Anthocyanin intramolecular interactions. A new mathematical approach to account for the remarkable colorant properties of the pigments extracted from Matthiola incana

Article abstract of DOI:10.1021/ja9535064

In the last few years, a series of investigations has brought to light a mechanism of stabilization of the colorant properties of certain anthocyanins. Intramolecular interactions take place between the chromophore moiety of the anthocyanin and one of its aromatic acid residues, which folds over the chromophore and thus confers protection against hydration and subsequent formation of colorless forms. In our continuing study of the physicochemical properties exhibited by acylated natural anthocyanins, we report here on a series of five structurally related pigments extracted from the violet flowers of Matthiola incana. These pigments all bear the same chromophore moiety, i.e., the cyanidin aglycon, but differ in the degree of glycosylation and acylation. Acidity constants for the deprotonation and hydration equilibria of the flavylium cation, together with rate constants for the hydration step alone were determination from UV-visible absorption measurements. The data support the existence of intramolecular, noncovalent interactions that strongly stabilize the colored forms of the pigments. However, none of the four more heavily substituted anthocyanins follows the above-mentioned mechanism that was previously successfully applied to the study of acylated anthocyanins. Consequently, a new mechanism with a different mathematical treatment is here developed to account for the different behavior exhibited by these distinctive pigments.

Full text of DOI:10.1021/ja9535064

4788
J. Am. Chem. Soc. 1996, 118, 4788-4793
Anthocyanin Intramolecular Interactions. A New Mathematical
Approach To Account for the Remarkable Colorant Properties
of the Pigments Extracted from Matthiola incana
Paulo Figueiredo,† Mourad Elhabiri,† Norio Saito,‡ and Raymond Brouillard*,†
Contribution from the Laboratoire de Chimie des Polyphe´nols, associe´ au CNRS,
UniVersite´ Louis Pasteur, Institut de Chimie, 1, rue Blaise Pascal, 67008 Strasbourg, France,
and the Chemical Laboratory, Meiji-Gakuin UniVersity, Totsuka-ku, Yokohama, Japan
ReceiVed October 19, 1995X
Abstract: In the last few years, a series of investigations has brought to light a mechanism of stabilization of the
colorant properties of certain anthocyanins. Intramolecular interactions take place between the chromophore moiety
of the anthocyanin and one of its aromatic acid residues, which folds over the chromophore and thus confers protection
against hydration and subsequent formation of colorless forms. In our continuing study of the physicochemical
properties exhibited by acylated natural anthocyanins, we report here on a series of five structurally related pigments
extracted from the violet flowers of Matthiola incana. These pigments all bear the same chromophore moiety, i.e.,
the cyanidin aglycon, but differ in the degree of glycosylation and acylation. Acidity constants for the deprotonation
and hydration equilibria of the flavylium cation, together with rate constants for the hydration step alone were
determined from UV-visible absorption measurements. The data support the existence of intramolecular, noncovalent
interactions that strongly stabilize the colored forms of the pigments. However, none of the four more heavily
substituted anthocyanins follows the above-mentioned mechanism that was previously successfully applied to the
study of acylated anthocyanins. Consequently, a new mechanism with a different mathematical treatment is here
developed to account for the different behavior exhibited by these distinctive pigments.
Introduction
Scheme 1
Anthocyanins, glycosylated polyhydroxy derivatives of 2-
phenylbenzopyrylium (flavylium) salts, are the pigments re-
sponsible for most of the wide variety of colors displayed by
flowers and fruits, where they are naturally produced.1,2
However, when extracted from plant material, the most common
anthocyanins (3-monoglucosides and 3,5-diglucosides) are
almost colorless in mildly acidic equilibrated aqueous solution
at room temperature, conditions similar to the ones observed
in plant cells. This color loss is due to an hydration reaction
(Scheme 1) of the colored flavylium cation, which produces
the colorless forms hemiacetal and chalcones.3-5
In order to explain the remarkable color stability of antho-
cyanins in nature, several studies were performed in model
solutions containing the pigments and some natural colorless
molecules, currently known as copigments.1,6-10 The results
† Universite´ Louis Pasteur.
‡ Meiji-Gakuin University.
X Abstract published in AdVance ACS Abstracts, May 1, 1996.
(1) Brouillard, R.; Dangles, O. In The FlaVonoids, AdVances in Research
Since 1986; Harborne, J. B., Ed.; Chapman and Hall: London, 1993; pp
565-588.
(2) Goto, T.; Kondo, T. Angew. Chem., Int. Ed. Engl. 1991, 30, 17-33.
(3) Brouillard, R.; Dubois, J. E. J. Am. Chem. Soc. 1977, 99, 1359-
1364.
(4) Brouillard, R.; Delaporte, B. J. Am. Chem. Soc. 1977, 99, 8461-
8468.
(5) Brouillard, R.; Delaporte, B.; Dubois, J. E. J. Am. Chem. Soc. 1978,
100, 6202-6205.
(6) Brouillard, R.; Mazza, G.; Saad, Z.; Albrecht-Gary, A. M.; Cheminat,
A. J. Am. Chem. Soc. 1989, 111, 2604-2610.
(7) Brouillard, R.; Wigand, M. C.; Dangles, O.; Cheminat, A. J. Chem.
Soc., Perkin Trans. 2 1991, 1235-1241.
(8) Mistry, T. V.; Cai, Y.; Lilley, T. H.; Haslam, E. J. Chem. Soc., Perkin
Trans. 2 1991, 1287-1296.
(9) Dangles, O.; Brouillard, R. Can. J. Chem. 1992, 70, 2174-2189.
(10) Wigand, M. C.; Dangles, O.; Brouillard, R. Phytochemistry 1992,
31, 4317-4324.
obtained, using UV-visible and 1H NMR spectrometries,
evidenced the existence of an intermolecular noncovalent
interaction (copigmentation) between the anthocyanin and
S0002-7863(95)03506-2 CCC: $12.00 © 1996 American Chemical Society

Anthocyanin Intramolecular Interactions
J. Am. Chem. Soc., Vol. 118, No. 20, 1996 4789
Chart 1
copigment molecules. Such hydrophobic complexation ef-
ficiently protects the chromophore against the nucleophilic water
attack, thus displacing the equilibria of Scheme 1 toward the
formation of more flavylium molecules.
now present a decrease in the value of Kh,13-15 and only minimal
changes in the deprotonation constant Ka, which signifies that
the interaction between the two moieties of the pigment, while
in the flavylium cation form, proceeds through hydrophobic
stacking and not through hydrogen bonding, since the existence
of free OH groups in the aglycon is a requisite for formation of
the quinonoidal bases, A (Scheme 1).
Since the last decade, with the improvement of extraction
and purification techniques, far more complex anthocyanin
structures, which gave stable colored solutions at mildly acidic
pH values, were discovered and characterized.1,11-15 Most of
these pigments possess cinnamic and/or malonic acid residues
esterified to the sugars. The flexible saccharide chains act as
linkers allowing the folding of the acyl aromatic rings over the
planar pyrylium ring, as evidenced by the existence of NOEs
The present work reports on the first known group of acylated
anthocyanins that represents a deviation to this, until now,
general behavior. The five pigments, which constitute a
structurally homogeneous series based on the cyanidin (3,5,7,3′,4′-
pentahydroxyflavylium) aglycon, were extracted from the violet
flowers of Matthiola incana.21 Their structures (Chart 1), fully
1
in H NMR between the aglycon protons and those of the
acids.16,17 The formation of such intramolecular copigmentation
complexes thus protects the chromophore against hydration in
an even more efficient way than that conferred by intermolecular
copigmentation.14
elucidated by H NMR techniques and FAB-MS, are 3-O-(2-
1
O-â-D-xylopyranosyl-â-D-glucopyranosyl)-5-O-â-D-glucopyra-
nosyl cyanidin (1), 3-O-(6-O-(trans-ferulyl)-2-O-(2-O-(trans-
sinapyl)-â-D-xylopyranosyl)-â-D-glucopyranosyl)-5-O-â-D-
glucopyranosyl cyanidin (2), 3-O-(6-O-(trans-p-coumaryl)-2-
O-(2-O-(trans-sinapyl)-â-D-xylopyranosyl)-â-D-glucopyranosyl)-
5-O-(6-O-(malonyl)-â-D-glucopyranosyl cyanidin (3), 3-O-(6-
O-(trans-caffeyl)-2-O-(2-O-(trans-sinapyl)-â-D-xylopyranosyl)-
â-D-glucopyranosyl)-5-O-(6-O-(malonyl)-â-D-glucopyranosyl
cyanidin (4), and 3-O-(6-O-(trans-ferulyl)-2-O-(2-O-(trans-
sinapyl)-â-D-xylopyranosyl)-â-D-glucopyranosyl)-5-O-(6-O-
(malonyl)-â-D-glucopyranosyl cyanidin (5). The thermody-
namic and kinetic data gathered are compared to the ones
obtained for cyanidin 3,5-diglucoside (cyanin), one of the most
common anthocyanins that can be considered a simpler structural
analogue, and reveal not only a marked increase in the value of
Ka for the more complex pigments but also an otherwise
unknown effect of acidity-dependent interaction, which prompted
us to discuss the results obtained in the light of a new
mechanistic and mathematical approach.
The following equations account for the transformations
undergone by anthocyanin molecules in mildly acidic aqueous
solutions:
Ka
\
AH+ y
z A + H+
(1)
Kh
AH+ + H2O y z (B + CE) + H+
\
(2)
Since the ring opening of B to form the E-chalcone occurs in
a very fast step,18,19 we write the equilibrium as a whole for
the sake of clarity; CZ, which is usually formed in very small
amounts20 is neglected. All the acylated molecules studied until
(11) Harborne, J. B.; Grayer, R. J. In The FlaVonoids, AdVances in
Research Since 1980; Harborne, J. B., Ed.; Chapman and Hall: London,
1988; pp 1-20.
(12) Strack, D.; Wray, V. In The FlaVonoids, AdVances in Research Since
1986; Harborne, J. B., Ed.; Chapman and Hall: London, 1993; pp 1-22.
(13) Dangles, O.; Saito, N.; Brouillard, R. Phytochemistry 1993, 34, 119-
124.
Experimental Section
(14) Dangles, O.; Saito, N.; Brouillard, R. J. Am. Chem. Soc. 1993, 115,
3125-3132.
(15) Figueiredo, P.; Elhabiri, M.; Toki, K.; Saito, N.; Dangles, O.;
Brouillard, R. Phytochemistry 1996, 41, 301-308.
(16) Goto, T.; Tamura, H.; Kawai, T.; Hoshino, T.; Harada, N.; Kondo,
T. Ann. N. Y. Acad. Sci. 1986, 471, 155-173.
(17) Yoshida, K.; Kondo, T.; Goto, T. Tetrahedron 1992, 48, 4313-
4326.
(18) McClelland, R. A.; Devine, D. B.; Sorensen, P. E. J. Am. Chem.
Soc. 1985, 107, 5459-5463.
(19) Brouillard, R.; Lang, J. Can. J. Chem. 1990, 68, 755-761.
Materials. Pigments 1-5 were isolated from the violet flowers of
Matthiola incana according to a published procedure.21 Their purity,
always superior to 98%, was checked by 1H NMR spectroscopy.
Cyanidin 3,5-diglucoside, henceforth referred to as cyanin, was a kind
gift of Prof. Sam Asen and was used without further purification, and
(20) Santos, H.; Turner, D. L.; Lima, J. C.; Figueiredo, P.; Pina, F. S.;
Mac¸anita, A. L. Phytochemistry 1993, 33, 1227-1232.
(21) Saito, N.; Tatsuzawa, F.; Nishiyama, A.; Yokoi, M.; Shigihara, A.;
Honda, T. Phytochemistry 1995, 38, 1027-1032.

4790 J. Am. Chem. Soc., Vol. 118, No. 20, 1996
Figueiredo et al.
Table 1. Maximum Visible Wavelength and Molar Absorption Coefficient for the Flavylium Cation Form of the Seven Anthocyaninsa
pigment
cyanin
antirrhinin
1
2
3
4
5
λmax/nm
508
510
7000
522
3600
528
15 100
536
19 000
538
21 200
538
20 100
ꢀAH+/mol-1 dm3 cm-1
a pH ) 0.9.
35 00024
cyanidin 3-rutinoside (antirrhinin) was synthesized following an
elsewhere described method.22 All other reagents used were of
analytical grade.
Absorption Spectra. Spectra were recorded with a Hewlett-Packard
diode-array spectrometer fitted with a quartz cell (d ) 1 cm) equipped
with a stirring magnet. A constant temperature of 25((0.1) °C was
obtained by use of a Lauda water-thermostated bath. Temperature was
measured with a Comark thermocouple.
Thermodynamic Measurements. Mother solutions of ca. 5 × 10-4
M of all the anthocyanins were prepared in 0.1 M HCl and left to
equilibrate in the dark for about 2 h. Then, for each pigment 10
solutions were prepared by 1:10 dilutions of the mother solutions with
different volumes of a NaOH 0.1 M solution and H2O so that the final
pH covered a range of 1.0-4.0. After equilibration in the dark, the
UV-visible spectra of these solutions were recorded. The values of
the hydration equilibrium constants (Kh) are gained from measuring
the relative hyperchromic shift at the visible absorption maxima as a
function of pH.
Figure 1. Electronic absorption spectra of 4 (4.05 × 10-5 M, T ) 25
°C) in aqueous solution. 1, pH 0.8; 2, pH 1.35; 3, pH 2.4.
here reported, there is one (pigment 1) where the hypochromic
effect is far more stronger, exhibiting an ꢀ ca. ten times smaller
than that published for cyanin24 (Table 1). Such a large drop
in the value of the absorption coefficient seems to be a general
characteristic of the molecules possessing a disaccharide as a
substituent group in position 3 of the chromophore, since a
similar pattern was already noticed in two other anthocyanins
presenting an analogous substitution pattern (delphinidin 3-gen-
tiobioside and antirrhinin).15,22 Although no other values for
molecules with a structure similar to 1 are of our knowledge,
this effect seems to be more intense when there is also a glycosyl
residue in position 5, since when comparing 1 with antirrhinin,
which shares the same chromophore, the first shows an ꢀ value
of roughly 50%25 that of the latter (Table 1).
When applied to anthocyanins in mildly acidic aqueous
solutions, a plot of D0/(D0 - D) Vs [H+], where D is the
maximum of the visible absorbance at a given pH and D0 is
the absorbance at pH < 1, gives a straight line that, through an
already established procedure,13-15 enables the obtention of a
global constant (K′ ) Kh + Ka) which allows the determination
of the equilibrium fractional amount of AH+ at a given pH value.
However, when this method is applied to the present set of
pigments a deviation from linearity is registered for molecules
2-5, while it gives good correlations when 1, antirrhinin, and
cyanin are concerned. This particular behavior is accompanied
by the observation of a sharp drop in intensity of the visible
absorption band of pigments 2-5 when the pH is increased from
a value around 0.7, where the flavylium cation is the sole
absorbing species, to a value of ca. 1.5, remaining then almost
unchanged until attainment of a pH superior to ∼2 as shown in
Figure 1 for pigment 4.
Kinetic Measurements. One milliliter of each equilibrated aqueous
solution of anthocyanin, at different pH values (1.0-2.5), is magneti-
cally stirred in the spectrophotometer cell. The concentrations of the
pigments are 6.4 × 10-5 M for 1, 1.7 × 10-5 M for 2, 1.8 × 10-5
M
for 3, 4.05 × 10-5 M for 4, 4.9 × 10-5 M for 5, 5.0 × 10-5 M for
cyanin, and 6.3 × 10-5 M for antirrhinin. To these solutions 1 mL of
phosphate buffer solutions, ranging in pH from 4.3 to 7.4, was quickly
added, and the visible absorbance at 520 nm (near the visible absorption
maxima for all the studied anthocyanins) is immediately recorded every
second over 120 s, to guarantee that the hydration equilibrium is
attained. The final pH was then measured and ranged from 2.3 to 4.4.
The exponential decay of the absorbance essentially reflects the
relaxation of the pH-dependent equilibrium depicted in eq 2 according
to an apparent first-order kinetics. The spectrophotometer software
automatically computes the first-order apparent rate constant of the
hydration reaction (k). The theoretical treatment that ensues for the
obtention of the equilibrium rate constants is given in full detail in ref
14.
Molecular Orbitals Calculations. Molecular mechanics, using the
MM+ force field,23 both in vacuo and in a simulated water solution
contained within a periodic box, were performed on an Escom Pentium
100 PC using the HyperChem program (Version 4, Hypercube, Inc.,
Ontario, Canada).
Results
Thermodynamic Data. The visible absorption band char-
acteristic of these pigments presents for these series of antho-
cyanins (1-5 plus cyanin and antirrhinin), which share the same
chromophore (cyanidin) but differ in the pattern of glycosylation
and acylation, a bathochromic shift (Table 1) when going from
the simpler (cyanin) to the structurally more complicated (5).
This shift is a property evidenced by the majority of the
complexes formed by the most common anthocyanins.1 Such
red shift is generally accompanied by a small decrease in the
molar absorption coefficient (ꢀ) which indicates that the
complexed forms, either resulting from inter- or intramolecular
associations, exhibit a slightly less intense coloration than their
corresponding free flavylium cations. However, this small
decrease in absorption intensity at very acidic pH values is
largely compensated by the gain of color obtained in mildly
acidic solutions. Nevertheless, in the present series of pigments
For solutions with a less acidic pH (∼2.1-3.5) it is clearly
noticed the appearance of a shoulder, on the right hand side of
the visible band, which increases with the pH and is ac-
companied by a gradual diminution of the flavylium cation
absorption (Figure 2a). This new band is characteristic of the
quinonoidal base form of the anthocyanins. Moreover, if the
flavylium contribution is subtracted from the whole spectra,
according to a procedure detailed elsewhere,26 the remaining
spectra show distinctively the formation of the quinonoidal base
at very low pH values (Figure 2b), which deeply contrasts with
(24) Brouillard, R.; El Hage Chahine, J. M. J. Am. Chem. Soc. 1980,
102, 5375-5378.
(25) Due to the low amount of the available sample, it was only possible
(22) Elhabiri, M.; Figueiredo, P.; Fougerousse, A.; Brouillard, R.
Tetrahedron Lett. 1995, 36, 4611-4614.
(23) Allinger, N. L. J. Am. Chem. Soc. 1977, 99, 8127-8134.
to estimate an approximate value of ꢀλ ≈ 3600 for 1.
max
(26) Figueiredo, P.; Pina, F.; Vilas-Boas, L.; Mac¸anita, A. L. J.
Photochem. Photobiol., A: Chemistry 1990, 52, 411-424.

Anthocyanin Intramolecular Interactions
J. Am. Chem. Soc., Vol. 118, No. 20, 1996 4791
a
b
Figure 3. Plot of D0/D as a function of 1/[H+] for pigment 5, according
to eq 10.
Table 2. Thermodynamic Parameters Obtained from Eq 10 for
Pigments 2-5a
pigment
2
3
4
5
K1
pK′
R
b
1.98
0.998
8.70
1.59
0.999
8.77
1.91
0.998
8.20
1.94
0.999
Figure 2. Electronic absorption spectra of 3 (1.8 × 10-5 M, T ) 25
°C) in aqueous solution, before (a) and after (b) the subtraction of the
flavylium cation absorption. 1, pH 0.8; 2, pH 1.6; 3, pH 2.0; 4, pH
2.8; 5, pH 3.5.
a T ) 25 °C. b Pigment 2 gives a K1 ) 1.15; see text for discussion.
D0 ) ꢀAH+CT ) ꢀAH+([AH+] + [CP] + [A] + [B]) (8)
we can rearrange eq 7 as
the, until now, generally observed formation of this form at a
pH closer to neutrality.2
D ) ꢀAH+([AH+] + ꢀCP/ꢀAH+[CP] + rA[A])
(9)
The observation of these distinctive characteristics for an-
thocyanins 2-5 together with the unapplicability of the model
postulated in ref 14 led us to bring forth the following hypothesis
of mechanism to account for the behavior of the four pigments,
as a function of pH, in acidic aqueous solutions
where rA stands for the ratio ꢀA/ꢀAH+. A combination of eqs 8
and 9, together with the expressions above defined for the
equilibrium constants K1, KCP, and KCP, gives eq 10.
h
a
1
K1
1
K1
\z CP
+ 1 +
(KCh P + KCa P)
AH+ y
(3)
(4)
[H+]
D0
D
)
(10)
ꢀCP
1
1
rAKCa P
KCh P
+
+
CP + H2O y z B + H+
[H+]
K1 ꢀAH
+
A plot of D0/D vs 1/[H+], for pigments 2-5, produces a fitting
KCa P
CP y z A + H+
(5)
of the experimental data (Figure 3), with very good correlations
(R), from where the values of K1 and K′ ) KCP + KaCP could be
with K1 ) [CP]/[AH+], KhCP ) [B][H+]/[CP], and KCa P
)
h
obtained (Table 2).
[A][H+]/[CP]. In eq 4 the fast equilibrating B and CE forms
are simply represented by B for the sake of clarity. Equation
3 accounts for the assumed equilibrium between two conforma-
tions of the flavylium cation, one with an acyl residue folded
over the chromophore in the form of an intramolecular complex
(CP), with a smaller ꢀ value than that of the other conformation
(AH+) which is not copigmented. AH+ has its domain of
existence only in very acidic media (pH < 1), while CP
dominates in the pH region 1.0-2.0. This would explain the
sudden drop of absorbance verified when going from very acidic
to slightly less acidic solutions. It will be the intramolecular
copigmentation complex thus formed that undergoes the usual
reactionsseqs 4 and 5sof hydration and deprotonation depicted
in Scheme 1.
Kinetic Data. The pH jumps conducted at constant tem-
perature for all the pigments studied produce, without exception,
an exponential decay of the visible absorbance, caused by the
pH-dependent hydration reaction. However, the ensemble of
kinetic data gathered, like the thermodynamic ones, should be
analyzed according to two different models. Thus, cyanin,
antirrhinin, and 1 follow the treatment, presented elsewhere,14
which uses eq 11 to fit the data gained from the spectropho-
tometric analysis.
(Ka + Kh + [H+])
Ka
k2
1
k2
1
)
+
(11)
+
k
[H ]
In eq 11, k2 represents the dehydration rate constant as defined
by the ratio Kh ) k1/k2, where k1 defines the rate constant for
the direct process (hydration). By plotting (Ka + Kh + [H+])/k
Vs 1/[H+] one can obtain directly k2 and Ka and, together with
the values obtained from the plot of D0/(D0 - D) Vs [H+], above
mentioned, also the values for Kh and k1.
Equations 6-8 represent the overall concentration of pigment
(CT), the visible absorption of an anthocyanin solution for a
given pH (D), and at pH < 1 (D0), where only AH+ absorbs
CT ) [AH+] + [CP] + [A] + [B]
D ) ꢀAH+[AH+] + ꢀCP[CP] + ꢀA[A]
(6)
(7)
For the remaining pigments (2-5) it is necessary to take into
account the reaction scheme proposed in eqs 3-5, which leads

4792 J. Am. Chem. Soc., Vol. 118, No. 20, 1996
Figueiredo et al.
Table 3. Thermodynamic and Kinetic Values Obtained for the Cyanidin Based Anthocyaninsa
pigment
antirrhininb
cyaninb
1b
2
3
4
5
pKCh P
2.93((0.02)
3.58((0.03)
1.01((0.01)
8.68((0.07)
2.07((0.02)
4.03((0.02)
2.92((0.03)
3.45((0.03)
1.29((0.02)
4.57((0.06)
4.93((0.06)
0.95((0.01)
2.04((0.01)
2.88((0.01)
7.85((0.03)
8.55((0.03)
1.78((0.01)
2.26((0.02)
38.52((0.27)
21.41((0.15)
2.84((0.01)
1.96((0.01)
4.26((0.02)
29.65((0.15)
2.75((0.02)
2.01((0.02)
3.49((0.03)
19.42((0.16)
pKCa P
kC1 P × 102/min-1
k2CP × 104/M-1 min-1
a T ) 25 °C. b The superscript CP does not apply in those cases.
to the following treatment for the experimental data obtained
through the spectrophotometric measurements.
Equation 12 describes the relaxation process of the hydration
equilibrium after a pH-jump
d[B]
dt
) kC1 P[CP] - kC2 P[B][H+]
(12)
with KhCP ) k1CP/kC2 P. If the principle of mass conservation is
expressed as
∆[AH+] + ∆[CP] + ∆[A] + ∆[B] ) 0
(13)
Figure 4. Electronic absorption spectra of the flavylium forms of 2
and 5 in aqueous solution. pH 0.9, T ) 25 °C.
one can combine eqs 12 and 13 to obtain the apparent first-
order rate constant kCP as defined by d∆[B]/dt ) -kCP∆[B]. It
comes in the form
values, thus hindering the folding of the acyl residue over the
chromophore.
From the analysis of the data presented in Table 2, it is evident
a common value of K1 ∼8.0-9.0 for pigments 3, 4, and 5 seems
to justify the mechanism conjectured above, eqs 3-5. Pigment
2 has a K1 of 1.15 and a spectrophotometric behavior intermedi-
ate between what is typically reported for other anthocyanins
and the spectral characteristics of compounds 3-5. Neverthe-
less, the theoretical interpretation of the absorption gain Vs pH,
for this molecule, is better achieved through the application of
eq 10 (R ) 0.9986) than by the use of the formalism reported
in refs 13-15 (R ) 0.9920). This predicts an important role
for malonyl residues on the formation of intramolecular
complexes by anthocyanins, since this is the sole difference
between pigments 2 and 5 (3 and 4 are also structurally closer
to 5). The influence of the malonyl group is already manifest
when comparing the spectra of the flavylium cations of 2 and
5 (Figure 4), notably the band at ca. 320 nm, characteristic of
the acylated anthocyanins, which is less intense than the one at
280 nm for 2, while the inverse happens with 3, 4, and 5. Such
increase in band intensity could be used as an empirical tool
for the characterization of simultaneously acylated and malo-
nylated anthocyanins.
The data reported in Table 3 allows the following assump-
tions: (i) The well-known increase in pKh when going from
3,5-diglucosylated molecules to 3-glucosylated ones,15,30 can
also be extended to antirrhinin (relatively to cyanin), which
possesses a particular type of disaccharide (a rutinose) bound
to position 3. (ii) A very low pKh value for 1, which can now
be considered as a distinguishing feature of some types of
3-disaccharides, which possess the ability of preferential
stabilization of the hemiacetal form by formation of intramo-
lecular hydrogen bonds between the hemiacetalic OH and one
of the free hydroxyls of the terminal sugar.15 Molecular
mechanics calculations, performed in a simulated aqueous
solution, with several possible structural conformations, yielded
for the conformation with the lower enthalpy value, a distance
of 3.18 Å between the above two hydroxyl groups, which is
generally considered as being capable of accommodating an
hydrogen bond.27-29 Similar calculations, performed with
antirrhinin, showed no such behavior, this being attributed to
1
K1
KCa P + KCh P + [H+] 1 +
(
)
k
CP ) kC2 P[H+]
(14)
(15)
1
K1
KCa P + [H+] 1 +
{
}
(
)
Equation 14 can be written in the following way
KCa P + KCh P + [H+](1 + 1/K1) 1 + 1/K1 KaCP
1
)
+
kCP
kC2 P
kC2 P [H+]
Thus, a plot of {KCa P + KhCP + [H+](1 + 1/K1)}/kCP as a
function of 1/[H+] yields a straight line with an intercept of (1
+ 1/K1)/kCP, from where the value of kCP can be readily
obtained if one takes the value for K1 obtained from eq 10, and
a slope equal to KCa P/k2CP, which leads immediately to the
values for KCa P, KhCP, and kC1 P. The values obtained for the
thermodynamic and kinetic constants of all the pigments studied
are presented in Table 3.
2
2
Discussion
Water, in its liquid state, is generally considered as a
structured network of hydrogen-bonded molecules27-29 respon-
sible for the specificity of most biological processes. It is also
known that even minor physical or chemical changes can disrupt
this particular three-dimensional organization and thus inactivate
these processes.28 Intermolecular copigmentation, a phenom-
enon driven by hydrophobic interactions, is likewise dependent
on solvent arrangement, being more strong at temperatures near
0 °C than at room temperature, since at the former water is a
quasi-crystalline medium which shows favor to the interaction
between anthocyanin and copigment.6,7 Given this reasoning,
it is possible to think of the “pH dependent” intramolecular
copigmentation of the flavylium cation form of anthocyanins
2-5, as associated to a change in the particular framework of
liquid water caused by the increase in acidity which will give
rise to a less organized solvent structure, at very acidic pH
(27) Rice, S. A. In Topics in Current Chemistry; Vol. 60, Structure of
Liquids; Springer-Verlag: Berlin, 1975; pp 109-200.
(28) Franks, F. Water; The Royal Society of Chemistry: London, 1984.
(29) Scheiner, S. Annu. ReV. Phys. Chem. 1994, 45, 23-56.
(30) Dangles, O.; Elhajji, H. HelV. Chim. Acta 1994, 77, 1595-1610.

Anthocyanin Intramolecular Interactions
J. Am. Chem. Soc., Vol. 118, No. 20, 1996 4793
the presence of a terminal CH3 group, instead of an hydroxyl,
in the sugar position favorable for the formation of an H bond.
(iii) The pKCh P values increase in the order 3, 2, 5, and 4.
Although kC1 P (hydration step) values are all larger (even
considerably larger in the case of pigment 3) than the one found
for cyanin (k1 in that case), this is somehow compensated by
the also larger values calculated for kC2 P. This behavior can be
interpreted through the analysis of the data collected from
molecular modelization of the four pigments, and the compari-
son of the structures with the minimum relative enthalpies
obtained for each compound. These results evidence the
existence, for all those four anthocyanins, of a “sandwich”
structure with the two aromatic acyl residues positioned on each
side of the chromophore, thus protecting it from hydration, the
difference in protection being given by the relative position of
the acid ring I relative to the B-ring of the aglycon, the closer
it is the lesser the efficiency of the nucleophilic attack.31 This
closeness also increases in the order 3, 2, 5, and 4. This increase
in vicinity of the two aromatic rings is accompanied, and
presumably caused, by an increasing proximity between the final
carbonyl group of the malonyl residue and the 7-OH of the
aglycon. (iv) Since the hydroxyl groups in positions 4′ and 7
are the ones that participate in the deprotonation equilibria to
form the quinonoidal bases,32,33 the relative proximity of the
malonyl to the 7-OH, with the possibility of H bond formation,
should increase the charge density in C-7 and thus facilitate
the deprotonation,34 with the concomitant decrease in pKa (Table
3). This assumption is further stressed by the inspection of the
values computed for the above mentioned interatomic distances,
which are respectively 3.43 Å for pigment 4, 3.60 Å for 5, and
4.72 for 3, following the increase in pKa. Since molecule 2
does not possess a malonyl residue, its pKa is higher than the
ones calculated for the above three pigments, although lower
than the ones obtained for cyanin or 1. This peculiar interaction
reveals an important role for malonyl residues in natural
colorants, since this lowering of the pKa, together with the
protection against color loss by hydration, allows the existence
of colored forms (flavylium cation or quinonoidal base) through
a very large range of acidic pH values. The cornerstone of the
mechanism of color stabilization and variation in the case of
this series of pigments rests on the formation of the quinonoidal
bases at low pH values. This is true for instance in the case of
compound 3 which has a pKhCP value lower than the one found
for cyanin. However, the loss of color stability, related to the
lowering of the pKCh P value is, in that case, largely compen-
sated by the decrease in the pKCa P value when going from
cyanin to pigment 3. Finally, it should be noted also the small
differences between pKCa P and pKCh P for pigments 2-5. This is
an important characteristic feature of color stabilization and
variation within the flowers of the Matthiola incana species.
Conclusions
The formation of intramolecular complexes, which prevent
the loss of color through hydration reactions, by natural
anthocyanins possessing at least one aromatic acyl residue linked
through sugar spacers at the aglycon moiety is here evidenced,
especially for compounds 4 and 5, which not only evidence the
already known effect of increase in pKh but, furthermore
constitute, from our knowledge, the two sole natural anthocya-
nins that present a value for their pKa lower than that of the
pKh. Moreover, a role for the simple, nonaromatic, malonyl
residue, related to this property, is also disclosed for the first
time in the present work. A new mathematical treatment of a
new equilibrium mechanism, complementary to the one already
employed for the characterization of similar pigments,13-15 is
also presented to account for the particular properties of the set
of molecules here discussed.
(31) Molecular mechanics calculations, performed in our laboratory, for
the intermolecular interactions between several simple (albeit with a
noncoplanar B ring as is the case with the present set) anthocyanins and
common copigments such as chlorogenic acid and caffeine, have demon-
strated a preferential interaction between the B ring of the pigment and the
aromatic residue(s) of the copigment.
(32) Kurtin, W. E.; Song, P.-S. Tetrahedron 1968, 24, 2255-2267.
(33) Costantino, L.; Rastelli, G.; Rossi, M. C.; Albasini, A. J. Chem.
Soc., Perkin Trans. 2 1995, 227-234.
(34) The molecular modelization, in AM1 parametrization,35 of a simple
polyhydroxylated anthocyanidin (3′,4′,3,7-tetrahydroxyflavylium) forming
an hydrogen bond, through its 7-OH, with a malonate, produces a relative
increase on the charge density of C-7 from 0.167 to 0.195, while the other
free hydroxyl groups of the cation, notably the 4′-OH, remain almost
unchanged.
Acknowledgment. The authors wish to express their grati-
tude to Prof. Sam Asen for the kind gift of a sample of cyanin.
Paulo Figueiredo wishes also to thank the European Union for
a post-doc grant ERBCHBICT941610.
(35) Dewar, M. J. S.; Zoebisch, E. G.; Healy, E. F.; Stewart, J. J. P. J.
Am. Chem. Soc. 1985, 107, 3902-3909.
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