Identification and Organoleptic Contribution of Vanillylthiol in Wines

Article abstract of DOI:10.1021/acs.jafc.5b05733

Vanillylthiol, a chemical compound reminiscent of clove and smoke, has been identified for the first time in young red and dry white wines. The chemical structure of this new aroma was confirmed by original chemical synthesis. Vanillylthiol was prepared by a two-step procedure from vanillin. The conversion of vanillin to divanillyl disulfide was easily achieved by treatment with an inorganic sulfur-donor reagent. Reduction of the disulfide gave the target thiol in good yield. The quantification of vanillylthiol in wine was performed by nonspecific liquid/liquid extraction (CH₂Cl₂), separation of the volatile compounds using gas chromatography, and specific detection using tandem mass spectrometry (triple quadrupole). Vanillylthiol was found particularly in young wines aged in new oak barrels. These wines contained between a few 50 ng/L to more than 8300 ng/L. The highest levels were found in red wines aged 12 months in new oak barrels. Given its perception threshold in a wine model solution (3.8 μg/L), vanillylthiol may contribute to the spicy, clove-like flavor of red wines aged in oak barrels.

Full text of DOI:10.1021/acs.jafc.5b05733

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Article
Identification and organoleptic contribution of vanillylthiol in wines
Alexandre Pons, Morgan Floch, Svitlana SHINKARUK, and Philippe Darriet
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05733 • Publication Date (Web): 23 Jan 2016
Downloaded from http://pubs.acs.org on January 24, 2016
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Page 1 of 31
Journal of Agricultural and Food Chemistry
Identification and Organoleptic Contribution of
Vanillylthiol in Wines
Morgan Flocha,d, Svitlana Shinkarukb,c, Philippe Darrieta ,
Alexandre Ponsa,d
a
Univ. Bordeaux, ISVV, EA 4577, Unité de recherche OENOLOGIE, Fꢀ33882 Villenave
d’Ornon, France
b
Univ. Bordeaux, CNRS, UMR 5255 ISM, 351 Cours de la Libération, Fꢀ33405 Talence
Cedex, France
c Bordeaux Sciences Agro, 1 Cours du Général de Gaulle, Fꢀ33175 Gradignan, France
d Seguin Moreau France, Z.I. Merpins, B.P. 94, Fꢀ16103 Cognac, France
Corresponding author: Alexandre Pons
alexandre.pons@uꢀbordeaux.fr
Tel : (0) 5 57 57 58 67
Fax : (0) 5 57 57 58 13
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Abstract
Vanillylthiol, a chemical compound reminiscent of clove and smoke, has been identified for
the first time in young red and dry white wines. The chemical structure of this new aroma was
confirmed by original chemical synthesis. Vanillylthiol was prepared by twoꢀstep procedure
from vanillin. The conversion of vanillin to divanillyl disulfide was easily achieved by
treatment with inorganic sulfurꢀdonor reagent. Reduction of the disulfide gave the target thiol
in good yield. The quantification of vanillylthiol in wine was performed by nonꢀspecific
liquid/liquid extraction (CH2Cl2), separation of the volatile compounds using gas
chromatography and specific detection using tandem mass spectrometry (triple quadrupole).
Vanillylthiol was found particularly in young wines aged in new oak barrels. These wines
contained between a few 50 ng/L to more than 8300 ng/L. The highest levels were found in
red wines aged 12 months in new oak barrels. Given its perception threshold in a wine model
solution (3.8 µg/L), vanillylthiol may contribute to the spicy, cloveꢀlike flavor of red wines
aged in oak barrels.
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Keywords: wine, aroma, thiol, oak barrel, aging
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INTRODUCTION
The use of oak wood is widely recognized as helpful for increasing the intrinsic quality of
wines. Contact with wood can occur during winemaking and aging or only during aging or
maturation. Indeed, the production of great wines often involves maturation in wood barrels.
During barrelꢀaging, wine flavor and taste are considerably modified by compounds released
directly from oak wood. Oak nonꢀvolatile compounds are nonꢀflavonoid polyphenols that
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include ellagitannins, phenolic acids, coumarins and polymeric compounds, some of which
1ꢀ3
contribute to the perception of astringency and bitterness.
Very recently, lignans such as
lyoniresinol have been shown to contribute to the bitterness taste of oakꢀmatured wines.4
Moreover, Marchal et al.5 identified new triterpenoids extracted from oak wood Quercus
petraea which can contribute to the increase in wine sweetness observed during oak aging. 6
Wines aged in oak barrels are marked by a complex mix of nuances reminiscent of coconut,
wood, vanilla, spice and toasted. Although hundreds of oakꢀderived volatile compounds have
been identified and are released during the contact between wine and oak wood, only some of
them are really involved in these nuances. For many years, the identification and quantitation
of odorant compounds released during barrel aging have received much attention. Vanillin
(vanilla), βꢀmethylꢀγꢀoctalactone (coconut), volatile phenols (spicy) and 2ꢀfuranmethanethiol
or furfurylthiol (FFT, toasted) are now considered to be the key molecules associated with
oak aging in barrels7, 8 and with oak chips.9 Volatile substance content is strongly affected by
natural factors as well as by botanical species (Quercus robur, Quercus petraea, Quercus
alba), geographical origins and cooperage techniques such as seasoning and toasting (also
called hydrothermolysis). Many other odorants are also associated directly or indirectly with
oak wood aging, thus increasing the complexity of wines aged in oak wood barrels.10ꢀ13
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These molecules of different chemical nature can be native in oak heartwood or appear during
the cooperage process (oak seasoning and toasting). When contact with oak occurs during
alcoholic fermentation, yeast metabolism may modify the intensity of “oak wood” flavor.14
For example, odorant vanillin can be reduced in flavorless vanillic alcohol. On the other hand,
yeast metabolism gives rise to new very odorant compounds, as evidenced by Blanchard.15
Following on from Tominaga8 who first identified FFT which is reminiscent of coffee in
wines, he demonstrated that furfural was a possible precursor of FFT. Furfural, which
presents a very high perception threshold (Table 1) is released by toasted wood when it comes
into contact with wine. In white wines, FFT was present only in barrelꢀfermented wines. The
formation of FFT by yeast in the presence of furfural increased when the conditions were
favorable for the production of HSꢀ anions in excess of the amounts required for protein
synthesis: high sulfur content (cysteine, sulfates) and relatively low nitrogen levels.15
It is now widely accepted that understanding the contribution of thiol compounds is essential
for investigating wine flavor at the molecular level. Indeed, compounds responsible for the
fruity character of young Sauvignon Blanc wines and others such as Petite Arvine, Semillon,
Petit and Gros Manseng, Koshu, have been identified as volatile thiols.16ꢀ18 The three most
important thiols are thought to be 3ꢀsulfanylhexanol (3SH, grapefruit/passion fruit), 3ꢀ
sulfanylhexyl acetate (3SHA, grapefruit/passion fruit) and 4ꢀmethylꢀ4ꢀsulfanylpentanꢀ2ꢀone
(4MSP, box tree and broom). More recently, other thiols such as 3ꢀsulfanylpentanꢀ1ꢀol,
3ꢀsulfanylheptanꢀ1ꢀol which evoke citrusꢀlike note, and the 2ꢀmethylꢀ3ꢀsulfanylbutanꢀ1ꢀol
reminiscent of raw onion, were identified in Sauternes wines.19 Absent from must, they were
found in wine after alcoholic fermentation and their concentrations were much higher when
Botrytis cinerea had developed on the grapes.
Other volatile thiols have also been identified in dry white wines and old champagnes20;
ethylꢀ3ꢀthiopropionate and benzenmethanthiol (smoke). More recently, a new odoriferous
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thiol associated with an unpleasant odor, ethyl 2ꢀsulfanylacetate (baked beans), was identified
in dry wine.21 Unlike other varietal thiols, it appears in wine during bottle aging and its
concentrations are related to oxygen levels during the preꢀfermentative procedures of
vinification.
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4ꢀHydroxyꢀ3ꢀmethoxybenzenemethanthiol (Vanillylthiol) is a sulfur organic compound (2)
described for the first time in 1949 by Kipnis.22 Its structure was validated with no convincing
instrumental results compared to the current analytical tools. Moreover, the presence of
vanillylthiol in wine and in general in food and beverages has never been reported. This paper
describes the synthesis, identification and a methodology for the quantitation of vanillylthiol
in red and white wines made with different grapes.
88 Materials and methods
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Chemicals. 4ꢀHydroxyꢀ3ꢀmethoxybenzaldehyde (Vanillin, 98 %) and sodium hydrogen
sulfide (98 %) were obtained from Alfa Aesar (Bischheim, France). Anhydrous sodium
sulfate (99 %), Ethanol (HPLC grade), dichloromethane (Rectapur grade) diethylether and
pentane were supplied by Prolabo (France). Lithium aluminum hydride (97 %), thiourea (99
%) and 3,4ꢀdimethylphenol (99 %) were purchased from SigmaꢀAldrich Chemicals (Lyon,
France). All organic solvents and inorganic salts used for organic synthesis were also
purchased from SigmaꢀAldrich Chemicals (Lyon, France). Acetoneꢀd6 was obtained from
EurisoꢀTop (SaintꢀAubin, France). L(+) tartaric acid (99.5 %) was from Fluka (France).
Tetrahydrofuran was dried by refluxing a solution containing sodium wires and
benzophenone under nitrogen and distilled immediately before use. The solution of sodium
hydrogen sulfide was prepared with distilled water immediately before use. All moistureꢀ
sensitive reactions were carried out in an argon atmosphere.
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Wines. A Sauvignon Blanc batch from the 2014 vintage was selected for this experiment.
After pressing of the grapes (pneumatic press, Vaslin Bucher, France), the must (25 HL) was
settled at 200 NTU (nephelometric turbidity unit) and inoculated with Saccharomyces
cerevisiae Zymaflore X5 strain (100 mg/L; Laffort SA, Floirac, France) and the assimilable
nitrogen content was adjusted to 200 mg/L. When one third of the alcoholic fermentation had
been completed, the must was placed in three barrels (225 L): two barrels made of new oak,
two 1ꢀyearꢀold oak barrels and two 2ꢀyearꢀold oak barrels. The barrels were made from
French oak wood (Quercus petraea) that was chosen by the cooperage (Seguin Moreau,
Cognac) as representative of its production. After the end of alcoholic fermentation, the wines
were aged on total lees (7 months). The level of free sulfur dioxide was maintained at 30
mg/L during the overall experiment to avoid oxidation and malolactic fermentation. Wine
samples were taken from each barrel at the end of alcoholic fermentation and during aging
where vanillylthiol were assayed.
Moreover, several red and white wines aged or not in oak barrels were provided from
wineries located in several appellations of Bordeaux and Burgundy.
Synthesis. Divanillyl disulfide (1). A solution of sodium hydrogen sulfide (10.8 g, 210 mmol)
in water (90 mL) was added dropwise to a solution of vanillin (5.0 g, 33 mmol) in ethanol (25
mL) at room temperature and stirred overnight. The mixture was then heated at 60°C for 3h,
cooled to room temperature and the resulting precipitate was filtered to give the
corresponding disulfide in 30 % yield as an odorless white powder (1.62 g; Rf= 0.52, Diethyl
ether/Pentane: 80/20). FDꢀMS (+): m/z 338.0647 for [M]+ (calculated for [C16H18O4S2]+,
338.0646) MS (EI, 70 eV) m/z (%) 170 (41), 138 (25), 137 (100), 136 (24), 122 (37), 107
1
(31), 106 (20), 105 (19), 94 (27), 78 (20), 77 (14), 65 (26), 66 (11). H NMR (Acetoneꢀd6) δ
6.88 (d, J = 1.5 Hz, 1H, CHꢀ2), 6.79 (d, J = 8.0 Hz, 1H, CHꢀ5), 6.76 (dd, J = 8.0 Hz, 1.5H,
CHꢀ6), 3.84 (s, 3H, CH3), 3.67 (s, 2H, CH2). 13C NMR (Acetoneꢀd6) δ 148.3 (CHꢀ4), 147.1
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(CHꢀ3), 129.5 (CHꢀ1), 123.1 (CHꢀ6), 115.7 (CHꢀ5), 113.8 (CHꢀ2), 56.3 (CH3), 43.7 (CH2). IR
(solid, KBr) cmꢀ1 3450 (s), 2979 (m), 1611 (s), 1510 (s), 1463 (s), 1436 (s), 1383 (s), 1368 (s),
1302 (s), 1263 (s), 1233 (s), 1150 (s), 1116 (s), 1028 (s).
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Vanillyl thiol (4ꢀHydroxyꢀ3ꢀmethoxybenzenemethanthiol, 2). A solution of divanillyl disulfide
1 (552 mg, 1.6 mmol) in dry tetrahydrofuran (20 mL) was added to a suspension of lithium
aluminum hydride (354 mg, 9.3 mmol) in dry tetrahydrofuran (20 mL) at ꢀ78°C under argon
atmosphere. The reaction was allowed to warm to room temperature for 12 h and then cooled
again (ice bath). The reaction was carefully quenched with iceꢀcold saturated aqueous
ammonium chloride solution (40 mL) and pH was adjusted to pH 2ꢀ3 with 1 M hydrochloric
acid. The resultant mixture was extracted with ethyl acetate (3 x 50 mL). The combined
organic phases were washed with saturated sodium chloride solution (2 x 50 mL), dried over
magnesium sulfate, filtered, and concentrated under reduced pressure to give the title
compound as a colorless oil (476 mg, 86 % yield, Rf=0.72, Diethyl ether/Pentane: 80/20). The
purity of the compound obtained, which was determined by GCꢀMS and 1H NMR, was > 95 %.
This compound was thus used without purification. FDꢀMS (+): m/z 170.0408 for [M]+
(calculated for [C8H10O2S]+, 170.0401). MS (EI, 70 eV) m/z (%) 170 (17), 137 (100), 138
1
(10), 122 (16), 107 (2), 94 (9), 78 (3), 65 (5), 66 (2). H NMR (Acetoneꢀd6) δ 7.51 (s, 1H,
OH), 6.95 (d, J = 1.4 Hz, 1H, CHꢀ2), 6.79 (dd, J = 8.0, 1.4 Hz, 1H, CHꢀ6), 6.75 (d, J = 8.0
Hz, 1H, CHꢀ5), 3.83 (s, 3H, CH3), 3.69 (d, J = 7.4 Hz, 2H, CH2), 2.10 (t, J = 7.4 Hz, 1H, SH).
13C NMR (Acetoneꢀd6) δ 148.2 (CHꢀ3), 146.3 (CHꢀ4), 133.8 (CHꢀ1), 121.4 (CHꢀ6), 115.6
(CHꢀ5), 112.5 (CHꢀ2), 56.2 (CH3), 28.9 (CH2). IR (liquid) cmꢀ1 3436 (s), 2963 (m), 2937 (s),
2842 (s), 2563 (s), 1726 (s), 1606 (s), 1513 (s), 1463 (s), 1432 (s), 1370 (s), 1271 (s), 1232
(s), 1152 (s), 1121 (s), 1032 (s).
Reaction of vanillyl alcohol with thiourea.23 A solution of vanillyl alcohol 3 (2.87 g, 18.6
mmol) in dry tetrahydrofuran (20 mL) was added to a solution of thiourea (1.5 g, 19.7 mmol)
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in 2 M hydrochloric acid (15 mL) at room temperature. The reaction was heated at 55 °C for 1
h and stirred at room temperature for 12 h. The resulting mixture was then treated with a 3 M
NaOH solution (11 mL) and heated at 50 °C for 4 h. The reaction mixture was cooled to
room temperature and then extracted with diethyl ether (3 x 50 mL). The pooled organic
phases were washed with brine (3 x 50 mL), dried over magnesium sulfate, and filtered. The
solvent was removed in vacuo to give a mixture of four compounds as a yellow oil (1.2 g).
The crude was purified by flash chromatography by using a polarity elution gradient
(Pentane/Et2O: 7/3 to 4/6).
Divanillyl sulfide 4 (white solid, 0.584 g, 44 % yield, Rf = 0.54, Diethyl ether/Pentane:
80/20). FDꢀMS (+): m/z 306.0934 for [M]+ (calculated for [C16H18O4S]+, 306.0925). MS (EI,
70 eV) m/z (%) 306 (40), 170 (25), 138 (53), 137 (100), 122 (29), 107 (23), 94 (19), 78 (10),
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65 (14). ESI (ꢀ): [MꢀH]ꢀ = 305.1; TOF (+): [M+Na]+ = 329.1; FDꢀMS (+): [M]+.= 306.1. H
NMR (Acetoneꢀd6) δ 7.48 (s, 1H, OH), 6.89 (s, 1H, CHꢀ2), 6.76 (m, 2H, CHꢀ5, CHꢀ6), 3.82
(s, 3H, CH3), 3.59 (s, 2H, , CH2). 13C NMR (Acetoneꢀd6) δ 148.3 (CHꢀ4), 146.5 (CHꢀ3),
130.7 (CHꢀ1), 122.5 (CHꢀ6), 115.4 (CHꢀ5), 113.2 (CHꢀ2), 56.2 (CH3), 36.2 (CH2). IR (solid,
KBr) cmꢀ1 3497 (s), 3439 (s), 3061 (m), 3034 (m), 2983 (s), 2950 (s), 2917 (s), 2843 (s), 1605
(s), 1513 (s), 1460 (s), 1446 (s), 1428 (s), 1358 (s), 1267 (s), 1227 (s), 1206 (s), 1157 (s),
1140 (s), 1113 (s), 1020 (s).
Bis(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)methane 5 (white solid, 0.284 g, 15 % yield, Rf = 0.55,
Diethyl ether/Pentane: 80/20). FDꢀMS (+): m/z 260.284 for [M]+ (calculated for [C15H16O4]+,
260.2851). MS (EI, 70 eV) m/z (%) 261 (62), 260 (100), 243 (40), 229 (100), 213 (30), 197
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(20), 185 (28), 157 (18), 137 (50), 124 (25), 107 (23), 94 (19), 77 (14), 65 (14). H NMR
(Acetoneꢀd6) δ 7.34 (s, 2H, OH), 6.82 (d, J = 1.8 Hz, 2H, CHꢀ2), 6.73 (d, J = 8.0 Hz, 2H, CHꢀ
5), 6.65 (dd, J = 8.0, 1.8 Hz, 2H, CHꢀ6), 3.78 (s, 8H, CH3, CH2). 13C NMR (Acetoneꢀd6) δ
148.2 (CHꢀ4), 145.6 (CHꢀ3), 134.2 (CHꢀ1), 122.0 (CHꢀ6), 115.5 (CHꢀ5), 113.2 (CHꢀ2), 56.2
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(CH3), 41.6 (CH2). IR (solid, KBr) cmꢀ1 3433 (s), 3052 (m), 3027 (m), 2961 (s), 2936 (s),
2904 (s), 2836 (m), 1604 (s), 1515 (s), 1454 (s), 1427 (s), 1374 (s), 1318 (m), 1265 (s), 1233
(s), 1206 (s), 1183 (s), 1142 (s), 1114 (s), 1035 (s), 1018 (s).
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Extraction. Solvent extraction. 50 mL of wine sample were spiked with 50 µL of 3,4ꢀ
dimethylphenol (10 mg/L, EtOH) as internal standard. The wines were also extracted at room
temperature with 10 mL of CH2Cl2 followed by 5 mL of CH2Cl2 under magnetic stirring (10
and 5 min at 700 rpm, respectively). Organic phases obtained were blended, dried over
anhydrous sodium sulfate, and concentrated to 500 µL under a nitrogen stream. The sample
was stable 5 days at ꢀ20°C.
SPME experiments. 65 ꢁm SPME fibers coated with Carboxen/Polydimethylsiloxane
(Carboxen/PDMS, StableFlex, Supelco) and Divinylbenzene/Carboxen/ Polydimethylsiloxane
(DVB/Carboxen/PDMS, StableFlex, Supelco) were used, and the extraction procedure was
carried out in the gas phase of the SPME vial without immersion in the sample solution, i.e.,
by HSꢀSPME method. During analysis of vanillylthiol by SPME, 10 mL of the wine sample
was injected into a 20 mL SPME vial. The SPME efficiency was evaluated using the
following parameters: (a) extraction temperature, 40 and 60 °C; (b) extraction time, 20 and 50
min; (c) ammonium sulfate addition, 0 and 5 g; (d) dilution factor 1 and 10.
Instrumentation. The chromatographic system comprised a gas chromatograph Trace GC
Ultra (Thermo Electron, USA) coupled to a triple quadrupole mass spectrometer TSQ
Quantum XLS operated in EI mode. The GC system was equipped with a Triplus RSH autoꢀ
sampler.
Gas chromatography method. GC separation was carried out on a nonꢀpolar ZBꢀ1MS
capillary column (100 % polydimethylsiloxane, 60 m; 0.25 mm; 1 µm) from Phenomenex
(France). The carrier gas was He (Linde gas, Bordeaux), 5.3 grade, with a flow rate of 1
mL/min. A 1 µL sample was injected via the autosampler into a split/splitless programmableꢀ
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temperature injector (closure time: 1 min, split flow 30 mL/min) and set as follows: 0.5 min at
230 °C and increasing to 250 °C at 14 °C/min and kept at that temperature for 10 min. Oven
temperature was initially set at 50 °C, held for 1 min, then increased to 160 °C at 6 °C/min,
increased to 222 °C at 3 °C/min and finally kept at 260 °C at 20 °C/min for 10 min. The MS
transfer line temperature was set at 250 °C.
Mass spectrometry. The mass spectrometer was used in electron impact mode (EI). Source
parameters were optimized as follows: source temperature, electron energy and emission
current were set at 230 °C, 70 eV and 30 µA, respectively. The mass spectrometer was
operated in Selected Reaction Monitoring (SRM) mode using argon as collision gas in cell
collision (1.5 mTorr). SRM conditions were optimized, as well as collision energy and gas
pressure values applied to each precursor ion and were dependent on each transition. PFTBA
(Perfluorotriꢀnꢀbutylamine) was used for mass calibration. Data acquisition and analyses were
performed using the Xcalibur software version 2.1 supplied by the manufacturer.
To determine the retention times of the analytes and their characteristic mass fragments, the
primary EI mass spectra were recorded in fullꢀscan mode (m/z 45–250). For selected reaction
monitoring (SRM) experiments, precursor ions were selected according to abundance and
product ion scans were investigated using the energy ramp function from 5 to 25 V. We also
repeated the analysis at different argon collision gas pressures (0.5, 1, 1.5 mTorr). SRM
conditions were set to a mass resolution of full width at half maximum height (FWHM) in Q1
and Q3 with a value of 0.7 Da and a scan time of 0.1 s. Collision energy was systematically
set to 10 V. The transitions were chosen with respect to optimized signalꢀtoꢀnoise (S/N) ratios
as well as absolute ion intensities for a standard solution of the analytes at 50 µg/L.
Vanillylthiol was monitored at m/z 170 → 137 (quantifier) and m/z 137 → 94 and 137 →
122 (qualifier). As the internal standard was detected in selected ion monitoring (SIM, m/z =
107), analyses were performed in two segments: IS (seg. 1), vanillylthiol (seg. 2). For
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quantitation, peak area ratios of the analytes and the IS were calculated as a function of the
concentration of the substances. Linear retention indices (LRI) were obtained by simultaneous
injection of samples and a series of alkanes (C7ꢀC23), detection was performed in scan mode.24
Validation of the method. The calibration curves were established between 0.02 and 20 ꢁg/L
of vanillylthiol in a white wine (8 points). The limit of detection (LOD) was defined as the
concentration which gave a signalꢀtoꢀnoise ratio (S/N) of 3. The limit of quantitation (LOQ)
was defined as the concentration which gave an S/N ratio of 10. These parameters were
experimentally calculated from the S/N obtained in wine sample analyses. Accuracy of the
method was assessed using replicates spiked at one level. Five replicate analyses were
performed for the determination of relative standard deviation (RSD).
Determination of detection thresholds. The detection threshold of vanillylthiol in model
solution (12 % ethanol, 5 g/L tartaric acid, pH 3.5) and wines was determined by using a
threeꢀalternative forced choice presentation (3ꢀAFC). The solutions were presented in glasses
corresponding to AFNOR (Association Française des Normes) standards. The odor detection
threshold corresponded to the minimum concentration below which 50 % the tasters
statistically failed to recognize the difference from the control.25 The sensory panel consisted
of 20 students and researchers (half of male and female) between 20 and 40 years old, from
the ISVV.
General features. Magnetic Resonance Spectroscopy (NMR): 1H and 13C NMR spectra were
recorded on a Bruker ACꢀ300 FT (1H: 300.13 MHz, 13C: 75.4 MHz), spectra referenced using
the lock frequency of deuterated solvent. Chemical shifts (δ) and coupling constants (J) are
expressed in ppm and Hz, respectively. Infrared (IR) spectra were recorded with a Perkin–
Elmer paragon 1000 FTꢀIR spectrophotometer in the 4000ꢀ600 cmꢀ1 range. Field desorption
mass spectra (FDꢀMS) were performed by the CESAMO (University of Bordeaux, France).
The measurements were carried out on a TOF mass spectrometer AccuTOF GCv by JEOL
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(Croissy sur Seine, France) using an FD emitter with an emission voltage of 10 kV. One to
two microliters of the compound solution in methanol were deposited on a 13ꢀmm emitter
wire. Thinꢀlayer chromatography (TLC) was performed on 60F TLC plates: thickness 0.25
mm, particle size 10 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
sodium nitroprussiate (1.5% in MeOH/NH3/HCl).
257 Results and discussion
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Chemical synthesis. Vanillylthiol was mentioned in 1949 by Kipnis et al.22, who studied the
preparation of aromatic and heterocyclic thiols by the interactions of aldehydes with hydrogen
disulfide. This was the only time when vanillylthiol was reported and it was characterized
simply by elemental analysis and refraction index, methods that are clearly insufficient for
strict structural identification.
In the current literature, thiols are classically synthesized in two steps via the formation of a
reactional intermediate either by addition to a multiple bond of thiol derivatives (RSH, ArSH,
RCOSH)19, 26ꢀ28 or by substitution with a sulfur containing nucleophiles.23, 29ꢀ31 Thioacetic acid
and potassium thioacetate are the most popular sulfur reagents used for the formation of stable
thioester intermediates, which can be easily isolated and purified before the next step. Target
31
thiols can then be obtained in good yield by mild saponification or reduction.30,
Alternatively, thiourea can also be used as an odorless sulfur reagent to convert alcohols to
the corresponding thiols via the basic hydrolysis of the unstable isothiouronium salt.23
As a single procedure described in literature for vanillylthiol preparation cannot be considered
as safe and environmentally friendly,22 using thiourea as sulfur atom source in reaction with
vanillyl alcohol 3, which was obtained by the reduction of vanillin with sodium borohydride
in methanol, as previously described by Feng,32 was chosen as the first approach (Figure 1).
In our case, the target thiol vanillylthiol 2 and its corresponding disulfide 1 were obtained
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only at trace amounts. The major disadvantage of this approach was the formation of a
number of coproducts, mainly divanillyl sulfide
4
and bis(4ꢀhydroxyꢀ3ꢀ
methoxyphenyl)methane 5. Compound 4 probably resulted from desulfurization of 1 or from
Cꢀsulfanylation of 3 by thiol 2.33 The presence of 5 can be explained by the selfꢀcondensation
of 3.34 Interestingly, our result differed from that of Kofod, who used thiourea for direct oneꢀ
pot conversion of furfuryl alcohol to the corresponding thiol,23 and from that of Firouzabadi,
who used thiourea to synthesize disulfides from halides.35 The identification and the structural
characterization of all coproducts became possible only after the meticulous purification of
the crude by flash chromatography using a polarity gradient. All attempts to convert isolated
divanillyl sulfide 4 to vanillylthiol using various reducing agents were unsuccessful.
Since a simpler procedure was needed, vanillin was directly treated by inorganic H2S donors,
sodium hydrogen sulfide (NaSH), in hydroꢀalcoholic solution under gentle heating. The
formation of a precipitate indicated the progress of the reaction. In these ecoꢀfriendly
conditions, divanillyl disulfide 1 was produced cleanly. Furthermore, no workꢀup or
purification procedures were needed and it was isolated in 30 % yield by simple filtration of
the reaction mixture. The disulfide 1 was then easily transformed into the target thiol 2 using
lithium aluminum hydride as reducing agent.
Identifying vanillylthiol in wine by GC-MS/MS. We first injected the pure compound in
order to determine its retention time and its linear retention indice (LRInon polar 1495) as well as
its mass fragmentation (Figure 2) in which we could observe the characteristic fragmentation
of thiol, with the loss of ꢀSH [Mꢀ33]. Volatile compounds of a young white wine and a red
wine both aged in oak barrels were extracted with CH2Cl2 and analyzed first by GCꢀMS/MS
in scan, SIM and finally in SRM mode. In white wine, a peak corresponding with the same
retention time (Rt 37,65 min) and the same mass fragmentation as that of pure vanillylthiol
was observed, thereby validating its identification (result not shown). A clean mass spectrum
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was not obtained in red wine owing to coelution, so its identification in scan (EI) mode was
not possible. Indeed, as depicted in Figure 3, a small overlapping peak was observed at 37.65
min for ion m/z 137, but no clear peak was detected at m/z 170. For this reason we decided to
validate its identification using an MS/MS, optimized first with pure vanillylthiol and using
two transitions: m/z 170 → 137 and m/z 137 → 120+94 (Figure 4). GCꢀMS/MS demonstrated
the existence of vanillylthiol in the same red wine extract with a peak at the retention time of
vanillylthiol (Figure 5). Furthermore, whatever the transition chosen, the peak shape formed
by the selected ions was not modified by coinjection of a comparable quantity of the reference
compound. To our knowledge, this is the first time that this new volatile thiol has been
reported in nature and consequently in wines.
Sensorial considerations. The olfactory impact of this new thiol on the overall aroma of
wines was examined by determining its odor threshold. Both in pure and diluted form (50
µg/L, 12 % EtOH), it smells cloveꢀlike with smoky notes (Table 1). As for other thiols found
in wines and resulting from the biotransformation of aldehydes, the presence of the –SH
function considerably modifies their organoleptic properties.8, 36
To illustrate this phenomenon, Table 1 shows the perception threshold as well as the common
descriptors of aldehydes and their thiol equivalents found in wines. In our case, the presence
of the –SH group in the vanillin structure gives rise to a more intense aroma with new
sensorial properties, ranging from a slightly intense vanilla flavor for vanillin to a more
intense odor of clove and smoke for vanillylthiol with a perception threshold which was lower
by the factor of 15.
Although various hypotheses have been proposed to explain some of these effects,37 the
mechanistic interpretation of the variability in odor perception threshold is still rather
uncertain owing to the multiꢀstep processes involved in olfaction: odorant diffusion in the
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mucus layer, effect of odorantꢀbinding proteins, biotransformation enzymes, and interaction
with a large repertoire of olfactory receptors.
Assay of vanillylthiol in wines. The determination of thiol compounds at low concentrations
in wines is known to be tricky owing to their propensity to undergo oxidative reactions during
extraction and preꢀconcentration, as well as to their highly fragmented mass spectrum (EI
mode) which leads to low inherent sensitivity and/or matrix effects. To overcome these
drawbacks, several means to remove matrix components and concentrate the sample have
been proposed.17, 39 The original method of Tominaga et al.17 was based upon the selective
extraction of thiols from an organic extract of wine using the reversible chelation between the
SH function and sodium pꢀhydroxymercuribenzoate. Later, other studies focused on sample
preparation development or derivatization.40ꢀ42
As quantitation of thiol compounds might be tricky with conventional GCꢀMS approaches
according to the compound, we decided to use a more sensitive apparatus based on GCꢀ
MS/MS triple quadrupole technology. This approach is widely used when the assay is
performed on LCꢀMS/MS, but very few studies have reported its use for quantitating odorants
in wines. However, Langen43 reported the usefulness of tandem mass spectrometry in the
specific detection of γꢀ and δꢀlactones and Mayr44 recently reported a similar approach for
quantitating oxidationꢀrelated flavors including aldehydes, ketones, furans and some alcohols.
Very recently, Thibon45 used this approach to assay at the same time thiols and oxidationꢀ
related compounds in white wine with LOD close to ng/L.
Two approaches were evaluated for the extraction of vanillylthiol from wine: liquid/liquid
extraction and solid phase microextraction (SPME). Two fibers with different coatings of
DVB/Carboxen/PDMS and Carboxen/PDMS and several preparation conditions including the
choice of temperature, extraction time, salt addition, and dilution factor of the wine were
tested. Unfortunately, whatever the conditions, the SPME technique was not able to extract
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this polar organic compound from the wine matrix. On the contrary, our preliminary
experiments showed that the liquid/liquid extraction technique with CH2Cl2 as solvent was
very suitable.
To optimize SRM detection, we had to consider the available mass fragments of vanillylthiol
before starting fragmentation. In the mass spectra, intense (base) peaks at m/z 137 [M – SH]+
or 170 M+ are the predominant signals (Figure 2). To evaluate the SRM experiments for
vanillylthiol analysis, we chose the transition m/z 170 → 137, which presents a higher signal
intensity compared to the other transition 137 → 122+94 (Figure 4).
Finally, the repeatability of the assay was confirmed by a series of five extractions of a non
wooded sample of a white wine spiked with 500 ng/L of vanillylthiol. Precision estimated in
terms of (RSD) was 9 %. The LOD and LOQ of the method were 24 ng/L and 76 ng/L,
respectively. The linearity of the method was determined by plotting the calibration curves of
the corresponding transition peak areas, normalized by that of the internal standard. In the
concentration range (LOQꢀ5000 ng/L), the control curve was linear ([Vanillylthiol (ng/L] =
0,334 H/Hei, R2 = 0.995). This standard curve was measured using the height of the peaks
rather than the peak areas because the chromatogram showed a small contaminated peak just
after the vanillylthiol (Figure 5).
Table 2 shows the results obtained for the vanillylthiol assay in six dry white wines and six
red wines aged or not in oak barrels, from different appellations, vines and from several
vintages and bottle aging. The levels found varied according to the type of wine, the age, and
mainly according to the aging period in oak barrels. According to the sample analyzed, white
wines generally contained lower levels of vanillylthiol than red wines. The odor activity value
(OAV) of vanillylthiol in these wines is systematically <1. The wines kept a long time in
barrels had the highest concentrations. The highest level of 8394 ng/L (OAV 2.2) was found
in a young merlot red wine (2013 vintage) kept for 12 months in oak barrels and analyzed at
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that time. It is therefore very likely that this thiol may contribute to the cloveꢀspicy flavor of
this red wine. For aged wines the levels were low (< 100 ng/L). This result is not surprising
owing to the high instability of thiol compounds during aging in bottles.21, 46
Incidence of barrel aging on vanillylthiol formation. Table 3 shows the evolution of
vanillylthiol concentrations in a white wine aged in different barrels. As can be seen, the
vanillylthiol level was strongly affected by the container used. The wines fermented and kept
with their lees in new oak barrels had a higher content than those fermented in used barrels.
Previous work on the formation of FFT in wine fermented and aged in barrels can shed light
on how vanillylthiol can be formed in wine. Blanchard15 found that high levels of furfural
released by toasted oak wood during aging were associated with the formation of FFT. The
substitution of the SH group for the furfural carbonyl group can be biochemical during
alcoholic fermentation (Saccharomyces cerevisiae) or malolactic fermentation (Oenococcus
oeni) and chemical during barrel aging, but it always occurs in the presence of H2S.47
Therefore, vanillylthiol might be formed in the same way but from vanillin, which is also
released by toasted oak wood. As vanillin accumulation is strongly linked with cooper
seasoning procedures,48 aging time and the volume of the barrel49 and heating intensity,50
vanillylthiol concentration might be modulated by this parameter.
Finally, given its odor properties and its levels found in wines, we postulate that it might
contribute to the spicy, cloveꢀlike flavor of wine, and particularly red wine, aged for a long
time in new oak barrels. Or course, further works will be necessary to have a better overview
of its distribution in wine and to understand its contribution to the overall aroma of wines
aged in oak barrels, this work is in progress. Moreover, it cannot be excluded that this new
thiol might contribute to the spicy flavor of wines in combination with other volatile
compounds reminiscent of similar flavors and extracted from oak wood such as eugenol and
isoeugenol.
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Chatonnet, P.; Boidron, J. N.; Pons, M., Effect of heat on oak wood and its chemical
composition. Part 2. Variations of certain compounds in relation to toasting intensity. Connaiss. Vigne
Vin 1989, 23, 223ꢀ250.
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527
528
529
530
531
532
533
534
535
536
537
538
Figure 1: Chemical pathways studied for vanillylthiol synthesis
Figure 2: Fragmentation spectrum of vanillylthiol under GCꢀMS in scan mode (EI, 70 eV)
Figure 3: Example of a chromatogram corresponding to the analysis by GC/QqQꢀMS in SIM
mode of liquid/liquid extract of a red wine for two selected ions: m/z 170 and m/z 137. The
retention time of vanillylthiol is indicated by an arrow
Figure 4: Product ion scan of m/z 136.8 (A) and 169.7 (B) obtained from vanillylthiol
fragmentation under GC/QqQꢀMS
Figure 5: Detection of vanillylthiol and verification of peak purity by coꢀinjection of a
comparable quantity of the reference compound in a merlot red wine (Medoc appellation,
2014 vintage) by GCꢀMS/MS in SRM mode by overlapping ions selected corresponding to
two transitions 170→137 (A); 137→122+94 (B), at the retention time of the reference
compound indicated by an arrow.
539
540
541
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Table 1: Example of perception thresholds and descriptors of important thiols and their
corresponding aldehydes identified in wines
Compounds
Perception threshold (µg/L)
Descriptors
Benzaldehyde
Benzenemethanthiol
Furfural
3000 38
0.00003 36
15000 14
0.00004 8
65 14
Almond
Gun flint
Roasted
Furfurylthiol (FFT)
Vanillin
Roasted bean, coffee
Vanilla
Vanillylthiol (VSH)
3.8
Clove, smoke
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Table 2: Vanillylthiol concentrations (ng/L) in young and old wines from various appellations and
vines. n =2.
Barrel
aging
period
(months)
Bottle
aging
period
(year)
Vanillylthiol
(ng/L)
Color Vine
Appellation
Vintage
OAV
W
W
W
W
W
W
R
Sauvignon
Bordeaux
2014
2013
2013
2013
2013
2013
2013
2013
2014
2014
2010
2010
0
0
< LOQ
<1
<1
<1
<1
<1
<1
2.2
<1
<1
<1
<1
<1
Sauvignon
Sauvignon
Semillon
Pessac Leognan
Pessac Leognan
Pessac Leognan
Burgundy
8
1
83 (22)
8
0
818 (38)
420 (28)
1006 (59)
1426 (89)
8394 (198)
867 (44)
1022 (31)
2011 (122)
< LOQ
8
0
Chardonnay
Chardonnay
Merlot
10
10
12
10
6
0
Burgundy
0
Pessac Leognan
0
R
Cabernet Sauvignon Medoc
Merlot Medoc
Cabernet Sauvignon Pessac Leognan
0.5
0
R
R
6
0
R
Merlot
Merlot
Saint Emilion
Saint Emilion
12
12
3.5
3.5
R
< LOQ
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Table 3: Evolution of vanyllylthiol concentrations in a Sauvignon Blanc wine during barrel aging. n=
2
Wines (aging period)
Must
3 months
< LOQ
7 months
422 (32)
New Barrels
1.y Barrels
nd
nd
nd
< LOQ
< LOQ
231 (28)
198 (34)
2.y Barrels
nd: not detected
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Figure 1
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Figure 2
ꢀSH
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Figure 3
100
50
m/z 170
100
50
0
m/z 137
37.0
38.0
39.0
40.0
Time (min)
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Figure 4
A
B
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