Acidity of hydroxyl groups: An overlooked influence on antiradical properties of flavonoids

Article abstract of DOI:10.1021/jo802716v

The reactions of 10 flavonoids with 2,2-diphenyl-1-picrylhydrazyl radical (dpph^.) carried out in alcohols always occur significantly faster than in acidified alcohols or in dioxane. These fast kinetics benefit from the contribution of the elect

Full text of DOI:10.1021/jo802716v

Acidity of Hydroxyl Groups: An Overlooked Influence on
Antiradical Properties of Flavonoids
†
‡
†
Malgorzata Musialik, Rafal Kuzmicz, Tomasz S. Pawłowski, and
,
†
Grzegorz Litwinienko*
Faculty of Chemistry, UniVersity of Warsaw, Pasteura 1, 02-093 Warsaw, Poland and Department of
Inorganic and Analytical Chemistry, Faculty of Pharmacy, Medical UniVersity of Warsaw, Banacha 1,
0
2-097 Warsaw, Poland
litwin@chem.uw.edu.pl
ReceiVed December 11, 2008
•
The reactions of 10 flavonoids with 2,2-diphenyl-1-picrylhydrazyl radical (dpph ) carried out in alcohols
always occur significantly faster than in acidified alcohols or in dioxane. These fast kinetics benefit from
the contribution of the electron transfer from a flavonoid anion to a radical, a mechanism known as
Sequential Proton-Loss Electron-Transfer (SPLET), which adds to the kinetics of single-step Hydrogen
Atom Transfer (HAT)/Proton Coupled Electron Transfer (PCET) processes (see Acc. Chem. Res. 2007,
4
0, 222.). The domination of SPLET over HAT/PCET in case of a flavonoid reacting with electron-
•
deficient radicals such as peroxyls or dpph in polar solvents explains the enhancement of antioxidant
activity of 3-hydroxyflavone. It also elucidates the great acceleration in the reactions of dpph with
•
quercetin, morin, galangin, and 7,8-dihydroxyflavone. The analysis of structure-acidity and structure-activity
relationships for 10 flavonoids clearly indicates that hydroxyl group at position 7 is the most acidic site.
Thus, in polar solvents this group can participate in radical reaction via SPLET. In nonpolar solvents the
most active site in quercetin (a flavonoid antioxidant commonly found in plants) is 3′,4′-dihydroxyl moiety
and HAT/PCET occurs. However, in ionization-supporting solvents an anion formed at position 7 is
•
responsible for very fast kinetics of quercetin/dpph reaction because both mechanisms participate: HAT
(from catechol moiety in ring B) and SPLET (from ionized 7-hydroxyl in ring A). Because of conjugation
of rings A, B, and C the final structure of the formed quercetin radical (or quercetin anion radical) is the
same for the SPLET and HAT/PCET mechanisms.
Introduction
structure of these compounds containing three rings designated
A, B, and C is depicted in Scheme 1. A presence of a double
bond, a carbonyl, and a hydroxyl group in the pyranyl ring C
serves as a basis for their classification into several classes and
subclasses. Substitution of A and B rings by hydroxyl groups
distinguishes individual members of each class.
Flavonoids are common components of plants. More than
4
000 chemically unique flavonoids identified in various plant
1
species have been isolated from almost all parts of the plant
such as leaves, stems, roots, fruits, or seeds. The general
†
University of Warsaw.
Medical University of Warsaw.
Flavonoids are among the major antioxidant constituents of
‡
2
our diet. Their daily intake (reported as above 100 mg) is almost
(
1) Beecher, G. R. In Antioxidant Food Supplements in Human Health;
Packer, L., Hiramatsu, M., Yoshikawa, T., Eds.; Academic Press: New York,
999.
at the same level as the sum of the daily doses of other
antioxidants including ꢀ-carotene (2-3 mg), vitamin C (70-100
1
1
0.1021/jo802716v CCC: $40.75  2009 American Chemical Society
J. Org. Chem. 2009, 74, 2699–2709 2699
Published on Web 03/10/2009

Musialik et al.
SCHEME 1
accepted that the excellent antioxidant properties of flavonoids
can be explained by the presence of catechol hydroxylic groups
in the B ring:
1
0,12,15
3
mg), and vitamin E (7 -10 mg). These data correspond to the
reports of many health benefits coming from dietary intake of
flavonoids. Among the most significant profits are the protection
4
The stability of the radical generated from catechol moiety
relies on the hydrogen bond (HB) formed between the hydroxyl
and oxygen possessing unpaired electron, reaction 1. The
presence of a CdC double bond in ring C conjugated with a
against oxidative diseases, ability to modulate the activity of
5
6,7
various enzymes, and interactions with specific receptors.
Since the mechanisms of action of flavonoids on human cells
and organism is not completely understood, extensive multi-
disciplinary studies covering the kinetics of antioxidant action
and enzyme binding in vitro, as well as their distribution,
absorption, excretion, and metabolism in vivo, still attract great
4
-oxo group is also of great importance for the delocalization
of an unpaired electron. Additional antioxidant activity is
assigned to the presence of a hydroxyl group at the 3- and
10
7-10
5-positions, and according to Bors et al., an internal hydrogen
bond to a 4-oxo group (if present) makes these positions
kinetically equivalent. The present knowledge does not explain,
however, the exact role of hydroxyl groups in rings A and C
attention.
In general, the ability of flavonoids to be effective
antioxidants depends on three factors: (i) the metal-chelating
potential that is strongly dependent on the arrangement of
hydroxyls and carbonyl group around the molecule, (ii) the
presence of hydrogen-/electron-donating substituents able to
reduce free radicals, and (iii) the ability of the flavonoid to
delocalize the unpaired electron leading to formation of a stable
phenoxyl radical. Both known modes of the antioxidant action,
i.e., preventive mechanism and chain-breaking mechanism, are
postulated to be responsible for the high activity of flavonoids.
However, although the first function, i.e., the ability to chelate
the iron and copper cations, is well understood, another one,
i.e., the ability of polyphenols to scavenge reactive oxygen
species such as peroxyl and hydroxyl radicals, is still far from
being fully understood.
(
Scheme 1) during the scavenging of radicals. Glycosylation
of the 3-OH group greatly reduces the antioxidant activity of
1
5
3
-hydroxyflavones. On the other side, it has been shown that
hydroxyl groups at positions 5 and 7 are not as potent as 3′,4′-
1
0
dihydroxyl (in the B ring).
The term “antioxidant activity” is not clearly defined, and
there are several definitions based on the nature of the oxidation
and reactive intermediates taking part in this process. For lipid
peroxidation the activity of an antioxidant can be considered
as the ability to suppress the propagation of the kinetic chain
of hydroperoxide formation:
As a result of their low redox potentials (EpH7 < 0.75 V vs
normal hydrogen electrode, NHE) flavonoids are able to reduce
free radicals with redox potentials higher than 0.8 V, such as
•
•
LOO + L-H f LOOH + L
(2)
(3)
•
-
+
•
•
superoxide (ca. 0.9 V vs NHE for O
2
, H /H
2
O
2
pair), alkoxyl
L +O f LOO
2
•
+
(
1.6 V vs NHE for RO ,H /ROH), peroxyl (0.77-1.44 V vs
•
+
NHE for ROO ,H /ROOH), and hydroxyl radicals (2.3 V vs
by the competitive reaction with phenolic/flavonoid antioxidant,
FlavOH:
•
+
11
2
NHE for HO ,H /H O). There are numerous studies devoted
to the importance of flavonoid structure for their antiradical
9
,10,12-15
activity as chain-breaking antioxidants.
It is generally
•
•
LOO + FlavOH f LOOH + FlavO
(4)
(
2) Dragsted, L. O.; Strube, M.; Leth, T. Eur. J. Cancer PreV. 1997, 6, 522–
28.
3) Pietta, P. G.; Simonetti, P. In Antioxidant Food Supplements in Human
As a result, assessment of a flavonoid as a potential radical
scavenger is frequently based on measurements of the rate
constants for its reaction with reactive oxygen species, i.e.,
oxygen-centered free radicals such as superoxide, hydroxyl, or
peroxyl radicals. Since the measurements of absolute rate
constants for reaction 4 are complicated and time-consuming,
some simple tests with model radicals such as 2,2-diphenyl-1-
5
(
Health.; Packer, L., Hiramatsu, M., Yoshikawa, T., Eds.; Academic Press: New
York, 1999.
(
4) (a) Rice Evans, C. A.; van Acker, S. A. B. FlaVonoids in Health and
Disease.; Marcel Dekker, Inc.: New York, 1998. (b) Bastianetto, S.; Quirion, R.
Neurobiol. Aging 2002, 23, 891–897.
(
5) Middleton, E. J. R.; Kandaswami, C. In The FlaVonoids AdVances in
Research Since 1986; Harborne, J. B., Ed.; Chapman & Hall/CRC: New York,
•
•+
1
993.
(
picrylhydrazyl radical (dpph ) or ABTS radical cation are
commonly applied.
6) (a) Ji, X. D.; Melman, N.; Jacobson, K. A. J. Med. Chem. 1996, 39,
7
81–788. (b) Medina, J. H.; Viola, H.; Wolfman, C.; Marder, M.; Wasowski,
C.; Calvo, D.; Paladini, A. C. Neurochem. Res. 1997, 22, 419–425. (c) Williams,
R. J.; Spencer, J. P.; Rice-Evans, C. Free Radical Biol. Med. 2004, 36, 838–
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Med. 1996, 20, 331–342. (b) Cao, G.; Sofic, E.; Prior, R. L. Free Radical Biol.
Med. 1997, 22, 749–760. (c) Arora, A.; Nair, M. G.; Strasburg, G. M. Free
Radical Biol. Med. 1998, 24, 1355–1363. (d) Teixeira, S.; Siquet, C.; Alves, C.;
Boal, I.; Marques, M. P.; Borges, F.; Lima, J. L.; Reis, S. Free Radical Biol.
Med. 2005, 39, 1099–1108.
(14) Jovanovic, S. V.; Steenken, S.; Tosic, M.; Marjanovic, M.; Simic, M. G.
J. Am. Chem. Soc. 1994, 116, 4846–4851.
(15) Zhou, B.; Miao, Q.; Yang, L.; Liu, Z. L. Chem. Eur. J. 2005, 11, 680–
691.
8
49.
(
(
(
(
7) Croft, K. D. Ann. N.Y. Acad. Sci. 1998, 854, 435–442.
8) Walle, T. Free Radical Biol. Med. 2004, 36, 829–837.
9) Pietta, P. G. J. Nat. Prod. 2000, 63, 1035–1042.
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1
86, 343–355.
(
11) Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and
Medicine; Oxford University Press Inc.: New York, 2007.
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996, 20, 933–956.
(
1
2
700 J. Org. Chem. Vol. 74, No. 7, 2009

Acidity Vs Antioxidant Action
In most papers on antioxidant activity of natural compounds,
the mechanism of reaction 4 is not discussed, thus giving the
impression that single-step hydrogen atom transfer (HAT)
occurs, which is not always true, because the kinetics and
mechanism of reaction 4 depends on the medium. In general,
the rate of HAT (including PCET) is governed by two major
factors. The first one is the strength of the phenolic O-H bond.
The higher the value of Bond Dissociation Enthalpy (BDEO-H),
the slower is the HAT process. The second factor is the
magnitude of the Kinetic Solvent Effect (KSE), i.e., the ability
of a phenol to be a HB donor and ability of a given solvent to
Previous studies of the SPLET mechanism allowed us to
successfully resolve a controversy over the mechanism of
25
curcumin antioxidant action and also to explain some anoma-
lies in the kinetics of the reaction of synthetic phenolic
24
antioxidant. Moreover, using this approach it has been proved
that kinetics of reaction occurring in alcohols can be affected
by participation of phenolate anion for weak acids such as
26
a
R-tocopherol, which is characterized by a pK two units greater
than that of phenol itself. Importantly, other groups of research-
ers also applied this reasoning in their studies. For example,
the occurrence of SPLET in methanol and ethanol has been
1
6,17
27
be a HB acceptor.
A phenol molecule that is hydrogen-
clearly demonstrated by Foti et al. and later confirmed in a
2
8,29
30
bonded to a solvent molecule does not react with radicals (for
number of experimental
and theoretical studies.
s
steric reasons). Thus, the rate constant k for HAT from a H
It is known that deprotonated flavonoids are more potent
atom donor to any attacking radical in all solvents can be
electron donors and are better radical scavengers than neutral
0
14,31,32
correlated with the rate constant in a non-HB solvent, k , via
molecules.
Thus, the exploration of the role of acidity
1
8
eq I:
and acid/base equilibria in the kinetics of phenol/radical
reactions can be helpful in the understanding of the structure-
activity relationship of natural antioxidants. In the current paper
we provide evidence supporting a hypothesis that the acidity
of OH groups in flavonoids has capital meaning for the kinetics
s
0
H
2
H
2
logk ) logk - 8.3R ꢀ
(I)
H
where R
HB (range 0 to ca. 1), and ꢀ
of the solvent to accept HB (range 0 to 1.00).
2
represents the relative ability of the substrate to donate
1
9
H
•
2
represents the relative ability
of their reactions with dpph . We decided to study 10 flavonoids
2
0
of various hydroxylation pattern (Chart 1) to explain the
mechanism of the antiradical action of quercetin, an example
of a flavonoid possessing a catechol group in ring B and other
hydroxyl groups in rings A and C. Quercetin modifies eicosanoid
biosynthesis, protects low-density lipoprotein from oxidation,
prevents platelet aggregation, and prevents or delays the
occurrence of age-related cognitive deficits and neurodegen-
erative diseases. Many of these benefits are correlated with anti-
In polar solvents that can support ionization of a phenol, the
HAT/PCET process still operates; however, some (sometimes
very significant) deviations from eq I can occur. These
divergences are caused by another mechanism affecting the
kinetics of reaction. According to this process a deprotonation
of a phenol in solvent S:
-
+
3
3
PhOH + S a PhO +HS
(5)
inflammatory and antioxidant properties of that flavonoid.
During the period from 2002 to the end of 2007 the SCOPUS
database lists more than 4900 scientific publications with
keyword “quercetin”, and in almost 30% of them the additional
keyword is “antioxidant”. The role of antioxidant action in
therapy is still discussed (redox-independent mechanisms can
be more important), and antiradical properties of quercetin attract
great attention. Therefore, the relationship between the acidity
and antiradical activity of quercetin, as well as of other
flavonoids, is particularly important.
is followed by fast electron transfer from phenolate to an
electron-deficient radical (Y , such as peroxyl or dpph ):
•
•
-
•
•
-
PhO +Y f PhO +Y
(6)
(7)
-
+
Y +HS f YH + S
This mechanism was named Sequential Proton-Loss Electron-
21,22
Transfer (SPLET).
The rate of SPLET mechanism depends
on the amount of phenolate anion. Thus, the ion-solvating ability
2
3
Results and Discussion
of a solvent (termed solvent acity and basity ), as well as the
acidity of a phenol, play a crucial role in HAT/SPLET
Acidity Constants of the Flavonoids. Figure 1 presents
absorbance plots for two monohydroxyflavones and two dihy-
2
1,22,24
competition/cooperation.
of phenols having low pK
yield product molecules having high pK
SPLET is favored for reactions
a
’s with electron-deficient radicals to
•
a
’s, e.g., dpph /dpph-H
(25) Litwinienko, G.; Ingold, K. U. J. Org. Chem. 2004, 69, 5888–5896.
(26) Musialik, M.; Litwinienko, G. Org. Lett. 2005, 7, 4951–4954.
•
21
and peroxyls ROO /ROOH.
(
27) Foti, M. C.; Daquino, C.; Geraci, C. J. Org. Chem. 2004, 69, 2309–
314.
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2
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1
(
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(
(
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(
J. Org. Chem. Vol. 74, No. 7, 2009 2701

Musialik et al.
CHART 1. Structures of the Flavonoids Studied in This
Work
As a result of this fact, the spectrophotometric titration is
frequently applied for the determination of pK . Although this
a
method is more time-consuming than potentiometric titrations,
it can be successfully applied for compounds of low solubility
(
below millimolar concentration) and for compounds having
extremely low or extremely high pK ’s.
a
To validate our method, we started our measurements with
the analysis of simple model phenols, obtaining values in
3
5
excellent agreement with literature ones. For example, for
-methylphenol we obtained pK ) 10.13 ( 0.06 (lit. 10.10
and 10.02), for 2-methoxyphenol pK ) 10.03 ( 0.05 (lit. 9.98
and 9.90), and for 3-nitrophenol pK ) 8.40 ( 0.02 (lit.
.23 and 8.36). After such validation of our methodology we
measured the pK ’s of the selected flavonoids. A typical plot of
4
a
a
a
3
5
8
a
the distribution of the neutral form of a flavonoid versus its
mono-, di-, and trianionic species within the entire pH interval
is presented in Supporting Information (Figure S1). The pK
a
values for 10 flavonoids calculated on the basis of spectropho-
tometric titrations are collected in Table 1, listed together with
the literature pK
a
’s for these flavonoids. With some exceptions
(
discussed below), the presented parameters that describe the
first step of deprotonation are in rather good agreement with
the previously published values. The less satisfying agreement
with literature data was obtained for second and third pK
Ionized flavonoids are unstable when exposed to air, and we
suppose that literature pK values for second, third, and higher
steps of deprotonation are contaminated by pK ’s of oxidation
a
’s.
a
a
products. To be sure that our results are free from such errors,
a continuous flow of nitrogen through the titration flask was
sustained to eliminate the traces of air.
Analysis of literature acidity constants reveals significant
variation among the published values. The most striking
examples are quercetin (differences for pKa1 are about 2.5 units)
and morin (the pKa1 values are within the range from 3.5 to
droxyflavones at pH range from 3 to 12. As can be seen, there
are two main bands: (i) a band with maximum at 320-400 nm
attributed to the ring B absorbance and (ii) a band with
maximum in the range 250-285 nm representing absorbance
3
4
of the A ring. Acid-base equilibria have a strong effect on
the UV-vis spectra of phenols and flavonoids, manifested by
the considerable changes of the band shapes and intensities with
changing pH values. For monohydroxylated compounds one or
more isosbestic points can be found corresponding to a neutral
form being in equilibrium with the deprotonated molecule. In
contrast, for polyhydroxylated phenols, when more than two
forms are present in the solution at the same pH, the clear
isosbestic points are not observed; see Figure 1D. Even small
changes in protonation status of any OH group are accompanied
by a strong increase or decrease in the intensity of the bands.
8
.2); see Table 1. The value obtained in our analyses for morin
pK ) 5.2) is located in the middle of that scattered range. In
the series of 10 compounds from Table 1 morin is the only
(
a
3
6
flavonoid with a 2′-hydroxyl group. Agrawal and Schneider
13
applied C NMR to follow the stepwise deprotonation of 14
(
34) Harborne, J. B., Marby, T. J., Mabry, H. The FlaVonoids; Academic
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Aqueous Solution.; Pergamon Press: Oxford, 1979.
(
(
36) Agrawal, P. K.; Schneider, H. Tetrahedron Lett. 1983, 24, 177–180.
37) Our results confirmed that morin is more acidic than other flavonoids
with a 7-OH group. The possible explanation of such exceptionally strong acidity
and unusual deprotonation site of this flavone is that deprotonation of the 2′-
hydroxyl group gives an anion additionally stabilized by a hydrogen bond with
the hydroxyl at position 3:
(
38) Thompson, M.; Williams, C. R.; Elliot, G. E. Anal. Chim. Acta 1976,
5, 375–381.
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8
(
Ges. Phys. Chem. 1984, 88, 759–767.
(
(
40) Georgievskii, V. P. Chem. Nat. Compd. 1980, 16, 136–141.
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2
005, 53, 2953–2960.
(
42) Escandar, G. M.; Sala, L. F. Can. J. Chem. 1991, 69, 1994–2001.
43) Sauerwald, N.; Schwenk, M.; Polster, J.; Bengsch, E. Z. Naturforsch.,
(
-4
FIGURE 1. Absorption spectra for 1.0 × 10 M flavonoids at various
pH values (water/methanol 1:1): (A) 3-hydroxyflavone, (B) 6-hydroxy-
flavone, (C) 5,7-dihydroxyflavone, (D) 3,6-dihydroxyflavone.
B: J. Chem. Sci. 1998, 53, 315-321.
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Chem. 2004, 12, 3627–3635.
(
2
702 J. Org. Chem. Vol. 74, No. 7, 2009

Acidity Vs Antioxidant Action
a
TABLE 1.
a
pK Values from the Literature and Measured in the Present Work, O-H Bond Dissociation Enthapies (BDE), and Ionization
b
Potentials (IP) for Neutral Molecules and for Anions
pK
a
IP (eV)
from anion
d
compound
lit. values
this work
9.4 ( 0.2
BDE (kcal/mol)
neutral
7.48
3
8
3
-hydroxyflavone
10.34
89.9
2.36
3
9
9
9
9
.6
.25
.99
4
4
0
1
6
7
-hydroxyflavone
-hydroxyflavone
9.0 ( 0.2
7.7 ( 0.1
3
3
8
9
8.48
.39
92.1
8.02
7.74
2.85
2.96
7
3
5
,6-dihydroxyflavone
9.0 ( 0.2;
1
0.7 ( 0.3
3
8
,7-dihydroxyflavone (chrysin)
8.37; 12.37
8.0 ( 0.2;
11.9 ( 0.4
94.9 (7)
4
0
9
7
7.6
.14
.9; 11.40
4
1
,
32 c
7
3
,8-dihydroxyflavone
,5,7-trihydroxyflavone (galangin)
7.4 ( 0.1; 11.4 ( 0.1
7.6 ( 0.1;
81.9 (7)
88.1 (3)
7.73
7.36
2.67
3.13
1
4
6.8; 9.4
9
1
.5 ( 0.1;
0.9 ( 0.3
4
0
0
5
,7,4′-trihydroxyflavanone (naringenin)
8.83
7.5 ( 0.1;
8
9
5.2 ( 0.1;
8.2 ( 0.2;
9
8.45 ( 0.1;
9.31 ( 0.2;
11.12 ( 0.2
89.5 (4′)
.4 ( 0.1;
.8 ( 0.1
4
3
,5,7,2′,4′-pentahydroxyflavone
8.04
1
4
(
morin)
,5,7,3′,4′-pentahydroxyflavone (quercetin)
3.46; 8.1
.9 ( 0.2
4
0
3
8.21
78.6 (4′)
7.03
2.69
,
42 e
5
7
7
.7; 7.1; 8.0; 9.9; 11.0
.03; 9.15
.7; 8.77; 9.8
43
4
4
a
b
In water/methanol, (1:1 v/v), measured by spectrophotometric titration. BDE and IP values in gas phase are predicted on the basis of DFT
3
2 c
d
e
calculations by Lemanska et al.
Predicted on the basis of QSAR, see ref 32. Values in parentheses refer to position of OH group. These values
were measured spectrophotometrically. Other pK
a
’s measured potentiometrically for quercetin are 5.54, 6.95, 8.21, 9.9, and 11.0; see ref 42.
CHART 2. Structures of 3,5,7-Trihydroxyflavone
phenols and 4 flavonoids (chrysin, apigenin, naringenin, morin)
in methanol or in DMSO mixtures with D O. They concluded
(
Galangin), Hydroxytetralones, and Hydroxyacetophenones
2
that in chrysin, apigenin, and naringenin the hydroxyl groups
are deprotonated in the range 7-OH > 4′-OH > 5-OH and,
exceptionally, deprotonation of morin starts at position 2′. Such
unusual deprotonation sequence in morin has never been
3
7
explained.
Most Acidic Site in Quercetin. Experimental data for
quercetin allow us to state that this pentahydroxyflavone is not
4
2
as acidic as reported previously by Escandar et al. Its acidity
4
0
is similar to that determined by Georgievskii, and the kinetic
data (discussed below) confirmed that observation. A correct
determination of the site of primary deprotonation of quercetin
is of great importance if reaction of quercetin anion with a
radical is considered. Spectrophotometric or potentiometric
titrations do not make it possible to distinguish the range of
acidity of particular OH groups in polyhydroxylated phenols.
Moreover, according to our knowledge, a limited number of
works actually discuss the site of primary dissociation. Harborne
et al. in their book noticed that the addition of sodium
methanolate caused ionization of hydroxyls at all positions, but
the addition of weaker base, i.e. sodium acetate, caused
ionization of hydroxyls only at positions 3, 7, and 4′. Values of
4
5
2, structure C) by Magnusson et al., as well as known
differences in acidities of 4-hydroxyacetophenone and 2-hy-
35
droxyacetophenone (Chart 2, structures D and E, respectively).
3
4
The presence of OH group at position 5 causes a rather slight
change of the acidity of the 7-OH group, and this observation
is in agreement with similar acidities of acetophenones (Chart
, compounds D and F). In general, with the exception of morin
discussed previously ), all of the compounds with a 7-OH
group have similar acidity (pK between 7.5 and 8.5) and this
group is the most acidic, despite the hydroxylation pattern in
other positions. Such observation corroborates with the results
of the work by Agrawal and Schneider, who ranged decreasing
acidities of OH groups in quercetin in the order 7 > 4′ > 3.
2
(
3
7
a
pK determined for monohydroxylated flavonoids can be helpful
for assignment of the acidity of particular hydroxyl groups in
polyhydroxylated flavonoids. The lower acidity of the 3-hy-
droxyl within the acidity range 7 > 6 > 3 (see Table 1) originates
from the formation of an intramolecular hydrogen bond. This
observation is in good agreement with the large difference in
acidities reported more than 40 years ago for 6-hydroxy-1-
tetralone (Chart 2, structure B) and 8-hydroxy-1-tetralone (Chart
a
36
(
45) Magnusson, L. B.; Postmus, C. J.; Craig, C. A. J. Am. Chem. Soc. 1963,
85, 1711–1715.
J. Org. Chem. Vol. 74, No. 7, 2009 2703

Musialik et al.
TABLE 2. Assignments of the 13C CP/MAS Signals and Chemical
a
Shifts of Neutral Quercetin
atom
δ, ppm
atom
δ, ppm
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
146.5 (146.2)
135.7 (135.3)
174.9 (174.3)
157.5 (157.1)
96.3 (96.1)
164.1 (164.0)
96.3 (96.0)
155.6 (155.0)
101.9 (101.6)
C-1′
C-2′
C-3′
C-4′
C-5′
C-6′
126.3 (125.0)
112.5 (112.3)
142.0 (141.6)
147.8 (148.0)
116.0 (115.7)
122.3 (121.9)
a
Literature 13C CP/MAS chemical shifts from ref 48 are in
parentheses.
we observed that signal for C-7 (164 ppm) was considerably
shifted toward higher frequency field and reached δ ) 168.7
ppm for the monosodium salt, Figure 2. Similar strong shifts
were also reported by Agrawal and Schneider for carbon atoms
FIGURE 2. 13C CP/MAS NMR spectra of quercetin and its monoso-
dium salt: neutral (black line, with peak assignments on the basis of
values from Table 3) and monosodium (red line). The spectra in color
and other spectra of neutralized quercetin (1 mmol of quercetin + 0.5
and 2 mmol of NaOH, respectively) are in Supporting Information.
13
adjacent to OH groups being deprotonated during C NMR
36
titration of phenols and flavonoids in solution. Deprotonation
of 7-OH produces significant shielding of C-4 and also some
changes in the ability of the carbonyl group to accept an
intramolecular hydrogen bond from hydroxyl groups at C3 and
C5. As a result, new split signals for C-4 appear in the range
Recently, that range of acidity has been questioned by Milane
et al., who claimed that “the pKa1 ) 7.7 could be attributed to
the deprotonation of hydroxyl on the 3-position, the pKa2
.77 to the hydroxyl on the 7-position (...). Finally, the pKa3
)
)
8
9
1
72-180 ppm. Taking into account the results of NMR
.8 corresponded to the hydroxyl in the 4′-position instead of
44,46
experiments together with our experimental pK data for the
a
the 7-position.”
However, our results for monohydroxylated
studied flavonoids, we can draw a conclusion that quercetin
initially deprotonates from the 7-OH group:
and dihydroxylated flavones collected in Table 1 clearly show
that pK for 3-OH group is almost two units higher than that
a
for 7-OH. Combination of two or three hydroxylic groups does
not increase the acidity of 3-OH, as was shown for 3,6-
dihydroxyflavone and galangin (Table 1). Thus, 3-OH group,
although more acidic than phenolic OH, cannot be more acidic
than 7-OH. The hypothesis that 3-OH is more acidic than 7-OH
3
6
is in surprising contradiction with other experimental results.
Importantly, it also contradicts with the results of our
structure-acidity relationship studies. To solve this contradiction
we decided to perform additional NMR measurements. Solid-
Our experimental results are in line with theoretical calcula-
tions for quercetin deprotonation in liquid phase.
Summarizing this part of our work, we can order the
investigated flavonoids with respect to their increasing acidity:
-hydroxyflavone < 3,6-dihydroxyflavone ≈ 6-hydroxyflavone
4
9,50
state 13C CP/MAS NMR technique is a valuable method of
analysis of powder solids that do not form crystals, so we
decided to apply this technique for quercetin and its partially
neutralized salts. Spectra of quercetin and its monosodium salt
3
13
< 3,5,7,3′,4′-pentahydroxyflavone (quercetin) < 5,7-dihydroxy-
flavone (chrisin) < 7-hydroxyflavone ≈ 3,5,7-trihydroxyflavone
(galangin) < 7,8-dihydroxyflavone < 5,7,4′-trihydroxyflavone
(naringenin) < 3,5,7,2′,4′-pentahydroxyflavone (morin). A com-
parison of the structures of these flavonoids with their acidity
leads to the following conclusions: (i) formation of an intramo-
lecular hydrogen bond causes a decrease in acidity of the OH
group being a HB-donor (5-OH and 3-OH), (ii) acidity of the
OH group being a HB-acceptor in catechol group does not
significantly increase in comparison to that of a non-H-bonded
group (acidity of 7,8-dihydroxyflavone is almost the same as
the acidity of 7-hydroxyflavone; the acidity of the 3′,4′-
dihydroxyl group is not stronger than the acidity of the 7-OH
in quercetin), (iii) with the exception of morin, the 7-OH group
are presented in Figure 2. Other C CP/MAS NMR spectra for
quercetin/NaOH ratios of 1:0.5 and 1:2, can be found in
13
Supporting Information. C NMR signals for neutral flavonoids
4
7
were carefully interpreted by Agrawal in his monograph.
13
Recently, detailed interpretation of C CP/MAS for a number
4
8
of flavonoids have been published by Wawer and Zielinska.
Analyzing these data, we were able to assign the solid-state
NMR signals for quercetin; see Table 2.
Comparison of the spectra shown in Figure 2 indicates that
the signals of carbon atoms C1′-C6′ in ring B of neutral
quercetin overlap with the C1′-C6′ signals for the monosodium
salt of quercetin (Figure 2). The signal for C-3 also remains in
the same field after neutralization of one OH group. In contrast,
(
46) It seems that Milane and collaborators misinterpreted their results of
1
50
H NMR analysis of partially neutralized quercetin. They reported that the first
(49) Recently, Leopoldini et al. calculated the acidity of some phenolic
deprotonation step is manifested by a decrease of the chemical shifts for H atoms
at C6 and C8 and (to a smaller extent) at the 2′ and 6′ positions, and they
concluded that the only explanation is that 3-OH was deprotonated first. In our
opinion these data do not entirely support this interpretation.
antioxidants in gas and liquid (water) phase. The solvation causes 30-40 kcal/
mol decrease of the deprotonation energies and can also change the acidity order
in a series of compounds. Importantly, their results showed that the most acidic
site in quercetin is 4′ (in the gas phase) and 7 (in water), in agreement with our
experimental results in a water/methanol system.
(50) Leopoldini, M.; Russo, N.; Toscano, M. J. Agric. Food Chem. 2006,
54, 3078–3085.
(
47) Agrawal, P. K. Carbon-13 NMR of FlaVonoids; Elsevier: Amsterdam,
1
989.
(
48) Wawer, I.; Zielinska, A. Magn. Res. Chem. 2001, 39, 374–380.
2
704 J. Org. Chem. Vol. 74, No. 7, 2009

Acidity Vs Antioxidant Action
TABLE 3. Comparison of Measured Bimolecular Rate Constants (k in M s-1) for Reactions of dpph with 10 Flavonoids and the
s
-1
•
a
b
Acceleration Ratio
solvent (ꢀH
)
2
+
e
+
e
EtOH, H EtOH, H+ ethyl acetate dioxane (0.47)
+
e
e
d
MeOH MeOH, H MeOH, H
+
c
e
e
c
e
e
c
c
kMeOH/kMeOH/H
flavonoid
-hydroxyflavone
-hydroxyflavone
-hydroxyflavone
,6-dihydroxyflavone
,7-dihydroxyflavone
(0.41)
10 mM
100 mM
EtOH (0.44)
10 mM
100 mM
(0.45)
(0.41)
f
f
3
6
7
3
5
47
0.88
0.040
0.083
1.7
0.45
0.012
0.022
1.3
7.0
ndf
ndf
0.055
0.0067
0.0053
0.21
0.061
104
26.7
5.5
17
0.22
0.12
23
0.083
0.43
8.0
ndf
ndf
nd
1.0
0.41
0.0053
0.0029
0.11
nd
1.6
0.46
f
2.7
0.15
nd
0.81
0.074
0.0030
18
(
chrisin)
7
3
,8-dihydroxyflavone
,5,7-trihydroxyflavone
40000
310
1500
4.6
190
3.4
14000
260
450
4.3
130
2.0
17.9
0.62
2.6
0.27
210
91
(
galangin)
,7,4′-trihydroxyflavanone
naringenin)
,5,7,2′,4′-pentahydroxyflavone 5200
morin)
,5,7,3′,4′-pentahydroxyflavone 3000
quercetin)
5
3
3
0.99
0.73
750
50
0.61
150
58
0.69
5400
5400
0.42
1500
41
0.32
150
25
0.034
0.011
8.9
1.6
35
51
(
15.0
9.3
(
3.0
(
a
In methanol, acidified methanol, ethanol, acidified ethanol, ethyl acetate, and dioxane. Full data with errors are given in Supporting Information.
b
c
d
e
Ratio of the rate constants in MeOH to the rate constants in acidified MeOH. Reference 20. Reference 25, statistically not corrected. Alcohols
containing acetic acid in 10 and 100 mM concentration. For acetic acid ꢀ
H
f
2
) 0.42 (ref 25). nd ) not determined.
is the most acidic site in all of the flavonoids studied in this
work, and the presence of other hydroxyls in position 3, 5, and
6
does not considerably change the acidity of 7-OH group.
Kinetic Measurements. Our present study of antiradical
abilities of mono- and polyhydroxylated flavonoids is based on
the measurements of kinetics of their reactions with dpph•
radical. The radical has been widely used to measure the
2
5
hydrogen atom donating abilities of natural antioxidants. The
•
rates of reaction of dpph with flavonoids (Flav-OH)
•
•
dpph + Flav-OH f dpph-H + Flav-O
(8)
•
were determined by monitoring the decay of dpph at 517 nm
2
1
FIGURE 3. Plot of bimolecular rate constant for reaction morin +
in a stopped flow apparatus, as described previously. Flav-
•
dpph in methanol versus increasing amount of acetic acid.
•
OH was always used in excess as compared with the dpph
-5
concentration, which was generally ca. 1-8 × 10 M. In all
H
•
in two hydroxylic solvents, i.e., methanol (ꢀ
2
) 0.41) and
measurements excellent pseudo-first-order decays of dpph were
H
ethanol (ꢀ ) 0.45). These two hydroxylic solvents have much
2
observed (with rate constant kex), and the bimolecular rate
s
higher dielectric constants, ε, than the two non-hydroxylic ones.
Hence, they have much greater abilities to support ionization
and, consequently, the SPLET reaction mechanism (eqs 5-7).
Comparison of the rate constants for reaction 8 carried out in
these four solvents can, therefore, explain the role of phenol
constants for reaction 8, k , were calculated from the slopes of
ex
plots of k vs [Flav-OH] as
ex
s
k
) constant + k [Flav-OH]
(II)
•
for dilute concentrations of flavonoids to exclude their self-
association.
ionization in dpph /flavonoid reactions. Results of measurements
s
are summarized in Table 3 together with k values measured in
It has to be emphasized that all flavonoids listed in Table 1
two alcohols acidified by the addition of acetic acid, as described
3
5
21,24,25
are more acidic than phenol (pK
a
) 10.0). Therefore, in
in our previous works.
•
ionization-supporting solvents all of these polyphenols should
exhibit increased reactivity toward peroxyls and dpph radicals,
In all of the solvents listed in Table 3, the dpph /flavonoid
•
reactions followed pseudo-first-order kinetics. Therefore, reac-
tion rate constants can be compared for pairs of solvents
characterized by similar HB-basicity. Indeed, the processes
carried out in methanol and ethanol are much faster than those
in dioxane and in ethyl acetate. For all compounds reacting in
methanol the addition of as little as 10 mM acetic acid
as predicted by SPLET mechanism. The participation of SPLET
in phenol-radical reaction is kinetically important, and the overall
rate of reaction can be hundreds of times faster as compared to
22
the pure HAT process. Equation I applies only to “pure” HAT/
PCET reactions. Thus, it can be employed as a valuable tool
for quantifying the importance of the SPLET process in different
s
dramatically decreases the k ; see Table 3. An increase of the
2
1
0
solvents and with different phenols. Unfortunately, k in eq I
concentrations of acetic acid caused the further decrease of the
•
could not be determined for our dpph /ArOH reactions because
flavonoids are insoluble in saturated hydrocarbons (ꢀ
We therefore measured k for this reaction in two non-hydroxylic
bimolecular rate constants (see Figure 3).
H
H
2
) 0.00).
Because ꢀ
2
parameters for acetic acid, methanol, and ethanol
s
are similar, we exclude additional HB formation with
CH COOH as a reason for the decreasing rate for flavonoid +
H
solvents, i.e., 1,4-dioxane (ꢀ
2
) 0.41, after statistical correction
is 0.47, see Table 3), and ethyl acetate (ꢀ
3
H
H
•
its ꢀ
2
2
) 0.45) and
dpph reaction. Thus, the decrease of reaction rate after addition
J. Org. Chem. Vol. 74, No. 7, 2009 2705

Musialik et al.
a
of acid is caused by a declining amount of phenolate anions,
which emphasizes the importance of dissociation to this electron-
rich form in the overall reactivity. In other words, in neat
alcohols all of the flavonoids are partially ionized and their
TABLE 4. Acceleration Ratios
kMeOH/kdioxane
flavonoid
7
,8-dihydroxyflavone
15384
1148
1000
900
770
584
209
90
galangin
quercetin
5,7-dihydroxyflavone
•
anions react rapidly with the dpph radical. Addition of the acid
suppresses ionization and the reaction is now governed only
by HAT instead of a combination of accelerated HAT and fast
SPLET mechanisms. Our previous experiments with the reaction
3
-hydroxyflavone
morin
,6-dihydroxyflavone
3
•
of simple phenols + dpph in the presence of base showed an
naringenin
6-hydroxyflavone
2
1
enormously high increase of rate constants. Indeed, in the
present experiments addition of a few drops of 0.1 mM sodium
methanolate to the quercetin solution caused a rate acceleration
too large to be measured by the stopped flow apparatus (reaction
was completed during the dead time of mixing).
60
41
7
-hydroxyflavone
a
•
Ratios of rate constants for reaction with dpph radicals carried out
in methanol and in dioxane.
Structure-Reactivity Relationship: Reactivity vs Acidity. As
21,24,25
galangin, a flavonoid that is 10 times less reactive toward dpph•
we reported previously,
the pure HAT mechanism occurs
in dioxane (ε ) 2.2, see Table 3). Assuming that all flavonoids
listed in Tables 1 and 2 have similar HB-donating ability and
neglecting the higher HB-donating ability of catechols, the same
magnitude of KSE should be observed. This assumption seems
but more acidic than quercetin. Since the acidity of some
flavonoids resembles the acidity of acetic acid (pK ) 4.8), the
a
more reliable quantity for comparison of the anion participation
MeOH dioxane
is the acceleration ratio expressed as k
/k
, which can
to be reasonable for unhindered hydroxyl groups in a series of
be applied as a parameter indicating the role of phenol
dissociation (and SPLET participation) in the overall kinetics
H
similar compounds (however, R
2
values are higher for cat-
5
1
•
echols, so catecholic flavonoids should be compared to each
other within their class). Therefore, in dioxane the range of the
of flavonoid + dpph in ionization-supporting solvents; see
Table 4.
•
dioxane
Reaction of Quercetin with dpph• Radical in Polar
flavonoid/dpph reactivity expressed as k
(collected in
Table 3) should be in opposite order to their BDE’s. Indeed,
quercetin is the most reactive (78.6 kcal/mol) followed by 7,8-
dihydroxyflavone (81.9 kcal/mol), which is more reactive than
galangin (88.1 kcal/mol) and 3-hydroxyflavone (89.9 kcal/mol),
and the least reactive are 7-hydroxyflavone (92.1 kcal/mol) and
Solvents. The astonishing value of the acceleration ratio
MeOH dioxane
k
/k
for 7,8-dihydroxyflavone shows the importance of
the acidity of the OH group in ring A. This is one of the most
prominent examples of the SPLET mechanism leading to
formation of stable phenoxyl radical. Quercetin, with a less
acidic catechol moiety in B ring, is not as efficiently activated
as 7,8-dihydroxyflavone. If the hydroxyl at position 3′ or 4′ of
quercetin was deprotonated first, the acceleration ratio for that
flavonoid should be of the same order as for 7,8-dihydroxyfla-
vone. Instead, the acceleration ratio for quercetin is similar to
5
2
chrisin (94.9 kcal/mol). This straightforward relationship
•
clearly shows that in nonpolar solVent a reaction between dpph
and 10 flaVonoids is controlled by the HAT mechanism
thermodynamically based on BDE Values. However, the rate
constants in neat methanol and ethanol are ranged in different
manner: 7,8-dihydroxyflavone . morin, quercetin > galangin,
followed by considerably less reactive: 3-hydroxyflavone . 3,6-
dihydroxyflavone > chrisin > naringenin > 6-hydroxyflavone >
that for galangin. Both flavones have an identical hydroxylation
MeOH dioxane
pattern in ring A and have almost the same k
/k
≈1000.
This is yet another (kinetic) proof showing that the 7-hydroxyl
group is the most acidic site in quercetin. However, the absolute
rate constant for reaction of dpph with quercetin is 10 times
7
-hydroxyflavone. If the presence of catechol moiety was the
•
most important factor affecting the rate of reaction, quercetin
should be as reactive as 7,8-dihydroxyflavone (and even more
reactive, as in dioxane), because both catecholic flavonoids can
form stable radicals where the remaining o-OH group stabilizes
the radical as a result of the intramolecular hydrogen bond-
•
larger than for the reaction of dpph with galangin. The same
(10-fold) difference in reactivity is also observed for the rate
constants in dioxane. Thus the 7-OH group in both flaVonoids
plays an important role as the site of ionization and of electron
transfer (according to SPLET). A significant difference in
5
3
ing:
absolute rate constants for the quercetin and galangin pair
MeOH dioxane
confronted with the same acceleration ratio k
/k
can
be explained by the presence of 3′,4′-dihydroxyl moiety in ring
•
B of quercetin. For the reaction of quercetin + dpph we
propose the mechanisms shown in Scheme 2.
In nonpolar solvents or in solvents that do not support
ionization, one-step HAT occurs from nonionized quercetin
(
structure I) to give quercetin radical (VI). In methanol (and
The nearly identical reactivity of morin (without catechol
moiety) and quercetin shows that the kinetics of the reactions
in methanol and ethanol are noticeably affected by the acidity
of a phenol and, as a result, by the amount of accessible
(51) Foti, M. C.; Barclay, L. R.; Ingold, K. U. J. Am. Chem. Soc. 2002, 124,
1
2881–12888.
(52) The same reactivity of 7-OH-flavone and 5,7-di-OH-flavone in dioxane
(
their O-H BDEs differ by ca. 3 kcal/mol; see Table 1) can be explained by
kinetic solvent effect: 7-OH-flavone is perhaps a slightly better HB donor than
5,7-diOH-flavone. We do not know the HB-donating ability of 7-OH group, but
the simple rule “the stronger acid, the better HB donor” should operate in this
+
MeOH MeOH/H
phenolate anions. The ratio (k
/k
) listed in the last
H
-1.32
column of Table 3 can be assumed to be a contribution of the
ionized form of flavonoid over the nonionized form to its overall
antiradical reactivity in methanol. This ratio is similar for
quercetin and morin and, surprisingly, two times larger for
case, in agreement with the equation R ) 23.41(pK )
(see: Mulder, P.;
2
a
Litwinienko, G.; Lin, S.; MacLean, P. D.; Barclay, L. R. C.; Ingold, K. U. Chem.
Res. Toxicol. 2006, 19, 79–85.
(
53) Wright, J. S.; Johnson, E. R.; DiLabio, G. A. J. Am. Chem. Soc. 2001,
123, 1173–1183.
2
706 J. Org. Chem. Vol. 74, No. 7, 2009

Acidity Vs Antioxidant Action
SCHEME 2. Possible Mechanisms for Quercetin Reaction with dpph Radicals
•
other polar solvents that can support ionization) the neutral
quercetin is in equilibria with monoanion form (structure II)
and two reaction pathways are possible. The first one is a HAT
from the catechol moiety to form the radical anion (Scheme 2,
step II f V). This reaction is strongly accelerated by increased
electron density in rings A and C. Furthermore, the alternative
route is a fast electron transfer from phenolate anion (structure
f V), which still is an efficient pathway of quercetin/radical
reaction (see also footnote 55). However, the SPLET starting
from deprotonation of the 7-OH group (more acidic than 3′,4′
hydroxyls) is the most reasonable explanation of the 1000-fold
5
4
acceleration of the reaction rate in methanol. Zielonka et al.
•-
•
reported the acceleration of the rates of Br
2
3
and N reactions
with deprotonated genistein versus its neutral form. They
confirmed their experimental observations with DFT calculations
of ∆IP ) -95 kcal/mol and ∆BDE ) -10 kcal/mol for
monoanionic form of genistein in comparison with its neutral
•
II) to dpph radical (step II f III). Since phenoxyl radicals
14,25
are considerably stronger acids than their parent compounds,
the acidity of all hydroxyl groups in structure III is dramatically
increased. Ring A is strongly electron-withdrawing, and as an
effect of conjugation, the catechol moiety in ring B is the most
probable site of deprotonation (step III f IV). After deproto-
nation of catechol the negative charge is immediately transferred
to strongly electron-deficient ring A, and finally a radical anion
with unpaired electron at ring B (catechol radical) and negative
charge at ring A is formed (structure V). A similar mechanism
was proposed by one of us to resolve the curcumin antioxidant
5
4
form. The results of our experiments (Table 4) show a strong
acceleration even for monohydroxylated flavonoids (their anions
cannot be a source of H atom donors, and thus SPLET
55
mechanism is responsible for the rate acceleration in this case).
It is not possible to distinguish the reaction pathways II f
V and II f III f IV f V on the basis of analysis of reaction
products (which is the same for both mechanisms and not
stable); however, the kinetic data clearly indicate that ionization
increases the phenol reactivity. Ionization, together with other
structural features (such as the presence of catechol groups,
conjugation, etc.) should be considered as important factors in
structure-activity relationship studies.
2
5
controversy.
In fact, radical III can cause deprotonation of any OH group
in quercetin, but such path will be not as productive as catechol
deprotonation leading to stable catechol radical anion (V). This
hypothesis is supported by the values of kinetic parameters for
Role of the 3-Hydroxyl Group. The SPLET mechanism and
acid-base equilibria can also be helpful in the explanation of
the role of the 3-hydroxyl group in flavonoids. In a few works
the hydroxyl group at position 3 was reported as the group that
galangin and 5,7-dihydroxyflavone. Although parameters
MeOH dioxane
k
/k
are about 1000 (proving the SPLET), the absolute
MeOH
rate constants k
than for quercetin (10 and 1000 times, respectively; see Table
), which is a consequence of the inability of galangin and 5,7-
are much smaller for these two compounds
9,15,57
increases the antioxidant activity of flavones.
Glycosylation
3
at 3-OH reduces the antioxidant activity of the compound.
dihydroxyflavone to form a stable semiquinone radical like
catechols. Another argument for favored fast deprotonation of
catechol moiety instead deprotonation of 3- and 5-OH groups
is that the latter are H-bonded to the carbonyl group, and thus
(
54) Zielonka, J.; Gebicki, J.; Grynkiewicz, G. Free Radical Biol. Med. 2003,
35, 958–965.
55) Values of oxidation-reduction potentials, pK
(
’s, and BDEs for dpph•
a
22
and several peroxyl radicals have been reviewed in ref. An example, how
deprotonation of a phenol can govern the reaction mechanism, was described
by Nakanishi et al.: reduction potential of 2,2-bis(4-tert-octylphenyl)-1-
they are weaker acids than 3′,4′-hydroxyls group.
5
6
MeOH dioxane
Comparing the values of k
/k
(listed in Table 4) for
0
picrylhydrazyl, dopph•, in acetonitrile Ered vs SCE ) 0.18 V, oxidation potential
quercetin () 1000) and for more acidic 7-hydroxyflavone ()
1), one can state that the acceleration of reaction rates is not
founded on the acidity of flavonoids. However, although
quercetin is 0.7 pK unit less acidic than 7-hydroxyflavone, the
IP value for the anionic form of the latter is 0.15 eV higher
2.85 eV, see Table 1), making the 7-hydroxyflavone anion a
0
of 2,2,5,7,8-pentamethylchroman-6-ol (PMHC, R-tocopherol analogue) Eox vs
4
SCE ) 0.77 V, and HAT process is preferred. However, deprotonation of the
0
0
OH group causes very large negative shift of Eox: for PMHC anion Eox )-0.47
V vs SCE, and thus electron transfer reduction becomes thermodynamically
0
a
possible. Similarly, a large decrease of Eox for catechin and deprotonated catechin
is observed (1.18 and 0.12 V vs SCE, respectively). All of these data describe
processes in acetonitrile. Reduction potentials for peroxyl radicals are higher
than for dpph , and thus the driving force for reaction between phenol anion
and peroxyl radical is larger.
(
Mochizuki, M.; Urano, S.; Okuda, H.; Ozawa, T.; Fukuzumi, S.; Ikota, N.;
Fukuhara, K. Org. Biomol. Chem. 2003, 1, 4085–4088.
(57) Ratty, A. K.; Das, N. P. Biochem. Med. Metab. Biol. 1988, 39, 69–79.
5
6
(
•
22
worse electron donor than the quercetin anion.
56) Nakanishi, I.; Miyazaki, K.; Shimada, T.; Iizuka, Y.; Inami, K.;
It should be stressed that the SPLET mechanism does not
exclude the conventional HAT/PCET process directly from the
site of the lowest BDEO-H (Scheme 2, step I f VI and step II
J. Org. Chem. Vol. 74, No. 7, 2009 2707

Musialik et al.
However, the primary reason for this phenomenon is unknown,
since the BDE for 3-OH group is quite high (see Table 1). Our
kinetic data solve this problem. An enolic OH at the position R
to the carbonyl group is more acidic than phenol itself. The
low electron affinity of the anion of 3-hydroxyflavone makes
this compound efficiently activated by ionization: in the alcohols
the enolic group is several hundred times more reactive (see
Table 4), and surprisingly, for that relatively less acidic
compound, the acceleration parameters are larger than for the
more acidic 7-hydroxyflavone. However, the IP value for
deprotonated 3-hydroxyflavone anion is 0.5 eV lower than for
dioxane, where pure HAT occurs from the catecholic hydroxyl
to radical. For 7,8-dihydroxyflavone, where the catechol group
is the most acidic site, the acceleration of the reaction rate in
methanol in comparison to dioxane reaches 4 orders of
magnitude. It can be only partially explained by smaller KSE
in methanol than in dioxane. Enormously high acceleration is
mainly due to flavonoid ionization.
As a result of the presence of hydroxyl groups, many
flavonoids (primarily existing in plants in glycosylated form)
58
are mainly located in the water phase of the biological systems,
and their solubility in a polar phase is additionally increased
by high acidity, which usually is stronger than for unsubstituted
phenol. Reactions of flavonoids with electron-deficient radicals
can be accelerated by the SPLET mechanism to effectively
minimize the accumulation of the reactive oxygen species in
the cell.
7
-hydroxyflavone anion (see Table 1), and regardless of the
smaller fraction of the anion, the driving force for electron
transfer is larger in the case of 3-hydroxyflavone than in
7
-hydroxyflavone. The same explanation is valid for polyhy-
droxyflavones containing 3-hydroxyl group. A very small
fraction of deprotonated 3-hydroxyl group with its smaller IP
is thermodynamically and kinetically important. Therefore, the
SPLET mechanism is a possible explanation of the enhancement
of antioxidant activity and the role of 3-hydroxyl group.
Naringenin is not as highly reactive in methanol or in dioxane
as the flavonoids containing enol group. It can be also
rationalized by the lack of a double bond between C-2 and C-3:
because ring A is not conjugated with ring B, the radical formed
during the HAT/SPLET process is not stable. If other hydroxyl
groups and rings A and B are not present, the 7-OH group alone
is not a sufficient condition for flavonoid to be a good
antioxidant.
Experimental Section
Materials. Flavonoids used in the experiments were of highest
purity. Their names and structures are listed in Chart 1. Since
•
phenol/dpph reaction kinetics in methanol and ethanol are highly
2
1
sensitive to traces of acids and bases, prior to the use these
•
solvents were fractionally distilled over a small amount of dpph
and a few beads of an acidic ion-exchange resin. Ethyl acetate was
fractionally distilled. 1,4-Dioxane was used as received from the
2 2
supplier. To remove dissolved O and CO , solvents were degassed
with dry nitrogen prior to use.
a
pK Measurements. Spectrophotometric titrations were per-
59
formed as described by Albert and Sergeant. Briefly, small
volumes of titrant (1 M KOH in water/methanol 1:1 mixture
Conclusions
2
containing a small amount of BaCl to remove traces of carbonates)
Analysis of the reaction rates of 10 flavonoids with dpph•
were added to 200 mL of flavonoid solution at a concentration of
-
4
1
0
M in water/methanol (1:1). A precision pH meter was used
radical in solvents of various polarity indicates a significant role
of their acidity in the overall reaction kinetics. The reactions
carried out in methanol and ethanol are faster than in acidified
alcohols or in dioxane. The ability of flavonoid to react quickly
and efficiently with electron-deficient radicals such as peroxyls
with a combined pH glass electrode calibrated on primary pH
60
standards for mixed solvents as recommended by IUPAC. After
each addition of titrant and when the pH value was stable, small
0
.5 mL samples of titrated solution were transferred to quartz
cuvettes (optical path 10 mm), and UV-vis spectra in the range
250-500 nm were recorded. Each time the samples were returned
to the main titrated solution. The titrant and titrated solutions were
•
or dpph depends on the acidity of phenolic hydroxyl groups
and on the stability of the formed radical. The observed kinetic
data are results of the interplay of several factors such as acidity,
polarity of the medium, ionization potentials of phenol anions,
ability of a flavonoid to be a hydrogen bond donor (and ability
of a solvent to be HB acceptor) and cannot be described by the
simple rule “the more acidic the flavonoid, the more active
radical scavenger”. However, the results of our work indicate
that the role of phenol acidity should attract greater attention.
For example, 3-hydroxyflavone containing an enolic hydroxyl
a
kept in an atmosphere free of carbon dioxide. The pK ’s were
calculated by the means of the SPECFIT (Bio-Logic Science) and
mainly by DATAN 3.1 (MultiD Analyses). The software analyzes
complete spectra (instead of single analytical wavelength) and
calculates protolytic equilibria as a function of pH and protolytic
constants on the basis of principal component analysis.
13
C CP/MAS NMR Measurements. Quercetin and its sodium
13
salts were analyzed by solid-state C NMR. The salts were obtained
by mixing oxygen-free methanolic solutions containing NaOH and
quercetin (the stoichiometric ratio was 0.5, 1.0, and 2.0 mol of
NaOH to 1.0 mol of quercetin). The mixture was then evaporated
under reduced pressure, and the resulting solid was dried under
vacuum. Solid-state cross-polarization under magic angle spinning
•
with high O-H BDE reacts very slowly with dpph , but even
minute deprotonation of the 3-hydroxyl makes such reaction
much faster: in methanol 3-hydroxyflavone is 100 times more
reactive than in acidified methanol.
1
3
(
C CP/MAS NMR) spectra were recorded at room temperature
A conjugation of ring A (usually containing more acidic
hydroxyls) with ring B improves the antioxidant potency of
flavonoids. In an optimal case, as for quercetin, flavones can
act via mixed HAT/PCET and SPLET mechanisms. In polar
solvents these flavonoids react much faster because the most
acidic OH group at position 7 in ring A is deprotonated and
phenolate anions react with radicals. Such a mechanism involv-
ing deprotonation, electron transfer from anion to a radical, and
intramolecular electron transfer to form stable flavonoid radical
anion with unpaired electron at catechol moiety (Scheme 2)
causes 1000-fold acceleration of the reaction rate in methanol
on a solid-state NMR spectrometer at the frequency of 100.6 MHz.
The experiments were carried out using a proprietary Bruker
probehead with 4 mm zirconia rotors driven by dry air at 8.0 kHz.
A contact time of 4 ms, repetition time of 30 s, and a spectral width
(58) (a) Rothwell, J. A.; Day, A. J.; Morgan, M. R. A. J. Agric. Food Chem.
2005, 53, 4355–4360. (b) Walgren, R. A.; Walle, U. K.; Walle, T. Biochem.
Pharmacol. 1998, 55, 1721–1727. (c) Hollman, P. C.; van Trijp, J. M.; Buysman,
M. N.; van der Gaag, M. S.; Mengelers, M. J.; de Vries, J. H.; Katan, M. B.
FEBS Lett. 1997, 418, 152–156.
(
59) Albert, A.; Sergeant, E. P. The Determination of Ionization Constants-
Laboratory Manual; Chapman and Hall: New York, 1984.
60) Inczedy, J.; Lengyel, T.; Ure, A. M. In Solution Thermodynamics, 3rd
ed,; Blackwell Science: Cambridge, MA, 1998; Chapter 3.
(
(
and perhaps in water) compared to the nonpolar solvent
2
708 J. Org. Chem. Vol. 74, No. 7, 2009

Acidity Vs Antioxidant Action
of 26 kHz were used for the accumulation of 200-900 scans.
Chemical shifts were calibrated indirectly through the adamantan
signal recorded at 38.3 ppm relative to TMS.
Acknowledgment. We thank Prof. Waclaw L. Kolodziejski
for his help with C CP/MAS NMR measurements and
interpretation of the spectra. Some of the pK measurements
a
were conducted by Sylwia Zera, M.Sc. The work was supported
by Ministry of Science and Higher Education (Grant No. 3T09A
109 29).
13
Kinetic Measurements. These were made following the pro-
2
4
•
cedure described previously. Decays of dpph (initial concentra-
-
5
tions 1-8 × 10 M) in the presence of a stoichiometric excess
(
for less reactive flavonoids at least 5-fold, for the most reactive
ones at least 3-fold excess) of a flavonoid at known concentrations
were monitored at 517 nm on an Applied Photophysics stopped-
flow spectrophotometer, SX 18 MV, equipped with a 150 W xenon
lamp at ambient temperature. Some of the measurements were
conducted using RX2000 Rapid Kinetic System Stopped Flow
Accessory (Applied Photophysics) connected to a UV-vis spec-
Supporting Information Available: Kinetic data (Tables
13
S1-S52), ionized forms distribution diagram (Figure S1),
C
CP/MAS NMR spectra (Figures S2-S4), and examples of
•
kinetic traces of quercetin + dpph reaction (Figure S5, S6).
•
trophotometer. The initial rates of reaction (usually 5-10% of dpph
This material is available free of charge via the Internet at
http://pubs.acs.org.
•
conversion measured as decrease of dpph absorbance at 517nm)
ex
were monitored, and pseudo-first-order rate constants, k , were
calculated as average values from at least two independent sets of
s
measurements. Values of k were calculated using eq II; see Results.
For additional details see Supporting Information.
JO802716V
J. Org. Chem. Vol. 74, No. 7, 2009 2709