Reducing activity of polyphenols with stable radicals of the TTM series. Electron transfer versus H-abstraction reactions in flavan-3-ols

Article abstract of DOI:10.1021/ol048015f

(Chemical equation presented) A new method to test the antioxidant activity of polyphenols by electron transfer reactions to a stable organic free radical, tris(2,4,6-trichloro-3,5-dinitrophenyl)methyl radical (HNTTM), is reported. Therefore, the activity of the natural flavanols, (-)-epicatechin, and two synthetic derivatives, 4β-(S-cysteinyl)epicatechin and 4β-(2- aminoethylthio)epicatechin, can be differentiated by their capacity to transfer hydrogen atoms to 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and to transfer electrons to HNTTM.

Full text of DOI:10.1021/ol048015f

ORGANIC
LETTERS
2004
Vol. 6, No. 24
4583-4586
Reducing Activity of Polyphenols with
Stable Radicals of the TTM Series.
Electron Transfer versus H-Abstraction
Reactions in Flavan-3-ols
Aurora Jime´nez,†,‡ Ariadna Selga,† Josep Llu´ıs Torres,† and Llu´ıs Julia`*,‡
Departament de Qu´ımica de Pe`ptids i Prote¨ınes and Departament de Qu´ımica
Orga`nica Biolo`gica, Institut d’InVestigacions Qu´ımiques i Ambientals (IIQAB-CSIC),
Jordi Girona 18-26, 08034 Barcelona, Spain
ljbmoh@cid.csic.es
Received September 29, 2004
ABSTRACT
A new method to test the antioxidant activity of polyphenols by electron transfer reactions to a stable organic free radical, tris(2,4,6-trichloro-
3,5-dinitrophenyl)methyl radical (HNTTM), is reported. Therefore, the activity of the natural flavanols, ( )-epicatechin, and two synthetic derivatives,
-(S-cysteinyl)epicatechin and 4 -(2-aminoethylthio)epicatechin, can be differentiated by their capacity to transfer hydrogen atoms to 2,2-
diphenyl-1-picrylhydrazyl radical (DPPH) and to transfer electrons to HNTTM.
−
4
â
â
Flavan-3-ols are natural polyphenols widely found in foods
that show the ability to act as antioxidants.1 Their free radical
scavenging activity lies in the transfer of the phenolic
H-atoms to oxygen free radicals such as hydroxyl and
peroxyl radicals, chain-carrying species in autoxidation and
initiators of many human degenerative diseases.2 Recently,
efforts have been made to obtain bio-based derivatives of
flavan-3-ols with improved ability to transfer hydrogen
atoms.3 DPPH, a stable organic nitrogen-centered free radical
with a high absorption in the visible part of the electronic
spectrum, has been used to test this antioxidant activity by
its ability to abstract hydrogens from polyphenols.4 Recently,
we have reported that the tris(2,4,6-trichloro-3,5-dinitrophe-
nyl)methyl radical (1), a stable organic carbon-centered free
radical of the TTM (tris(2,4,6-trichlorotriphenyl)methyl radi-
cal) series, is a good sensor to test the activity of polyphenols
measuring their capacity to participate in electron transfer
reactions.5 Therefore, the activity of polyphenols may be
† Departament de Qu´ımica de Pe`ptids i Prote¨ınes′.
‡ Departament de Qu´ımica Orga`nica Biolo`gica′.
(1) (a) Harbone, J. B. The FlaVonoids: AdVances in Research Since 1986;
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Vin˜a-Ribes, J. Br. J. Nutr. 1998, 80 Suppl 1, S77. (c) Bravo, L. Nutr. ReV.
1998, 56, 317. (d) Yang, C. S.; Lee, M.-J.; Chen, L.; Yang, G. EnViron.
Health Perspect. 1997, 105 Suppl 4, 971. (e) Soleas, G. J.; Diamandis, E.
P.; Goldberg, D. M. J. Clin. Lab. Anal. 1997, 11, 287. (f) Ruf, J. C. Drug
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V.; Brossaud, F.; Moutounet, M. Phytochemistry 1996, 43, 509.
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(c) Torres, J. L.; Lozano, C.; Julia´, L.; Sa´nchez-Baeza, F. J.; Anglada; J.
M.; Centelles, J. J.; Cascante, M. Bioorg. Med. Chem. 2002, 10, 2497.
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10.1021/ol048015f CCC: $27.50
© 2004 American Chemical Society
Published on Web 10/29/2004

measured either by their ability to donate H-atoms or by their
electron transfer properties, and, in general, both processes
are correlated. Along with the antioxidant properties against
destructive radicals, a disadvantage of many natural polyphe-
nols such as catechins is that their low ionization potentials
make them easily oxidized by atmospheric oxygen by
electron transfer, generating O2•-, a very active, electron-
rich radical, and hydrogen peroxide under certain conditions.6
This is why, nowadays, efforts are focused to prepare new
polyphenols, more stable to oxygen but keeping the high
tendency to transfer H-atoms.7
spectroscopy. The π*-π electronic transition characteristic
of radical 1 and the broad and less energetic transition of
anion 1- in chloroform-methanol (10%) appear at 384 and
497 nm, respectively (Figure 1).
Radicals of the series of TTM are a kind of organic carbon-
centered free radicals whose great persistence is mainly
due to steric hindrance of six chlorine atoms around the
trivalent carbon.8 All these radicals are completely disas-
sociated and very stable both in solid and in solution. Their
inefficiency to abstract H-atoms from hydrogen-labile species
is accounted for by steric hindrance, and therefore they are
inoperative in these processes. However, they are very
sensitive to electron transfer reactions; in the presence of
electron donor species they are easily reduced to carbanions
with stabilities comparable to their precursors, and their
electrochemical behavior by cyclic voltammetry shows
reversible reduction processes. It is worth noting the pos-
sibility of modulating the redox properties of these radicals
by simply introducing different substituents into their
aromatic structure.5 Therefore, we have prepared radical 1,9
an oxidant (E°) 0.58 V vs NaCl-saturated calomel elec-
trode), stronger than TTM and sensitive to the presence of
flavan-3-ols.
Figure 1. Absorption spectra of radical 1 in CHCl3 (π*-π band
at 387 nm) and anion 1- (band at 497 nm) from a solution of
triphenylmethane 1H and Bu4NOH in THF.
When two equimolecular solutions of radical 1 and
catechol are mixed together, the electronic spectrum of the
mixture displays peaks of the radical 1 and the negatively
charged species 1-, which denotes the electron transfer from
polyphenol to radical. Figure 2 shows the evolution of both
To get more insight into the electron transfer mechanism
of the oxidation of natural polyphenols by radical 1, the
activity of this oxidant magnetic species was tested with two
simple models, catechol (1,2-dihydroxybenzene) and resor-
cinol (1,3-dihydroxibenzene), as the presumably active
moieties in the efficient antioxidant (-)-epicatechin, and the
course of these reactions was monitored by electronic
Figure 2. Evolution of the vis spectrum of a ∼10-4 M solution of
radical 1 and catechol (1:1) in CHCl3.
absorptions with time. After 1 min of reaction, ∼35% of
the radical was reduced, and ∼68% had reacted after 30 min.
The decrease of the intensity of the peak at 497 nm corre-
sponding to the carbanion during the reduction suggests that
the electron transfer is followed by protonation of the anion
in the course of the reaction, 1H being the final product of
the process. However, if experiments are carried out with
resorcinol, as an alternative active moiety of (-)-epicatechin,
the reaction practically does not take place. So, only ∼3%
of the radical was reduced after 30 min of reaction and ∼15%
after 4 h. This is in agreement with the results of other
authors stressing the preponderant role of the catechol
moiety.10
(5) Torres, J. L.; Varela, B.; Brillas, E.; Julia´, L. Chem. Commun. 2003,
74.
(6) (a) Nakayama, T.; Enoki, Y.; Hashimoto, K. Food Sci. Technol. Int.
1995, 1, 65. (b) Miura, Y. H.; Tomita, I.; Watanabe, T.; Hirayama, T.;
Fukui, S. Biol. Pharm. Bull. 1998, 21, 93.
(7) Pratt, D. A.; DiLabio, G. A.; Brigati, G.; Pedulli, G. F.; Valgimigli,
L. J. Am. Chem. Soc. 2001, 123, 4625.
(8) (a) Armet, O.; Veciana, J.; Rovira, C.; Riera, J.; Castan˜er, J.; Molins,
E.; Rius, J.; Miravitlles, C.; Olivella, S.; Brichfeus, J. J. Phys. Chem. 1987,
91, 5608. (b) Carilla, J.; Fajar´ı, Ll.; Julia´, L.; Riera, J.; Viadel, Ll.
Tetrahedron Lett. 1994, 35, 6529. (c) Teruel, L.; Viadel, Ll.; Carilla, J.;
Fajar´ı, Ll.; Brillas, E.; San˜e´, J.; Rius, J.; Julia´, L. J. Org. Chem. 1996, 61,
6063. (d) Carilla, J.; Fajar´ı, Ll.; Julia´, L.; San˜e´, J.; Rius, J. Tetrahedron
1996, 52, 7013.
4584
Org. Lett., Vol. 6, No. 24, 2004

Scheme 1
The two-step mechanism of reduction is consistent with
the results found when radical 1 reacts with catechol (see
Scheme 1).
Table 1. Free Radical Scavenging Power (Hydrogen Transfer)
and Electron Transfer of Polyphenols
DPPH methoda
radical 1 method
electrons
The oxidant ability of radical 1 in the presence of
polyphenolic antioxidants has also been evaluated by electron
paramagnetic resonance (EPR),11 measuring the decrease of
the intensity of the radical signal from diluted solutions (60
µM) in chloroform-methanol (2:1) with variable concentra-
tions of (-)-epicatechin (2) and two different synthetic
derivatives, 4â-(S-cysteinyl)epicatechin3c (3) and 4â-(2-
aminoethylthio)epicatechin3b (4). The results were expressed
as the efficient dose ED50 standing for the necessary
micromoles of polyphenol to react with half the amount of
free radical divided by micromoles of initial radical 1. The
stoichiometric value is obtained by multiplying ED50 by two,
and the inverse of this value represents the moles of 1
reduced by 1 mol of antioxidant or, in other words, the
number of transferred electrons per molecule of polyphenol.12
A comparison of the parameters of the free radical scaveng-
ing power of these polyphenols, measured by the DPPH
method3b,c (number of transferred hydrogens per molecule
of polyphenol), and the parameters of their reduction power,
measured by the radical 1 method (number of transferred
electrons per molecule of polyphenol), is summarized in
H atoms
stoichiometric
value
per
molecule
stoichiometric
value
per
molecule
compound
catechol
0.34
0.4
0.24
0.22
2.9
2.8
4.2
4.5
0.44
0.42
0.42
0.43
2.3
2.4
2.4
2.3
2
3
4
a Results reported in refs 3b,c.
Table 1. Parameters from catechol are also enclosed in Table
1 for comparative purposes.
(9) Preparation of 1H, as a precursor of radical 1, and 1 are as follows:
Synthesis of Tris(2,4,6-trichloro-3,5-diphenyl)methane. A mixture of
tris(2,4,6-trichlrophenyl)methane (0.780 g), fuming nitric acid (40 mL),
and sulfuric acid (30% SO3) (10 mL) was stirred (80 °C) (72 h) and
then poured into an excess of glass-water. The precipitate, separated by
filtration and dried under reduced pressure, was tris(2,4,6-trichloro-3,5-
diphenyl)methane (1.095 g; 94%). Selected data can be found in ref 5.
Synthesis of Tris(2,4,6-trichloro-3,5-dinitrophenyl)methyl Radical. An
aqueous solution of tetrabutylammonium hydroxide (1.5 M) (0.71 mL)
was added to a solution of tris(2,4,6-trichloro-3,5-dinitrophenyl)meth-
ane (0.770 g) in acetone (40 mL) and stirred at room temperature (20
min). Solid chromium(VI) oxide (0.450 g) was added, and the mixture was
stirred in argon and in the dark for a period of time (20 h). The mix-
ture was poured into an excess of hydrochloric acid aqueous solution, and
the precipitate was separated by filtration. The solid in chloroform was
column chromatographed (silica gel), affording tris(2,4,6-trichlro-3,5-
dinitrophenyl)methyl radical (0.541 g; 70%). Selected data can be found in
ref 5.
(10) Kondo, K.; Kurihara, M.; Miyata, N.; Suzuki, T.; Toyoda, M. Free
Rad. Biol. Med. 1999, 27, 855. Dangles, O.; Fargeix, G.; Dufour, C. J.
Chem. Soc., Perkin Trans. 2 2000, 1653.; Sang, S. M.; Cheng, X. F.; Stark,
R. E.; Rosen, R. T.; Yang, C. S.; Ho, C. T. Bioorg. Med. Chem. 2002, 10,
2233.
(11) EPR spectrum with spectral parameters of tris(2,4,6-trichloro-3,5-
dinitrophenyl)methyl radical in CH2Cl2 is provided in ref 5.
(12) Analogous methodology has been used when the antioxidant activity
of these polyphenols by H-abstraction was analyzed in the presence of DPPH
(see ref 3c).
Catechol, as a simple model, was able to transfer two
electrons and reduce two molecules of radical 1 per molecule
and is also suitable to transfer two hydrogens and reduce
two DPPH molecules per molecule. In such a case, the
stoichiometry of both processes is the same and the reactions
proceed as follows (see Scheme 2).
The reactivity of (-)-epicatechin is quite similar to that
of catechol (Table 1), corroborating that the more reactive
moiety of the molecular structure in both electron transfer
and hydrogen abstraction is the catechol ring. This is a first
approximation of the reactivity of the polyphenols with 1.
Org. Lett., Vol. 6, No. 24, 2004
4585

Table 2. Observed Rate Constants, k1 (M-1 s-1), for the
Reaction of Radical 1 (60 µM) with Antioxidants at 25 °C in
Methanol
Scheme 2
antioxidant
radical 1/
concentration antioxidant
(µM)
molar ratio catechol
2
3
4
5
10
20
12.0
6.0
3.0
301 ( 61 815 ( 138 282 ( 27 214 ( 14
207 ( 21 669 ( 20 264 ( 23 260 ( 33
210 ( 3 659 ( 21 300 ( 16 300 ( 3
The actual scavenging mechanism is clearly more complex,
as evidenced by the stoichiometric values in Table 1 and
the literature.13,14 When (-)-epicatechin and derivatives such
as 3 and 4 were examined, the results in Table 1 revealed
remarkable differences among them. While the stoichiom-
etries for both chemical processes with (-)-epicatechin and
for the electron transfer process with 3 and 4 show similarity,
the presence of mercapto substituents in position 4 of the
heterocycle in 3 and 4 seems to affect the hydrogen
abstraction process in both derivatives. These flavanols are
capable of reducing at least one mole more of DPPH per
mol of antioxidant relative to (-)-epicatechin. This increase
in the number of labile hydrogens of the molecule may be
ascribed to the presence of the long-chain substituent at C4.
However, to evaluate the effectiveness of the antioxidants,
it is of more interest to provide the kinetics of their reactions
with radical species. Thus, some preliminary results on
kinetic studies of the reaction of radical 1 with the reported
antioxidants have been carried out following the general
kinetic model 1 reported by Dangles et al. to assess the ability
of antioxidants to transfer H atoms to DPPH.14 In our
experiments, the electron transfer reaction to radical 1 from
polyphenols in methanol was monitored at 25 °C by
following the decrease of the absorbance (λmax ) 385 nm)
in the electronic spectrum of 1 after the addition of variable
concentrations of polyphenols. In contrast with what happens
when DPPH is used, the two steps of the decay of the
absorbance of DPPH, one fast and the other slow, in the
course of the reaction with the antioxidant are hardly
distinguishable in the case of using radical 1. Values of the
kinetic constants at three different concentrations of the
reported polyphenols are shown in Table 2. An analysis of
these results suggests that (-)-epicatechin is the more active
electron-donating antioxidant, since the activity of the
catechol and of the derivatives of (-)-epicatechin, 3 and
4, are very similar. Consequently, although the stoichio-
metric factors of 3 and 4 are nearly twice the value of
(-)-epicatechin, this antioxidant has shown to be more
effective as electron donor to radical 1. An estimation of
the kinetics of 2 with DPPH as a stable radical in methanol
at 25 °C was also performed. At the DPPH/2 molar ratio of
12, the same as one of the molar ratios in Table 2, the
observed rate constant is 3468 ( 311, much higher than the
value reported for radical 1. Detailed results of the rate
constants of the other polyphenols with DPPH recorded under
similar conditions are now in progress.
What has been said above is a first approximation of the
reactivity of these polyphenols, because the overall mech-
anism of their antioxidant behavior is clearly complex, as
evidenced by the stoichiometric values in Table 1, which in
some cases do not fit to the required values. As reported
before,3c the higher than theoretical number of hydrogens
or electrons involved in these processes would be regarded
as the result of the antioxidant activity of the oligomeric
fractions derived from the first generated phenolic reactive
radicals. It should also be underlined that the reported kinetic
results are only indicative of the relative efficacy of the
different antioxidants, as they refer to a stable carbon-
centered radical instead of a very reactive oxygen-centered
radical like those actually involved in autoxidation.
In summary, this manuscript reports a new method to
distinguish between hydrogen- and electron-donating prop-
erties in polyphenols. Therefore, while polyphenols 3 and
4 preserve the same power as electron donors as
(-)-epicatechin (2), they provide more hydrogens to be
transferred to radical species. The ability of radicals of the
TTM series and in particular of radical 1 to be reduced by
electron transfer reactions to very stable negatively charged
species detected by spectroscopy has made it possible to
corroborate the reducing capacity of natural and synthetic
polyphenols by electron transfer reactions. Kinetics and stoi-
chiometric factors of the reactions of the reported polyphe-
nols with this new stable radical 1 in different solvents and
conditions are currently underway in our laboratories.
Acknowledgment. Financial support for this research
from the Spanish Ministry of Science and Technology
through Project PPQ2003-06602-C04-01 is gratefully
acknowledged.
(13) Kondo, K.; Kurihara, M.; Miyata, N.; Suzuki, T.; Toyoda, M. ArchiV.
Biochem. Biophys. 1999, 362, 79. Hotta, H.; Sakamoto, H.; Nagano, S.;
Osakai, T.; Tsujino, Y. Biochim. Biophys. Acta General Subjects 2001, 1526,
159.
(14) Goupy, P.; Dufour, C.; Loonis, M.; Dangles, O. J. Agric. Food
Chem. 2003, 51, 615.
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Org. Lett., Vol. 6, No. 24, 2004