Hydrogen Sulfide Reactivity with Thiols in the Presence of Copper(II) in Hydroalcoholic Solutions or Cognac Brandies: Formation of Symmetrical and Unsymmetrical Dialkyl Trisulfides

Article abstract of DOI:10.1021/jf9602582

In the presence of copper(II), hydrogen sulfide reacts with thiols (methanethiol and ethanethiol) to yield symmetrical or unsymmetrical trisulfides (dimethyl trisulfide, diethyl trisulfide, and ethyl methyl trisulfide). In alcoholic beverages, these compounds are known for their nauseous character, reminiscent of onion smell, and for their low detection levels (dimethyl trisulfide: 0.1 μg/L). A mechanism for trisulfide formation is proposed that involves a two-step redox reaction.

Full text of DOI:10.1021/jf9602582

J. Agric. Food Chem. 1996, 44, 3935
−3938
3935
Hyd r ogen Su lfid e Rea ctivity w ith Th iols in th e P r esen ce of
Cop p er (II) in Hyd r oa lcoh olic Solu tion s or Cogn a c Br a n d ies:
F or m a tion of Sym m etr ica l a n d Un sym m etr ica l Dia lk yl Tr isu lfid es
Mustapha Nedjma*,† and Norbert Hoffmann‡
Laboratoire d’Œnologie and Laboratoire de Re´arrangements Thermiques et Photochimiques, Associe´ au
CNRS, UFR Sciences Exactes et Naturelles, “Moulin de la Housse”, B.P. 1039,
F-51687 Reims Cedex 2, France
In the presence of copper(II), hydrogen sulfide reacts with thiols (methanethiol and ethanethiol) to
yield symmetrical or unsymmetrical trisulfides (dimethyl trisulfide, diethyl trisulfide, and ethyl
methyl trisulfide). In alcoholic beverages, these compounds are known for their nauseous character,
reminiscent of onion smell, and for their low detection levels (dimethyl trisulfide: 0.1 µg/L). A
mechanism for trisulfide formation is proposed that involves a two-step redox reaction.
Keyw or d s: Symmetrical and unsymmetrical disulfides; symmetrical and unsymmetrical trisulfides;
copper catalysis
INTRODUCTION
alcoholic beverages. But, the origin and formation
mechanism of trisulfide are still unknown. S-Methyl-
cysteine sulfoxide is the DMTS precursor in plants such
as cabbage, turnip, or broccoli, and different mecha-
nisms of trisulfide formation have been discussed.
In Brassica and Allium plants, the action of C-S
lyases on S-methylcysteine sulfoxide has been proposed
as a mechanism of trisulfide formation by Chin and
Lindsay (1994). According to Boelens et al. (1971), these
trisulfides might arise from the reaction of disulfides
with elemental sulfur. Murayama (1970), however,
proposed that the second step of the mechanism results
from the reaction of the unstable methanesulfenic acid
with H2S, which leads to DMTS. It has also been
reported that broccoli florets stored in the absence of
oxygen produced higher amounts of DMTS (Hansen et
al., 1992). Nevertheless, S-methylcysteine sulfoxide is
not found in cognac brandies or cognacs.
In many cases, yeast cells are responsible for the
appearence and the biotransformation of sulfur com-
pounds. The presence of yeast strains causes the
formation of hydrogen sulfide (H2S), depending on the
fermentation temperature and the medium pH value
(Rankine, 1968; Eschenbruch, 1973; Vos and Gray,
1979; Wenzel and Dittrich, 1983; Monk, 1986; De Mora
et al., 1986). H2S formation is, however, also observed
during malolactic fermentation. This latter phenom-
enon remains unexplained (Monk, 1986). The large
quantities of the H2S generated in young wines after
fermentation may lead to nauseous sulfur-linked smells.
Sulfur-based phytoprotecting substances are also known
as sources of volatile sulfur compounds and, in particu-
lar, hydrogen sulfide (Eschenbruch, 1974; Wenzel et al.,
1980; Schu¨tz and Kunkee, 1979; Maujean et al., 1993;
Rauhut and Ku¨rbel, 1994).
The nucleophilic character of the sulfur atom should
cause a high reactivity of H2S with the major products
of alcoholic fermentation (ethanol, carbon dioxide), with
carbonyl compounds (aldehyde and ketone), or with
other constituents present in wines and brandies.
Because of this reactivity, H2S may be the precursor of
nauseous volatile products with lower detection levels
(Maujean et al., 1993; Rauhut et al., 1993).
Hitherto, among the dialkyl trisulfides, only dimethyl
trisulfide (DMTS) has been detected in cognac, whisky,
wine, and beer samples (Peppard, 1978; Leppa¨nen et
al., 1979). The quantities of DMTS found in cognac and
whisky samples by Leppa¨nen et al. (1979) were, respec-
tively, 1 and 5.4 µg/L. The perception level was found
by these authors to be extremely low (≈0.1 µg/L), with
a noxious onion-like smell in synthetic media [hydroal-
coholic solution containing 10% ethanol (v/v) at pH 3.2]
and in beer samples. Moreover, Ronkainen (1973)
observed trisulfide formation during the distillation of
In this study, we present results concerning the
formation of symmetrical and unsymmetrical dialkyl
trisulfides in hydroalcoholic media via the reactivity of
H2S with thiols (methanethiol and ethanethiol) in the
presence of copper (II).
EXPERIMENTAL PROCEDURES
Chromatograms were obtained by a static headspace method
with a gas chromatograph (Shimadzu GC 17A) coupled to a
chemiluminescence detector (SCD 355 from Sievers). The
separation of sulfur products was carried out on a 30 m × 0.32
mm poly(dimethylsiloxane) (4-µm film thickness) capillary
column (SPB1-sulfur from Supelco); Nedjma and Maujean,
1995). The operating conditions were as follows. Column
temperature program, 1 min step at 35 °C followed by a 10
°C/min temperature gradient to reach 55 °C and a 25 °C/min
temperature gradient to 250 °C; pressure program, 1 min step
at 82 kPa followed by a 30 kPa/min pressure gradient to reach
320 kPa; injector temperature, 115 °C. The carrier flow rate
was 3 mL/min at the beginning of the analysis. Hydrogen and
air input in the burner were, respectively, set at 100 and 40
mL/min. The oxygen pressure generator was maintained at
48 kPa (7 psig).
* Author to whom correspondence should be ad-
dressed at 20 rue du Danube, F-51100 Reims, France
(telephone (00 33) 26 05 32 30; fax (00 33) 26 05 32 79).
† Laboratoire d’Œnologie.
Two methods were used for gas volume sampling. Hamilton
Teflon luer-lock 5-mL gastight (1005 LTN) and solid-phase
microextraction (SPME; from Supelco) with a fused silica fiber
coated with a stationary phase [poly(dimethylsiloxane), 100
µm o.d.) to adsorb volatile and semivolatile sulfur compounds.
‡ Laboratoire de Re´arrangements Thermiques et Pho-
tochimiques.
S0021-8561(96)00258-0 CCC: $12.00
© 1996 American Chemical Society

3936 J. Agric. Food Chem., Vol. 44, No. 12, 1996
Nedjma and Hoffmann
1
The H and 13C NMR spectra were recorded in CDCl3 with
Ta ble 1. Con cen tr a tion of Tr a n sition Meta ls in Cogn a c
Br a n d y a n d Cogn a c
a Bruker AC 250 spectrometer.
Rea gen ts a n d Ch em ica ls. Normapur ethanol (analytical
grade), L(+)-tartaric acid (TH2), and potassium hydrogeno-
tartrate (THK) from Prolabo were used. Only distilled and
deionized water (resistivity equal to 18.2 MΩ/cm) were used.
Ethanethiol and gaseous methanethiol were obtained from
Aldrich. Sodium sulfide from Merck was dissolved in aqueous
solution buffered at pH 3.2 (TH2/THK) to generate H2S. The
chloroform used for the preparation of the standards was
distilled from calcium hydride (CaH2). All of these compounds
were at least 97% pure.
concentration (mg/L)
beverage
cognac brandy 0.5-3 < 0.1
cognac 0.3-5 0.01-0.65 0.01-0.45 0.01-1.00
copper
iron
manganese
zinc
0.03-0.06 0.01-0.22
P r ep a r a tion of Sta n d a r d s. Solution A. Hydroalcoholic
solution contained 20% ethanol (v/v) was buffered at pH 3.2
(wine pH) by adding TH2 (3 g/L) and THK (3 g/L).
Trisulfides Formation. The solutions of H2S, methanethiol,
and ethanethiol were prepared according to the method of
Nedjma et al. (1995).
A 250-mL flask was filled with 200 mL of the solution A
containing copper chloride (CuCl2‚2H2O, 500 µg/L). This
solution was stored at 4 °C overnight. Then, 500 µL of the
solutions of 4 × 10-2 g/L of H2S, methanethiol, or ethanethiol
were added in different conbinations to this medium. The
flask containing the resulting mixture was then hermetically
capped with silicone rubber septa (sleeve stoppers), and the
mixture was allowed to warm to room temperature. The
reaction was monitored by gas chromatography. The products
of the reaction were identified by comparison with authentic
standards that were synthesized according to Capozzi et al.
(1989a,b).
Ethyl Methyl Disulfide (EMDS). A solution of 500 mg (3.25
mmol) of S-ethyl ethyl thiosulfonate and 902 mg of (methyl-
thio)trimethylsilane (7.5 mmol) in 20 mL of chloroform was
heated under reflux until there was total conversion of the
initial compound (2 h). The solvent was eliminated by distil-
lation. The residue was chromatographed by thin-layer chro-
matography (TLC; petroleum ether/AcOEt at 95/5) to provide
122 mg (35%) of EMDS: 1H NMR (CDCl3 , 250 MHz) δ 1.4
(3H, t, J ) 7 Hz), 2.4 (3H, s), 2.7 (2H, q, J ) 7 Hz); 13C NMR
14.43 (CH2), 22.1 (CH3), 32.84 (CH3); GC; tR ) 6.28 min.
Dimethyl Trisulfide (DMTS). First, 1.36 g (7.64 mmol) of
bis(trimethylsilyl) sulfide was added to a solution of 840 mg
(7.64 mmol) of S-methyl methylthiosulfonate in 20 mL of
chloroform. The reaction mixture was then heated and
refluxed for 16 h. The solvent was removed by distillation
(atmospheric pressure). The crude product was purified by
TLC (petroleum ether/AcOEt at 95/5) to give 625 mg (65%) of
pure DMTS: 1H NMR (CDCl3 , 250 MHz) δ 2.6 (6H, s); 13C
NMR δ 22.62 (2 CH3); GC tR ) 7.39 min.
F igu r e 1. Chromatogram obtained after 24 h of reaction at
room temperature in the absence of copper(II). (1) H2S; (2)
MeSH; (3) EtSH; (4) DMDS.
Sch em e 1. Su lfu r P r od u cts Obta in ed fr om th e
Rea ction of H2S w ith Th iols in th e P r esen ce of
Cop p er (II) (* In d ica tes P r op osed Str u ctu r e)
Diethyl Trisulfide (DETS). The preceding procedure was
also used for the preparation of DETS. First, 1 g (6.49 mmol)
of S-ethyl ethylthiosulfonate and 1.18 g (6.49 mmol) of bis-
(trimethylsilyl)sulfide were converted. Flash chromatography
with petroleum ether/AcOEt (95/5) gave 718 mg (72%) of pure
DETS: 1H NMR (CDCl3, 250 MHz) δ 1.35 (6H, t, J ) 7 Hz);
2.9 (4H, q, J ) 7 Hz), 13C NMR δ 14.3 (2 CH2), 32.6 (2 CH3);
GC tR ) 8.62 min.
methanethiol leads to DMDS and DMTS. Reaction with
ethanethiol under the same conditions leads to DEDS
and DETS.
If methanethiol and ethanethiol were treated under
these conditions, symmetrical and unsymmetrical di-
sulfides and trisulfides were formed, whereas no reac-
tion was observed when copper was absent (Figure 1).
Among these products, we have identified two sym-
metrical disulfides (DMDS and DEDS), two symmetrical
trisulfides (DMTS and DETS), and one unsymmetrical
disulfide [ethyl methyl disulfide (EMDS)]. The chro-
matographic behavior of the remaining unidentified
compound indicates it is ethyl methyl trisulfide (EMTS;
Scheme 1).
Different modifications of the conditions were tested
to increase the formation of trisulfides from H2S, thiols,
and copper. The optimal conditions were obtained with
5 equivalents of copper(II), 1 equivalent of thiols, and 5
equivalents of H2S. Under these conditions and after
RESULTS AND DISCUSSION
In beer, the concentration of transition metals are
generally in the range 0.01-1.6 mg/L (Binns et al., 1978;
Hough et al., 1982). The presence of copper and H2S
leads to copper sulfide (CuS) precipitation (Thorne et
al., 1970). Thus, the nauseous hydrosulfurous smells
are diminished in beer. In the case of cognac brandies
and cognacs, the copper concentration is also the most
important one (Table 1). The concentration of these
metals are even higher and exceed the concentration of
sulfur products, in particular thiols (methanethiol and
ethanethiol) that are in the µg/L range. Our results
obtained with hydroalcoholic solutions and cognac bran-
dies show that after 24 h at room temperature, the
reaction of H2S in the presence of copper(II) with

Dialkyl Trisulfides from H2S in Hydroalcoholic Solutions and Cognac Brandies
J. Agric. Food Chem., Vol. 44, No. 12, 1996 3937
F igu r e 4. Gas chromatogram of disulfides and trisulfides
obtained from the reaction of H2S with thiols in the presence
of copper sulfate by the gas sample method. (2) MeSH; (3)
EtSH: (4) DMDS; (5) EMDS; (6) DEDS; (7) DMTS; (8) EMTS;
(9) DETS.
F igu r e 2. Gas chromatogram of disulfides and trisulfides
obtained from the reaction of H2S with thiols in the presence
of copper chloride by the gas sample needle method. (4) DMDS;
(5) EMDS; (6) DEDS; (7) DMTS; (8) EMTS; (9) DETS.
Sch em e
2.
Tr isu lfid e
F or m a tion
Step
fr om
S-Meth ylcystein e Su lfoxid e â-Elim in a tion (Mu r a ya m a ,
1970; P ep p a r d , 1978)
because of the low adsorption of thiols and H2S. In this
reaction, copper appears to be an absolute requirement
for formation of disulfides and trisulfides. We also
showed that when H2S is increased from 1 to 5 equiva-
lents, with methanethiol and ethanethiol (at 1 equiva-
lent each and in the presence of 5 equivalents of
copper(II), trisulfide formation becomes the major reac-
tion. The reaction yield is lower when copper chloride
(CuCl2‚2H2O) is replaced by copper sulfate (CuSO4) at
the same concentration (Figure 4). Indeed, an incom-
plete conversion was observed.
Murayama (1970) proposed a mechanism for DMTS
formation in beer from S-methylcysteine sulfoxide â-
elimination during the cooking of several brassicaceous
vegetables leading to methanesulfenic acid, an unstable
intermediate product. This intermediate reacts with
H2S giving DMTS (Scheme 2). It has also been observed
that the presence of sulfur dioxide in the reaction
medium diminishes the S-methylcysteine sulfoxide con-
centration (DMTS-precursor; Peppard, 1978). Muraya-
ma’s mechanism cannot be envisaged in cognac brandies
or cognacs because of the absence of S-methylcysteine
sulfoxide. Moreover, we observed trisulfide formation
in hydroalcoholic solutions without this compound. The
mechanism presented in Scheme 3 appears to be the
most reasonable one. This mechanism is based on the
two coupled redox reactions, where copper is the oxi-
dant. However, the hydroalkyl disulfide intermediate
has not been found by chromatographic analysis, per-
F igu r e 3. Gas chromatogram of disulfides and trisulfides
obtained from the reaction of H2S with thiols in the presence
of copper chloride by the SPME method. (4) DMDS; (5) EMDS;
(6) DEDS; (7) DMTS; (8) EMTS; (9) DETS.
24 h at room temperature, no H2S and thiols were
observed (Figure 2). These optimized conditions could
be used in synthetic organic chemistry for the prepara-
tion of trisulfides.
Two chromatograms obtained by two different sam-
pling techniques (gas needle and SPME) are displayed
in Figures 2 and 3. With SPME, the responses of
disulfides and trisulfides increased. However, this
technique is not suitable for the follow-up of reactions

3938 J. Agric. Food Chem., Vol. 44, No. 12, 1996
Nedjma and Hoffmann
Sch em e 3. P r op osed Mech a n ism for th e F or m a tion of
Sym m etr ica l or Un sym m etr ica l Tr isu lfid es
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making. Am. J . Enol. Vitic. 1974, 25, 157-161.
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during the fermentation of grape must by Saccharomyces
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L. C. Broccoli storage under low-oxygen atmosphere: iden-
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haps because of its low stationary concentration.
A
radical mechanism has to be considered. Further
experimental study is needed to verify the validity of
each step of the proposed mechanism.
CONCLUSION
The results reported here give new insight into the
origin of symmetrical and unsymmetrical trisulfides and
their mechanism of formation in cognac brandies.
Indeed, for the first time, it has been shown that
trisulfide formation was possible from the reaction of
H2S with thiols in the presence of copper(II). Concern-
ing preparative organic chemistry, these observations
may lead to a new method of synthesis of trisulfides.
Further studies in this field are in progress in our
laboratory.
ABBREVIATIONS USED
DMDS, dimethyl disulfide; EMDS, ethyl methyl di-
sulfide; DEDS, diethyl disulfide; DMTS, dimethyl trisul-
fide; EMTS, ethyl methyl trisulfide; DETS, diethyl
trisulfide; GC, gas chromatography; SCD, sulfur chemi-
luminescence detector.
ACKNOWLEDGMENT
The study was performed in the laboratory of Profes-
sor Alain Maujean, who is gratefully acknowledged. We
are grateful to Alexa Thille for English corrections.
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Wenzel, K.; Dittrich, H. H.; Seyffardt, H. P.; Bohnert, J .
Schwefelru¨cksta¨nde auf Trauben und im Most und ihr
Einfluâ auf die H2S-Bildung. Wein Wissenscaft 1980, 35,
414-420.
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Received for review April 15, 1996. Revised manuscript
received September 10, 1996. Accepted September 11, 1996.X
This work was supported by the Ministe`re de l’Agriculture,
R. Cantagrel, and J . P. Vidal of BNIC (Bureau National
Interprofessionnel de Cognac).
J F9602582
X Abstract published in Advance ACS Abstracts, No-
vember 1, 1996.