Identification of adducts between an odoriferous volatile thiol and oxidized grape phenolic compounds: Kinetic study of adduct formation under chemical and enzymatic oxidation conditions

Article abstract of DOI:10.1021/jf204295s

HPLC-MS and ¹H, ¹³C, and 2D NMR analyses were used to identify new addition products between 3-sulfanylhexan-1-ol (3SH) and o-quinones derived from (+)-catechin, (-)-epicatechin, and caftaric acid. The kinetics of formation of these adducts were monitored in a wine model solution and in a must-like medium by HPLC-UV-MS with the aim of understanding the chemical mechanism involved in reactions between volatile thiols and o-quinones. One o-quinone-caftaric acid/3SH adduct, three o-quinone-(+)-catechin/3SH adducts, and three o-quinone-(-)-epicatechin/3SH adducts were characterized. Caftaric acid was oxidized faster than (-)-epicatechin and (+)-catechin when these phenolic compounds were incubated in a one-component mixture with polyphenoloxidase (PPO) in the presence of 3SH. Consequently, o-quinone-caftaric acid formed adducts with 3SH more rapidly than o-quinone-(+)-catechin and o-quinone-(-)-epicatechin in the absence of other nucleophilic species. Furthermore, o-quinone-(-)-epicatechin reacted faster than o-quinone-(+)- catechin with 3SH. Sulfur dioxide decreased the yield of adduct formation to a significant extent. Under chemical oxidation conditions, the rates and yields of adduct formation were lower than those observed in the presence of PPO, and o-quinone-caftaric acid was slightly less reactive with 3SH, compared to oxidized flavan-3-ols. The identification of o-quinone-caftaric acid/3SH and o-quinone-(+)-catechin/3SH adducts in a must matrix suggests that the proposed reaction mechanism is responsible for 3SH loss in dry wines during their vinification and aging process.

Full text of DOI:10.1021/jf204295s

Article
pubs.acs.org/JAFC
Identification of Adducts between an Odoriferous Volatile Thiol and
Oxidized Grape Phenolic Compounds: Kinetic Study of Adduct
Formation under Chemical and Enzymatic Oxidation Conditions
Maria Nikolantonaki,† Michael Jourdes,† Kentaro Shinoda,‡ Pierre-Louis Teissedre,† Stephane Quideau,§
́
and Philippe Darriet*,†
†Unite
́ ́
de recherche Oenologie, EA 4577, USC INRA 1219, Institut de Sciences de la Vigne et du Vin, Universite Bordeaux Segalen,
210 chemin de Leysotte, CS 50008, 33882 Villenave d’Ornon Cedex, France
‡Suntory Wine International Limited, 2-3-3 Daiba, Minato-ku, Tokyo 135-8631, Japan
§Institut des Sciences Molec
́ ́ ́
ulaires (CNRS-UMR 5255) and Institut Europeen de Chimie et Biologie, Universite de Bordeaux, 2 rue
Robert Escarpit, 33607 Pessac Cedex, France
S
* Supporting Information
1
ABSTRACT: HPLC−MS and H, 13C, and 2D NMR analyses were used to identify new addition products between 3-
sulfanylhexan-1-ol (3SH) and o-quinones derived from (+)-catechin, (−)-epicatechin, and caftaric acid. The kinetics of formation
of these adducts were monitored in a wine model solution and in a must-like medium by HPLC−UV−MS with the aim of
understanding the chemical mechanism involved in reactions between volatile thiols and o-quinones. One o-quinone-caftaric
acid/3SH adduct, three o-quinone-(+)-catechin/3SH adducts, and three o-quinone-(−)-epicatechin/3SH adducts were
characterized. Caftaric acid was oxidized faster than (−)-epicatechin and (+)-catechin when these phenolic compounds were
incubated in a one-component mixture with polyphenoloxidase (PPO) in the presence of 3SH. Consequently, o-quinone-caftaric
acid formed adducts with 3SH more rapidly than o-quinone-(+)-catechin and o-quinone-(−)-epicatechin in the absence of other
nucleophilic species. Furthermore, o-quinone-(−)-epicatechin reacted faster than o-quinone-(+)-catechin with 3SH. Sulfur
dioxide decreased the yield of adduct formation to a significant extent. Under chemical oxidation conditions, the rates and yields
of adduct formation were lower than those observed in the presence of PPO, and o-quinone-caftaric acid was slightly less reactive
with 3SH, compared to oxidized flavan-3-ols. The identification of o-quinone-caftaric acid/3SH and o-quinone-(+)-catechin/3SH
adducts in a must matrix suggests that the proposed reaction mechanism is responsible for 3SH loss in dry wines during their
vinification and aging process.
KEYWORDS: volatile thiols, phenolic compounds, reactivity, oxidation, polyphenoloxidase, oxygen
compounds under wine oxidation conditions.15−18 During
INTRODUCTION
■
barrel and bottle aging of red and white wines, a significant
decrease of volatile odoriferous thiols such as 3SH has been
noticed due to the presence of dissolved oxygen.19,20 Moreover,
experiments carried out under controlled oxidation conditions,
in red wines, have shown that the disappearance of 3SH in
oxygenated wine was not concomitant with the oxygen
consumption, but occurred 48 h later.19 Consequently, the
degradation of a volatile thiol such as 3SH could not be
exclusively related to its direct oxidation, but probably also to
its reactivity with other species present in wine. Blanchard et
al.19 showed that oxidized (+)-catechin reacted with 3SH,
resulting in a loss of the fruity varietal character, due to this
compound, in red wines. Recently, under winelike oxidation
conditions, Nikolantonaki et al.17 established a clear difference
between (+)-catechin and (−)-epicatechin in their reactivity
toward aromatic volatile thiols such as 3SH, 4MSP, and 2FMT.
Volatile thiols are well-known to be relevant to the character-
istic aroma of several white and red wines. Volatile thiols are
very potent odorant molecules that contribute to the
complexity of the flavors of wines.1−9 During the past 20
years, several volatile thiols have been identified. For example,
4-methyl-4-sulfanylpentan-2-one (4MSP),1 which was first
isolated in Sauvignon blanc wines, exhibits a strong box-tree
odor that turns into a cat urine odor at higher concentrations.
Together with 3-sulfanylhexan-1-ol (3SH)4 and 3-sulfanylhexyl
acetate (3SHA),10 4MSP can contribute to the typicality of
varietal wines such as Sauvignon blanc, Semillon, Scheurebe,
Petite Arvine, Gewurztraminer, and Muscat d’Alsace.1,2,4,7,11
̈
3SH is the most abundant polyfunctional thiol in wine, present
in relatively high concentrations (i.e., 100−3500 ng L−1) as
compared to 4MSP (i.e., 0.3−97 ng L−1) and 3SHA (i.e., 0.1−
777 ng L−1).6,12 Other thiols such as 2-furanmethanethiol
(2FMT), 2-methyl-3-furanthiol, and benzenemethanethiol,
which are formed during wine aging, can contribute to the
empyreumatic nuances in a wine “bouquet”.3,13,14
Received: October 20, 2011
Revised: February 11, 2012
Accepted: February 12, 2012
Published: February 12, 2012
Thiols are reactive molecules, and they can be transformed
into disulfides and/or consumed by oxidized phenolic
© 2012 American Chemical Society
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system (Millipore, Molsheim, France). The crude PPO extract was
prepared as a powder from merlot grapes, harvested in 2008, as
described by Singleton et al.31
Reactions in Model Must. (+)-Catechin, (−)-epicatechin, and
caftaric acid (9 mM) were incubated with 3SH in excess (12 mM) at
30 °C with 5 g L−1 of crude enzymatic extract. Enzymatic reactions
were carried out in an aqueous potassium hydrogen tartrate aerated
medium (5 g L−1, pH = 3.5, 5 mg L−1 dissolved oxygen, LDO HQ10,
The presence of (+)-catechin and (−)-epicatechin, together
with Fe(III) catalyzing their oxidation into o-quinones, favors
the disappearance of such thiols.17 However, the presence of
free SO2 slows down volatile thiol consumption.17
Indeed, volatile thiols can undergo nucleophilic addition
reactions with certain electrophiles. One of the most abundant
and chemically compatible electrophiles in wine and must is the
o-quinones formed from phenolic compounds by enzymatic or
chemical oxidation. Chemical oxidation generally occurs in
fermenting grape must and in aging wine, whereas enzymatic
oxidation occurs primarily in freshly crushed grapes, but may
also take place in fermenting must in which the amount of SO2
added is insufficient to inactivate PPO completely.21,22 As
volatile thiols are nucleophiles, they can thus add to the
electrophilic sites of o-quinones in a conjugate addition fashion
according to the Michael-type addition scenario,23 which leads
to the formation of adducts differing from one another by the
quinonoid carbon center(s) at which the thiol function has
reacted. These adducts are nonvolatile, and their formation
could likely underpin the diminishing of the wine aroma
intensity. The efficacy of the addition reaction between volatile
thiols and o-quinones derived from the oxidation of phenolic
(catecholic) compounds strongly depends upon the nucleo-
philic strength of the thiol and the oxidation rate of each
phenolic substrate. The nucleophilicity of thiols is mainly
modulated by their steric hindrance, primary thiols being more
reactive than tertiary thiols.24 Such nucleophilic additions have
already been reported in the literature concerning the addition
of thiols to o-quinones derived from caftaric acid,23,25 caffeic
acid,26 gallic acid,27 (+)-catechin,28 (−)-epicatechin,29 and 4-
methylcatechol.30 Most of these precedents relate to reactions
performed in an alkaline buffer26,28 or organic medium.27
However, in acidic white grape must, Singleton et al.31 observed
the reaction of a caftaric acid-derived o-quinone with the
tripeptide thiol, glutathione (GSH), which led to the formation
of what they called the “grape reaction product” (GRP, 2-S-
glutathionyl caffeoyl tartrate). They also studied the reaction of
this same o-quinone, generated using the PPO enzyme, with
other thiols (i.e., cysteine, 2-mercaptoethanol).31
Hach, Dusseldorf, Germany). Aliquots of 10 mL of each reaction were
̈
prepared in amber vials. Samples were added with 50 mg L−1 of
potassium bisulfate solution in order to stop the reactions. All
reactions were performed in triplicate.
Reactions in Model Wine. (+)-Catechin, (−)-epicatechin, and
caftaric acid (9 mM) were dissolved in one component mixture in an
aqueous potassium hydrogen tartrate (5 g L−1, pH 3.5) containing
12% (v/v) of ethanol. The model solution was aerated (5 0.1 mg
L−1 dissolved oxygen, LDO HQ10, Hach, Dusseldorf, Germany),
̈
before the addition of each substrate, with medical grade compressed
air (Air Liquide, France) for 10 min using a diffuser (Porex) with a
pore diameter of 12 μm. Aliquots of 10 mL of each substrate were
prepared in amber vials. Stock solutions of Fe(II) sulfate, SO2, H2O2,
and 3SH were prepared in degassed water immediately before use. The
Fe(II) (50 mM) and SO2 (0.78 mM) were introduced to the reaction
medium with a syringe. In all cases, reactions were initiated upon
addition of hydrogen peroxide (300 mM), followed by addition of
3SH (12 mM), to the model wine solution with a syringe. The
temperature was set at 35 °C, and all reactions were performed in
triplicate. Samples were added with 50 mg L−1 of potassium bisulfate
solution in order to stop the reactions.
Identification of Adducts between 3-Sulfanylhexan-1-ol and
o-Quinones Derived from (+)-Catechin, (−)-Epicatechin, and
Caftaric Acid by Liquid Chromatography−Electrospray Ioniza-
tion Mass Spectrometry. HPLC analyses were performed on a
HPLC Finnigan system constituted of a Surveyor autosampler, a
Finnigan ternary pump and a UV−vis 200 detector (Finnigan)
coupled to a 1.2 Xcalibur data treatment system. Samples (20 μL)
were injected on a reversed-phase Agilent Nucleosil C18 column (250
mm × 4 mm, 5 μm). The mobile phase was composed of solvent A
[H2O−HCOOH (996:4, v/v)] and solvent B [MeOH-HCOOH
(996:4, v/v)]. The elution gradient was different for the three reaction
media. The elution gradient used to monitor the reactions of caftaric
acid/3SH and (+)-catechin/3SH was 0−5 min, 0−31% solvent B; 5−
25 min, 31−38% solvent B; 25−40 min, 38−48% solvent B; and 40−
53 min, 100% solvent B, followed by washing and reconditioning of
the column. The elution gradient used to monitor the reactions of
(−)-epicatechin/3SH was 0−5 min, 0−35% solvent B; 5−25 min, 35−
45% solvent B; 25−50 min, 45−70% solvent B; and 50−55 min, 100%
solvent B, followed by washing and reconditioning of the column. The
gradient was applied at a flow rate of 1 mL min−1.
The ion trap mass spectrometer was a LCQ Deca (Thermo
Finnigan) equipped with an electrospray ionization source. 200 μL of
the total solvent flow was injected into the mass spectrometer source
with a split. Analyses were carried out in the negative ion mode. The
source parameters were as follows: spray voltage (3.7 kV), capillary
voltage (70.0 V), sheath gas (65 arbitrary units), auxiliary gas (20
arbitrary units), and capillary temperature (250 °C). Nitrogen was
used as the nebulizing gas. Helium was used as the damping gas. The
model solutions were first analyzed in full MS mode (m/z 100−1000)
and then in selected ion mode using the following molecular ion mass
m/z: 289 for (+)-catechin and (−)-epicatechin; 311 for caftaric acid;
421 for adducts 1a/b, 2, 4a/b, and 5; 553 for adducts 3 and 6; and 443
for adduct 7.
The main purpose of this study was to progress in the
understanding of mechanisms underpinning the evolution of
volatile thiols in dry white wines and their must matrix through
the monitoring of their reactions with oxidized phenolic
compounds formed under chemical and enzymatic pathways.
Adducts between the major appropriate oxidized phenolic
compounds in white must and wine [i.e., caftaric acid,
(+)-catechin, and (−)-epicatechin] and 3SH were identified
and characterized. The influence of the presence of transition
metal ions (Fe(II) sulfate) and SO2 on the formation of these
adducts was also investigated.
MATERIALS AND METHODS
■
Reagents, Chemicals, and Materials. (+)-Catechin hydrate
(98%), (−)-epicatechin (98%), formic acid, reagent grade, and L-
tartaric acid were purchased from Sigma-Aldrich (Saint-Quentin
Fallavier, France). 3-Sulfanylhexan-1-ol (>95%) was obtained from
Lancaster (Bischheim, France). L-Caftaric acid was purchased from
Dalton (Toronto, Canada). Sulfur dioxide (potassium bisulfate form)
was at a concentration of 10% (w/v) in water (Laffort, Bordeaux,
France). Hydrogen peroxide solution 35% in water and methanol
(HPLC grade) were purchased from Merck (Darmstadt, Germany).
Iron(II) sulfate and HPLC-grade methanol were purchased from VWR
(Fontenay-sous-Bois, France). Pimaricin (natamycin) was purchased
from DMS (Heerlen, Holland). Water was purified using a Milli-Q
Synthesis of Adducts between 3-Sulfanylhexan-1-ol and o-
Quinones Derived from (+)-Catechin, (−)-Epicatechin, and
Caftaric Acid. 250 mg of (+)-catechin, (−)-epicatechin, or caftaric
acid in 500 mL of a model medium, which consisted of aerated water
(5 0.1 mg L−1 dissolved oxygen, LDO HQ10, Hach, Dusseldorf,
̈
Germany), tartaric acid 5 g L−1 at pH 3.50, was treated with 10 g L−1
of crude polyphenoloxidase in the presence of 1 g L−1 of 3SH. The
reaction mixtures were kept at room temperature for 48 h until the
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Figure 1. Structures of adducts between 3-sulfanylhexan-1-ol and o-quinones derived from (A) (+)-catechin [6′-(7′-sulfanylhexan-9′-ol)−catechin
(1a/b); 5′-(7′-sulfanylhexan-9′-ol)−catechin (2); 5′-6′-(7′-sulfanylhexan-9′-ol)−catechin (3)], (B) (−)-epicatechin [2′-(7′-sulfanylhexan-9′-ol)−
epicatechin (4a/b); 5′-(7′-sulfanylhexan-9′-ol)−epicatechin (5); 2′-5′-(7′-sulfanylhexan-9′-ol)−epicatechin (6)], and (C) caftaric acid [2′-(3″-
sulfanylhexan-1″-ol)−caftaric acid (7)] formed under chemical and enzymatic oxidation in acidic conditions.
total consumption of the substrate, as monitored by HPLC−UV
analysis. The crude reaction mixtures, after filtration on a fiber glass
membrane, were washed three times with 250 mL of chloroform in
order to eliminate the residual 3SH. After washing, the extracts were
concentrated to dryness under vacuum. The residues were then
dissolved to 100 mL of ultrapure water, and the aqueous solutions
were frozen and freeze-dried.
Semipreparative HPLC Purification. The purification of each
reactional medium was carried out on a HP 1090 (Agilent
Technologies, Palo Alto, CA) HPLC system equipped with an HP
1090 UV detector (Agilent Technologies, Palo Alto, CA) fixed at 280
nm. Acquisitions were performed using Chemstation software.
Separations were performed using a Nucleosil C-18 column (21.4 ×
250 mm, 5 μm) with a guard column filled with the same phase. The
solvents and the gradient elution were the same described above. The
flow rate was set at 4 mL min−1, and the injection volume was set to
250 μL of each crude reaction mixture (80 mg L−1).
Kinetic Study in White Native Must Matrix by HPLC/ESI-MS.
50 mL of samples were taken at regular intervals (every hour for the
first 12 h and once per day for the next four days) throughout the
incubations and immediately extracted once with ethyl acetate (50
mL). The organic phases were evaporated under reduced pressure, the
extracts were dissolved in 2 mL of methanol, and the solution was
filtered through a 0.45 μm filter. 20 μL of samples were injected in
HPLC/ESI-MS using the analytical conditions described above.
RESULTS AND DISCUSSION
■
Our experimental approach was based on first evaluating the
outcome of the reactions between the selected phenolic
compounds [i.e., caftaric acid, (+)-catechin, and (−)-epicate-
chin] and 3SH on an analytical scale in an acidic aqueous
solution under enzymatic oxidation conditions. The reaction
progress was monitored by high-performance liquid chroma-
tography coupled to electrospray ionization mass spectrometry
(HPLC/ESI-MS) analysis. Products were identified on the
basis of their retention time and molecular mass. Reactions
were also performed on a semipreparative scale under
otherwise identical conditions in order to obtain pure products
in sufficient amount for their full structural characterization by
NMR spectroscopy. All these reactions were also carried out in
a model wine system, consisting of a 12% (v/v) aqueous
ethanol solution containing 5 g L−1 of tartaric acid at pH 3.5, in
order to analytically determine the kinetics of adduct formation
in a winelike medium under chemical oxidation conditions.
Adducts between 3-Sulfanylhexan-1-ol and o-Qui-
nones derived from (+)-Catechin and (−)-Epicatechin.
To determine the structure of the products formed through a
chemical reaction between flavan-3-ols and 3SH in wine and
must during fermentation, (+)-catechin and (−)-epicatechin
were separately incubated with 3SH in an aqueous acidic
medium in the presence of crude PPO. The reaction progress
was monitored by HPLC/ESI-MS analysis. One hour after
incubation, the HPLC/ESI-MS results showed the formation of
four major products, 1a/b, 2, and 3 (tR = 26.0, 27.1, 34.8, and
NMR. Detailed descriptions of NMR spectra of all the new isolated
compounds are given in the Supporting Information.
Kinetic Study in Model Wine and in Must-like Solutions by
HPLC−UV. The equipment used for the HPLC monitoring conditions
for the incubation in a model wine solution or in must solution was
similar to that used for the identification of the adducts. The
formations of the o-quinone-(+)-catechin/3SH and o-quinone-
(−)-epicatechin/3SH adducts were monitored at 280 nm, whereas
that of the o-quinone-caftaric acid/3SH adducts was monitored at 320
nm. External calibration curves for the synthesized adducts in
methanol (50%), in the range of 0.05−2 mM, were run and recorded
in triplicate. Calibration curves for the quantitative analyses of 1a, 1b,
2, 3, 4a, 4b, 5, and 6 were established at 280 nm and at 320 nm for 7.
Calibration curves were determined using a methanolic solution (50%)
spiked with dilute methanol solutions containing 1a, 1b, 2, 4a, 4b, 5,
and 7 at final concentrations ranging from 0 to 10 mM. The
quantification limit was calculated as the minimum concentration that
generated a peak signal 10 times higher than the signal from
background noise. 1a, 1b, 2, 4a, 4b, 5, and 7 concentrations were then
determined from the regression equations, and the results were
expressed in mM. 3 and 6 concentrations were expressed in mM
equivalent 2 and 5, respectively.
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46.5 min, respectively). After 20 h, when the total consumption
of the (+)-catechin substrate had occurred, the reaction was
stopped in order to prevent the oxidation of the newly formed
products. Thus, the products from the (+)-catechin/3SH
reaction mixture were directly separated by semipreparative
HPLC and the 1a/b, 2, and 3 products were obtained in a
18:18:59:4 ratio and with respective yields of 17, 17, 55, and
4%. The analogous compounds 4a/b, 5, and 6 (tR = 28.8, 30.8,
37.6, and 44.8 min, respectively) were obtained from the
reaction using (−)-epicatechin and 3SH in a 18:18:59:4 ratio
and with a yield of 17%:17%:56%:4%, respectively.
unambiguously established the structure of 1a as 6′-(7′-
sulfanylhexan-9′-ol)−catechin, which features a 3SH moiety
connected to the (+)-catechin moiety through a thioether
linkage at position 6′ of the (+)-catechin aromatic B-ring.
Similar correlations were also observed on the HMBC
spectrum of 1b, hence revealing that 1b also has a 3SH moiety
linked to the (+)-catechin moiety through a thioether linkage at
position 6′ of the (+)-catechin aromatic B-ring. Overall, 1a and
1b are two 6′-(7′-sulfanylhexan-9′-ol)−catechin diaster-
eoisomers resulting from the addition of a (R)- or (S)-3-
sulfanylhexan-1-ol enantiomer on the oxidized (+)-catechin
moiety.
The HPLC/ESI-MS analysis of the purified compounds 1a/
b, 2, 4a/b, and 5, performed in negative ion mode, showed a
molecular ion mass of m/z = 421 ([M − H]−) indicating that
these compounds could result from the nucleophilic addition of
3SH onto the o-quinone species derived from the oxidation of
(+)-catechin or (−)-epicatechin (Figures 1A and 1B) in a
similar manner as that previously reported for the addition of
glutathione or cysteine onto the oxidized forms of caftaric acid,
caffeic acid, (−)-epicatechin and (−)-epigallocatechin.23,26,28−30
The presence of three different products with the same
molecular ion mass (i.e., m/z = 421) in each reaction mixture
could result from a nucleophilic attack by 3SH on three
different electrophilic carbon centers (i.e., C2′, C5′, and C6′) of
the B-ring of the o-quinones derived from (+)-catechin and
(−)-epicatechin. Moreover, high resolution ESI-MS analyses
showed that 1a/b, 2, 4a/b, and 5 have the same molecular
formula, i.e., C21H26O7S. The HPLC/ESI-MS analysis of the
other two products, 3 and 6, revealed a molecular ion mass of
m/z = 553, which indicated a double addition of 3SH onto the
oxidized flavan-3-ols (Figures 1A and 1B). Thus, the formation
of 3 and 6 results from a second nucleophilic addition of 3SH
onto the o-quinones deriving from the oxidation of the initially
formed adducts 1a/b, 2, 4a/b, and 5. Also, in this case,
according to the HPLC−UV chromatogram, peaks 3 and 6
represented a mixture of products with the same molecular
formula, i.e., C27H38O8S2. However, the HPLC analysis did not
allow the separation of the two diastereoisomers resulting from
the use of racemic 3SH.
The NMR spectra of 2 and 5 also revealed important
similarities, and both spectra appeared to be more complex with
more signals than those of 1a/b. Nevertheless, a closer analysis
showed that most of the signals were duplicated, especially
those corresponding to the carbons and protons around the
chiral center of 3SH (i.e., C-7′). In fact, after having assigned all
the signals observed in the proton and carbon spectra using
two-dimensional NMR analyses (HMQC, HMBC, and COSY),
it appeared that 2 was not a single compound, but an
inseparable mixture of the two diastereoisomers resulting from
the use of racemic 3SH. Despite the presence of these two
diastereoisomers, it was possible to determine the connecting
link between the 3SH and (+)-catechin moieties using the long-
range proton−carbon correlations. The HMBC map showed an
intense correlation between 3SH proton H-7′ at 3.18 ppm and
carbon C-5′ of the (+)-catechin aromatic B-ring resonating at
120.6 ppm. Moreover, the presence of two doublets with a
small coupling constant at 6.81 ppm (J = 1.67 Hz) and at 6.91
ppm (J = 1.67 Hz), which were attributed to the resonances of
protons H-2′ and H-6′ of the (+)-catechin aromatic B-ring,
confirmed the absence of a proton on carbon C-5′ of this ring.
In conclusion, the compound mixture 2 was identified as a
mixture of the two 5′-(7′-sulfanylhexan-9′-ol)−catechin diaster-
eoisomers that resulted from the nucleophilic addition of the
two 3SH enantiomers onto carbon-C-5′ of the (+)-catechin
aromatic B-ring. Similar correlations were also observed on the
HMBC spectrum of 5, hence revealing that 5 is also a mixture
of the two 5′-(7′-sulfanylhexan-9′-ol)−epicatechin diaseter-
oisomers in which the 3SH moiety is connected to the
(−)-epicatechin moiety through a thioether linkage at position
5′ of the (−)-epicatechin aromatic B-ring.
The complete structural elucidation of compounds 1a/b, 2,
4a/b, and 5 was achieved by 1D and 2D NMR analysis. The
quantities of the HPLC purified compounds, 3 and 6, were
limited, and their low yield precluded further characterization
by NMR. The structure of 1a was determined by one-
The complete structural elucidation of compounds 4a/b was
also achieved by 1D and 2D NMR analyses. The aromatic
1
dimensional H and 13C and two-dimensional HMQC and
1
HMBC NMR experiments. The key point in this structural
determination was the identification of the connection between
the 3SH and (+)-catechin moieties that was established using
long-range proton−carbon HMBC correlation data. The
HMBC spectrum shows an intense correlation between proton
H-7′ (δH 3.32 ppm) of the 3SH unit and carbon C-6′ (δC 121.4
ppm) of the (+)-catechin aromatic B-ring. The latter was
assigned from its HMBC correlation with proton H-2 (δH 6.86
ppm) of the (+)-catechin C-ring, which was itself identified by
its characteristic chemical shift. Moreover, the identification of
this connecting link between the 3SH and (+)-catechin
moieties was also supported by the presence of a broad singlet
integrating for two protons in the aromatic region at 6.86 ppm.
This resonance signal was attributed to protons H-2′ and H-5′
of the (+)-catechin aromatic B-ring. The other protons and
carbons of the aliphatic chain of the 3SH moiety and those of
the (+)-catechin moiety were all assigned from HMQC and
HMBC correlation data. Thus, these 1D and 2D NMR analyses
region in the H NMR spectrum of 4a revealed two mutually
coupled doublets, as confirmed by COSY experiments, at 6.86
ppm (J = 8.40 Hz) and 7.16 ppm (J = 8.40 Hz). These signals
were assigned to protons H-5′ and H-6′ of the (−)-epicatechin
aromatic B-ring. These strictly mutually coupled aromatic B-
ring protons are a consequence of the absence of protons on
carbon C-2′ of the same ring, which indicates that the 3SH
moiety is, in this case, connected to this position of the
(−)-epicatechin unit. Moreover, this linkage was confirmed by
a three-bond correlation between proton H-7′ (δH 3.36 ppm) of
the 3SH moiety and carbon C-2′ (δC 118.5 ppm) of the
aromatic B-ring in the HMBC map. This latter carbon was
unambiguously assigned from its diagnostic three-bond
correlations with protons H-2 (δH 5.48 ppm) and H-6′ (δH
7.16 ppm). The other protons and carbons were assigned to the
aliphatic chain of the 3SH moiety and to those of the
(−)-epicatechin moiety by HMQC and HMBC experiments.
These NMR analyses unambiguously established the structure
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Figure 2. Kinetic formation rate of adducts between 3-sulfanylhexan-1-ol and o-quinones derived from (+)-catechin, (−)-epicatechin and caftaric
acid in model must under enzymatic oxidation conditions. (A) 1a/b, 2, and 3 formation in the presence of polyphenoloxidase (PPO); (B) 1a/b, 2,
and 3 formation in the presence of PPO and SO2; (C) 4a/b, 5, and 6 formation in the presence of PPO; (D) 4a/b, 5, and 6 formation in the
presence of PPO and SO2; (E) 7 formation in the presence of PPO; (F) 7 formation in the presence of PPO and SO2. The compound numbering is
defined in Figure 1. 1a/b and 4a/b present the summary of the 1a and 1b and the 4a and 4b diastereoisomers, respectively.
of 4a as 2′-(7′-sulfanylhexan-9′-ol)−epicatechin, which features
a 3SH moiety connected to the (−)-epicatechin moiety
through a thioether linkage at position 2′ of the (−)-epicatechin
aromatic B-ring. Similar NMR data were also observed for 4b,
thus revealing that 4b also has a 3SH moiety connected to the
(−)-epicatechin moiety through the same type of thioether
linkage. Overall, 4a/b are two 2′-(7′-sulfanylhexan-9′-ol)−
epicatechin diaseteroisomers resulting from the addition of
the (R)- or (S)-3-sulfanylhexan-1-ol enantiomer on the
oxidized (−)-epicatechin.
Adducts between 3-Sulfanylhexan-1-ol and o-Qui-
nones Derived from Caftaric Acid. The procedure used for
the hemisynthesis of adducts between o-quinone-caftaric acid
and 3SH was identical to the procedure used for the oxidized
flavan-3-ols, o-quinone-(+)-catechin and o-quinone-(−)-epica-
techin. The progress of the reaction was monitored by HPLC/
ESI-MS. After 1 h of incubation, the reaction was stopped while
the total consumption of the phenolic substrate occurred and
the reaction mixture was separated by semipreparative HPLC
to furnish one reaction product, 7, as an amorphous yellowish
powder with a 75% yield. HPLC/ESI-MS analysis of 7, in
negative ion mode, showed a molecular ion mass of m/z = 443
([M − H]−) indicating that 7 could result from the
nucleophilic addition of 3SH onto oxidized caftaric acid in a
manner similar to what was observed for o-quinone-
(+)-catechin and o-quinone-(−)-epicatechin (Figure 1C).
Moreover, high resolution ESI-MS analyses supported the
evidence for the formation of such an adduct, since the
molecular formula of 7 was thus established to be C19H24O10S.
The full characterization of 7 revealed that it was also a
mixture of two diastereoisomers. However, it was again possible
to easily determine the connecting link between the 3SH and
caftaric acid moieties. The 1H NMR spectrum of 7 revealed two
mutually coupled doublets in the aromatic region, as confirmed
by a COSY experiment, at 6.95 ppm (J = 8.32 Hz) and at 7.38
ppm (J = 8.32 Hz). These resonances were attributed to
protons H-5′ and H-6′ of a 2′-substituted caftaric acid aromatic
ring. This substitution was also supported by the observation of
long-range proton−carbon correlations on the HMBC map
between proton H-3″ (δH 3.15 ppm) of the 3SH moiety and
carbon C2′ (δC 117.1 ppm) of the caftaric acid aromatic ring.
This carbon resonance was assigned from its correlations with
the characteristic aromatic proton H-6′ (δH 7.38 ppm) and the
ethylenic proton H-7′ (δH 8.47 ppm). The other protons and
carbons of the aliphatic chain of the 3SH moiety and those of
the caftaric acid moiety were assigned using HMQC and
HMBC correlation data. Thus, 7 was confirmed to be a mixture
of the two diaseteroisomers of 2′-(3″-sulfanylhexan-1″-ol)−
caftaric acid that resulted from the nucleophilic addition of
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racemic 3SH at the C-2′ center of the o-quinone derived from
the oxidation of caftaric acid.
consumption was 10% and 45% greater than that of
(+)-catechin and (−)-epicatechin, respectively. The above
results could be related to the fact that caftaric acid is known
to be a better substrate than (+)-catechin and (−)-epicatechin
for grape PPO (Figure 3), and thus its oxidation rate was well
correlated with the yield of adduct 7 (80%).
Reactivity of 3-Sulfanylhexan-1-ol with (+)-Catechin,
(−)-Epicatechin, and Caftaric Acid under Enzymatic
Oxidation Conditions. In order to gain a better under-
standing of the mechanism of 3SH addition onto oxidized
(+)-catechin, (−)-epicatechin, and caftaric acid, the kinetics of
formation of all the adducts characterized by NMR were
examined by HPLC analysis with UV detection. The addition
mechanism involved production of the o-quinone from either
(+)-catechin, (−)-epicatechin, or caftaric acid by PPO-catalyzed
oxidation of their catechol unit, followed by the conjugate
addition of 3SH. Among these three substrates that were
incubated with 3SH in equal concentrations (9 mM) in the
presence of PPO, o-quinone-caftaric acid led to the greater
production of adducts with 3SH (5 mM; Figure 2E), followed
by o-quinone-(+)-catechin (4.5 mM of adduct with 3SH;
Figure 2A) and then by o-quinone-(−)-epicatechin (2.2 mM of
adduct with 3SH; Figure 2C). Indeed, and in agreement with a
previously published article,32 the caftaric acid consumption
was about 69% after 3 days of incubation (Figure 3). This
For the three substrates, no lag period was observed at the
beginning of the reaction (Figure 2). The addition of 3SH to
both o-quinone-(+)-catechin and o-quinone-(−)-epicatechin
followed a linear trend, whereas the o-quinone-caftaric acid/
3SH adduct formation kinetics could be divided into two time
periods. During the first period (phase I), from T0 to T1day, the
formation rate of 7 was high and reached 80% of its final yield,
and during phase II, from T1day to T3days, the reaction rate was
two times slower than in phase I. In both kinetic phases, the
formation of 7 followed a linear regression. The HPLC−UV
monitoring of the reaction mixtures containing the starting
flavan-3-ols showed that the formation of adducts between 3SH
and either o-quinone-(+)-catechin or o-quinone-(−)-epicate-
chin had a similar rate. The rates of addition of 3SH to the 5′ or
6′ position of the B-ring of the oxidized (+)-catechin were also
similar (≈2 mM). In the case of o-quinone-(−)-epicatechin, the
attack of 3SH at the 5′ position (5, 1.2 mM) was favored over
the 2′ position (4a/b, 0.7 mM). The formation rate of 7 was
slightly higher than that of 2 and double that of 5. In all flavan-
3-ol model solutions, (+)-catechin/3SH and (−)-epicatechin/
3SH, we observed the multiple addition of 3SH with no
difference to the yield of the double adducts after three days of
incubation (≈0.4 mM). The absence of multiple adducts in the
case of o-quinone-caftaric acid could be explained by the fact
that 7 is no longer a substrate for PPO, as previously observed
with 2-S-glutathionylcaftaric acid (GRP).31
The addition of sulfur dioxide in the reaction media directly
influenced the rates and yields of all adducts formed (Figures
2B; 2D; 2F). Under enzymatic oxidation conditions,
SO2reduces PPO activity, but at the same time, reacts with o-
quinones to form o-dihydroxyphenols and sulfones.33,34 When
SO2 was introduced in the must model system containing 3SH,
we observed the suppression of every phenolic compound's
Figure 3. (+)-Catechin, (−)-epicatechin, and caftaric acid con-
sumption (expressed in percentages) under enzymatic oxidation
conditions after 3 days of incubation with 3-sulfanylhexan-1-ol and
either in the presence or in the absence of SO2.
Figure 4. Kinetic formation rate of 1a/b, 2, and 3 under chemical oxidation conditions: (A) control; (B) with SO2; (C) with iron(II) sulfate; (D)
with iron(II) sulfate and SO2. The compound numbering is defined in Figure 1. 1a/b presents the summary of the 1a and 1b diastereoisomers.
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Figure 5. Kinetic formation rate of 4a/b, 5, and 6 under chemical oxidative conditions: (A) control; (B) with SO2; (C) with iron(II) sulfate; (D)
with iron(II) sulfate and SO2. The compound numbering is defined in Figure 1. 4a/b presents the summary of the 4a and 4b diastereoisomers.
consumption (Figure 3). Consequently, the formation rates and
yields of all adducts decreased significantly. In the case of the
flavanol substrates, the addition of SO2 significantly slowed
down the beginning of the reactions, with an equal duration in
both cases (T0 to T1day). After one day of incubation, the
production of the o-quinone-(−)-epicatechin/3SH adducts
(4a/b, 5) followed an exponential regression and that of the
o-quinone-(+)-catechin/3SH adducts (1a/b, 2) followed a
linear curve (Figures 2B and 2D). The addition of SO2 into the
caftaric acid/3SH reaction mixture affected the kinetic profile of
the reaction which displayed a linear regression fitting. In that
case, the final yield of adduct 7 was two times less abundant
than when obtained in the absence of SO2, and this diminution
was proportional to that of the caftaric acid consumption
(Figure 2F).
Reactivity of 3-Sulfanylhexan-1-ol with (+)-Catechin,
(−)-Epicatechin, and Caftaric Acid under Chemical
Oxidation Conditions. The reactions of o-quinone-(+)-cat-
echin, o-quinone-(−)-epicatechin, and o-quinone-caftaric acid
with 3SH were also investigated in an air-saturated winelike
solution. In all cases, the reactions were initiated by the
addition of hydrogen peroxide into the reaction mixtures in
order to initiate the Fenton reaction. Under these chemical
oxidation conditions, the yields and rates of adduct formation
were significantly lower than those observed under enzymatic
oxidation conditions. It has been stated35 that the production of
o-quinones is faster under enzymatic oxidation than under
autoxidation conditions, but once o-quinones were formed,
their reactivity toward thiol addition was the same whether they
originated from enzymatic or chemical oxidation. In wine acidic
conditions (Figures 4 and 5), the rate of formation of the o-
quinone-(−)-epicatechin/3SH adducts (4a/b, 5, and 6) was
13% higher than the one observed for the formation of the o-
mM for 4a/b) (Figure 5A). 3SH bisadducts were produced
with significant difference (56%) in terms of yields after ten
days of reaction (i.e., 3, 0.13 mM, and 6, 0.31 mM). The kinetic
study of o-quinone-(−)-epicatechin/3SH adduct formation was
stopped after T10day since (−)-epicatechin autoxidation
products dominated over the adduct formation (data not
shown). This phenomenon was observed and amplified in the
presence of iron(II) for both the (+)-catechin and (−)-epi-
catechin reaction mixtures.
The effect of SO2 addition was also examined under these
chemical oxidation conditions as shown in Figures 4 and 5.
Clearly, the presence of SO2 did not prevent the formation of
3SH adducts. However, the rate of the o-quinone-(+)-catechin/
3SH reaction measured at T10day was approximately 16% slower
than that in the absence of SO2 (Figures 4A and 4B). This
result could explain the presence of a lag phase at the beginning
of this reaction, in which the consumption of (+)-catechin was
limited. Surprisingly, the presence of SO2 did not modify the
rate and yield of the o-quinone-(−)-epicatechin/3SH reaction
at all (Figure 5B). According to the literature, catechols (i.e., 4-
methylcatechol, caffeic acid, (+)-catechin) that are used/
present at concentrations similar to those of our model
reactions (i.e., 9 mM) appeared to be oxidized at a faster rate
than SO2, which then reacted with the hydrogen peroxide
produced during the oxygen reduction.36,37
However, the kinetic study of the production of 3SH adducts
derived from the oxidation of caftaric acid (Figure 6) again
revealed the special and mutual reactivity of 3SH and the
caftaric acid-derived o-quinone, as previously observed under
enzymatic oxidation conditions. Under the control chemical
oxidation conditions (H2O2, at 35 °C), the formation of 7 was
observed, with a maximum yield at T2day. This result also
indicated a rate of formation for 7 that was much greater than
those observed for 2 and 5 (i.e., 5-fold greater than for 5 and
15-fold greater than for 2). Moreover, once the maximum yield
of 7 was reached, no further conversion was observed.
quinone-(+)-catechin/3SH adducts (1a/b, 2, and 3) at T10day
.
Using equimolar amounts of each flavan-3-ol (9 mM) with
an excess of 3SH (12 mM), the thiol function addition to the B-
ring of the oxidized (+)-catechin occurred equally at both the 2′
and 5′ positions (i.e., 1a/b, 0.81 mM; 2, 0.73 mM, Figure 4A).
However, 3SH preferentially added to the 5′ position of the
(−)-epicatechin-derived o-quinone (i.e., 0.35 mM for 5 vs 0.19
The effect of iron(II) on the formation rates and yields of
1a/b, 2, 3, 4a/b, 5, 6, and 7 in the wine model media was then
investigated. Similar experiments to those described above were
performed in an oxygen saturated system, in which a 6-fold
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of caftaric acid oxidation surprisingly dropped sharply in the
presence of SO2 and a lag phase was observed for the formation
of 7, with the consequence of a maximum yield noticed at T6day
instead of T2day (Figure 6).
Identification of Adducts between 3-Sulfanylhexan-1-
ol and o-Quinones Derived from (+)-Catechin and
Caftaric Acid in Must Supplemented with 3-Sulfanyl-
hexan-1-ol. After the structural characterization of the adducts
1a/b, 2, 3, 4a/b, 5, 6, and 7, and the mechanistic study
performed in the model solution, the identification of these
adducts in an enological matrix was then investigated.
Considering the fact that under enzymatic oxidation conditions
the yield of adducts was higher than under chemical oxidation
conditions, the analysis was performed in a white native
Sauvignon blanc must (Graves, 2008 vintage) freshly extracted
at an industrial scale during grape pressing with a conventional
white vinification process. Aliquots of 300 mL of must,
containing 22 mg L−1 of (+)-catechin, 2.7 mg L−1 of
(−)-epicatechin, 124 mg L−1 of caftaric acid, and 20 mg L−1
of free SO2 were either supplemented or not with 3SH (100 μg
L−1) and 100 mg L−1 of pimaricine (natamycin), as an alcoholic
fermentation inhibitor. Then, the mixtures were incubated at
room temperature under stirring and while exposed to air for a
period of 5 days. Each experiment was performed in duplicate.
The formation of the adducts of interest in this native must
matrix was first verified and the kinetics of their formation was
then monitored by HPLC coupled to mass spectrometry
analysis. Acquisitions were carried out in negative ion mode
with a full scan on the mass range 100−1000. After 24 h of
incubation, adducts 2 and 7 were indeed generated as the major
HPLC/ESI-MS-detected known products. The monitoring of
their production in this complex matrix permitted us to observe
evidence of a level of formation of the o-quinone-(+)-catechin/
3SH adduct which was higher than that of the o-quinone-
caftaric acid/3SH adduct, even though the initial concentration
of (+)-catechin (i.e., 22 mg L−1) was six times lower than that
of caftaric acid (i.e., 120 mg L−1) in the must matrix (Figure 7).
Figure 6. Kinetic formation rate of 7 under chemical oxidation
conditions. Impact of sulfur dioxide and iron(II) sulfate. The
compound numbering is defined in Figure 1.
molar excess of H2O2 was used relative to the quantity of
iron(II) sulfate added. The presence of iron increased the final
yields of all 3SH monoadducts derived from the oxidation of
(+)-catechin (i.e., 1a/b and 2) (Figure 4C), (−)-epicatechin
(i.e., 4a/b and 5), (Figure 5C) and caftaric acid (i.e., 7) (Figure
6). However, the reaction rate was dependent upon the nature
of the starting phenolic compound. The formation rates of 2
and 5 were increased about 2-fold and those of 7 and 1a/b 2.5-
and 4-fold, respectively. The kinetic profile of the formation of
7 was similar to that observed in the absence of iron salts
(Fe(II) sulfate). However, iron increased the yield of 7, by
promoting o-quinone-caftaric acid production, but not the rate
of thiol addition. In the presence of iron(II) sulfate, the
addition of 3SH at position 6′ of the o-quinone-(+)-catechin
was favored compared to that at position 5′ (Figure 4C). In
contrast, no preference for the addition site of 3SH on o-
quinone-(−)-epicatechin was observed in the presence of
iron(II) sulfate (Figure 5C). Under these oxidative conditions
(Fe(II), H2O2) a major production of multiple substitution
adducts appeared with both o-quinone-(+)-catechin and o-
quinone-(−)-epicatechin. Single adducts between 3SH and o-
quinone-(+)-catechin or o-quinone-(−)-epicatechin appeared
to be easily oxidized in the presence of Fenton reagents (Fe(II),
H2O2), resulting in the formation of multiadducts. Moreover,
the production of 6 was 17% higher than that of 3 after 10 days
of incubation, indicating that o-quinone-(−)-epicatechin/3SH
adducts could undergo coupled oxidation reactions more easily
than the adducts of o-quinone-(+)-catechin/3SH.
However, the catalytic role played by iron(II) in oxidizing
polyphenolic flavanoids was clearly demonstrated during the
incubation period. In the presence of SO2, the concentrations
of 1a/b, 2, and 3 dropped slightly (Figure 4D), whereas the
rates of formation of 4a/b, 5 and 6 appeared unchanged
(Figure 5D). In the presence of iron(II) and H2O2 and
(−)-epicatechin, SO2 became partially ineffective as an
antioxidant.17 This phenomenon might be related to the
rapid decrease of free SO2 and the resulting increase in sulfate
in the medium due to the production of hydrogen peroxide:
Figure 7. Kinetic formation rate of 2 and 7 as well as (+)-catechin and
caftaric acid consumption in white must under enzymatic oxidative
conditions. The compound numbering is defined in Figure 1.
H2O2 + HSO3 → SO4 + H+ + H2O.36 Moreover, as
indicated in the literature, the autoxidation of SO2 is initiated
by coordination to a transition metal, such as Fe(III), to form a
sulfite complex.36 Electron transfer within the complex results
in the formation of a sulfite radical with the release of Fe(II).
The sulfite radical is then able to add to oxygen in order to
produce the highly oxidizing peroxomonosulfite radical, which
is capable of oxidizing sulfite to regenerate sulfite radicals and
so continue the radical chain reaction.36 Nevertheless, the level
−
2−
Caftaric acid was also noticed to be oxidized more rapidly than
(+)-catechin in the must, but its consumption was not
proportional to the formation of 7. It would thus seem that
the caftaric acid-derived o-quinones in such a matrix have a
greater affinity to other nucleophiles than to 3SH. The
disappearance of caftaric acid could alternatively be related to
the formation of different products with other phenolic
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ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed descriptions of NMR spectra of all the new isolated
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
*Tel: +33-557575860. Fax: +33-557575813. E-mail: philippe.
darriet@oenologie.u-bordeaux2.fr.
Funding
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We thank the Bordeaux Wine Council (CIVB) and Aquitaine
Council for their financial support.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We express our thanks to Dr. Ced
Lefeuvre, and Laura Montero for contributing to some
́
ric Saucier, Dr. Dorothee
́
preliminary data for this study.
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