web 05/08/2008

Synthesis of Individual Cysteine Conjugates
J. Agric. Food Chem., Vol. 56, No. 10, 2008 3759
Figure 1. Structures of 3-MH (1), 3-MHA (2), 4-MMP (3), and the cysteinylated conjugates of 3-MH (4).
MATERIALS AND METHODS
This compound (0.32 g, 1.0 mmol) was then stirred in dichlo-
romethane containing TFA (3.42 g, 30.0 mmol) at room temperature
for 2 h. The solvents were removed, and the residue was passed through
a short ion-exchange resin column (Amberlite IRA-400), eluting with
water, to give, after evaporation, the desired cysteinylated compound
(3R)-4 as its hydrochloride salt (0.18 g, 70%): [R]D -14.1 (c 0.32,
H2O); 1H NMR (D2O) δ 4.25 (1H, dd, J ) 6.5, 4.7 Hz, H8), 3.77-3.65
(2H, m, H1), 3.23-3.02 (2H, m, H7), 2.88 (1H, app quintet, J ∼ 6.2
Hz, H3), 1.96-1.30 (6H, m, H2,4,5), 0.86 (3H, t, J ) 7.2 Hz, H6); 13C
NMR (D2O) δ 170.7, 59.3, 53.0, 43.0, 36.6, 36.2, 29.8, 19.4, 13.3.
(3S)-2-Amino-3-[(3-hydroxy-1-propylpropyl)sulfanyl]propanoic
Acid (3S)-4. (3S)-8 (0.26 g, 0.77 mmol) was demethylated exactly as
above using sodium hydroxide (29 mg, 0.95 equiv) to give the
monoprotected intermediate (0.21 g, 83%): [R]D +97.8 (c 0.45, CHCl3).
NMR spectra were recorded as solutions in chloroform-d and were
obtained on a Varian Gemini spectrometer operating at either 300 MHz
(1H) or 75.5 MHz (13C). Specific rotations were recorded on a PolAAr
21 polarimeter. All reagents were purchased from Sigma-Aldrich. All
solvents were of the highest commercial grade available. Diethyl ether
and THF were distilled from sodium/benzophenone immediately prior
to use. All organic solutions were dried over anhydrous sodium sulfate
prior to filtration.
Methyl N-Butoxycarbonyl-L-cysteine (6). L-Cysteine methyl ester
(5) (5.05 g, 29.4 mmol) in dichloromethane (30 mL) was stirred with
di-tert-butyl dicarbonate (6.42 g, 29.4 mmol) and triethylamine (2.97
g, 29.4 mmol) at room temperature for 18 h. The solvent was removed
and the residue chromatographed on silica (20:80 EtOAc/CHCl3) to
give the N-protected compound (7.01 g, 100%), the spectral parameters
of which were identical with those reported previously (25).
Methyl 2-Butoxycarbonylamino-3-[(3-hydroxy-1-propylpropyl)sul-
fanyl]propanoate (8). (E)-2-Hexenal (0.75 g, 7.6 mmol), triethylamine
(1.55 g, 15.3 mmol), and the diprotected compound 6 (1.80 g, 7.6 mmol)
in acetonitrile (35 mL) were stirred at room temperature for 3 days.
Removal of the solvent and silica chromatography gave the desired
aldehyde adduct 7 (2.17 g, 85%) as an inseparable mixture of
diastereomers.
This adduct, 7 (1.69 g, 5.08 mmol), in methanol (10 mL) at 0 °C
was treated with sodium borohydride (0.09 g, 2.38 mmol), which was
added in two portions separated by 5 min. The reaction was stirred at
room temperature for 3 h before being quenched with ammonium
chloride solution. The solution was extracted with dichloromethane,
dried, and concentrated to give the desired alcohol 8 (1.31 g, 77%).
Separation into the component diastereomers was achieved on silica
using a ternary eluant comprising EtOAc (35%), CHCl3 (52.5%), and
hexane (12.5%). Obtained were pure samples of the first eluting, or
“front” diastereomer (0.565 g), the second eluting “rear” diastereomer
(0.38 g), and a fraction containing both (0.315 g).
This product (0.20 g, 0.63 mmol) was then treated with TFA (2.14
g, 18.8 mmol) exactly as above to give (3S)-3 as its hydrochloride salt
(0.12 g, 73%): [R]D +20.8 (c 0.14, H2O); 1H NMR (D2O) δ 4.29 (1H,
app t, J ∼ 5.6 Hz, H8), 3.69 (2H, t, J ) 6.6 Hz, H1), 3.24-3.04 (2H,
m, H7), 2.86 (1H, app quintet, 3J ∼ 6.4 Hz, H3), 1.94-1.29 (6H, m,
H
2,4,5), 0.86 (3H, t, J ) 7.0 Hz, H6); 13C NMR (D2O) δ 170.6, 59.3,
52.8, 43.1, 36.5, 36.2, 29.6, 19.4, 13.2.
Ethyl d8-Hexenoate (d8-11). Commercially available d10-butanol (9)
(5.0 g, 59.5 mmol) was oxidized to the corresponding aldehyde 10 by
the procedure outlined in Mancuso and Swern (21). To the final
dichloromethane solution of 10 (used directly because of its high
volatility) was added (carbethoxymethylene)triphenylphosphorane (23.0
g, 66 mmol), and the mixture was allowed to stir at room temperature
for 7 days. The solvent was then removed, and hexane (200 mL) was
added to the residue and allowed to stir for 30 min. The mixture was
filtered and the filtrate concentrated and distilled (60-70 °C at 8 mmHg)
to give d8-(11) as a clear oil (4.8 g, 54%).
(()-d10-3-Mercaptohexanol (1). To labeled ester d8-11 (4.8 g, 32
mmol) in THF (25 mL) was added triethylamine (4.04 g, 40 mmol)
and thiolacetic acid (4.84 g, 64 mmol). After 3 days of stirring at room
temperature, the mixture was diluted with dichloromethane (100 mL)
and washed with 1 M HCl solution and brine. The solvent was removed
and the residue, (()-d10-12, was diluted in dry ether (50 mL). This
ether solution was added dropwise to another ether solution containing
LiAlD4 (1.74 g, 41.6 mmol) at 0 °C. After 2 h of stirring at room
temperature, the reaction was cooled in ice and quenched by dropwise
addition of a saturated aqueous solution of sodium sulfate. The aqueous
layer was acidified with 1 M HCl (to pH 3) and extracted with ether.
The combined ether extracts were washed with brine, dried, concen-
trated, and distilled (70-80 °C at 15 mmHg) to give the target (()-
d10-(1) (1.27 g, 32%). The very high degree of isotopic substitution
made characterization by NMR problematic. However, the compound
showed appropriate GC retention time and mass spectral fragmentation
pattern when compared with the unlabeled compound: d10-(1) m/z (%)
144 (30), 143 (<1), 109 (32), 91 (31), 62 (100), 60 (78), 46 (47); d0-
(1) m/z (%) 134 (17), 100 (50), 82 (37), 57 (63), 55 (100), 41 (52).
Preparation of Samples for Analysis of 1. An aliquot (70 µL) of
(3R)-8 (front diastereomer): [R]D -16.4 (c 0.86, CHCl3); 1H NMR
(CDCl3) δ 5.43 (1H, br s, NH), 4.53 (1H, m, H8), 3.90-3.64 (2H, m,
H1), 3.75 (3H, s, OMe), 3.04-2.76 (3H, m, H3,7), 2.39 (1H, br s, OH),
1.91-1.82 (2H, m, H2), 1.62-1.34 (4H, m, H4,5), 1.44 (9H, s, tBu),
0.91 (3H, t, J ) 7.0 Hz, H6); 13C NMR (CDCl3) δ 171.9, 155.1, 80.6,
59.9, 53.3, 52.8, 42.6, 38.5, 37.3, 33.8, 28.5, 20.0, 14.2.
1
(3S)-8 (rear diastereomer): [R]D +47.3 (c 1.1, CHCl3); H NMR
(CDCl3) δ 5.40 (1H, br s, NH), 4.52 (1H, m, H8), 3.88-3.64 (2H, m,
H1), 3.74 (3H, s, OMe), 3.02-2.84 (2H, m, H7), 2.82-2.66 (1H, m,
H3), 2.32 (1H, br s, OH), 1.89-1.78 (2H, m, H2), 1.67-1.35 (4H, m,
H
4,5), 1.42 (9H, s, tBu), 0.89 (3H, t, J ) 7.0 Hz, H6; 13C NMR (CDCl3)
δ 171.7, 155.5, 80.2, 60.1, 53.4, 52.5, 43.4, 37.9, 37.3, 32.9, 28.2, 19.9,
13.9.
(3R)-2-Amino-3-[(3-hydroxy-1-propylpropyl)sulfanyl]propanoic
Acid (3R)-4. (3R)-8 (0.41 g, 1.24 mmol) was treated with sodium
hydroxide (47 mg, 0.95 equiv) in methanol (20 mL) at room temperature
for 3 h and then allowed to stand in the refrigerator overnight. The
solution was diluted with water and extracted with dichloromethane.
The aqueous layer was acidified (to pH 2), saturated with solid sodium
chloride, and thoroughly extracted with EtOAc. The EtOAc extracts
were dried and concentrated to give the product (0.34 g, 84%): [R]D
-23.5 (c 0.34, CHCl3).
2
a solution containing racemic H10-1 (14.32 µg/mL in ethanol) was
added, as an internal standard (IS) using a glass syringe (100 µL SGE),
to a fermented sample (10 mL) containing salt (2 g, NaCl, BDH) and
ethylenediaminetetraacetic acid (EDTA, approximately 20 mg) in a 20
mL SPME vial with a magnetic crimp cap (Gerstel). A similar sample

3760 J. Agric. Food Chem., Vol. 56, No. 10, 2008
Pardon et al.
Figure 2. Syntheses of (3R)-4 and (3S)-4.
was prepared without added IS. For analysis of enzyme hydrolysates,
samples were prepared in the same manner except that IS (7 µL) was
added to the hydrolysate sample (1 mL) diluted in Milli-Q water (4
mL) containing salt (200 mg, NaCl, BDH) and EDTA (approximately
2 mg, added to suppress sample oxidation catalyzed by metal ions).
Standard samples of aqueous ethanol (10%) spiked with H10-1 and
with or without unlabeled 1 were prepared and analyzed at the same
time to calibrate the method.
Enzyme Assay. The assay mixture was prepared by adding (3S)-4
(10 µL, 1 mg/mL) or (3R)-4 (12.8 µL, 0.78 mg/ml), pyridoxal
5′-phosphate (20 µL, 1 mM), and assay buffer (1.88 or 1.852 mL, 20
mM phosphate buffer, pH 7.0; 1 mM EDTA) to total 2 mL. The reaction
was initiated by adding 1 mg of Apotryptophanase (Sigma-Aldrich,
St. Louis, MO), and the reaction mixture was incubated at 37 °C for
1 h. The reaction was stopped by placing it on ice, and the mixture
was then stored at -20 °C prior to analysis. Assays were conducted in
duplicate.
2
GC-MS Analysis. Samples were analyzed with a Hewlett-Packard
(HP) 6890N gas chromatograph fitted with a Gerstel MPS2 autosampler
and coupled to a HP 5973N mass spectrometer. The autosampler was
fitted with an automated 65 µm carbowax/divinylbenzene (CW/DVB)
SPME fiber (Supelco). The gas chromatograph was fitted with an
approximately 30 m × 0.25 mm i.d. AlphaDex 120 (Supelco) fused
silica capillary column having 0.25 µm film thickness. The carrier gas
was helium (BOC gases, ultrahigh purity), and the flow rate was 1.1
mL/min. The oven temperature started at 50 °C, was held at this
temperature for 1 min, then increased to 140 at 2 °C/min, then to 260
at 40 °C/min, and held at this temperature for 10 min. The injector
was held at 220 °C and the transfer line at 260 °C. The sample was
extracted at 40 °C for 30 min and desorbed in the inlet for 15 min.
The splitter, at 45:1, was opened after 36 s. Fast injection was done in
pulsed splitless mode with an inlet pressure of 25.0 psi maintained
until splitting. The injection sleeve (Supelco, 0.75 mm i.d.) was
borosilicate glass. Positive ion electron impact spectra at 70 eV were
recorded in the range m/z 35-350 for scan runs. For quantification of
1, mass spectra were recorded in the selective ion monitoring (SIM)
mode. The ions monitored in SIM runs were m/z 60, 62, 92, 109, and
144 for [2H10]-1 and 55, 82, 88 100, and 134 for 1, respectively. Selected
fragment ions were monitored for 30 ms each. The underlined ion for
each compound was the ion typically used for quantitation, having the
best signal-to-noise and the least interference from other wine
components. The other ions were used as qualifiers. The limit of
detection for 1 was 5 µg/L, and the coefficient of determination (r2)
was 0.994.
Yeast Ferment. Saccharomyces cereVisiae strain AWRI 1655 was
grown in 5 mL of YPD overnight at 30 °C. A total of 1.5 mL of culture
was inoculated into two separate 250 mL SCD liquid media [6.7 g/L
yeast nitrogen base (Difco, Detroit, MI); 6% glucose] spiked with 2.5
mL (3S)-4 (1.0 mg/mL) or 3.2 mL (3S)-4 (0.78 mg/mL), and contained
in a fermentation flask with an airlock and sidearm septum for sampling.
Duplicate ferments were conducted at 27 °C for 3 days, after which
samples were centrifuged to remove yeast and the supernatant was
stored at -20 °C prior to analysis.
RESULTS AND DISCUSSION
Tominaga et al. (12) recently reported a preparation of a
cysteinylated conjugate of 1. They treated hexenal with L-
cysteine and reduced the crude product with sodium borohy-
dride. However, there was no direct assessment of the compo-
sitionofthefinalproductobtainedotherthanbytrimethylsilylation
and GC-MS analysis. Subsequently, and independently of each
other, both Starkenmann (26) and Wakabayashi et al. (18)
conducted this same reaction and found, in both cases, that the
major product of the first step was in fact a substituted 1,3-
thiazolidine-4-carboxylic acid, with the desired 1,4-cysteine
adduct being only a minor component in the product mixture.
Under these circumstances, the observation of 1 in the enzymatic
reactions conducted by Tominaga et al. (12), using their

Synthesis of Individual Cysteine Conjugates
J. Agric. Food Chem., Vol. 56, No. 10, 2008 3761
Figure 3. Portions of 13C NMR spectra indicating diastereomeric purity of (a) front diastereomer of 8 (from left to right, the peaks correspond to C3, C4,
C2, and C7); (b) the product of treating the front diastereomer of 8 with 2.5 equiv of NaOH; and (c) the product of treating the front diastereomer of 8
with 0.95 equiv of NaOH.
Figure 4. Synthesis of racemic d10-1.
substrate, cannot be assigned unambiguously to the action of
the microbiological agent on the cysteinylated analogue. The
problem of cyclization was overcome by the use of N-Boc-
protected L-cysteine, which produced, after chromatography, a
diastereomeric mixture of ∼90% purity (26). More recently,
Luisier et al. described a synthesis of a diastereomeric mixture
of the cysteinylated analogues by addition of cysteine to ethyl
hex-2-enoate followed by reduction of the ester (27).
For our synthesis of the precursors 4 (Figure 2), we chose
to begin with commercially available L-methylcysteine hydro-
chloride (5), which we felt would give rise to products that
would be soluble in organic solvents and would therefore be
amenable to a standard purification regimen using normal phase
silica gel. In our hands, the final products proved to be of higher
purity (>95% by NMR) than those prepared earlier using more
water soluble reagents. L-Methylcysteine hydrochloride (5) was
converted into its N-Boc analogue 6 as described elsewhere (25).
Addition of 6 in a conjugate fashion to (E)-2-hexenal provided
an inseparable diastereomeric mixture of the cysteinylated
aldehydes 7. The mixture was reduced with sodium borohydride
to give a mixture of the cysteinylated alcohols 8, which were
able to be separated on silica gel. The two diastereomers were
originally designated “front”-8 and “rear”-8, in reference to
the order in which they eluted off the column: front-8 and rear-8
were subsequently shown to be (3R)-8 and (3S)-8, respectively,
after the enzyme and ferment experiments.
The esterifying methyl group in each of the two isomers of
8 was removed by treatment with sodium hydroxide in aqueous
methanol. However, the amount of base used proved to be
crucial; when treated with 2.5 equiv of hydroxide, following a
procedure from the literature (28), there was observed almost
total epimerization of the amino acid stereocenter, as indicated
in Figure 3b. This problem was overcome by the use of just
less than one full equivalent in base, as shown in Figure 3c.
Following removal of the N-Boc group, (3R)-4 and (3S)-4 (from
the front-8 and rear-8 isomers, respectively) were isolated. NMR
spectral analysis indicated that the sample of (3S)-4 contained

3762 J. Agric. Food Chem., Vol. 56, No. 10, 2008
Pardon et al.
(4) Werkhoff, P.; Guntert, M.; Krammer, G.; Sommer, H.; Kaulen,
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1-2% of the (3R)-4 diastereomer. No (3S)-4 was detected in
the sample of the (3R)-4 isomer.
To determine the absolute stereochemistry of the two isomers
of 4, solutions of each in a model medium were fermented to
dryness using a yeast strain developed in our laboratory that
yields relatively high proportions of 1 (22). The ferments were
then analyzed using an R-cyclodextrin chiral GC column and
deuterium-labeled 1 as the internal standard. Previously, Kot-
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2
seridis and Baumes described a preparation of H2-1 that they
used for their quantification method (23). However, we chose
to use a d10 analogue, which has the advantage of giving
complete baseline separation from the unlabeled analyte on both
chiral and achiral chromatography. The synthesis of d10-1 is
outlined in Figure 4. d10-Butanol (9) was oxidized under Swern
conditions (24) to produce d8-butanal (10), which, because of
its extreme volatility, was not isolated but rather obtained as a
solution in dichloromethane. This solution was then treated with
the stabilized phosphorane (carbethoxymethylene)triphe-
nylphosphorane to produce the d8-hexenoate ester 11. Addition
of thiolacetic acid proceeded as described earlier (16) to produce
d8-12, which was reduced with lithium aluminum deuteride to
give the required d10-1.
Chromatography of racemic 1 on the R-cyclodextrin column
gave good separation of the enantiomers (R ) 1.3). The first
eluting isomer was the 3R form and the second isomer, the 3S
form, of 3-MH, as shown by comparison with enantiomerically
pure samples synthesized according to published methods (21).
On treatment with Apotryptophanase enzyme, the front isomer
of 4 gave exclusively the 3R isomer of 1 (82% yield) with no
trace of the 3S enantiomer present. Conversely, the rear isomer
of 4 gave the 3S enantiomer of 1 (43%) accompanied by a trace
of the 3R form (S/R ) 98.5:1.5), reflecting the diastereomeric
purity of the starting conjugate. The same stereochemical
outcome was observed when the two isomers of 4 were added
separately to fermentations conducted by the S. cereVisiae
AWRI 1655 yeast strain, which gave 1.0 and 1.1% yields of
the 3R or 3S isomers of 1, respectively. No 1 was observed in
control samples (no fermentation). Accordingly, the front isomer
of 4 was assigned the 3R stereochemistry and the rear isomer
the 3S stereochemistry.
These results show that, under these experimental conditions
and with this particular yeast strain, there is little difference in
the efficiency of 1 release from the two isomeric forms of the
cysteine conjugates 4. The availability of the isomerically pure
samples of 4 described here will enable further studies with
different yeast strains and at various concentrations. They will
also allow the development of analytical methods for determin-
ing the concentration of each isomer in grape samples.
(16) Howell, K. S.; Swiegers, J. H.; Elsey, G. M.; Siebert, T. E.;
Bartowsky, E. J.; Fleet, G. H.; Pretorius, I. S.; de Barros Lopes,
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G. M.; Fleet, G. H.; Høj, P. B.; Pretorius, I. S.; de Barros Lopes,
M. Genetic determinants of volatile thiol release by Saccharo-
myces cereVisiae during wine fermentation. Appl. EnV. Microbiol.
2005, 71, 5420–5426.
ACKNOWLEDGMENT
(18) Wakabayashi, H.; Wakabayashi, M.; Eisenreich, W.; Engel,
K.-H. Stereochemical course of the generation of 3-mercap-
tohexanal and 3-mercaptohexanol by ꢀ-lyase-catalyzed cleavage
of cysteine conjugates. J. Agric. Food Chem. 2004, 52, 110–
116.
We thank Prof. I. S. Pretorius and Dr. M. J. Herderich for
their helpful feedback and encouragement.
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Received for review January 7, 2008. Revised manuscript received
March 11, 2008. Accepted March 15, 2008. This project is supported
by Australia’s grapegrowers and winemakers through their investment
body, the Grape and Wine Research and Development Corporation,
with matching funds from the Australian Government, and by the
Commonwealth Cooperative Research Centres Program. The work was
conducted by The Australian Wine Research Institute and Flinders
University as part of the research program of the Cooperative Research
Centre for Viticulture.
JF8000444