Physicochemical studies of new anthocyano-ellagitannin hybrid pigments: About the origin of the influence of oak C-glycosidic ellagitannins on wine color

Article abstract of DOI:10.1002/ejoc.200901133

Kinetic and thermodynamic UV/Vis and structural NMR spectroscopic investigations on the anthocyano-ellagitannin hybrid 1-deoxyvescalagin- (1β→8)-oenin revealed that its enhanced color stability relative to that of the red-colored pigment oenin and resulti

Full text of DOI:10.1002/ejoc.200901133

FULL PAPER
DOI: 10.1002/ejoc.200901133
Physicochemical Studies of New Anthocyano-Ellagitannin Hybrid Pigments:
About the Origin of the Influence of Oak C-Glycosidic Ellagitannins on Wine
Color
Stefan Chassaing,[a,b] Dorothée Lefeuvre,[a,b,c] Rémi Jacquet,[a,b] Michael Jourdes,[a,b,c]
Laurent Ducasse,[b] Stéphanie Galland,[d] Axelle Grelard,[a] Cédric Saucier,[c]
Pierre-Louis Teissedre,[c] Olivier Dangles,*[d] and Stéphane Quideau*[a,b]
Dedicated to Prof. Raymond Brouillard on the occasion of his retirement
Keywords: Natural products / Dyes/Pigments / Anthocyanins / Polyphenols / Ellagitannins
Kinetic and thermodynamic UV/Vis and structural NMR
spectroscopic investigations on the anthocyano-ellagitannin
derived oenin chromophore and the oak-derived vescalagin
4,6-hexahydroxydiphenoyl moiety. The results led us to sug-
hybrid 1-deoxyvescalagin-(1βǞ8)-oenin revealed that its en- gest that this bathochromism-inducing molecular association
hanced color stability relative to that of the red-colored pig-
ment oenin and resulting bathochromism can be rationalized
in terms of an intramolecular π-stacking between the grape-
could be the first ellagitannin-based molecular-level expla-
nation of the red-to-purple color change that takes place dur-
ing the aging of wine in oak barrels.
a result of various chemical reactions that slowly lead to
more stable wine-specific pigments.[2,3] Several chemical re-
actions involving other compounds found in wine, such as
(oligo)flavanols (i.e., from flavan-3-ols to proanthocyanid-
ins),[4] acetaldehyde,[5] pyruvic acid,[6] or vinylphenols,[7]
have been shown to form compounds exhibiting either bluer
or more orangey tints (Scheme 1).
Few investigations have taken into account the influence
of the container on the modulation of wine color. The faster
disappearance of grape anthocyanins in wines aged in oak-
made casks compared with those aged in hermetic cement/
inox vats has generally been attributed to wood porosity
favoring oxidation.[8] However, enabling dioxygen (O2) dif-
fusion is far from being the only contribution of oak wood
to the wine chemical profile, as evidenced for example by
the acceleration of the color evolution upon the simple ad-
dition of oak chips.[9] Indeed, an aqueous alcoholic solution
like wine not only extracts carbohydrate-, lignin-, and fatty
Introduction
Anthocyanins are plant pigments widely found in flowers
and fruits, the pigmentation of which varies from red to
blue depending on various factors, such as pH, complex-
ation with metal ions, π-stacking interactions with colorless
aromatic compounds (i.e., copigmentation), as well as the
structure of the anthocyanins themselves.[1] The bright red
color of young wines is the result of the release of antho-
cyanins from red grape skins during the maceration stage
of the wine-making process. These native grape pigments, in
particular oenin (1), the major anthocyanin in Vitis vinifera
species, progressively disappear during wine maturation as
[a] Institut Européen de Chimie et Biologie,
2 rue Robert Escarpit, 33607 Pessac Cedex, France
E-mail: s.quideau@iecb.u-bordeaux.fr
[b] Institut des Sciences Moléculaires (UMR-CNRS 5255), Uni- acid-derived fragments from toasted oak barrels or chips,
but also hydrolyzable tannins.[10] Among the latter figure C-
versité de Bordeaux,
351 cours de la Libération, 33405 Talence Cedex, France
glycosidic ellagitannins such as vescalagin (3),[11] which is
[c] Institut des Sciences de la Vigne et du Vin, Bordeaux-Aquitaine
(UMR-INRA 1219), Université de Bordeaux,
210 Chemin de Leysotte CS 50008, 33882 Villenave d’Ornon,
France
involved in stereoselective condensation reactions with wine
nucleophiles, including grape anthocyanins.[12] We wish to
report herein our results on the physicochemical characteri-
zation of the novel pigments 4 and 5 generated from 3 and
the anthocyanin oenin (1), as well as from its aglycone mal-
vidin (2; Scheme 2).
[d] Université d’Avignon et des Pays de Vaucluse, INRA, UMR
408, 84000 Avignon, France
E-mail: olivier.dangles@univ-avignon.fr
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200901133.
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O. Dangles, S. Quideau et al.
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Scheme 1. From red grape oenin (1) to either bluer or more orangey wine genuine pigments.[2–7]
HMBC correlations between its 1-H atom and some of the
aromatic carbon atoms of the anthocyani(di)n unit A-ring,
Results and Discussion
The condensation reaction between vescalagin (3), isolated and the β-orientation of the anthocyani(di)n unit at C-1
from Quercus robur heartwood, and oenin (1) was per- was deduced from a Karplus interpretation of the small
3
formed in an acidic organic solution (i.e., 1.5% TFA/THF, coupling constant (i.e., J = 2.0 Hz) between the 1-H and
v/v) and furnished the anthocyano-vescalagin 4, as de- 2-H protons of the glucose moiety of the vescalagin unit,[12]
scribed previously (Scheme 2).[12a] The feasibility of this (ii) the question of whether the anthocyani(di)n unit is
condensation reaction in wine had previously been con- linked through either its C-6Ј or C-8Ј atom to the vescalagin
firmed by observing the formation of 4 in a standard wine unit was answered on the basis of our interpretation of di-
model reaction mixture (i.e., 5 g/L of tartaric acid in 12% agnostic HMBC correlations exhibited by the flavylium A/
EtOH/H2O, v/v, adjusted to pH 3.2; Figure 1).[12a]
C-ring fusion carbon atoms (i.e., C-4Јa and C-8Јa).[13] These
The anthocyano-vescalagin 5 was prepared from 3 and carbon atoms were easily assigned on the basis of their
commercially available malvidin (2) in a manner similar to characteristic chemical shifts and HSQC/HMBC corre-
that followed for 4. Both of these compounds resulted from lations within the flavylium moiety (see Tables S1 and S2 in
an acid-catalyzed nucleophilic substitution at the C-1 center the Supporting Information). The strong HMBC corre-
of 3 with retention of the configuration (Scheme 2). Their lation between C-4Јa and either 6’-H or 8’-H combined
regio- and stereochemistries were determined by NMR with the lack of correlation between C-8Јa and either 6’-H
analyses: (i) the C-1 connectivity with the vescalagin unit or 8’-H enabled the assignment of the correlating proton to
was determined on the basis of the observation of strong 6’-H and not 8’-H. Furthermore, the observation of strong
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Influence of Oak C-Glycosidic Ellagitannins on Wine Color
Figure 1. HPLC detection of the formation of adducts 4 and 5
starting from vescalagin (3) and oenin (1) in a wine model solution
(i.e., 5 g/L of tartaric acid in 12% EtOH/H2O, v/v, adjusted to
pH 3.2, room temp.).[12a]
being structurally fulfilled only in the case of a C-8Ј (and
not a C-6Ј!) linkage.[4b,5] Hence, the C-8Ј/C-1 connectivity
between the anthocyani(di)n and the vescalagin moieties of
the anthocyano-ellagitannins 4/5 have here been unambigu-
ously confirmed.
Scheme 2. Acid-catalyzed formation of the anthocyano-vescalagin
condensation products 4 and 5 starting from vescalagin (3) and
either oenin (1) or malvidin (2) in an acidic organic solution (i.e.,
1.5% TFA/THF, v/v, 60 °C, 1–3 d).
Figure 2. Diagnostic portion of the ROESY correlation map of 1-
deoxyvescalagin-(1βǞ8)-oenin (4) recorded in 1% TFA/MeOD
(v/v). For an estimation of through-space interatomic distances, see
Table 4.
two-bond HMBC correlations between this shielded proton
and both C-5Ј and C-7Ј on the flavylium A-ring is in agree-
ment with this assignment of 6’-H. We also performed a
ROESY experiment which revealed through-space connec-
tivities between B-ring protons (i.e., 2ЈЈ-H and 6ЈЈ-H) and
The most intriguing and wine-related aspect of the for-
the 1-H/2-H protons of the vescalagin moiety (Figure 2). mation of these anthocyano-ellagitannins is their remark-
Such connectivities are known to be characteristic of a C-8Ј able change in color compared with that of their precursors.
linkage of the anthocyani(di)n moiety, the observed spatial Indeed, in the course of these hemisyntheses, the initial red
proximity between the B-ring protons and 1-H/2-H protons reaction mixture gradually turned to deep purple, both in
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the 1.5% TFA/THF medium and in the wine model solu-
tion at pH 3.2 (Figure 1). It is important to recall that, in
mildly acidic aqueous solutions, anthocyanins, such as
oenin (1), typically undergo reversible transformations lead-
ing to mixtures of colored and colorless species.[14] Thus,
the addition of water to the colored flavylium ion AH+
yields a colorless hemiketal B that is in fast cycle–chain
equilibrium with the (E)-chalcone CE, itself in slow equilib-
rium with the (Z)-chalcone CZ (Scheme 3).
Scheme 4. Putative regioisomerization process of 1-deoxyvescala-
gin-(1βǞ8)-oenin (4) to 1-deoxyvescalagin-(1βǞ6)-oenin (6) in
mildly acidic aqueous solutions [Vesca = vescalagin (3)-derived
moiety].
ing an explanation for the change of color. From the pH
dependence of the apparent rate constant of hydration and
of the visible absorbance at the end of the hydration process
(i.e., before CZ formation) and after complete equilibration,
the following kinetic and thermodynamic parameters can
be determined:[15]
Their values are summarized in Table 1 for pigment 4
and its precursor 1. A comparative interpretation of these
data shows that the vescalagin moiety does not alter the
acidic properties of the flavylium cation because the pKa
value is essentially unaltered. However, it appears that 4 is
less prone than its precursor (lower pKh) to forming a col-
orless hemiketal. With respect to hydration, 4 can thus be
claimed to be a more stable pigment than 1. This enhanced
stability is in fact due to a faster dehydration of the hemike-
tal B derived from 4 (see k–h values in Table 1). Although
the pKh difference is rather weak, it is worth noting that its
impact on the percentage of colored species at wine pH
(e.g., 3.2) is significant (i.e., 50% for 4 vs. 30% for 1).
Scheme 3. Structural transformations of anthocyanins 1 and 4 in
mildly acidic aqueous solutions [Vesca = vescalagin (3)-derived
moiety].
Alternatively, proton transfer from AH+ yields colored
tautomeric quinonoid bases A. One can assume with confi-
dence that our novel pigment 1-deoxyvescalagin-(1βǞ8)-
oenin (4) behaves similarly. In this context, rotation around
the C-4Ј–C-4Јa bond of the (E)-chalcone CE followed by C-
ring reclosure and dehydration is a conceivable process that
could cause the regioisomerization of 4 to the less sterically
hindered 1-deoxyvescalagin-(1βǞ6)-oenin (6), however, this
C-6 regioisomer was not isolated (Scheme 4).
Table 1. Kinetic and thermodynamic parameters for the structural
transformation of the selected anthocyanins (0.1  citrate/phos-
phate buffer, ionic strength set at 1  by NaCl, T = 25 °C).[a]
We then examined whether any modulation of such a
pH-dependent equilibrium could be brought about by the
presence of the vescalagin unit with a view to perhaps find- [a] For details of the calculations, see the Supporting Information.
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Influence of Oak C-Glycosidic Ellagitannins on Wine Color
The visible spectra of 1 and 4, as well as their corre- ety of another. However, such a self-association process
sponding aglycones 2 and 5, were first recorded at pH 1 does not seem to be responsible for the modulation of color
(i.e., in the pure flavylium form). The visible absorption because dilution of a solution of 4 (pH 1) from 1.2ϫ10–4 to
band of both 4 and 5 showed a bathochromic shift of about 6.9ϫ10–6 mol/L had no effect on its λmax value (Figure 4).
25 nm with respect to 1 and 2, thus corroborating the pur-
ple hue observed for 4 and 5 (Figure 3, Table 2). The same
observation was made at wine pH.
Figure 4. Visible spectra of 1-deoxyvescalagin-(1βǞ8)-oenin (4) at
different concentrations in aqueous 1  HCl (pH 1). a)
1.2ϫ10–4 mol/L; b) 6.0ϫ10–5 mol/L; c) 3.1ϫ10–5 mol/L; d)
6.9ϫ10–6 mol/L (1 cm optical pathlength).
Furthermore, we evaluated the intermolecular copigmen-
tation ability of vescalagin (3) towards oenin (1) at pH 1
and 3.2 (Figure 5). Based on the fact that the flavylium
form of 1 far from confering much color to a solution in
the pH range of about 3 to neutrality (see Table 1), vescala-
gin (3) could indeed act as a copigment of 1 because the
addition of 3 clearly enhances the color of the solution
when [copigment]/[pigment] Ͼ 5 (Figure 5, b). However, as
ano-ellagitannins 4 and 5 in aqueous 1  HCl solutions (pH 1). can be seen in Figure 5, no significant bathochromic shift
Figure 3. Visible spectra of oenin (1), malvidin (2) and the anthocy-
Estimated molar absorption coefficients (ε [L/mol·cm], 1 cm optical
was observed, thus confirming that the purple hue of the
pathlength): 1: ε = 26000, c = 4.8ϫ10–5 mol/L; 2: ε = 18400, c =
anthocyano-vescalagin adduct 4 (and 5) is not a result of
6.7ϫ10–5 mol/L; 4: ε = 16300, c = 9.4ϫ10–5 mol/L; 5: ε = 15700,
intermolecular interactions between the vescalagin and the
flavylium units of different molecules of 4 (or 5).
c = 7ϫ10–5 mol/L.
Having thus ruled out the possibility of intermolecular
copigmentation, we turned our attention to similar but in-
tramolecular interactions. The geometrical and electronic
spectral criteria of our anthocyano-ellagitannins were first
examined by computational means (see the Supporting In-
formation for details). Because the observed bathochromic
shift appeared to be quasi-independent of the presence of
the 3-O-glucosyl unit on the anthocyanidin chromophore
(Figure 3), calculations were performed only on the agly-
cones malvidin (2) and 1-deoxyvescalagin-(1βǞ8)-malvidin
(5). Geometry optimizations were carried out by using
AM1 parameters[17] with H2O as the solvent. UV/Vis spec-
tra were then calculated from the resulting minimum-energy
conformations using the ZINDO method. To check the reli-
ability of this semi-empirical approach, the ZINDO-calcu-
lated spectrum of 2 was compared with that obtained by
using time-dependent density functional theory (TD-DFT)
at the B3LYP/6-31G(d) level of theory. Both calculations
correctly predicted the experimental λmax value of 517 nm
for 2 [i.e., 516 and 520 nm by ZINDO and B3LYP/6-
Table 2. Maximum absorbance wavelengths for oenin (1), malvidin
(2), 1-deoxyvescalagin-(1βǞ8)-oenin (4), and 1-deoxyvescalagin-
(1βǞ8)-malvidin (5) in four different acidic solutions.[a]
[a] A: Aqueous 1  HCl solution adjusted to pH 1 by the addition
of 1  NaOH (see Figure 3). B: Aqueous 0.02  HCl and 0.2 
KOH buffer solution (pH 1). C: Wine model solution composed of
12% EtOH/H2O (v/v) and tartaric acid (5 g/L) adjusted to pH 3.2
by the addition of 1  NaOH. D: Aqueous 0.1  HCl and 0.1 
potassium hydrogen phthalate buffer solution (pH 3.2). n.d.: not
determined.
A bathochromic shift is often observed when antho- 31G(d), respectively]. However, the first-allowed electronic
cyanins are involved in copigmentation with colorless phen- transition (i.e., single excitation mainly from HOMO to
olic compounds.[1] The bathochromism observed here could LUMO) calculated for 5 using the ZINDO method also
therefore simply be due to intermolecular interactions be- gave a value of 517 nm (see the Supporting Information for
tween the anthocyanidin chromophore of one molecule of details on these calculations). This failure to predict the ba-
4 (or 5) and the galloyl-derived unit of the vescalagin moi- thochromism of the visible band of 5 led us to evaluate the
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Figure 6. Evolution of the ZINDO-calculated first-allowed transi-
tion for malvidin (2, malv) and 1-deoxyvescalagin-(1βǞ8)-malvi-
din (5, vesca-malv) as a function of the size of the active space (n
= m).
tochromism seems to be a general property of anthocyanins
that has been interpreted in terms of the low-energy excited
state of the flavylium nucleus being less polar than the
ground state.[18]
Figure 5. a) Visible spectra of oenin (1; 2.5ϫ10–5 ) and vescalagin
(3) at 0, 1, 2, 5, 10, and 20 [copigment]/[pigment] molar ratios (pH
= 1.0, T = 25 °C, optical pathlength = 1 cm, solvent = aqueous
HCl/KCl buffer, ionic strength = 0.40 ). b) Visible spectra of oenin
(1; 3.8ϫ10–4 ) and vescalagin (3) at 0, 1, 2, 5, 10, and 20 [copig-
ment]/[pigment] molar ratios (pH = 3.2, T = 25 °C, optical
pathlength = 1 cm; solvent = aqueous KH2PO4 buffer, ionic
strength = 0.20 ).
ZINDO procedure by modifying the size of its active space,
which involves every single excitation from a set of n occu-
pied molecular orbitals (MOs) to a set of m unoccupied
MOs (the m + n MO defines the active space).
Calculations were initially performed by arbitrarily set-
ting n and m at 80. Examination of the dependence of the
size of this active space (n = m) on calculated spectra re-
vealed that the first-allowed transition for 2 reaches an as-
ymptotic value of 517 nm for n Ն 60, whereas the value
of λ for the hybrid 5 appears to follow a slow but steady
progression towards bathochromically shifted values for n
Ͼ 110 (Figure 6). Unfortunately, we could not calculate any
value of λ for n Ͼ 110 because the ZINDO procedure does
not permit the size of the active space to be extended (see
the Supporting Information).
Figure 7. Evaluation of solvent effects on a) the visible spectra of
1-deoxyvescalagin-(1βǞ8)-oenin (4; 2.4ϫ10–5 ) in four different
solvents containing 1% TFA (v/v) (T = 25 °C, optical pathlength
= 1 cm) and b) the visible spectra of oenin (1; 2.0ϫ10–5 ) in four
different solvents containing 1% TFA (v/v) (T = 25 °C, optical
pathlength = 1 cm).
Notwithstanding such a failure to predict the bathochro-
mism of the visible band of 5, we examined the effect of
solvent by recording the visible spectra of 4 in H2O,
It has been claimed that such a change in electronic dis-
CH3CN, MeOH, and THF (2.4ϫ10–5 mol/L). A trend tribution in the flavylium nucleus could strengthen its apti-
towards an increase in λmax with decreasing solvent polarity tude for stacking phenolic rings, thereby providing a con-
was observed (Figure 7 and Table 3). Such a negative solva- ceivable rationale for the bathochromic effect typically ob-
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Influence of Oak C-Glycosidic Ellagitannins on Wine Color
Table 3. Maximum absorbance wavelengths and molar absorption much weaker or absent in the hemiketal B such that the
coefficients for oenin (1) and 1-deoxyvescalagin-(1βǞ8)-oenin (4)
activation energy barrier for dehydration is lower for 4
in four different solvents containing 1% TFA (v/v). Pigment con-
(higher k–h, see Table 1 and Schemes 3 and 4).
centrations: [1] = 2.0ϫ10–5  and [4] = 2.4ϫ10–5  (T = 25 °C,
optical pathlength = 1 cm).
Conclusions
The bathochromism of the visible band of the anthocy-
ano-ellagitannin 4 (and 5) relative to that of the corre-
sponding free anthocyani(di)n component 1 (or 2) can arise
either from stronger intramolecular interactions between
their flavylium and vescalagin 4,6-HHDP moieties in the
low-energy excited state of the flavylium ion[18] or from a
charge-transfer contribution.[21] Although the exact cause
[a] Expressed in 103 L/mol·cm.
of this bathochromism is still hypothetical at the fundamen-
tal level, the results described herein constitute a first plaus-
ible molecular-level explanation of the red-to-purple color
served upon copigmentation.[18] To corroborate the plausi-
bility of such a claim in the case of pigments 4 and 5, we
had to find evidence of an intramolecular interaction in-
volving their flavylium units. A closer look at the 1H NMR
spectroscopic data of 4 and 5 indicates an abnormally up-
field chemical shift for the 2Ј-H of the galloyl-derived V-
ring (i.e., 5.65 and 5.68 ppm), whereas the 2Ј-H atoms of
the III- and IV-rings resonate in the range 6.50–6.80 ppm
evolution observed during the early stages of wine-maturing
processes in oak barrels (or with the addition of oak chips),
and possibly involving oak ellagitannins.
Experimental Section
(see Figure 2 and Tables S1 and S2 in the Supporting Infor- General: Oenin (1) was obtained by extraction from Merlot grapes
and purification by centrifugal partition chromatography[22] (see
the Supporting Information for details). Malvidin (2) was pur-
chased from Extrasynthese as its chloride salt. (–)-Vescalagin (3)
mation). Such a large upfield shift (ca. 1 ppm) of 2ЈV-H in
4 and 5 suggests that this proton is under the influence of
an anisotropic effect putatively emanating from an interac-
tion with the flavylium moiety, as previously observed, inter
was extracted from Quercus robur heartwood and purified as de-
scribed previously.[17,23] The Amberlist XAD-7 HP resin was pur-
alia, in the intramolecular face-to-face stacking conforma-
chased from Supelco. Tetrahydrofuran (THF) was purified by dis-
tion of the dicaffeoylated anthocyanin gentiodelphin.[19]
tillation from sodium/benzophenone under N2 immediately before
Here, the possibility of such a stacking was confirmed by
use. Methanol (MeOH), acetonitrile (CH3CN), ethyl acetate
the observation on the ROESY correlation map of 4 of
through-space connectivities between 2ЈV-H and the 2ЈЈ-H
(EtOAc), and n-butanol (BuOH) were of HPLC quality and Milli-
Q (Millipore) water was used for HPLC analyses and purifications.
and 6ЈЈ-H atoms of the flavylium B-ring (see Figure 2). The different mobile phases used for all HPLC analyses and sepa-
rations were composed of solvent A [H2O/H3PO4 (999:1)] and sol-
vent B [MeOH/H3PO4 (999:1)], or solvent C [H2O/HCOOH
(990:10)] and solvent D [MeOH/HCOOH (990:10)], or solvent E
[H2O/HCOOH (950:50)] and solvent F [MeOH/HCOOH (950:50)],
or solvent G [H2O/HCOOH (996:4)] and solvent H [MeOH/
HCOOH (996:4)] (see the Supporting Information for details of the
HPLC equipment used). IR spectra were recorded with a FT-IR
Perkin–Elmer Paragon 1000 PC spectrometer. Low- and high-reso-
lution liquid secondary ion mass spectra (LSIMS, HRMS) were
obtained from the Centre d’Etudes Structurales et d’Analyses des
Calibration of this data map revealed a mean distance be-
tween these protons of 3.0Ϯ0.4 Å (Table 4), which is less
than the equilibrium van der Waals’ separation of 3.4 Å.
Taken together, these NMR spectroscopic data demonstrate
that the galloyl-derived V-ring and the flavylium B-ring of
4 experience intramolecular contacts, possibly by van der
Waals or charge-transfer interactions.[19] These interactions
seem largely to be retained in the transition state of the
hydration process because the corresponding rate constant
is very close to that of oenin (1), but they are probably Molécules Organiques (CESAMO), Université Bordeaux 1, and
Table 4. Interatomic distances between 2ЈЈ-H/6ЈЈ-HB and 2Ј-HV, and 1-H, and 2-H.[a,b]
[a] See Figures 2 and S8 in the Supporting Information for details. [b] The intensity Vij of a ROESY cross-peak is related to the inverse
sixth power of the interatomic distance dij between two spins i and j (isolated spin pair approximation ISPA).[20] The cross-peaks in the
spectra were integrated separately for each side of the diagonal and an average was taken with the different values. The cross-peak
intensity that corresponds to the covalently fixed distance of 1.8 Å between the two geminal protons 6-H was used for internal calibration.
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from the Centre Régional de Mesures Physiques de l’Ouest
(CRMPO), Université Rennes 1.
Hemisynthesis of 1-Deoxyvescalagin-(1βǞ8)-oenin (4)[12a]
Visible Absorbance Experiments: Visible absorption spectra were
recorded with a Kontron Instrument-Uvikon 922 spectrophotome-
ter or a Biotek Instrument-Uvikon XL spectrophotometer fitted
with a plastic or quartz 10 mm cell width.
In Organic Solution: Oenin chloride (1; 55 mg, 0.104 mmol) and
(–)-vescalagin (3; 84 mg, 0.09 mmol) were dissolved in an anhy-
drous THF solution (10 mL) containing trifluoroacetic acid
(150 µL). This reaction mixture was stirred at 60 °C for 3 d, after
which time HPLC monitoring using a gradient elution (0–20 min:
0–20% solvent B; 20–35 min: 20–100% solvent B; 35–40 min: 100%
solvent B) with a flow rate of 1.0 mL/min indicated no further for-
mation of 4. The reaction progress was also monitored by HPLC/
ESIMS using a different gradient elution (0–20 min: 0–20% solvent
D; 20–35 min: 20–100% solvent D; 35–40 min: 100% solvent D)
with a flow rate of 1.0 mL/min. The mixture was then evaporated
and the residue was dissolved in water (10 mL) and freeze-dried to
furnish a reddish purple powder (144 mg). This powder was sepa-
rated by semi-preparative HPLC using a gradient elution (0–
20 min: 0–30% solvent F; 20–24 min: 30–100% solvent F; 24–
36 min: 100% solvent F) with a flow rate of 16 mL/min–1 to fur-
nish, after freeze-drying of the corresponding fractions, 1 (10 mg,
19%), 3 (60 mg, 71%), 4 (4 mg, 3%), and 5 (7 mg, 6%). This reac-
tion was repeated four times to afford 14 mg of 4 as an amorphous
Molecular Modeling: The geometry of malvidin (2) was optimized
by using the semi-empirical AM1 parametrization available in the
AMPAC package[24,25] and by ab initio DFT with the three-param-
eter hydrid functional B3LYP and the 6-31G(d) basis set available
in the Gaussian 03 package.[26] Similar molecular parameters were
obtained by either calculation protocol. The main difference be-
tween the AM1 and DFT methods was the value of the dihedral
angle between the phenolic B and the pyrylium C rings of the mole-
cule, values of –16 or +3° were obtained by AM1 or DFT, respec-
tively. The true minima were checked by vibrational calculations.
The vescalagin unit of 1-deoxyvescalagin-(1βǞ8)-malvidin (5) was
first fully minimized by a Monte-Carlo search using the MM3*
force field.[17,27] As a result of the large number of atoms in this
anthocyano-ellagitannin hybrid, further geometry optimization was
only performed at the AM1 level. This was accomplished both in
the gas phase and in water by using the SM5.2 protocol. The re-
sulting geometries were qualitatively similar (see the Supporting
Information for the Cartesian coordinates and details of the
ZINDO calculations).
deep-purple powder. IR (KBr): ν = 3410, 1742 cm–1. 1H NMR
˜
[400 MHz, [D4]MeOH/TFA (99:1)], see Table S2 in the Supporting
Information. 13C NMR [100 MHz, [D4]MeOH/TFA (99:1)], see
Table S2 in the Supporting Information. LSIMS: m/z (%) = 1409
(100) [M+], 1247 (52). HRMS (LSIMS): calcd. for C64H49O37
1409.1953; found 1409.1988.
NMR Analysis: Spectra were recorded in the indicated solvent with
a Bruker DPX 400NB NMR spectrometer equipped with a 5 mm
direct QNP probe with gradient capabilities. Rotating frame nu-
clear Overhauser spectroscopy (ROESY) was performed at 283 and
298 K with 1024(t2)ϫ256(t1) data points in States-TPPI mode with
presaturation of the water signal. A relaxation delay of 3 s and 32
scans per increment; a sweep width of 6000 Hz in two dimensions,
and a ROESY spin-lock of 300 ms were applied. Data processing
was performed with Topspin software using a sine-bell multipli-
cation in two dimensions.
In the Wine Model Solution: Oenin chloride (1; 1.1 mg,
0.002 mmol) and (–)-vescalagin (3, 2 mg, 2.1 µmol) were dissolved
in a standard wine model solution (1.5 mL) composed of a 12%
(v/v) aqueous EtOH solution containing 5 g/L tartaric acid and
adjusted to pH 3.2 by addition of a 1  aqueous NaOH solution.
This mixture was stirred at room temperature and kept in the dark
for 4 months. The reaction progress was monitored by HPLC using
a gradient elution (0–20 min: 0–20% solvent B; 20–35 min: 20–
100% solvent B; 35–40 min: 100% solvent B) with a flow rate of
1.0 mL/min and indicated the formation of 4 and 5 (see Figure 1).
Supporting Information (see also the footnote on the first page of
this article): Experimental and analytical procedures, molecular
modeling details and full NMR spectroscopic data for compounds
4 and 5.
Hemisynthesis of 1-Deoxyvescalagin-(1βǞ8)-malvidin (5)[12a]
Acknowledgments
In Organic Solution: Malvidin chloride (2; 16.5 mg, 0.045 mmol)
and (–)-vescalagin (3; 42 mg, 0.045 mmol) were dissolved in an an-
hydrous THF solution (20 mL) containing trifluoroacetic acid
(300 µL). The reaction mixture was stirred at 60 °C for 3 d, after
which time HPLC monitoring using a gradient elution (0–20 min:
0–30% solvent B; 20–35 min: 30–100% solvent B; 35–40 min: 100%
solvent B) with a flow rate of 1.0 mL/min indicated quasi-comple-
tion of the reaction. The reaction progress was also monitored by
HPLC/ESIMS using a different gradient elution (0–20 min: 0–20%
solvent H; 20–35 min: 20–100% solvent H; 35–40 min: 100% sol-
vent H) with a flow rate of 1.0 mL/min. The mixture was evapo-
rated and the residue was dissolved in water (30 mL) and freeze-
dried to furnish a red powder (67 mg). This powder was separated
by semi-preparative HPLC using a gradient elution (0–20 min: 0–
10% solvent F; 20–35 min: 10–100% solvent F; 35–40 min: 100%
solvent F) with a flow rate of 16 mL/min to furnish, after freeze-
drying, pure 5 (14 mg, 25%) as an amorphous red powder. IR
The authors thank the Conseil Interprofessionnel du Vin de Bor-
deaux (Grant No. 8383/8384) and the Conseil Régional d’Aqui-
taine (Grant Nos. 20020306001F and 20000305002) for financial
support.
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Received: October 6, 2009
Published Online: November 20, 2009
Eur. J. Org. Chem. 2010, 55–63
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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