Pyranoanthocyanin dimers: A new family of turquoise blue anthocyanin-derived pigments found in port wine

Article abstract of DOI:10.1021/jf9044414

In the present work, several compounds bearing similar spectroscopic features were found to occur in aged Port wines and respective sediments (lees). The data obtained revealed two new families of compounds with unique spectroscopic characteristics, displaying a wavelength of the maximum absorption at high wavelength in the visible spectrum at ～730 and ～680 nm. The structure of these pigments was elucidated by liquid chromatography/diode array detector-mass spectrometry (LC/DAD-MS) and nuclear magnetic resonance (NMR), and their formation pathway in wines was established. Their structure is constituted by two pyranoanthocyanin moieties linked together through a methyne bridge. This new family of compounds displays an attractive and rare turquoise blue color at acidic conditions and has never been reported in the literature.

Full text of DOI:10.1021/jf9044414

5154 J. Agric. Food Chem. 2010, 58, 5154–5159
DOI:10.1021/jf9044414
Pyranoanthocyanin Dimers: A New Family of Turquoise Blue
Anthocyanin-Derived Pigments Found in Port Wine
†
´
J
OANA
O
LIVEIRA,† JOANA
A
ZEVEDO,† ARTUR M. S. SILVA,‡ NATERCIA
T
EIXEIRA
,
†
,†
L
UIS
C
RUZ,† NUNO
MATEUS
,
AND
VICTOR DE
F
REITAS
*
†
~
^
Centro de Investigac-ao em Quı
´
mica, Departamento de Quı
´
mica, Faculdade de Ciencias, Universidade do
Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal, and ‡Departamento de Quı
´
mica e Quımica
´
^
Organica de Produtos Naturais e Agroalimentares (QOPNA), Universidade de Aveiro, 3810-193 Aveiro,
Portugal
In the present work, several compounds bearing similar spectroscopic features were found to occur
in aged Port wines and respective sediments (lees). The data obtained revealed two new families of
compounds with unique spectroscopic characteristics, displaying a wavelength of the maximum
absorption at high wavelength in the visible spectrum at ∼730 and ∼680 nm. The structure of these
pigments was elucidated by liquid chromatography/diode array detector-mass spectrometry (LC/
DAD-MS) and nuclear magnetic resonance (NMR), and their formation pathway in wines was
established. Their structure is constituted by two pyranoanthocyanin moieties linked together
through a methyne bridge. This new family of compounds displays an attractive and rare turquoise
blue color at acidic conditions and has never been reported in the literature.
KEYWORDS: Pyranoanthocyanins; wine; lees; unusual spectroscopic features
INTRODUCTION
liquid chromatography/diode array detector-mass spectrometry
(LC/DAD-MS), and studies performed in model solutions
revealed that these latter pigments were formed from the reaction
of carboxypyranoanthocyanins with flavanols mediated by acet-
aldehyde (19, 20). Similar compounds were also detected and
identified, with the flavanol moiety replaced by a phenolic
molecule in their structure (catechol, phenol, syringol, or
guaiacol) (21). The detection and identification of portisins point
to new chemical pathways that can occur in red wines and belong
to the second generation of compounds, where the main pre-
cursors involved are no longer anthocyanins but anthocyanin-
derived pigments.
The detection and identification of new pigments in wines help
to better understand the color changes observed during matura-
tion. The present work deals with the detection and identification
of two new families of compounds in a 9-year-old Port wine and
respective sediments (lees).
Red wine is a very complex matrix because of the extraction of
a wide variety of compounds from red grape skins. Among red
wines, Port wine has a higher complexity, owed to the addition of
wine spirit (to stop the fermentation), which confers them great
potential for the formation of new compounds (1, 2). Anthocya-
nins are the main pigments present in grapes, being responsible
for the red-violet color observed in young red wines. During
wine aging and maturation, anthocyanins participate in several
reactions leading to the formation of new pigments. The trans-
formation mechanisms involved in the formation of new pig-
ments were first described as condensation reactions of antho-
cyanins with flavanols mediated or not by acetaldehyde (3-10).
However, over the past decade, new anthocyanin-derived pig-
ments have been detected and identified, namely, pyranoantho-
cyanins that are known to be formed from the reaction of
anthocyanins with small molecules, such as acetaldehyde (11),
acetoacetic acid (12), pyruvic acid (13), vinylphenol (14), vinyl-
guaiacol (15), vinylcatechol (16), and vinylcatechin (17). Pyrano-
anthocyanins are thought to contribute to the orange hues
observed during wine maturation. Among the different pyra-
noanthocyanins present in red wines, carboxypyranoanthocya-
nins are the main pigments detected by high-performance liquid
chromatography (HPLC) after only 1 year of aging (18). A few
years ago, a new family of anthocyanin-derived compounds
(portisins) had been detected and isolated and was found to
display unusual spectroscopic features, presenting a bluish color
in acidic conditions (19). Nuclear magnetic resonance (NMR),
MATERIALS AND METHODS
Reagents. 4-Vinylphenol was purchased from Sigma-Aldrich
(Madrid, Spain). TSK Toyopearl gel HW-40(S) was purchased from
Tosoh (Tokyo, Japan). 8-Vinylcatechin, vinylcatechol, carboxypyrano-
malvidin-3-glucoside, and methylpyranomalvidin-3-glucoside were
synthesized and purified as previously described (12, 22, 23).
Wine and Lees Samples. The studied wine was a commercial red Port
wine (vintage style from the year 1998) aged in a bottle for 9 years [pH 3.7,
20% alcohol (v/v), 6.3 g/L total acidity, and 22 mg/L total SO2]. The lees
were obtained from the bottom of the bottle by filtration.
Wine and Lees Fractionation. The compounds present in lees were
extracted with a solution of acidulated methanol (100 mL) by stirring for 2
h. After that, the mixture was filtered and the methanol was evaporated in
a rotator evaporator at room temperature.
*To whom correspondence should be addressed. Telephone: þ351-
220402558. Fax: þ351-220402658. E-mail: vfreitas@fc.up.pt.
pubs.acs.org/JAFC
Published on Web 03/05/2010
© 2010 American Chemical Society

Article
J. Agric. Food Chem., Vol. 58, No. 8, 2010 5155
Figure 1. HPLC-DAD chromatograms of the fraction of the lees extract eluted from TSK Toyopearl gel with 60% (v/v) aqueous methanol, recorded at 730
and 680 nm.
The lees extract and the Port wine filtered were fractionated by TSK
Toyopearl HW-40(S) column chromatography using different aqueous
solutions with increasing percentages of methanol up to 80% (v/v). The
fractions (20, 40, 60, and 80% methanol) obtained from wines and lees
were analyzed by HPLC-DAD-MS.
Reaction of Carboxypyranomalvidin-3-glucoside with Other
Wine Phenolics. Five solutions of carboxypyranomalvidin-3-glucoside
(4 mM) were incubated in an aqueous solution of ethanol 20% (v/v) at pH
4.0 with malvidin-3-glucoside (4 mM), vinylphenol (4 mM), 8-vinylcate-
chin (4 mM), vinylcatechol (4 mM), and vinylpyranomalvidin-3-gluco-
side-catechin (4 mM). All of the reaction mixtures were left to react at
30 °C. The formation of the new compounds was monitored by
HPLC-DAD.
Reaction of Methylpyranomalvidin-3-glucoside with Carboxy-
pyranomalvidin-3-glucoside and Purification of the New Formed
Compound. Methylpyranomalvidin-3-glucoside (1.4 μM) and carboxy-
Figure 2. UV-vis spectra of compounds 4 and 7 recorded from the
HPLC-DAD.
pyranomalvidin-3-glucoside (2 μM) were incubated in 40 mL of an
most intense ion in the first scan, and a MS-MS of the most intense ion
aqueous solution of 20% ethanol (v/v) at pH 4.0. The reaction mixture
using a relative collision energy of 30 and 60 V.
was left to react at 30 °C. The formation of new compounds was monitored
by HPLC-DAD. A similar reaction was performed between the coumar-
oyl derivative of methypyranomalvidin-3-glucoside (1.4mM) and carboxy-
pyranomalvidin-3-glucoside (2 mM) in 5 mL of an aqueous solution of
ethanol 20% (v/v). After the maximum formation of the new compound
(14 days) was reached, the reaction mixtures were pre-purified by TSK
Toyopearl HW-40(S) gel column chromatography, with the major
compound formed being eluted with a 50% (v/v) methanol aqueous
solution. After that, each compound was isolated by semi-preparative
HPLC using the conditions described above. The structures of the
compounds were characterized by LC/DAD/electrospray ionization
(ESI)-MS and NMR.
HPLC Analysis. The fractions and reaction mixture were analyzed
with Knauer K-1001 HPLC on a 250 ꢀ 4.6 mm inner diameter reversed-
phase C18 column (Merck, Darmstadt); detection was carried out at 676
and 730 nm using a Knauer K-2800 DAD. The solvents were A, H2O/
HCOOH (9:1), and B, HCOOH/H2O/CH3CN (0.5:19.5:80). The gradient
consisted of 20-85% B for 70 min at a flow rate of 1.0 mL/min. The
column was washed with 100% B for 20 min and then stabilized with the
initial conditions for another 20 min.
LC-MS Analysis. All of the fractions and the reaction mixture were
analyzed by LC-MS to detect the presence of new polyphenolic com-
pounds. A Finnigan Surveyor series liquid chromatograph, equipped with
a 150 ꢀ 4.6 mm inner diameter, 5 μm LicroCART reversed-phase C18
column thermostatted at 25 °C, was used. The mass detection was carried
out by a Finnigan LCQ DECA XP MAX (Finnigan Corp., San Jose, CA)
mass detector with an atmospheric pressure ionization (API) source of
ionization and an ESI interface. Solvents were A, H2O/TFA (99.9:0.1),
and B, HCOOH/H2O/CH3CN (0.5:19.5:80). The HPLC gradient usedwas
the same reported above for the HPLC analysis. The capillary voltage was
4 V, and the capillary temperature was 300 °C. Spectra were recorded in
positive-ion mode between m/z 300 and 1500. The mass spectrometer was
programmed to do a series of three scans: a full mass, a zoom scan of the
NMR Analysis. 1H NMR (500.13 MHz) and 13C NMR (125.77 MHz)
spectra of compound 8 was measured in dimethylsulfoxide (DMSO)/
trifluoroacetic acid (TFA) (9:1) on a Bruker-Avance 500 spectrometer at
293 K with tetramethylsilane (TMS) as the internal standard. 1H chemical
shifts were assigned using 1D and 2D 1H NMR (gCOSY), while 13C
resonances were assigned using 2D NMR techniques (gHMBC and
gHSQC) (24, 25). The delay for the long-range C/H coupling constant
was optimized to 7 Hz.
RESULTS AND DISCUSSION
Detection of New Polyphenolic Compounds in Port Wine and
Respective Lees. The presence of new polyphenolic compounds
was assessed in a 9-year-old red Port wine and the respective lees.
The wine fractions obtained from TSK Toyopearl gel column
chromatography revealed compounds already described in the
literature, such as carboxypyranoanthocyanins (13, 26-29),
portisins (17), and other pyranoanthocyanins (data not shown)
(6, 30, 31). However, in the fraction eluted with 60% (v/v)
methanol, several pigments were detected with spectroscopic
features never observed before in red wines or lees (Figure 1).
Most ofthe pigments detected in this fraction displayed a UV-vis
spectrum with a λmax around 730 and 680 nm (Figure 2), which
probably correspond to two new families of compounds. These
λmax values are quite bathochromically shifted compared to that
of other pigments present in wines (anthocyanins, around
525 nm; carboxypyranoanthocyanins, 510 nm; and portisins,
570 nm). The molecular weight and mass spectra of those new
pigments are shown in Table 1. Compounds 6 and 7 are present in
wines only in trace amounts in comparison to compounds 1-5.
For all compounds, the fragmentation patterns are consistent

5156 J. Agric. Food Chem., Vol. 58, No. 8, 2010
Oliveira et al.
Table 1. Molecular Ion and Respective Fragments MS2 and MS3 Obtained by LC-ESI-MS of Several Compounds Detected in Wine Lees (L) and Port Wine (W)
(with λmax Recorded from the LC-DAD)
peak (compound)
λmax (nm)
m/z [Mþ]
m/z (MS2)
fragment
m/z (MS3)
fragment
source
1
2
3
4
5
6
7
730
730
727
733
727
676
676
1205
1205
1337
1351
1321
1191
1337
1043
1043
1029
1043
1013
1029
1029
glucose
glucose
735
735
721
735
705
721
721
coumaroylglucose
coumaroylglucose
coumaroylglucose
coumaroylglucose
coumaroylglucose
coumaroylglucose
coumaroylglucose
W þ L
W þ L
W þ L
W þ L
W þ L
Wa þ L
Wa þ L
coumaroylglucose
coumaroylglucose
coumaroylglucose
glucose
coumaroylglucose
a Trace amounts.
Figure 3. HPLC-DAD of the reaction of mv-3-glc-py with 8-vinylcatechin after 2 days and UV-vis spectrum recorded from the LC-DAD detector.
with the loss of two sugar moieties, acylated or not with coumaric
acid. These data point that the structure of the new pigments
detected includes a double anthocyanin-derived arrangement.
The LC-MS data point to some similarity between the structures
of compounds 1-5, differing by 16 and 30 amu from each other,
which probably correspond to different hydroxylation and meth-
oxylation patterns. These structural differences are particularly
visible in the MS3 spectrum, where the sugar moietiesare released.
This could mean that this family of pigments results from the
different anthocyanins present in wine, with a different pattern of
substitution of ring B, and differing from the presence or absence
of acylated sugars. Additionally, as shown in Table 1, compounds
3 and 7 have the same ion mass and same fragmentation pattern
but they displayed different spectroscopic features (λmax ∼ 727
and 676 nm, respectively). Therefore, these compounds are likely
to have some structural differences and belong to different
families of pigments.
The isolation of the referred compounds from wines and lees is
very difficult because of the complexity of the compounds present
in these matrixes. Therefore, the amounts ofcompounds obtained
were very low, which made their structural identification by
NMR difficult. Thus, the synthesis of these new polyphenolic
compounds was attempted in model solutions to better under-
stand their formation in wines and to obtain sufficient amounts
for NMR structural characterization.
Tentative Synthesis of the New Detected Compounds in Model
Solutions. Because it was assumed that these newly formed
pigments are likely to bear a double anthocyanin-derived ar-
rangement, several reactions were undertaken in the wine model
solution involving anthocyanins, different classes of pyra-
noanthocyanins [carboxypyranoanthocyanins, methylpyranoan-
thocyanins and vinylpyranoanthocyanins (portisins)], and other
compounds present in wines, such as vinylphenol, vinylcatechol,
and vinylcatechin. Compounds displaying similar HPLC-
MS-DAD spectroscopic features than some of the new pigments
were detected from different reactions involving necessarily
carboxypyranomalvidin-3-glucoside and pyranoanthocyanins
(portisins and methylpyranoanthocyanins) or the vinyl com-
pounds referred to above.
However, a single chromatographic peak 8 with a λmax at
672 nm and with an ion mass at m/z 1045 was observed after
2 days of reaction between carboxypyranomalvidin-3-glucoside
with vinylphenol, vinylcatechol, or vinylcatechin (Figure 3).
The UV-vis spectrum and mass fragmentation pattern of this
newly formed pigment 8 is consistent with those of two of the new
compounds detected in Port wine lees (pigments 6 and 7). Thus,
the compound obtained in model solutions and these ones
detected in Port wine and respective lees are likely to have a
related structure. Unfortunally, the reaction yields were very
low, and the formation of this new compound was found to be
very limited because the major pigments formed from the
reaction of carboxypyranomalvidin-3-glucoside with vinyl-
phenolics were the respective portisins already described in the
literature (19-21, 28) (Figure 3).
However, in the reaction of carboxypyranomalvidin-3-gluco-
side with methylpyranomalvidin-3-glucoside, pigment 8 started
to appear in less than 1 day and attained the maximum forma-
tion after 14 days in a much higher quantity (∼12 mg; yield
of 20%).
Compound 6 was obtained from the reaction of the coumaroyl
derivative of methylpyranomalvidin-3-glucoside with carboxy-
pyranomalvidin-3-glucoside with a λmax at 676 nm. The mole-
cular weight of this later compound has more 146 amu than
compound 8, corresponding to the presence of a coumaroyl
group. The LC-MS data of this compound showed an ion mass
at m/z 1191 and the same two fragments at m/z 1029 and 721 than
compound 6 detected in Port wine and lees. Furthermore, the
HPLC retention time and the visible spectra are in agreement with
the results obtained for pigment 6. After purification by Toyo-
pearl gel column chromatography and isolation by semi-prepara-
tive HPLC, compounds 6 and 8 were characterized by NMR
spectroscopy.

Article
NMR Spectroscopy. The structural characterization of com-
J. Agric. Food Chem., Vol. 58, No. 8, 2010 5157
expected. Indeed, for compound 8, protons 20,60 and 30,50-(OMe)2
of B rings appeared as singlets at 7.55 and 3.87 ppm, respectively.
The chemical equivalence was also observed for protons 9 of both
D rings that were assigned at 6.78 ppm. Protons 8 and 6 of A rings
also appeared as a singlet at the same chemical shift at 6.91 ppm.
The fifth signal situated at 5.89 ppm is consistent with the
chemical shift of the proton of the methyne bridge. The assign-
ment of the carbon resonances was possible using 2D NMR
techniques (gHSQC and gHMBC). Carbons C-20,60, C-9, C-6, C-
8, C-11, and OCH3 were assigned through their direct correlation
1H-13C in the heteronuclear single-quantum coherence (HSQC)
spectrum at 107.3, 99.5, 99.5, 95.3 and 55.7 ppm, respectively. All
of the glucose carbons were attributed through their direct
1H-13C correlation with the glucose protons. Carbons C-2, C-
3, C-10, and C-40 were attributed through their long-range
correlation with protons H-20,60. Carbons C-30,50 were assigned
at 147.3 ppm from the correlation with protons H-20,6 and OCH3
in the heteronuclear multiple-bond correlation (HMBC) spec-
trum. The correlation of protons H-8 and H-6 in the HMBC
spectrum led to the assignment of carbons C-4a, C-5, C-7, and C-
8a. Carbons C-10 from D rings were attributed to 160.1 ppm by
their long-range correlation with the methyne proton H-11.
Because of the small amount of material obtained for com-
pound 6, its NMR data only allowed for a partial structural
characterization. Although all protons were assigned, only the
carbons attached to the protons of the molecule were assigned by
HSQC (Table 3).
pounds 6 and 8 was performed by NMR analysis in DMSO/TFA
(9:1). The assignment of the proton and carbon chemical shifts
(Table 2) of compound 8 led to the proposed structures shown in
Figure 4. The double pyranoanthocyanin moieties are linked
through a methyne bridge. Because this structure has a symmetry
plan that passes through the methyne carbon-11, only five signals
in the proton spectrum (excluding the sugar moieties) were
Table 2. 1H and 13C Chemical Shifts of the New Compound 8, Determined in
DMSO/TFA (9:1)a
position
δ 1H (ppm); J (Hz)
δ 13
C
HSQC
HMBC
Pyranomalvidin Moieties
2C
3C
155.3
131.9
na
H-20,60
H-20,60
4C
4aC
5A
103.3
150.0
H-6,8
H-6,8
6A
7A
6.91; s
6.91; s
6.78
98.8
163.7
98.8
H-6
H-8
H-6,8
H-6,8
8A
8aA
9D
150.0
99.5
H-9
10D
10B
20,60B
30,50B
40B
160.1
119.5
107.3
147.3
153.2
55.7
H-11
H-20,60
7.55; s
3.89; s
H-20,60
OCH3
H-20,60; OCH3
H-20,60
Similar to the previously described compound 8, an overall
symmetry was observed. Indeed, the chemical shifts of protons
20B and 60B, protons 30,50-(OMe)2, and protons 9D appeared as
only one singlet at 7.48, 3.87, and 6.88 ppm, respectively. Also,
protons 8 and 6 of rings A appeared as a singlet at the same
chemical shift at 6.85 ppm. The chemical shift of the proton of the
methyne bridge was assigned at 5.83 ppm.
30,50-OMe
Methyne Linkage
95.3
11
5.89; brs
=CH-
Glucose Moiety
The major difference between this compound 6 and compound
8 regards the sugar moieties. The region of the signals of the sugar
moieties of compound 6 is much more complex, displaying a
duplication of signals. These data suggest that the two sugar
moieties are not equivalent, as confirmed by the detection of two
anomeric protons at 4.71 and 4.66 ppm. One of the sugar moieties
is acylated with a coumaroyl group, which yielded more signals
attributed to the coumaroyl protons; protons 2000,6000 and 3000,
5000 were attributed to two doublets (J = 8.3 Hz) at 7.00 and
100
4.76; d, 7.6
3.44; /
3.29; t, 8.5
103.0
74.1
76.2
69.8
77.6
60.7
60.7
H-100
200
H-200
300
H-300
400
3.15; /
H-400
500
3.11; /
H-500
6a00
6b00
3.35; /
H-600a, 600b
H-600a, 600b
3.57; /
a s, singlet; brs, broad singlet; d, doublet; t, triplet; /, unresolved; na, not
assigned.
Figure 4. General structure of the new pyranoanthocyanin dimers 6, 7, and 8 detected in wine, lees, and model solutions.

5158 J. Agric. Food Chem., Vol. 58, No. 8, 2010
Oliveira et al.
Table 3. 1H and 13C Chemical Shifts of the New Compound 6, Determined in
DMSO/TFA (9:1)a
position
δ 1H (ppm); J (Hz)
δ 13
C
Pyranomalvidin Moieties
6A
8A
9D
6.85
6.85
6.88
7.48
3.87; s
99.0
99.0
99.2
Figure 6. Color of pyranoanthocyanin dimers in ethanol aqueous solution
at pH 2.0.
20,60B
107.6
56.2
30,50-OMe
Methyne Linkage
5.83
11
100
96.1
Glucose Moiety
4.71
102.9
Coumaroylglucose Moiety
100
4.66
5.95
102.9
113.7
144.9
127.7
115.4
CHdCHRCO2R
CHβdCHCO2R
2000,6000
7.25
7.00; d, 8.3
6.66; d, 8.3
3
000,5000
a s, singlet; d, doublet.
Figure 7. Hypothetic general structure for compounds 1-5 detected in
wine and lees. R1, R2, R4, and R5 = H, OH, or OMe. R3 and R6 = glucose or
coumaroylglucose.
In conclusion, two new families of anthocyanin-derived pig-
ments were detected in a 9-year-old red Port wine and the
respective lees, displaying unusual spectroscopic features. One
group of these newly formed pigments (compounds 6 and 7)
displayed a λmax at 676 nm in the UV-vis spectrum and was
detected in higher levels in wine lees probably because of their
lower solubility in 20% aqueous ethanol, characteristic of Port
wine. In fact, during the synthesis in a 20% (v/v) ethanol aqueous
Port wine model solution, the formation of a sediment corre-
sponding to that compound was observed. The structure of these
compounds was found to correspond to a double pyranoantho-
cyanin arrangement linked by a methyne bridge. These pigments
may arise from the reaction of carboxypyranoanthocyanins with
vinylphenolics and mainly with other pyranoanthocyanins occur-
ring in the wine, such as methylpyranoanthocyanins. Carboxy-
pyranoanthocyanins had already been found to be the major
precursors for the formation of new wine pigments, such as
portisins (16), after only 1 year of aging. Therefore, it is not
surprisingly that they were found to be once again at the origin of
some of these new pigments. At acidic and neutral pH, these
compounds revealed a very attractive unusual turquoise blue
color, appearing as blue sky pigments in acidic conditions
(Figure 6).
Another group of these newly formed pigments was also
detected in both Port wine and lees (compounds 1-5). The
LC-MS data of these compounds also suggested that they are
likely to be characterized by a double anthocyanin-derived
arrangement, as previously discussed. The difference between
both families of compounds seems to be an unsaturated carbon
involved in the conjugation system, which would also explain the
higher λmax. However, to fit the mass data, this is only possible if
the corresponding compound has no charge (Figure 7). A
comparison of the MS spectra of the two families indicates that
they probably have the same substitution pattern on the ring B
Figure 5. Proposed chemical pathway for the formation of compounds 6,
7, and 8 in red wine and model solutions.
6.66 ppm, respectively. Protons R and β were situated at 5.95 and
7.25 ppm.
Mechanism of Formation. Figure 5 shows a proposal for the
formation mechanism of compounds 6, 7, and8. AtwinepH(∼3.5),
deprotonation of the methyl group of the methylpyranoanthocya-
nin may occur, giving rise to the formation of a methylene group at
carbon C-10. Therefore, these new pigments may result from the
nucleophilic attack of the double bond of this methylene group to
the electrophilic carbon C-10 of the carboxypyranoanthocyanin
molecule. The last step should involve the loss of a formic acid
molecule, leading to the formation of a structure with two pyr-
anoanthocyanin moieties linked through a methyne group.
Attending to the yield obtained (∼20%), this mechanism
probably explains the great part of those compounds formed in
Port wines. The mechanism involving the vinyl-phenolics to give
these compounds seems to be more complex, difficult to explain,
and hence, still unknown.

Article
J. Agric. Food Chem., Vol. 58, No. 8, 2010 5159
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Because most of these pigments were found to occur in wine
lees, probably because of their low solubility, their contribution
to the overall color of red wine is thought to be negligible.
Nevertheless, unravelling their formation mechanism allows us
to establish new chemical pathways involving different wine
pigments and, by this way, contributing indirectly to the color
evolution of red wine. After portisins, pyranoanthocyanin dimers
constitute a second group found in wines belonging to the second
generation of anthocyanin-derived pigments in which grape
anthocyanins are no longer involved directly in their formation.
ACKNOWLEDGMENT
The authors thank Dr. Zelia Azevedo for the LC/DAD-MS
´
(20) Mateus, N.; Oliveira, J.; Santos-Buelga, C.; Silva, A. M. S.; de
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from the Symington Family Estates Company for supplying the
wine samples.
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Received for review December 15, 2009. Revised manuscript received
February 9, 2010. Accepted February 20, 2010. This research was
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supported by a Ph.D. grant from Fundac-ao para a Ciencia e a
~
Tecnologia (FCT, Praxis BD/22622/2005) and grants from Fundac-ao
^
para a Ciencia e a Tecnologia (FCT, PTDC/AGR-ALI/65503/2006,
SFRH/BD/30915/2006, and PTDC/AGR-ALI/67579/2006) all from
(14) Fulcrand, H.; Benabdeljalil, C.; Rigaud, J.; Cheynier, V.; Moutounet,
M. A new class of wine pigments generated by reaction between
Portugal and FEDER funding.