Quercetin (=2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one) Glycosides and Sulfates: Chemical Synthesis, Complexation, and Antioxidant Properties

Article abstract of DOI:10.1002/1522-2675(20010516)84:5<1133::AID-HLCA1133>3.0.CO;2-Z

Glycosides, acylated glycosides, and sulfates of Quercetin (=3,3',4',5,7-pentahydroxyflavone; 1), which is, together with its derivatives, among the most common polyophenols found in plants and in the human diet, were prepared and quantitatively investiga

Full text of DOI:10.1002/1522-2675(20010516)84:5<1133::AID-HLCA1133>3.0.CO;2-Z

Helvetica Chimica Acta ± Vol. 84 (2001)
1133
Quercetin (ˆ2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-
benzopyran-4-one) Glycosides and Sulfates: Chemical Synthesis,
Complexation, and Antioxidant Properties
by Bertrand Alluis and Olivier Dangles1)*
Â
Â
Universite Claude Bernard-Lyon I, UMR-CNRS 5078, Laboratoire des Polyphenols,
Ã
Bat. 303, 43, bld du 11 Novembre 1918, F-69622 Villeurbanne
Glycosides, acylated glycosides, and sulfates of quercetin (ˆ 3,3',4',5,7-pentahydroxyflavone; 1), which is,
together with its derivatives, among the most common polyphenols found in plants and in the human diet, were
prepared and quantitatively investigated for their ability to bind metal ions (AlIII, FeII, FeIII), enhance and vary
natural colours (anthocyanin copigmentation), and trap potentially damaging radicals (antioxidant activity).
Introduction. ± Flavonoids and other polyphenols are ubiquitous secondary
metabolites in plants [1] which, as food components, enjoy increasing interest from
researchers, food manufacturers, and consumers. Indeed, polyphenol-rich diets (e.g.,
the regular consumption of fruits, vegetables, red wine, and tea) have been repeatedly
correlated with a low risk of developing cardiovascular diseases and cancers [2], the
two major causes of mortality in occidental countries. Because of their antioxidant
properties and their ability to bind to a wide range of proteins including enzymes
[1][3], polyphenols may be partially responsible for these protective effects.
Besides phenolic acids (benzoic and cinnamic acid derivatives), flavonols (3-
hydroxyflavones) are one of the main classes of dietary polyphenols [1]. Their
antioxidant properties essentially stem from their ability to trap reactive O-species
(ROS) and bind transition-metal ions, which, if not suitably complexed, could enter
redox cycles that produce ROS [3]. This work focuses on quercetin (3,3',4',5,7-
pentahydroxyflavone; 1), the most representative dietary flavonol (abundant in onions,
apples, and tea) and a potent antioxidant. In most naturally occurring flavonols, at least
one of the OH groups of the polyphenolic nucleus (aglycone) is glycosylated by mono-
and oligosaccharides involving neutral sugars such as d-glucose, d-galactose, l-
rhamnose, and d-xylose. Quercetin b-d-glucopyranosides are particularly interesting
because their biodisposability, irrespective of the location of the sugar moiety on the
polyphenolic nucleus, is better than that of quercetin and other quercetin glycosides,
possibly as a consequence of a facilitated intestinal absorption via glucose transporters
[4]. Moreover, quercetin sulfates deserve particular attention, not only because of their
natural occurrence [1][5], but also because the quercetin conjugates found in the
plasma of human volunteers after consumption of a meal rich in plant products are
actually glucuronides and sulfates [6].
1
Â
)
New address: Universite dꢀAvignon et des Pays de Vaucluse, UFR Sciences, UMR INRA A 408, 33, rue
Louis Pasteur, F-84000 Avignon (e-mail: Olivier.Dangles@univ-avignon.fr).

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In this work, simple methods for the preparation of quercetin glycosides, via a
phase-transfer glycosidation step, are described, quercetin sulfates being prepared
following already published procedures [7]. The quercetin derivatives are then tested
for their ability to bind metal ions (Al3‡, Fe3‡, Fe2‡) and trap DPPH (diphenylpicryl-
hydrazyl), a coloured free radical commonly used for assessing the antioxidant (H-
atom-donating) activity. In addition, the highly H2O-soluble sulfates are also
demonstrated to form exceptionally stable molecular complexes with polyphenolic
plant pigments (anthocyanins), thereby allowing natural colours to be deeply
intensified and varied (copigmentation). Quite frequently in plants, some sugar OH
groups in flavonoid glycosides are acylated by a variety of aromatic and aliphatic acids
[1] including succinic acid (ˆ butanedioic acid). Some easily available and highly H2O-
soluble succinylated quercetin glycosides are also investigated for their ability to bind
metal ions with the possible participation of their free carboxy groups.
Results and Discussion. ± 1. Synthesis (see Scheme 1). The direct glycosidation of
quercetin (1) by 2,3,4,6-tetra-O-acetyl-a-d-glucopyranosyl bromide (2) under mildly
alkaline to neutral phase-transfer conditions that have proved suitable for the
glycosidation of flavones and other acidic phenols [8] did not allow us to isolate any
glycoside. Since the most acidic OH groups (OH C(7), OH C(4')) are dissociated
even in neutral aqueous solutions [9], one may infer that the corresponding polyanions
are much too polar to be transferred into the organic phase to further react with the
glycosyl donor. To facilitate the phase transfer of quercetin anions and also to better
control the regioselectivity, quercetin (1) was partially protected. After a failed attempt
to prepare the methylene acetal, 1 was converted to its diphenylmethylene acetal 3 in
33% yield by directly heating it with Ph2CCl2 (4 equiv.) at 1708 (Scheme 1). The
reaction in refluxing pyridine (5 equiv. of Ph2CCl2) gave slightly lower yields (25%).
Glycosidation of 3 by 2 under phase-transfer conditions (CH2Cl2, sat. KHCO3 solution,
tris[2-(2-methoxyethoxy)ethyl]amine (TMEA), 408) resulted in the regioselective
formation of the 7-O-b-d-glucopyranoside 4 in moderate yields (ca. 30% after
saponification of the acetate groups). The regioselectivity is in agreement with the
preferential dissociation of OH C(7) in the aqueous phase. Subsequent hydro-
genolysis of the diphenylmethylene acetal (yield 60%) led to 7-O-b-d-glucopyrano-
sylquercetin (5) or quercimeritrin, whose spectral characteristics are in agreement with
those of a sample extracted from plants [10].
The strategy of partial benzylation and subsequent glycosidation has already been
described as a means to prepare quercetin glucosides [11]. When a solution of 1 in
t
DMF was treated with benzyl bromide (10 equiv.) and BuOK (5 equiv.) at room
temperature, a mixture of 3,4',7-tri-O-benzyl- and 3,3',4',7-tetra-O-benzylquercetin
(resp., 6 and 7) was obtained. Flash chromatography (silica gel) of the mixture yielded
6 and 7 in 40 and 14% yields, respectively. Glycosidation of 6 by 2 under the usual
conditions led to the 3'-O-b-d-glucoside 8 in 48% yield after saponification of the
acetate groups. The choice of a sat. KHCO3 solution for the aqueous phase seems the
best compromise since it allowed the dissociation of the weakly acidic OH C(3') and
at the same time minimized the hydrolysis of 2. With 1m KHCO3 or sat. K2CO3 solution,
the yields were significantly lower (ca. 30%). The absence of reactivity of OH C(5) is
attributable to the strong intramolecular H-bond it forms with C(4)ˆO (d 12.67 in

Helvetica Chimica Acta ± Vol. 84 (2001)
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Scheme 1. Chemical Synthesis of Quercetin Derivatives

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Helvetica Chimica Acta ± Vol. 84 (2001)
(D)6DMSO for OH C(5) in 6). Subsequent hydrogenolysis of the benzyl groups of 8
led to 3'-O-b-d-glucopyranosylquercetin (9). Alternatively, 7 could be glycosylated by
2,3,4,6-tetra-O-acetyl-a-d-galactopyranosyl bromide under more alkaline conditions
(sat. K2CO3 solution) to deprotonate the weakly acidic OH C(5) proton (yield 35%,
after saponification of the acetate groups); after debenzylation, 5-O-b-d-galactopyr-
anosylquercetin (10) was obtained. Our method is a significant improvement over the
previously reported synthesis of 3'- and 5-O-glucosides 9 and 10 of quercetin (yields 30
and 8%, respectively, for the glycosidation step under Koenigs-Knorr conditions) [11a].
The partial or total succinylation of flavonoid sugar moieties has been shown to be a
very efficient way to increase the water solubility of flavonoids for application as colour
enhancers [12]. The reaction is regioselective for the sugar OH groups and does not
even require the preliminary protection of the aglycone OH groups. Thus, when 8 was
treated with succinic anhydride (6 equiv.) in pyridine in the presence of N,N-
dimethylpyridin-4-amine (DMAP; 1 equiv.) at 708, the corresponding 2'',3'',4'',6''-
tetrakis(hydrogen succinate) was isolated in quantitative yield; subsequent hydro-
genolysis led to 11 (Scheme 1).
Acylation of the chrysin (5,7-dihydroxyflavone) 7-O-glucoside by aromatic acyl
chlorides has been shown to occur regioselectively (yield ca. 30%) at the sugar primary
OH group [8a]. Similarly, treatment of 8 by 3,4,5-trimethoxybenzoyl chloride (2 equiv.
added in several portions within 2 h) in pyridine in the presence of DMAP (0.5 equiv.)
afforded the 6-O-acyl derivative as the only product (Scheme 1). The low yield (16%)
suggests that the steric hindrance exerted by the aglycone on the sugar moiety is
stronger with 3'-O-b-d-glucosides than with 7-O-b-d-glucosides. Subsequent debenzyl-
ation gave 3'-O-[6-O-(3,4,5-trimethoxybenzoyl)-b-d-glucopyranosyl]quercetin (12).
The signals of some aglycone protons in 12 are significantly shielded with respect to the
corresponding signals in non-acylated 9 ( 0.11 and 0.32 ppm for H C(6) and
H
C(8), resp.). Similarly, the signal of the equivalent Hortho protons of the acyl group
are shielded by 0.30 ppm with respect to the corresponding signals in methyl 3,4,5-
trimethoxybenzoate. These observations suggest intramolecular stacking interactions
between both aromatic moieties in 12, as already observed in the acylated 7-O-b-d-
glucosides of chrysin [8a].
Quercetin sulfates 13 and 14 were prepared upon treatment by tetrabutylammo-
nium hydrogen sulfate ((Bu4N)HSO4) and dicyclohexylcarbodiimide (DCC) in
pyridine according to an already published procedure [7]. Their spectral characteristics
are in agreement with the literature.
2. Metal Complexation. Metal complexation by polyphenols is a phenomenon with
many biological implications. Formation of stable H2O-soluble polyphenol complexes
with AlIII may, e.g., contribute to general mechanisms of AlIII uptake in plants and AlIII
toxicity in humans in relation to neurological and bone disorders [13]. AlIII Complex-
ation by polyphenols may also inhibit the antioxidant activity of dietary polyphenols
circulating in plasma [14] and participate in colour expression in plants [15]. Moreover,
polyphenols can take part in reductive mechanisms of iron uptake from ferritin or soil
oxides [16]. They can, however, decrease the absorption of iron from the diet [17].
Finally, polyphenols, especially flavonoids, can prevent ROS generation (e.g., the Fenton
reaction) by sequestering iron ions under inactive complexes [18] although pro-oxidant
effects have been evidenced, depending on the flavonoid structure and iron concentration.

Helvetica Chimica Acta ± Vol. 84 (2001)
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Quercetin is able to chelate metal ions via the 3-hydroxy-4-oxo, 5-hydroxy-4-oxo,
and 3',4'-dihydroxy (catechol) groups [9][19]. In quercetin derivatives, one of the
binding sites may be cancelled because of derivatization. The complexation reaction
may be written as:
‡
M ‡ LHn ! [ML] ‡ nH
LHn, M, and [ML] stand for the flavonol (neutral form), metal ion, and 1:1 chelate,
respectively. Upon complexation, the metal ion displaces n protons from the flavonol
nucleus. Except in the case of succinylated flavonol glycosides, for which n may be
higher than 2 because of the participation of free carboxy groups in the binding, n is
equal to 1 when chelation occurs at the 3-hydroxy-4-oxo or 5-hydroxy-4-oxo group
(deprotonation of OH C(3) or OH C(5)), and to 2 when chelation occurs at the
catechol group (deprotonation of OH C(3') and OH C(4')). The equilibrium
‡
constant is K ˆ ([[ML]] ´ [H ]n)/([M] ´ [LHn]). Since the pH is fixed in the complex-
ation experiment, the apparent affinity of the flavonol for M can also be quantified by
‡
K' ˆ K/[H ]n ˆ [[ML]]/([M] ´ [LHn]).
The plots of the absorbance (at a fixed wavelength of the chelate absorption band)
as a function of the total metal concentration can be fitted against Eqns. 1 and 2 with
the sole assumption of 1:1 complexation (A, A0 ˆ absorbances in the presence and
absence of metal ion, respectively; [M] ˆ free metal concentration; Mt ˆ total metal
concentration; c: total flavonol concentration). Therein, r stands for the ratio of molar
absorption coefficients e([ML])/e(LHn).
1 ‡ rK'‰MŠ
A ˆ A0
(1)
(2)
1 ‡ K'‰MŠ
K'c
ꢀ
ꢁ
Mt ˆ [M] 1 ‡
1 ‡ K'‰MŠ
Complexation of Al3‡ by quercetin derivatives was investigated in MeOH/0.2m
acetate buffer 1:1 (pH 5.0, 258) to allow comparisons with the poorly H2O-soluble
quercetin. Upon addition of Al3‡, new absorption bands in the range 420 ± 430 nm
appear that correspond to the (flavonolato)aluminium complexes (Fig. 1). Values for
parameters r and K' deduced from the curve-fitting procedure are reported in Table 1.
If Al3‡ complexation primarily occurred with the catechol group, one would expect a
sharp decrease in the K' value upon derivatization of the catechol group (glycosidation,
sulfation). This is not the case. For instance, the K' value is slightly higher for 3'-b-d-
glucopyranosylquercetin (9) than for quercetin itself (1). In addition, the 3-hydroxy-4-
oxo group must be a better chelating site for Al3‡ than the 5-hydroxy-4-oxo group since
complexation on the latter site is accompanied by the removal of the OH C(5) proton,
which is strongly stabilized by H-bonding with C(4)ˆO. Consistently, the affinity of
quercetin for Al3‡ is higher than that of chrysin (5,7-dihydroxyflavone) under the same
conditions [8a]. Hence, binding is very likely to occur via the 3-hydroxy-4-oxo group in
this series, in agreement with previous investigations of AlIII complexation by flavonols
in MeOH [19]. Al3‡ Complexation is strongest for 11, a result suggesting that one or
two carboxylate groups take part in the binding. Although remote from the binding site,

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Helvetica Chimica Acta ± Vol. 84 (2001)
the 7-O-sulfate group of 13 also exerts a favourable influence on the complexation
(Table 1), possibly by long-range electrostatic interactions. The observation that the
second sulfate group of 14 does not further increase the apparent binding constant may
be explained by conflicting electronic effects. Indeed, sulfation of both OH C(7) and
OH C(4') is expected to lower the electron-donating properties of both groups and
thus the electron-density at C(4)ˆO (via conjugation) and, to a lesser degree, at
OH C(3).
Fig. 1. UV/VIS Spectra of 11 (5 ´ 10 5 m) and of its Al3‡ complex at pH 5.0 and 258 (MeOH/0.2m acetate buffer
1:1). Al3‡/11 molar ratio 0 (1), 0.25 (2), 1 (3), 2.5 (4), and 5 (5)
Table 1. Complexation of Al3‡ by Quercetin Derivatives (pH 5.0, 258). For the definition of r and K', see text.
1
K' ´ 10 3 [m
]
r(420 nm)
a
quercetin
9a)
)
4.6 (Æ 0.2)
7.0 (Æ 0.8)
39.7 (Æ 8.1)
18.8 (Æ 3.8)
13.4 (Æ 3.0)
12.2 (Æ 0.1)
10.7 (Æ 0.2)
7.9 (Æ 0.2)
21.0 (Æ 0.7)
23.4 (Æ 1.0)
11a)
13a)
14a)
rutinb)
15b)
12.0 (Æ 1.2)
36.9 (Æ 0.6)
5.1 (Æ 0.5)
5.1 (Æ 0.1)
a) MeOH/0.2m Acetate buffer 1 : 1. b) MeOH/0.2m Acetate buffer 5 : 95.
Aluminium complexation was also investigated with rutin (ˆ 3-O-(a-l-rhamnopyr-
anosyl-1 ! 6-b-d-glucopyranosyl)quercetin), a commercially available and ubiquitous
quercetin glycoside, and its hexakis(hydrogen succinate) 15 [12]. Rutin is a better AlIII
ligand than 15 (Table 1). Hence, polysuccinylation of a sugar moiety may increase or
decrease the affinity of the aglycone for Al3‡ depending on the location of the
glycosyloxy group at the polyphenolic nucleus.
The well-known affinity of polycarboxylate ligands for iron drove us to test the
flavonoid poly(hydrogen succinates) 11 and 15 as iron ligands. However, the general
propensity of iron complexes with polyphenols to precipitate required adaptation of
the experimental procedure. The complexation kinetics was monitored after addition
of a small volume of concentrated metal solution in the spectrometer cell. The

Helvetica Chimica Acta ± Vol. 84 (2001)
1139
6'''
OH
Me
OR
OH
O
2'
OR
OR
1'''
7
2
HO
O
O
5'
O
6''
1''
OR
O
O
3
4
5
OR
RO
OH
rutin R = H
15 R = C(=O)CH2CH2CO2H
absorbance values used in the estimation of K' correspond to the plateau typically
reached in a few minutes (Fig. 2). This method allowed us to ensure that the
complexation equilibrium was reached and that precipitation did not begin yet, two
conditions to be met for a rigorous analysis.
Fig. 2. Time dependence of the absorbance at 420 nm of 15 (5 ´ 10 5 m) after addition of Fe2‡ (0.5 equiv.) at pH 5
and 258 (MeOH/0.2m acetate buffer 5 :95)
Complexation of Fe2‡ and Fe3‡ by quercetin, rutin, and their succinylated
derivatives 11 and 15 (Figs. 3 and 4) could be satisfactorily investigated by means of
the 1:1 binding model (Eqns. 1 and 2) from which values for parameters r and K' were
estimated (Tables 2 and 3). For each metal ion, the affinities of quercetin and 11 are of
the same order of magnitude. Hence, the possible participation of the carboxylate
groups of 11 may compensate for the introduction of a bulky, electron-withdrawing
sugar moiety at the 3' position, which, in addition, suppresses the ability of the B ring to
complex metal ions. The affinity of 15 for both Fe2‡ and Fe3‡ is very significantly higher
than that of rutin, an observation clearly pointing to the efficient participation of the
free carboxylate groups of 15. Remarkably, the affinity of 15 for Fe3‡ is so high that the
pH must be lowered to 1 for an accurate estimation of the corresponding K' value.

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Helvetica Chimica Acta ± Vol. 84 (2001)
Under such acidic conditions, the binding of Fe3‡ by rutin is not even detectable. In a
pH 1 solution of FeIII and 15 in equal concentrations (10 3 m), it can thus be estimated
that ca. 80% of FeIII is bound, whereas FeIII remains in its free form in the presence of
rutin under the same conditions. Since the ROESY spectrum of 15 displays several
long-range correlations between the B ring and the rhamnose moiety [12], it may be
assumed that the ligand can adopt a folded conformation in which the catechol group
and two succinate groups (probably at Rha) are in close proximity, thus providing a
very efficient multidentate binding site for Fe3‡.
3. Anthocyanin Complexation. Anthocyanins are polyphenolic pigments mainly
responsible for the red, purple, and blue colours displayed by flowers and fruits [1]. In
Fig. 3. a) UV/VIS Spectra of 15 (5 ´ 10 5 m) and its Fe2‡ complex at pH 5.0 and 258 (MeOH/0.2m acetate buffer
5 :95; Fe2‡/15 molar ratio 0 (1), 0.4 (2), 1 (3), and 2 (4); b) Plot of the absorbance at 420 nm as a function of the
&
*
total metal concentration for rutin ( ) and 15 ( ). The solid lines are the results of the curve-fitting procedures
according to Eqns. 1 and 2.

Helvetica Chimica Acta ± Vol. 84 (2001)
1141
Fig. 4. UV/VIS Spectra of 11 (5 ´ 10 5 m) and of its Fe3‡ complex at pH 5.0 and 258 (MeOH/0.2m acetate buffer
1:1; Fe3‡/11 molar ratio 0 (1), 0.1 (2), 0.2 (3), 0.3 (4), 0.5 (5), 1 (6), and 2 (7)).
Table 2. Complexation of Fe2‡ by Quercetin Derivatives (pH 5, 258). For the definition of r and K', see text.
3
1
K' ´ 10 [m
]
r(420 nm)
a
quercetin )
151 (Æ 48)c)
4.8 (Æ 0.2)
2.0 (Æ 0.1)
11a)
183 (Æ 97)c)
rutinb)
15b)
10.2 (Æ 0.9)
71 (Æ 13)
28.9 (Æ 1.1)
8.7 (Æ 0.2)
a) MeOH/0.2m acetate buffer 1 : 1. b) MeOH/0.2m acetate buffer 5 : 95. c) Large standard deviations because of
quasi-irreversible binding.
Table 3. Complexation of Fe3‡ by Quercetin Derivatives (258). For the definition of r and K', see text.
1
K' ´ 10 3 [m
]
r(420 nm)
quercetina)
25.8 (Æ 3.4)
59 (Æ 14)
6.3 (Æ 0.2)
2.7 (Æ 0.1)
11a)
rutinb)
15c)
22.5 (Æ 1.9)
5.2 (Æ 0.1)
24.0 (Æ 2.1)
6.0 (Æ 0.2)
a) MeOH/0.2m acetate buffer 1 : 1, pH 5. b) MeOH/0.2m acetate buffer 5 : 95, pH 4. No detectable binding in
c
MeOH/0.1m HCl 5 : 95. ) MeOH/0.1m HCl 5 : 95.
the vacuoles where anthocyanins are stored, the mildly acidic aqueous conditions
favour the reversible conversion of the colored forms (flavylium ions) to colorless
hemiacetals via water addition at C(2) (Scheme 2). Fortunately, phenolic acids and
flavonoid glycosides, which are also abundant in vacuoles, form stable stacking
molecular complexes with the large planar polyphenolic nuclei of flavylium ions [20].
This phenomenon, known as copigmentation, efficiently competes with H2O addition
on the pyrylium moiety and thus helps maintain anthocyanins in their colored forms.
Since conversion of anthocyanins into colorless forms may be the first step toward
pigment degradation [21], copigmentation may be viewed as an efficient color-

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Helvetica Chimica Acta ± Vol. 84 (2001)
Scheme 2. Water Addition to Malvin
stabilizing process, and there is great interest in plant extracts rich in both anthocyanins
and potent water-soluble copigments for use as food colourings [22].
The efficiency of water-soluble quercetin derivatives 11, 13, and 14 in copigmenta-
tion was tested with malvin (Scheme 2), one of the most common naturally occurring
anthocyanins. In strongly acidic solution (pH 1), where malvin is in a pure flavylium
form, copigmentation is manifested by bathochromic shifts in the VIS absorption band
of the pigment (Fig. 5) that may reflect a stronger interaction of the copigment with the
pigment in its first excited state than with the ground-state pigment [12]. In mildly
acidic solutions (pH 3.5), closer to those of natural media, copigmentation of malvin
was investigated by monitoring the loss in VIS absorbance in malvin/copigment
solutions (copigment/pigment molar ratios 200 for 11, 20 for 13 and 14) induced by
increasing the temperature as a consequence of the partial (endothermic) dissociation
of the copigmentation complexes (Fig. 5) [12][23]. Under such conditions, the relative
hyperchromic shift at the isosbestic point of the free and bound flavylium ions
(determined at pH ca. 1 where the pigment is in the pure flavylium form) can be very
simply related to the binding constant K according to Eqn. 3 (A, A0 ˆ values for the
VIS absorbance in the presence and absence of copigment, respectively; c ˆ total
copigment concentration) [23].
A
A0
ˆ cK
(3)
A0
From the linear plots of ln [(A A0)/A0] as a function of 1/T (Vanꢀt Hoff
behaviour), values for the temperature-independent enthalpy and entropy changes of
copigmentation could be estimated (Table 4). Whereas 11 binds to malvin with an
affinity close to that measured with analogous succinylated rutin derivatives [12],
quercetin sulfates 13 and 14 were found to be exceptionally potent copigments. Hence,
at pH 1, bathochromic effects of ca. 40 nm are reached with a copigment/pigment molar
ratio of 20, i.e., 10 times lower than with the most efficient rutin derivatives. Moreover,
the values for the copigmentation binding constant K (on the order of 104 m 1 at 258)
are by far the highest recorded until now. At first sight, the strong affinity of sulfates 13
and 14 for malvin could stem from an additional electrostatic attraction between the
negatively charged sulfate group(s) and the positively charged flavylium nucleus of

Helvetica Chimica Acta ± Vol. 84 (2001)
1143
Fig. 5. Copigmentation of malvin (5 ´ 10 5 m) by 13 (10 3 m): a) At pH 3.5 (0.2m acetate buffer), VIS spectra of
malvin/13 at 15.48 (1), 20.58 (2), 25.18 (3), 29.78 (4), 34.98 (5), 39.88 (6) and 44.88 (7); VIS spectrum of malvin
alone at 25.18 (8). b) At pH 0.9 and 258, VIS spectra of malvin alone (1) and of malvin/13 (2).
Table 4. Thermodynamic Parameters for Copigmentation of Malvin by Quercetin Derivatives Deduced from
Temperature Variations in the Range 15 ± 458 (0.2m acetate buffer, pH 3.5).
11
23.6 (Æ 0.1)
18.0 (Æ 0.4)
1560
27
13
14
34.7 (Æ 0.3)
40.0 (Æ 0.8)
8940
36
1
DH8[kJ mol
]
37.3 (Æ 0.6)
45.6 (Æ 1.5)
DS8[J K 1 mol
]
1
1
a
K [M
]
)
14470
39
b
Dlmax [nm]
)
a) Determined at 258 from DH8 TDS8 ˆ RTInK. b) Determined at pH ca. 1 and 258 with the copigment/
pigment molar ratios 100 (11) and 20 (13, 14).
malvin. However, since disulfate 14 binds to malvin less strongly than monosulfate 13
mainly for enthalpic reasons, it is not clear that favourable electrostatic interactions
make an important contribution to the stability of copigmentation complexes.
Comparisons with quercetin (virtually insoluble in weakly acidic buffers) being
impossible, we just point out that the malvin-13 complexation is as exothermic as the
malvin-rutin complexation [12]. The more favourable entropy term in the case of the
malvin-13 complexation may indicate that, being quite rigid, 13 binds to malvin with

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Helvetica Chimica Acta ± Vol. 84 (2001)
relatively little reorganization. Although less potent than the quercetin sulfates, 11 is
still a good copigment. Its binding to malvin, although weakly exothermic, displays a
relatively favourable entropy term. Similar thermodynamic behaviors were already
observed with the succinylated rutin derivatives and were analyzed in terms of
compensating structure-breaking and structure-making contributions that accompany
the desolvation of carboxy/carboxylate groups upon binding [12].
From the 1H-NMR spectra of malvin, copigment 14, and a solution of malvin and 14
in equimolar concentrations (Fig. 6, Table 5), it can be seen that copigmentation
induces very large shieldings of all protons at the interacting polyphenolic nuclei.
Hence, each partner is located inside the cone of diamagnetic anisotropy of the other, in
agreement with a face-to-face arrangement in the complex (stacking).
Table 5. Complexation-Induced Shieldings Dd ( ˆ d(complex) d(free form)) [ppm] of the Proton Signals of
Malvin and 14 in D2O/CD3OD/CF3CO2D 90 : 9 : 1. Copigment/pigment molar ratio 1, 258.
D ˆ d(complex) d(free form)[ppm]
H
C(4)
H
C(6)
0.48
0.56
H
C(8)
0.57
0.26
H
C(2')
0.81
0.62
H
C(5')
0.45
H
C(6')
0.59
0.62
14
malvin
±
0.51
The influence of the solvant on the pigment-copigment interaction was studied with
the malvin-13 couple. A solution of malvin (5 ´ 10 5 m) and 13 (5 ´ 10 4 m) in 0.1m
aqueous HCl and the same solution in MeOH acidified by a concentrated HCl
solution to a final concentration of 0.1m, were mixed in different proportions. The VIS
spectra of the resulting solutions were recorded and the wavelength of VIS absorption
maximum plotted as a function of the percentage of MeOH (Fig. 7). The experiment
was repeated with the pigment malvin alone. In the absence of copigment, the lmax
value raises monotonously with the percentage of MeOH, as expected from the
negative solvatochromism of flavylium ions (first excited state less polar than ground
state). In the presence of the copigment, the lmax value first sharply decreases and then
slowly increases when the amount of MeOH increases. Such variations reflect the
combination of two factors when the percentage of MeOH increases: the dissociation
of the copigmentation complex which tends to lower the lmax value (dominant factor in
the range 0 ± 40%) and the negative solvatochromism of malvin, which tends to elevate
the lmax value (dominant factor in the range 50 ± 100%). Both plots are superimposed
beyond a percentage of MeOH of 40 ± 50%, thus suggesting that, despite its stability in
H2O, the copigmentation complex is completely dissociated in MeOH/H2O mixtures in
which MeOH is the main component. Hence, although stronger in MeOH (er ˆ 32.6)
than in H2O (er ˆ 78.5), electrostatic interactions do not allow maintenance of the
pigment-copigment association under such conditions. The hydrophobic effect requires
a highly organized network of H-bonded H2O molecules around the nonpolar parts of
organic solutes and must be severely weakened when MeOH molecules gradually
displace H2O molecules from the solvation shells. Hence, unlike electrostatic
interactions, the hydrophobic effect, which is maximal in pure H2O, appears as a
crucial contribution to the copigmentation driving force, in agreement with previous
investigations [24].

Helvetica Chimica Acta ± Vol. 84 (2001)
1145
Fig. 6. 1H-NMR Spectra (300 MHz) of 14 (1), malvin (2) and malvin/14 (3; copigment/pigment molar ratio 1) at
278 in D2O/CD3OD/CF3CO2D 90 :9 :1).

1146
Helvetica Chimica Acta ± Vol. 84 (2001)
Fig. 7. Plot of the wavelength of the VIS absorption maximum of malvin (5 ´ 10 5 m) as a function of the
&
*
percentage of MeOH in MeOH/H2O mixtures. ˆ malvin alone; ˆ malvin ‡ 13 (10 equiv.). The acidity was
kept constant (0.1m HCl), 258.
4. Antioxidant Properties. One of the main mechanisms by which flavonoids exert
their antioxidant activity consists in transferring electrons and/or H-atoms to biological
ROS (superoxide, hydroxyl, lipid alkoxyl, and peroxyl radicals) [3][25]. Investigating
the reaction of antioxidants with DPPH (diphenylpicrylhydrazyl), a highly coloured
commercially available radical, provides a simple way to compare antioxidants for their
H-atom-donating ability [12][26]. In this work, the decay of the DPPH VIS absorbance
(lmax 515 nm in MeOH) following the addition of the antioxidant was monitored over
one minute and quantitatively analysed (see below). The kinetic runs were repeated
with different DPPH/antioxidant molar ratios. In most cases, the solutions were kept in
the spectrometer cell for 1 h to ensure the complete conversion of the antioxidant to
inert oxidation-degradation products. From the overall amplitude of VIS absorbance,
the stoichiometry (n) of the antioxidant (number of DPPH radicals trapped per
molecule of antioxidant) was estimated by Eqn. 4 (Af ˆ absorbance 1 hour after
addition of the antioxidant to the DPPH solution, A0 ˆ initial absorbance, c ˆ initial
antioxidant concentration, c0 ˆ initial DPPH concentration). Of course, the initial
DPPH/antioxidant molar ratio c0/c must be higher than n for Eqn. 4 to apply.
ꢀ
ꢁ
c0
c
Af
A0
n ˆ
1
(4)
Flavonols displaying a free 1,2-dihydroxybenzene (catechol) moiety such as
quercetin (1) may be assumed to react with DPPH according to the following
mechanism (Scheme 3) [27]: i) H-Abstraction from the flavonol (FH2) to DPPH occurs
with simultaneous formation of aryloxyl radicals FH (rate constant k1). ii) Fast
disproportionation of FH into the parent flavonol and its quinone follows. The latter

Helvetica Chimica Acta ± Vol. 84 (2001)
1147
NO2
Ph
DPPH
O2N
N
N
Ph
NO2
subsequently undergoes fast solvent addition to yield F 2). Like FH2, F also displays a
catechol group with labile H-atoms and can further react with DPPH. iii) H-
Abstraction from F to DPPH occurs with simultaneous formation of aryloxyl radicals
(rate constant k2), which subsequently disproportionate (f ˆ 1/2).
This mechanism, which requires free OH groups at positions 3, 3', and 4', is assumed
to hold for quercetin derivatives 5, 10, and 13. It is consistent with the evidence of
quinone intermediates, the structural identification of the quinone-solvent adduct in
the case of quercetin, the high stoichiometries (n ꢀ 4), usually measured with flavonols
having a 1,2-dihydroxybenzene moiety [27], and investigations of polyphenolic radicals
by pulse radiolysis [29]3). Assuming a quasi-stationary state for the radicals, Eqns. 5 ± 7
were derived and used in the curve-fitting of the kinetic traces to give optimized k1 and
k2 values in reasonable agreement for different DPPH/antioxidant molar ratios
(Table 6).
d
dt
[DPPH] ˆ k1[FH2][DPPH] ‡ k2[F][DPPH]
(5)
(6)
(7)
d
dt
k
2
[FH2] ˆ 1[FH2][DPPH]
d
dt
k
[F] ˆ 1[FH2][DPPH] fk2[F][DPPH]
2
In the case of rutin, the glycosidation of OH C(3') cancels the o-quinone/p-
quinonoid tautomerism so that the rutin quinone (F) is much less prone to solvent
addition and is actually detectable by 1H-NMR [27]. Its residual antioxidant activity is
simply accounted for by postulating a H-atom transfer to DPPH with rate constant k2
(f ˆ 1 in Eqn. 7).
Glycosidation of OH C(5) (10) and OH C(7) (5) does not significantly lower the
antioxidant stoichiometry, in agreement with the above-mentioned mechanism, in
which neither OH C(5) nor OH C(7) plays a role. However, both glycosides react
with DPPH more slowly than the aglycone (quercetin). The steric hindrance of the
glycosyl groups as well as their probable electron-withdrawing effect on the
polyphenolic nucleus may account for this observation. In contrast, glycosidation of
OH C(3') (9) and sulfation of OH C(4') (14) both result in a dramatic decrease in
the antioxidant stoichiometry and the apparent rate of H-atom abstraction. The
2
)
F is represented as a mixture of pyran- and furan-containing forms (in equilibrium via a non-detected
triketone form), since the latter structure has been proposed for the product isolated after electrochemical
oxidation of quercetin [28].
The fast second-order decay of quercetin radicals in aqueous solution (2k ˆ 3.4 ´ 106 m 1 s 1) [29] suggests
that their recombination via disproportionation may be faster than their reaction with a second DPPH
radical.
3
)

1148
Helvetica Chimica Acta ± Vol. 84 (2001)
Scheme 3. Mechanism for the Reaction of Quercetin with DPPH
OH
HO
O
O
OH
OH
Quercetin
FH2
OH
k1
- 1 H
.
OH
OH
OH
O
HO
O
O
HO
O
O
HO
O
O
.
O
OH
.
OH
O
OH
OH
OH
OH
FH
fast
O
O
O
HO
O
HO
O
O
OH
OH
O
+ Quercetin
OH
O
OH
fast
ROH (R = H, Me)
OH
OH
OH
OR
HO
HO
O
O
OH
O
OH
O
O
OH
OH
O
F

Helvetica Chimica Acta ± Vol. 84 (2001)
1149
Table 6. H-Abstraction from Quercetin Derivatives by DPPH (MeOH, 258). For the definition of parameters
k1, k2 and n, see text.
DPPH [equiv.]
k1 [m 1 s 1]a)
k2 [m 1 s 1]a)
nb)
quercetin
4
8
4
8
6.4
9.4
4 ± 8
6.2
9
2519 (Æ 77)
2800 (Æ 71)
1481 (Æ 11)
1840 (Æ 17)
639 (Æ 6)
617 (Æ 18)
776 (Æ 14)
43 (Æ 2)
50 (Æ 1)
213 (Æ 4)
250 (Æ 5)
±
364 (Æ 9)
433 (Æ 22)
126 (Æ 3)
91 (Æ 1)
±
±
6.8
±
5.9
±
ca. 7
ca. 1
±
6.3
±
4.6
ca. 0.4
rutin
5
728 (Æ 9)
c
9
10
)
437 (Æ 4)
505 (Æ 11)
13d)
4
6
4 ± 8
6190 (Æ 180)
5610 (Æ 150)
c
14
)
a) From the curve-fitting of the kinetic traces (time interval: 60 s) according to Eqns. 5 ± 7. b) From Eqn. 4; final
absorbance Af recorded 1 h after addition of the antioxidant. c) Slow decay, no valid kinetic model. d) A
1
transient absorption of the quinone intermediate at the wavelength of detection (e at 515 nm ca. 8000 m 1 cm
)
must be assumed for a correct curve-fitting.
radicals derived from 9 and 14 are probably much less stable than those of quercetin
and may quickly recombine to form inert dimers4). These results confirm that the 1,2-
dihydroxy substitution at the B ring is essential to the antioxidant activity. It may thus
be assumed that the OH C(4') and OH C(3') are primarily involved in H-atom
transfer reactions to DPPH and/or that each OH group strongly facilitates H-
abstraction from the other by stabilizing the corresponding radical by a combination of
electronic and H-bonding effects. The favourable role of the catechol group in the
antioxidant (H-atom-donating) activity can be rationalized from in vacuo semi-
empirical quantum-mechanics calculations (AM1 parametrization) with simple
phenols such as 3,4-dihydroxybenzaldehyde (16), 4-hydroxy-3-methoxy-benzaldehyde
(17), and 3-hydroxy-4-methoxybenzaldehyde (18), in which the aldehyde moiety
models the electron-withdrawing enone moiety of flavones and flavonols (Scheme 4).
The rotamer of 16 in which the most acidic OH group (OH C(4)) acts as the H-bond
1
donor (16a) is more stable by 0.8 kcal mol than the one in which OH C(3) is the
donor (16b), in agreement with a more stable intramolecular H-bond (3.7 vs. 2.8 kcal
1
mol ). Although dissociation of the OH group at C(3) is favoured in 16a since it
preserves the most stable H-bond, dissociation of the OH group at C(4) in 16b is less
1
endothermic by 1.1 kcal mol , probably because of an extended electron-delocaliza-
tion in the radical with respect to the parent phenol, but also because the intra-
1
molecular H-bond involving the OH C(3) donor is stronger by 0.4 kcal mol in the
radical than in the parent phenol. Despite a probable gain in electron-delocalization
upon dissociation of the OH group at C(4) in 17, the corresponding energy is higher by
3.5 kcal mol 1 than that of 16b, a gap roughly matching the inevitable loss in H-bonding
upon H-abstraction from 17. Although accompanied by the loss of a weaker H-bond,
4
)
The second-order decay of flavonoid radicals in aqueous solution is very significantly faster with flavonoids
devoid of a 1,2-dihydroxy substitution at the B-ring [29].

1150
Helvetica Chimica Acta ± Vol. 84 (2001)
dissociation of the OH group at C(3) in 18 is slightly more endothermic than
dissociation of the OH group at C(4) in 17, suggesting that electron-delocalization in
the radicals is the prevailing factor.
Scheme 4. Semi-Empirical Quantum-Mechanics Calculations with Simple Phenols (see text)a)
O
O
O
H
H
76.4
H
H
3.7
3.7
O
H
16a
O
O
- 0.2
+ 0.8
H
O
O
O
3.2
2.8
H
H
75.3
H
H
O
O
16b
O
O
O
H
78.8
H
H
3.3
O
OCH3
17
O
CH3
O
CH3
O
OCH3
O
2.8
H
79.3
H
H
O
18
O
O
1
a) Energy values in kcal mol
.
The quercetin conjugates detected in the plasma of human volunteers after a meal
rich in plant products are sulfoglucuronides of quercetin and 3'-O-methylquercetin [6],
the exact structures of which are still unknown. Models of quercetin conjugates
(quercetin 3-O-sulfate, and enzymatically prepared glucuronides of quercetin and 3'-O-

Helvetica Chimica Acta ± Vol. 84 (2001)
1151
methylquercetin), although less potent than quercetin, still significantly retard lipid
peroxidation in plasma low-density lipoproteins (LDL), probably via regeneration of
the endogenous amphiphilic antioxidant a-tocopherol at the LDL surface (H-atom
transfer from the flavonols to the a-tocopheryl radical). In addition, the plasma of rats
intragastrically administered with quercetin contains the same type of conjugates as
those found in man and is more resistant to peroxidation than control plasma [30].
Finally, glucosides and methyl ethers of quercetin have been demonstrated to inhibit
lipid peroxidation in LDL and liposomes, 7-O-methylquercetin and the 3- and 7-O-b-d-
glucosides being much more potent than 3'-O-methylquercetin and the 4'-O-b-d-
glucoside [31]. The present work dealing with quercetin sulfates and glycosides (as
models of glucuronides) is consistent with the latter investigations. When glycosidation
or sulfation does not take place at the catechol moiety (B ring), which displays the most
labile H-atoms, it, however, induces a slowing down of the H-atom transfer reactions
(with the possible exception of quercetin 7-O-sulfate). When glycosidation or sulfation
takes place at the catechol moiety, it results in the suppression of one of the most labile
H-atoms and makes the residual H-atom (OH C(3') or OH C(4')) less reactive. It
also cancels out the formation of o-quinones that provide an efficient way for
subsequent degradations (high stoichiometries). Consequently, the antioxidant activity
is drastically reduced. Hence, one may speculate that the residual antioxidant activity
of the circulating conjugates is due to a fraction in which the B ring is spared.
Experimental Part
Á
General. Malvin (Extrasynthese, Genay, France), and quercetin (1) and DPPH (Aldrich) were used as
received. Quercetin 7-O-sulfate (13), quercetin 4',7-di-O-sulfate (14), and rutin 2'',3'',4'',2''',3''',4'''-hexakis(hy-
drogen succinate) (15) were synthesized according to already published procedures [7][12]. UV/VIS Spectra:
Hewlett-Packard-8453 diode-array spectrometer, equipped with a magnetically stirred quartz cell (optical path
length 1 cm), with a thermostated water bath; lmax in nm. NMR Spectra: 300-MHz- and 500-MHz-Bruker
1
apparatus at 278; chemical shifts l in ppm, coupling constants J in Hz; H signals assigned from 1D and 2D-
COSY spectra; 13C signals assigned from DEPT and comparisons with [32].
2-(2,2-Diphenyl-1,3-benzodioxol-5-yl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one (3). A mixture of querce-
tin (1; 2 g, 5.91 mmol) and Ph2CCl2 (4.54 ml, 23.65 mmol) under N2 is heated at 1708 for 15 min under vigorous
stirring. After cooling in an ice bath and dilution in a few ml of CH2Cl2, purification by FC (silica gel, AcOEt/
light petroleum 1:9 ! 1:4) affords 3 (920 mg, 33%). Beige solid. TLC (silica gel, AcOEt/light petroleum ether
1
1:4): Rf 0.16. UV/VIS (MeOH): 251, 359. H-NMR ((D6)DMSO, 300 MHz): 11.80 (s, OH C(5)); 7.79 ± 7.76
(m, H C(4'), H C(6')); 7.62 ± 7.58 (m, Hm of Ph); 7.40 ± 7.39 (m, Ho and Hp of Ph); 7.00 (d, J ˆ 8.1, H C(7'));
6.42 (s, H C(8)); 6.30 (s, H C(6)). FAB-MS (neg. mode): 465.0 ([M H] ).
2-(2,2-Diphenyl-1,3-benzodioxol-5-yl)-3,5-dihydroxy-7-[(tetra-O-acetyl-b-d-glucopyranosyl)oxy]-4H-1-
benzopyran-4-one. A soln. of 2,2,4,6-tetra-O-acetyl-a-d-glucopyranosyl bromide (2; 530 mg, 1.29 mmol) in
CH2Cl2 (20 ml) is added to a soln. of 3 (500 mg, 1.07 mmol) and tris[2-(2-methoxyethoxy)ethyl]amine (TMEA;
343 ml, 1.07 mmol) in sat. KHCO3 soln. (20 ml) under N2. After heating at 408 for 24 h, a second portion of 2
(530 mg) is added and the mixture heated for 24 more h. After cooling and addition of H2O (100 ml), the
mixture is extracted with CH2Cl2 (3 Â 100 ml) and washed with 0.1m HCl (100 ml) and H2O (2 Â 100 ml). After
drying (MgSO4), evaporation, and purification by FC (silica gel, light petroleum ether/AcOEt 4 :1 ! 3 :2), the
protected glucoside (222 mg, 26%) is obtained as a yellow oil. TLC (silica gel, AcOEt/light petroleum ether
2 :3): Rf 0.21. UV/VIS (MeOH): 301, 362. 1H-NMR (CDCl3, 300 MHz): 11.74 (s, OH C(5)); 7.79 (dd, J ˆ 8.8,
1.5, H C(6')); 7.76 (d, J ˆ 1.5, H C(4')); 7.61 ± 7.54 (m, Hm of Ph); 7.40 ± 7.38 (m, Ho and Hp of Ph); 7.01 (d, J ˆ
8.8, H C(7')); 6.58 (d, J ˆ 2.2, H C(8)); 6.44 (d, J ˆ 2.2, H C(6)); 5.39 ± 5.28 (m, H C(2''), H C(4'')); 5.19
(d, J ˆ 7.4, H C(1'')); 5.15 (t, J ˆ 9.5, H C(3'')); 4.29 (dd, J ˆ 11.7, 5.1, 1 H C(6'')); 4.21 (dd, J ˆ 11.7, 2.9, 1
H
C(6'')); 3.95 (ddd, J ˆ 9.5, 5.1, 2.9, H C(5'')); 2.11 ± 2.05 (4 s, 4 Ac). 13C-NMR (CDCl3, 75 MHz): 175.7
(C(4)); 171.6 (MeCO); 171.0 (MeCO); 170.6 (MeCO); 169.7 (MeCO); 162.6 (C(7)); 161.4 (C(5)); 156.6

1152
Helvetica Chimica Acta ± Vol. 84 (2001)
(C(8a)); 149.5 (C(3'a)); 148.1 (C(2)); 146.4 (C(7'a)); 140.1 (C(3)); 136.4 (Cipso Ph); 129.8 (Cp (Ph)); 128.9 (Cm
(Ph)); 126.6 (Co (Ph)); 124.8 (Ph2C); 123.5 (C(7')); 118.3 (C(5')); 109.1 (C(6')); 108.3 (C(4')); 105.6 (C(4a));
99.7 (C(1'')); 98.6 (C(6)); 95.7 (C(8)); 73.0 (C(5'')); 72.8 (C(3'')); 71.3 (C(2'')); 68.6 (C(4'')); 62.4 (C(6'')); 21.5 ±
20.6 (Me (Ac)).
2-(2,2-Diphenyl-1,3-benzodioxol-5-yl)-3,5-dihydroxy-7-(b-d-glucopyranosyloxy)-4H-1-benzopyran-4-one
(4). A soln. of the protected glucoside (20 mg, 0.024 mmol) and KOH (27 mg, 0.48 mmol) in EtOH (5 ml) is
‡
refluxed for 4 h, then cooled, and neutralized with Dowex (H form). After filtration and concentration, 4
(16 mg, 99%) is obtained as a white solid. TLC (silica gel, AcOEt/butan-2-one/HCO2H/H2O 20 :2 :1:1): Rf 0.67.
1
UV/VIS (MeOH) ˆ 256, 371. H-NMR ((D6)DMSO, 300 MHz): 7.85 (s, H C(4')); 7.74 (d, J ˆ 8.8, H C(6'));
7.59 ± 7.53 (m, Hm (Ph)); 7.50 ± 7.45 (m, Ho and Hp (Ph)); 7.25 (d, J ˆ 8.8, H C(7')); 6.85 (d, J ˆ 1.5, H C(8));
6.43 (d, J ˆ 1.5, H C(6)); 5.08 (d, J ˆ 7.4, H C(1'')); 3.71 (d, J ˆ 10.3, 1 H C(6'')); 3.51 ± 3.16 (m, H C(2''),
H
C(3''), H C(4''), H C(5''), H C(6'')). 13C-NMR ((D6)DMSO, 75 MHz): 177.1 (C(4)); 163.7 (C(7)); 161.2
(C(5)); 156.7 (C(8a)); 148.8 (C(3'a)); 147.6 (C(2)); 147.1 (C(7'a)); 140.2 (C(3)); 137.8 (Cipso (Ph)); 130.4 (Cp
(Ph)); 129.5 (Cm (Ph)); 126.6 (Co (Ph)); 124.8 (Ph2C); 124.1 (C(7')); 118.0 (C(5')); 109.8 (C(6')); 108.7 (C(4'));
105.6 (C(4a)); 100.7 (C(1'')); 99.8 (C(6)); 95.3 (C(8)); 78.0 (C(5'')); 77.3 (C(3'')); 73.9 (C(2'')); 70.4 (C(4'')); 61.4
(C6'')). FAB-MS (neg. mode): 627.0 ([M H] ).
3,5-Dihydroxy-2-(3,4-dihydroxyphenyl)-7-(b-d-glucopyranosyloxy)-4H-1-benzopyran-4-one (5). A mix-
ture of 4 (60 mg, 0.10 mmol) and 10% Pd/C (102 mg, 0.10 mmol) in THF/EtOH 1:1 (4 ml) is vigorously stirred
under H2 (1 bar) for 24 h at r. t., then purged with N2, filtered on Celite, and washed with EtOH. Evaporation
and purification by FC (silica gel, AcOEt/light petroleum ether 4 :1 ! AcOEt/MeOH 9 :1), gave 5 (27 mg,
60%). Yellow solid. TLC (silica gel, AcOEt/butan-2-one/HCO2H/H2O 20 :2 :1:1): Rf 0.31. UV/VIS (MeOH):
256, 367. 1H-NMR ((D6)DMSO, 300 MHz): 12.51 (s, OH C(5)); 9.66 (s, OH C(4')); 9.51 (s, OH C(3')); 9.31
(s, OH C(3)); 7.72 (d, J ˆ 2.2, H C(2')); 7.55 (dd, J ˆ 8.8, 2.2, H C(6')); 6.90 (d, J ˆ 8.8, H C(5')); 6.76 (d, J ˆ
2.2, H C(8)); 6.42 (d, J ˆ 2.2, H C(6)); 5.07 (d, J ˆ 6.6, H C(1'')); 3.66 (d, J ˆ 10.3, 1 H C(6'')); 3.52 ± 3.16
(m, H C(2''), H C(3''), H C(4''), H C(5''), C(6'')). 13C-NMR ((D6)DMSO, 75 MHz): 175.7 (C(4)); 162.3
(C(7)); 160.0 (C(5)); 157.4 (C(8a)); 147.6 (C(4')); 147.4 (C(2)); 144.8 (C(3')); 136.0 (C(3)); 121.8 (C(1')); 119.9
(C(6')); 115.7 (C(5')); 115.3 (C(2')); 104.2 (C(4a)); 99.8 (C(1'')); 98.7 (C(6)); 94.1 (C(8)); 77.2 (C(5'')); 76.4
(C(3''); 73.5 (C(2'')); 69.6 (C(4'')); 60.7 (C(6'')). FAB-MS (neg. mode): 463.0 ([M H] ).
3,7-Bis(benzyloxy)-2-[4-(benzyloxy)-3-hydroxyphenyl]-5-hydroxy-4H-1-benzopyran-4-one (6) and 3,7-
Bis(benzyloxy)-2-[3,4-bis(benzyloxy)phenyl]-5-hydroxy-4H-1-benzopyran-4-one (7). tBuOK (3.32 g,
29.6 mmol) is added to a soln. of 1 (2.0 g, 5.9 mmol) in anh. DMF (30 ml) under N2. After stirring for 2 h at
r.t., a first portion of benzyl bromide (3.52 ml, 29.6 mmol) is added and a second one (3.52 ml) after 12 h. After
24 h, H2O (200 ml) and 1m HCl (20 ml) are added, and the mixture is extracted with AcOEt (3 Â 100 ml),
washed with H2O (2 Â 100 ml), dried (MgSO4) and evaporated. Purification by FC (silica gel, light petroleum
ether ! light petroleum ether/AcOEt 85 :15) affords 6 (1.33 g, 40%) and 7 (577 mg, 14%).
Data of 6: Yellow solid. TLC (silica gel, AcOEt/light petroleum ether 1:4): Rf 0.33. UV/VIS (MeOH): 354.
1H-NMR ((D6)DMSO, 300 MHz): 12.67 (s, OH C(5)); 9.47 (s, OH C(3')); 7.56 (d, J ˆ 1.5, H C(2')); 7.52 ±
7.29 (m, H C(6'), 3 Ph); 7.13 (d, J ˆ 8.8, H C(5')); 6.80 (d, J ˆ 1.5, H C(8)); 6.47 (d, J ˆ 1.5, H C(6)); 5.23 ±
5.22 (br. s, PhCH2); 5.02 (s, 1 PhCH2). 13C-NMR ((D6)DMSO, 75 MHz): 179.0 (C(4)); 165.0 (C(7)); 161.9
(C(5)); 157.2 (C(8a)); 150.1 (C(3')); 147.5 (C(2)); 147.4 (C(4')); 137.6 (Cipso (Bn)); 137.5 (Cipso (Bn)); 137.4 (Cipso
(Bn)); 136.9 (C(3)); 129.4 ± 128.4 (CH (Bn)); 123.3 (C(1')); 121.4 (C(5')); 116.5 (C(6')); 114.1 (C(2')); 106.2
(C(4a)); 99.3 (C(6)); 94.0 (C(8)); 74.2 (PhCH2); 70.8 (PhCH2); 70.6 (PhCH2).
Data of 7: Yellow solid. TLC (silica gel, AcOEt/light petroleum ether 1:4): Rf 0.52. UV/VIS (MeOH): 352.
‡
FAB-MS (pos. mode): 663.2 ([M ‡ H] ). 1H-NMR ((D6)DMSO, 300 MHz): 12.63 (s, OH C(5)); 7.75 (d, J ˆ
1.5, H C(2')); 7.65 (dd, J ˆ 8.8, 1.5, H C(6')); 7.49 ± 7.27 (m, 4 Ph); 7.22 (d, J ˆ 8.8, H C(5')); 6.85 (d, J ˆ 2.2,
H
C(8)); 6.48 (d, J ˆ 2.2, H C(6)); 5.24 (br. s, 2 PhCH2); 5.06 (s, 1 PhCH2); 4.97 (s, 1 PhCH2). 13C-NMR
((D6)DMSO, 75 MHz): 179.0 (C(4)); 165.0 (C(7)); 161.8 (C(5)); 157.0 (C(8a)); 156.5 (C(4')); 151.5 (C(3'));
148.4 (C(2)); 137.8 (Cipso (Ph)); 137.7 (Cipso (Bn)); 137.6 (Cipso (Bn)); 137.4 (Cipso (Bn)); 136.9 (C(3)); 129.4 ±
128.4 (CH (Bn)); 123.2 (C(1')); 123.1 (C(5')); 115.0 (C(6')); 114.3 (C(2')); 106.2 (C(4a)); 99.3 (C(6)); 94.4
(C(8)); 74.3 (PhCH2); 71.0 (PhCH2); 70.9 (PhCH2); 70.7 (PhCH2). FAB-MS (neg. mode): 571.3 ([M H] ).
3,7-Bis(benzyloxy) 2-{4-(benzyloxy)-5-hydroxy-3-[(tetra-O-acetyl-b-d-glucopyranosyl)oxy]phenyl}-4H-1-
benzopyran-4-one. A soln. of 2 (99 mg, 0.24 mmol) in CH2Cl2 (10 ml) is added to a soln. of 6 (90 mg,
0.16 mmol) and TMEA (50 ml, 0.16 mmol) in sat. KHCO3 soln. (10 ml). The mixture is heated at 408 for 96 h,
3 portions of 2 (66 mg, 0.16 mmol) being added each 24 h to complete the reaction. After cooling and addition
of H2O (20 ml), the mixture is extracted with CH2Cl2 (3 Â 20 ml) and washed with 1m HCl (30 ml) and H2O
(50 ml). After drying (MgSO4), evaporation, and purification by FC (silica gel, light petroleum ether/AcOEt

Helvetica Chimica Acta ± Vol. 84 (2001)
1153
4 :1 ! 3 :2), the protected glucoside (70 mg, 48%) is obtained as a yellow solid. TLC (silica gel, AcOEt/light
1
petroleum ether 2 :3): Rf 0.46. H-NMR (CDCl3, 300 MHz): 12.66 (s, OH C(5)); 7.95 (d, J ˆ 2.2, H C(2'));
7.71 (dd, J ˆ 8.8, 2.2, H C(6')); 7.43 ± 7.36 (m, 3 Ph); 7.02 (d, J ˆ 8.8, H C(5')); 6.54 (d, J ˆ 1.5, H C(8)); 6.45
(d, J ˆ 1.5, H C(6)); 5.33 ± 5.24 (m, H C(2''), H C(4'')); 5.16 ± 5.14 (3 s, 3 PhCH2); 5.03 (d, J ˆ 9.5, H C(3''));
4.77 (d, J ˆ 10.3, 1 H C(6'')); 4.70 (d, J ˆ 7.4, H C(1'')); 4.05 (m, H C(6'')); 3.06 (m, H C(5'')); 2.07 ± 1.98 (4
s, 4 Ac). 13C-NMR (CDCl3, 75 MHz): 179.1 (C(4)); 171.0 (MeCO); 170.7 (MeCO); 169.8 (MeCO); 169.7
(MeCO); 165.0 (C(7)); 162.5 (C(5)); 157.1 (C(8a)); 156.2 (C(3')); 151.6 (C(2)); 146.6 (C(4')); 138.2 (C(3));
137.1 (Cipso (Bn)); 136.6 (Cipso (Bn)); 136.2 (Cipso (Bn)); 129.6 ± 127.6 (CH (Bn)); 124.2 (C(5')); 123.6 (C(1'));
119.3 (C(6')); 114.8 (C(2')); 106.6 (C(4a)); 100.2 (C(1'')); 99.1 (C(6)); 93.5 (C(8)); 74.9 (PhCH2); 72.8 (C(5''));
72.2 (C(3'')); 71.2 (C(2'')); 71.1 (PhCH2); 70.9 (PhCH2); 68.4 (C(4'')); 62.0 (C(6'')); 21.5 ± 20.8
(4 MeCO).
3,7-Bis(benzyloxy)-2-[4-(benzyloxy)-3-(b-d-glucopyranosyloxy)phenyl]-5-hydroxy-4H-1-benzopyran-4-
one (8). Saponification of the acetate groups of the protected glucoside gives 8 (48 mg, 99%). White solid. TLC
(silica gel, AcOEt): Rf 0.17. UV/VIS (MeOH): 348. 1H-NMR ((D6)DMSO, 300 MHz): 12.63 (s, OH C(5));
7.79 (d, J ˆ 1.5, H C(2')); 7.70 (dd, J ˆ 8.8, 1.5, H C(6')); 7.52 ± 7.29 (m, 3 Ph); 7.19 (d, J ˆ 8.8, H C(5')); 6.90
(d, J ˆ 1.5, H C(8)); 6.48 (d, J ˆ 1.5, H C(6)); 5.27 (s, 1 PhCH2); 5.23 (s, 1 PhCH2); 5.05 (s, 1 PhCH2); 4.89 (d,
J ˆ 8.1, H C(1'')); 3.69 (d, J ˆ 11.8, 1 H C(6'')); 3.46 (dd, J ˆ 11.8, 5.2, 1 H C(6'')); 3.35 ± 3.13 (m, H C(2''),
H
C(3''), H C(4''), H C(5'')). 13C-NMR ((D6)DMSO, 75 MHz): 179.0 (C(4)); 165.0 (C(7)); 161.8 (C(5));
157.1 (C(8a)); 156.8 (C(3')); 151.3 (C(2')); 147.6 (C(4')); 137.7 (Cipso (Bn)); 137.6 (Cipso (Bn)); 137.2 (Cipso (Bn));
136.9 (C(3)); 129.4 ± 128.3 (CH (Bn)); 124.4 (C(5')); 123.4 (C(1')); 116.9 (C(6')); 115.0 (C(2')); 106.2 (C(4a));
101.7 (C(1'')); 99.4 (C(6)); 94.3 (C(8)); 78.1 (C(5'')); 77.6 (C(3'')); 74.1 (C(2'')); 74.0 (PhCH2); 71.2 (PhCH2);
‡
71.1 (PhCH2); 70.5 (C(4'')); 60.7 (C(6'')). FAB-MS (pos. mode): 735.5 ([M ‡ H] ).
2-[3-(b-d-Glucopyranosyloxy)-4-hydroxyphenyl]-3,5,7-trihydroxy-4H-1-benzopyran-4-one (9). Hydrogen-
olysis of the benzyl groups of 8 and purification by FC (silica gel, AcOEt/light petroleum ether 9 :1 ! AcOEt/
MeOH 9 :1) give 9 (11 mg, 37%). Yellow solid. TLC (silica gel, AcOEt/butan-2-one/HCO2H/H2O 20 :2 :1:1): Rf
0.35. UV/VIS (MeOH): 269, 366. 1H-NMR ((D6)DMSO, 300 MHz): 12.45 (s, OH C(5)); 10.78 (s, OH C(7));
9.45 (s, OH C(4')); 9.32 (s, OH C(3)); 7.96 (d, J ˆ 2.2, H C(2')); 7.84 (dd, J ˆ 8.8, 2.2, H C(6')); 6.97 (d, J ˆ
8.8, H C(5')); 6.49 (d, J ˆ 1.5, H C(8)); 6.19 (d, J ˆ 1.5, H C(6)); 4.78 (d, J ˆ 6.6, H C(1'')); 3.77 (d, J ˆ 11.0,
1 H C(6'')); 3.57 (dd, J ˆ 11.0, 5.1, 1 H C(6'')); 3.37 ± 3.15 (m, H C(2''), H C(3''), H C(4''), H C(5'')).
13C-NMR ((D6)DMSO, 75 MHz): 179.0 (C(4)); 161.5 (C(7)); 161.4 (C(5)); 157.0 (C(8a)); 156.8 (C(3')); 151.1
(C(2)); 146.3 (C(4')); 136.6 (C(3)); 124.2 (C(5')); 123.0 (C(1')); 117.0 (C(6')); 116.6 (C(2')); 106.2 (C(4a)); 103.5
(C(1'')); 99.3 (C(6)); 94.6 (C(8)); 78.1 (C(5'')); 76.8 (C(3'')); 74.1 (C(2'')); 70.4 (C(4'')); 61.5 (C(6'')). FAB-MS
(neg. mode): 463.1 ([M H] ).
3,7-Bis(benzyloxy)-2-[3,4-bis(benzyloxy)phenyl]-5-[(tetra-O-acetyl-b-d-galactopyranosyl)oxy]-4H-1-ben-
zopyran-4-one. A soln. of 2,3,4,6-tetra-O-acetyl-a-d-galactopyranosyl bromide (372 mg, 0.91 mmol) in CH2Cl2
(15 ml) is added to a soln. of 7 (400 mg, 0.6 mmol) and TMEA (193 ml, 0.6 mmol) in sat. K2CO3 soln. (15 ml).
After heating at 408 for 24 h, a second portion of glycosyl donor (250 mg, 0.6 mmol) is added and the mixture
further heated for 24 h. After the usual treatment and purification by FC (silica gel, light petroleum ether/
AcOEt 4 :1 ! 3 :2), the protected glucoside (209 mg, 35%) is obtained as a yellow oil. TLC (silica gel, AcOEt/
light petroleum ether 2 :3): Rf 0.45. 1H-NMR (CDCl3, 300 MHz): 7.73 (s, H C(2')); 7.55 (d, J ˆ 8.8, H C(6'));
7.45 ± 7.16 (m, 4 Ph); 6.94 (d, J ˆ 8.8, H C(5')); 6.92 (d, J ˆ 2.2, H C(8)); 6.72 (d, J ˆ 2.2, H C(6)); 5.20 (s, 2
PhCH2); 5.11 (s, 1 PhCH2); 5.12 ± 4.99 (m, H C(3''), H C(4'')); 4.93 (t, J ˆ 8.1, H C(2'')); 4.91 (s, 1 PhCH2);
4.74 (d, J ˆ 8.1, H C(1'')); 4.29 ± 4.16 (m, H C(5''), 2 H C(6'')); 2.15 (s, Ac); 2.09 (s, Ac); 2.02 (s, Ac); 1.98 (s,
Ac).
3,7-Bis(benzyloxy)-2-[3,4-bis(benzyloxy)phenyl]-5-(b-d-galactopyranosyloxy)-4H-1-benzopyran-4-one.
Saponification of the acetate groups of the protected galactoside gives a white solid (99 mg, 100%). TLC (silica
gel, AcOEt/butan-2-one/HCOOH/H2O 20 :2 :1 :1): Rf 0.57. UV/VIS (MeOH): 253, 352. 1H-NMR ((D6)DMSO,
300 MHz): 7.76 (d, J ˆ 1.5, H C(2')); 7.65 (dd, J ˆ 8.8, 1.5, H C(6')); 7.52 ± 7.28 (m, 4 Ph); 7.22 (d, J ˆ 8.8,
H
C(5')); 7.08 (d, J ˆ 2.2, H C(8)); 6.92 (d, J ˆ 2.2, H C(6)); 5.25 (s, 2 PhCH2); 4.96 (s, 2 PhCH2); 4.81 (d, J ˆ
8.1, H C(1'')); 3.77 ± 3.45 (m, H C(2''), H C(3''), H C(4''), H C(5''), 2 H C(6'')). FAB-MS (neg. mode):
823.1 ([M H] ).
3,7-Dihydroxy-2-(3,4-dihydroxyphenyl)-5-(b-d-galactopyranosyloxy)-4H-1-benzopyran-4-one (10). Hy-
drogenolysis of the benzyl groups of the partially protected galactoside and purification by FC (silica gel,
AcOEt/light petroleum ether 4 :1 ! AcOEt/MeOH 4 :1) give 10 (25 mg, 49%). Yellow solid. TLC (silica gel,
AcOEt/butan-2-one/HCO2H/H2O 20 :2 :1 :1): Rf 0.11. UV/VIS (MeOH): 368. 1H-NMR ((D6)DMSO,
300 MHz): 11.11 (s, OH C(7)); 9.61 (s, OH C(4')); 9.34 (s, OH C(3')); 8.90 (s, OH C(3); 7.66 (d, J ˆ 2.2,

1154
Helvetica Chimica Acta ± Vol. 84 (2001)
H
H
C(2')); 7.52 (dd, J ˆ 8.8, 2.2, H C(6')); 6.90 (d, J ˆ 8.8, H C(5')); 6.80 (d, J ˆ 2.2, H C(8)); 6.71 (d, J ˆ 2.2,
C(6)); 4.70 (d, J ˆ 7.4, H C(1'')); 3.65 ± 3.24 (m, H C(2''), H C(3''), H C(4''), H C(5''), 2 H C(6'')).
FAB-MS (neg. mode): 463.0 ([M H] ).
3,7-Bis(benzyloxy)-2-{4-(benzyloxy)-3-{[2,3,4,6-tetra-O-(3-carboxy-1-oxopropyl)-b-d-glucopyranosyl]-
oxy}phenyl}-5-hydroxy-4H-1-benzopyran-4-one. A soln. of 8 (200 mg, 0.27 mmol), DMAP (33 mg, 0.27 mmol),
and succinic anhydride (163 mg, 1.63 mmol) in anh. pyridine (10 ml) is heated at 708 under N2 for 16 h. After
evaporation of pyridine under vacuum and dilution with acetone (10 ml), the mixture is neutralized with Dowex
‡
(H form) to pH 2 ± 3 (wet pH paper), filtered, and evaporated. Yellow solid (300 mg, 98%). TLC (silica gel,
AcOEt/butan-2-one/HCO2H/H2O 20 :2 :1 :1): Rf 0.88. 1H-NMR ((D6)DMSO, 300 MHz): 12.61 (s, OH C(5));
12.16 (s, CO2H); 7.83 (d, J ˆ 2.2, H C(2')); 7.78 (dd, J ˆ 8.8, 2.2, H C(6')); 7.46 ± 7.31 (m, 3 Ph); 7.27 (d, J ˆ 8.8,
H
C(5')); 6.84 (d, J ˆ 2.2, H C(8)); 6.49 (d, J ˆ 2.2, H C(6)); 5.37 (t, J ˆ 9.5, H C(3'')); 5.24 ± 5.21 (m, 3
PhCH2); 5.20 (d, J ˆ 8.1, H C(1'')); 5.13 (dd, J ˆ 8.1, 9.5, H C(2'')); 5.00 (t, J ˆ 9.6, H C(4'')); 4.97 (d, J ˆ 11.0,
1 H C(6'')); 4.11 ± 4.01 (m, 1 H C(6''), H C(5'')); 2.42 (s, 8 CH2 (suc)). FAB-MS (neg. mode): 1133.0
([M H] ).
3,5,7-Trihydroxy-2-{4-hydroxy-3-{[2,3,4,6-tetra-O-(3-carboxy-1-oxopropyl)-b-d-glucopyranosyl]oxy}phen-
yl}-4H-1-benzopyran-4-one (11). Hydrogenolysis of the benzyl groups of the preceding compound gives a crude
mixture, which is diluted with H2O (50 ml), washed with CHCl3 (3 Â 50 ml) and lyophilized: 11 (210 mg, 90%).
Yellow-green solid. TLC (silica gel, AcOEt/butan-2-one/HCO2H/H2O 20 :2 :1:1): Rf 0.82. UV/VIS (MeOH):
368. 1H-NMR ((D6)DMSO, 300 MHz): 7.92 (d, J ˆ 1.5, H C(2')); 7.83 (dd, J ˆ 8.1, 1.5, H C(6')); 7.00 (d, J ˆ 8.1,
H
H
C(5')); 6.47 (d, J ˆ 1.5, H C(8)); 6.17 (d, J ˆ 1.5, H C(6)); 5.48 (t, J ˆ 9.5, H C(4'')); 5.42 (d, J ˆ 8.1,
C(1'')); 5.10 (dd, J ˆ 9.5, 8.1, H C(2'')); 5.06 (t, J ˆ 9.5, H C(3'')); 4.23 ± 4.05 (m, H C(5''), 2 H C(6''));
2.38 (s, 8 CH2 (suc)). 13C-NMR ((D6)DMSO, 75 MHz): 176.6 (C(4)); 174.8 ± 174.7 (4 CO2H); 173.1 ± 172.1 (4
CO2R); 161.3 (C(7)); 157.5 (C(5)); 157.1 (C(8a)); 151.2 (C(2)); 146.2 (C(4')); 145.4 (C(3')); 136.8 (C(3)); 125.7
(C(5')); 122.8 (C(1')); 117.0 (C(6')); 116.0 (C(2')); 103.0 (C(4a)); 100.4 (C(1'')); 99.7 (C(6)); 94.6 (C(8)); 72.2
(C(5'')); 71.7 (C(3'')); 71.5 (C(2'')); 68.8 (C(4'')); 62.7 (C(6'')); 31.5 ± 30.1 (8 CH2 (suc)). FAB-MS (neg. mode):
863.1 ([M H] ).
3,7-Bis(benzyloxy)2-{4-(benzyloxy)-3-{[6-O-(3,4,5-trimethoxybenzoyl)-b-d-glucopyranosyl]oxy}phenyl}-
5-hydroxy-4H-1-benzopyran-4-one. In small portions, 3,4,5-trimethoxybenzoyl chloride (95 mg, 0.41 mmol) is
added to a soln. of 8 (100 mg, 0.14 mmol) and DMAP (9 mg, 0.07 mmol) in anh. pyridine under N2. After
stirring for 72 h at r.t., evaporation under vacuum, and dilution in AcOEt (50 ml), the mixture is washed with
0.1m HCl (50 ml), sat. NaHCO3 (50 ml), and sat. NaCl soln. (2 Â 50 ml), dried, and evaporated and the residue
purified by FC (silica gel, AcOEt/light petroleum ether 3 :2 ! 4 :1): Yellow solid (21 mg, 16%). TLC (silica gel,
AcOEt): Rf 0.33. UV/VIS (MeOH): 268, 345. 1H-NMR ((D6)DMSO, 300 MHz): 12.49 (s, OH C(5)); 7.83 (dd,
J ˆ 8.8, 1.5, H C(6')); 7.77 (d, J ˆ 1.5, H C(2')); 7.53 ± 7.30 (m, 3 Ph); 7.18 (d, J ˆ 8.8, H C(5')); 6.86 (s,
H
C(2'''), H C(6''')); 6.58 (d, J ˆ 2.2, H C(8)); 6.40 (d, J ˆ 2.2, H C(6)); 5.26 ± 5.22 (m, 3 PhCH2); 5.04 (d,
J ˆ 11.0, 1 H C(6'')); 4.97 (d, J ˆ 7.4, H C(1'')); 4.92 (br. d, J ˆ 11.0, 1 H C(6'')); 3.55 (s, 1 MeO); 3.44 (s, 2
‡
MeO); 3.39 ± 3.20 (m, H C(2''), H C(3''), H C(4''), H C(5'')). FAB-MS (pos. mode): 929.7 ([M ‡ H] ).
3,5,7-Trihydroxy-2-{4-hydroxy-3-{[6-O-(3,4,5-trimethoxybenzoyl)-b-d-glucopyranosyl]oxy}phenyl}-4H-1-
benzopyran-4-one (12). Hydrogenolysis of the benzyl groups of the preceding compound and purification by FC
(C18 silica gel, H2O ! H2O/MeOH 1:1) gave 12 (4 mg, 64%). Yellow solid. TLC (silica gel, AcOEt/butan-2-
one/HCO2H/H2O 20 :2 :1:1): Rf 0.72. UV/VIS (MeOH): 265, 371. FAB-MS (neg. mode): 657.5 ([M H] ).
1H-NMR ((D6)DMSO, 300 MHz): 7.89 (d, J ˆ 8.8, H C(6')); 7.84 (s, H C(2')); 6.96 (d, J ˆ 8.8, H C(5')); 6.88
(s, H C(2'''), H C(6''')); 6.16 (s, H C(8)); 6.08 (s, H C(6)); 4.95 (d, J ˆ 7.4, H C(1'')); 4.89 (d, J ˆ 10.3, 1
H
H
C(6'')); 4.18 (dd, J ˆ 10.3, 6.6, H C(6'')); 3.56 (s, 1 MeO); 3.55 (s, 2 MeO); 3.42 ± 3.28 (m, H C(2''),
C(3''), H C(4''), H C(5'')). 13C-NMR ((D6)DMSO, 125 MHz): 166.1 (C(4)); 166.0 (CO (acyl)); 165.1
(C(7)); 156.6 (C(5)); 153.6 (C(8a)); 153.1 (C(3'''), C(5''')); 146.1 (C(3')); 145.7 (C(2)); 142.8 (C(4')); 142.2
(C(4''')); 125.6 (C(1')); 125.5 (C(1''')); 124.3 (C(5')); 117.1 (C(6')); 115.8 (C(2')); 107.5 (C(4a)); 107.0 (C(2'''),
C(6''')); 103.2 (C(1'')); 102.2 (C(6)); 94.6 (C(8)); 76.5 (C(5'')); 75.0 (C(3'')); 73.8 (C(2'')); 71.3 (C(4'')); 65.0
(C(6'')); 60.8 (1 MeO); 56.8 (2 MeO).
Data Analysis. The curve-fittings were carried out on a Pentium-120 PC (Scientist program, MicroMath,
Salt Lake City, Utah, USA) by least-squares regression. Optimized values for the parameters and the
corresponding standard deviations are reported.
Copigmentation of Malvin. A given copigment is added to a 5 ´ 10 5 m soln. of malvin in a 0.2m acetate buffer
at pH 3.5 (copigment/pigment molar ratio 20 for 13 and 14 and 200 for 11). An aliquot of the soln. is placed in
the spectrometer cell and UV/VIS spectra are recorded every 58 in the temp. range 15 ± 458. The soln. is kept ca.
30 min at the selected temp. before each measurement to allow the structural transformations of the pigment

Helvetica Chimica Acta ± Vol. 84 (2001)
1155
(H2O addition) to reach complete equilibrium. The experiment is repeated with the soln. of malvin without
copigment. Both solns. are then acidified to pH 0.9 ± 1.0 by conc. HCl soln. (no significant dilution) for the
determination of the isosbestic point of malvin (flavylium form) and its copigmentation complex.
H-Abstraction by DPPH. To 2 ml of freshly prepared 2 ´ 10 4 m DPPH in MeOH placed in the spectrometer
cell, 50 ± 100 ml of a freshly prepared 10 3 m soln. of flavonol in MeOH were added. UV/VIS spectra were
recorded every 0.5 s over 1 min for the determination of the rate constants of H-atom abstraction. Runs over 1 h
were used for the determination of the total antioxidant stoichiometry.
Aluminium Complexation by Flavonols. In a typical experiment, freshly prepared 10 4 m flavonol in MeOH
was diluted twice with a 0.2m acetate buffer (final pH 5.0). In one part of this soln., AlCl3 ´ 6 H2O was added to a
final concentration of 5 ´ 10 3 m. If necessary, the pH was brought back to 5.0 by addition of conc. NaOH soln.
(without significant dilution). The flavonol and flavonol/aluminium solns. were then mixed in different ratios to
prepare samples having intermediate Al3‡ concentrations for spectral measurements.
Iron Complexation by Flavonols. In a typical experiment, a few ml of 3 ´ 10 3 m FeSO4 ´ 7 H2O or Fe(NO3)3 ´
9 H2O in MeOH was added at time zero to a sample (2 ml) of flavonol (5 ´ 10 5 m) in a given buffer placed in the
thermostated cell of the UV/VIS spectrometer. Spectra were recorded over a few min until equilibrium was
reached. Absorbance values measured at equilibrium were used for the determination of the apparent iron-
flavonol binding constants. No precipitation occurred under such conditions.
Semi-Empirical Quantum-Mechanics Calculations. Energy optimizations were run in vacuo on a Pentium-
90 PC with the HyperChem program (Autodesk, Sausalito, California, USA) and the AM1 parametrization. The
unrestricted Hartree-Fock method was selected for both the open-shell (aryloxyl radicals) and close-shell species
(phenols). The bond-dissociation energies were identified to the energy differences between the aryloxyl
radicals and the corresponding phenols. The H-bond dissociation energies in phenols and aryloxyl radicals were
identified to the energy differences between non-H-bonded and H-bonded rotamers (1808 rotation about the
C
OH bond).
REFERENCES
[1] E. Haslam, ꢁPractical Polyphenolicsꢀ, University Press, Cambridge, 1998; ꢁThe Flavonoids, Advances in
Research since 1986ꢀ, Ed. J. B. Harborne, Chapman and Hall, London, 1994; J. B. Harborne, C. A.
Williams, Phytochemistry 2000, 55, 481; A. Scalbert, G. Williamson, J. Nutr. 2000, 130, 2073S.
[2] M. G. L. Hertog, E. J. M. Feskens, P. C. H. Hollman, M. B. Katan, D. Kromhout, Lancet 1993, 342, 1007; S.
Renaud, M. de Lorgeril, Lancet 1992, 339, 1523; K. A. Steinmetz, J. D. Potter, J. Am. Diet Assoc. 1996, 96,
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Weisgerber, M. B. Katan, Crit. Rev. Food Sci. Nutr. 1997, 37, 771.
[3] P.-G. Pietta, J. Nat. Prod. 2000, 63, 1035; ꢁFlavonoids in Health and Diseaseꢀ, Eds. C. A. Rice-Evans and L.
Packer, M. Dekker, New York, 1997.
[4] P. C. H. Hollman, J. H. M. De Vries, S. D. van Leeuwen, M. J. B. Mengelers, M. B. Katan, Am. J. Clin. Nutr.
1995, 62, 1276; M. R. Olthof, P. C. H. Hollman, T. B. Vree, M. B. Katan, J. Nutr. 2000, 130, 1200.
[5] A. Yagi, T. Uemura, N. Okamura, H. Haraguchi, T. Imoto, K. Hashimoto, Phytochemistry 1994, 35, 885.
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Received February 7, 2001