A New Method to Study Antioxidant Capability: Hydrogen Transfer from Phenols to a Prefluorescent Nitroxide

Article abstract of DOI:10.1021/ol035589w

(Equation presented) Rate constants for hydrogen abstraction from phenols by a prefluorescent-TEMPO probe are reported. The nitroxide is employed as a potential model of peroxyl radicals. The probe works by nitroxide suppression of the fluorescence of the

Full text of DOI:10.1021/ol035589w

ORGANIC
LETTERS
2003
Vol. 5, No. 22
4145-4148
A New Method to Study Antioxidant
Capability: Hydrogen Transfer from
Phenols to a Prefluorescent Nitroxide
Carolina Aliaga, Alexis Aspe´e, and J. C. Scaiano*
Department of Chemistry, UniVersity of Ottawa, Ottawa, Canada K1N 6N5
tito@photo.chem.uottawa.ca
Received August 23, 2003
ABSTRACT
Rate constants for hydrogen abstraction from phenols by a prefluorescent-TEMPO probe are reported. The nitroxide is employed as a potential
model of peroxyl radicals. The probe works by nitroxide suppression of the fluorescence of the chromophore. The fluorescence is restored
when the nitroxide abstracts a hydrogen atom to produce the diamagnetic hydroxylamine. The phenols studied in this project exhibited rate
constants between 0.003 and 0.2 M-1 s-1. A deuterium isotope effect of 10 for TROLOX confirms that the mechanism is dominated by hydrogen
transfer.
Phenols have been widely used as scavengers for free radicals
involved in oxidative stress in living organisms. Their chain-
breaking antioxidant action is based on the trapping of
radicals involved in the oxidative chain by efficient hydrogen
transfer.1-3 Rate constants for hydrogen abstraction from
phenols by peroxyl-, alkoxyl-, nitrogen-, and carbon-centered
radicals have been reported.4-7 However, due to the high
reactivity of some of these radicals, they present low
selectivity toward compounds with similar hydrogen-donat-
ing properties. This is particularly true in the evaluation of
hydrogen-transfer reactions of highly reactive polyphenols
such as flavonoids.
Different methodologies have been developed to evaluate
antioxidant capability of individual compounds or in complex
mixtures rich in polyphenols, such as wines and infusions.8
Those methodologies are based on (i) indirect measurement
of oxygen consumption,9,10 or competitive reactions with a
highly reactive reference compound, (e.g., c-phycocyanin)
employing peroxyl radicals,11 or (ii) direct monitoring of the
consumption of a persistent free radical such as DPPH or
ABTS.12,13 The information obtained has allowed the de-
(1) Burton, G. W.; Ingold, K. U. J. Am. Chem. Soc. 1981, 103, 6472.
(2) Rice-Evans, C. A.; Miller, N. J.; Paganda, G. Free Rad. Biol. Med.
1996, 20, 933.
(3) Burton, G. W.; Ingold, K. U. Acc. Chem. Res. 1986, 19, 194.
(4) Burton, G. W.; Doba, T.; Gabe, E. J.; Hughes, K.; Lee, F. L.; Prasad,
L.; Ingold, K. U. J. Am. Chem. Soc. 1985, 107, 7053.
(5) Das, P. K.; Encinas, M. V.; Steenken, S.; Scaiano, J. C. J. Am. Chem.
Soc. 1981, 103, 4162.
(8) Perez, D. D.; Leighton, F.; Aspe´e, A.; Aliaga, C.; Lissi, E. A. Biol.
Res. 2000, 33, 71.
(9) Wayner, D. D.; Burton, G. W.; Ingold, K. U.; Locke, S. FEBS Lett.
1985, 187, 33.
(10) Romay, C.; Pascual, C.; Lissi, E. A. Braz. J. Med. Res. 1996, 29,
175.
(11) Cao, G.; Alessio, H. M.; Cutler, R. G. Free. Rad. Biol. Med. 1993,
303.
(6) Ingold, K. U. In Radical Reaction Rates in Liquids; Fisher, H., Ed.;
Landolt-Borstein, New Series; Springer-Verlag: Berlin, 1984; Vol. 13, Part
C.
(12) Goupy, P.; Dufour, C.; Loonis, M.; Dangles, O. J. Agric. Food
Chem. 2003, 51, 615.
(7) Franchi, P.; Lucarini, M.; Pedulli, G. F.; Valgimigli, L.; Lunelli, B.
J. Am. Chem. Soc. 1999, 121, 507.
(13) Rice-Evans, C.; Miller, N. J.; Bolwell, P. G.; Bramley, P. M.;
Pridham, J. B. Free Rad. Res. 1995, 28, 1051.
10.1021/ol035589w CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/10/2003

scription of the quality (reactivity) and the quantity (stoi-
chiometry) for the added antioxidant reacting with the
radicals used. However, those studies do not provide
information about the type of radical trapped and the
antioxidant mechanism involved. This is particularly true in
studies employing DPPH and ABTS, where reactions of
hydrogen abstraction and electron transfer have been
proposed.12-14
Fast reaction of nitroxide radicals with ubiquinol-9,
phenolic, and thiol antioxidants have been reported.15,16
Recently, Aliaga et al.17 using ESR spectroscopy and
product studies have shown a very slow reaction between
TROLOX and tempol in aqueous solvents and suggested that
traces of metal ions could catalyze the reaction. In the
absence of metal ions, the kinetics of this process are believed
to reflect hydrogen transfer.
detect and quantify directly primary radical concentrations
generated by pulsed laser photolysis in homogeneous sol-
vents.20 These studies have allowed the quantification of very
low concentrations of radicals that usually are not detected
by conventional spectroscopic techniques or difficult by ESR.
We propose here the use of QT as a probe mimicking
peroxyl radical reactivity21 in order to evaluate the antioxidant
activity of phenolic compounds and to obtain kinetic
parameters involved in the hydrogen-transfer process. Ni-
troxide compounds are orders of magnitude less reactive than
peroxyl radicals;4 this characteristic is perceived as an
advantage since it allows evaluation of the selectivity for
highly reactive phenols. A similar methodology has previ-
ously proposed to quantify vitamin C in biological fluids,22
where an electron-transfer mechanism was proposed. In our
systems (vide infra), the mechanism is believed to be
hydrogen transfer; further, the enhancement of fluorescence
for QT is much larger than for the dansyl probe reported. 22
Most reactions were carried out under an inert atmosphere
in methanol, water, and phosphate buffer (50 mM, pH 7.0).
The aqueous solvents were pretreated with Chelex100 in the
presence of 0.1 mM diethylenetriaminepentaacetic acid
(DTPA) in order to minimize the concentration of free metal
ions. The rate constants observed (kobs) were obtained by
monitoring the growth of the fluorescence according to eq
1, where I∞, I0, and It represent the fluorescence intensities
in the plateau region, initially and at time “t”, respectively.
In this paper, we evaluate the ability of 4-(3-hydroxy-2-
methyl-4-quinolinoyloxy)-2,2,6,6-tetramethylpiperidine-1-
oxyl free radical (QT), a prefluorescent TEMPO probe, to
monitor hydrogen transfer from reactive phenols (Scheme
1)18 The mechanism by which this prefluorescent nitroxide
Scheme 1. Phenols and Polyphenols Studied and Probe
Structure
I∞ - I0
ln
) kobs
t
(1)
(
)
I∞ - It
Kinetic analysis, under pseudo-first-order reaction condi-
tions, leads to the rate constant for hydrogen abstraction
directly from the slope of Figure 1B (eq 2). The rate
expression derived under these experimental conditions is
shown in eq 3
R-NO• + PhOH f R-NOH + PhO•
(2)
(3)
xe
xe
ln
) k1t
probe19 works is based on the suppression of the intra-
molecular quenching of the fluorescent chromophore by the
nitroxide group. The fluorescence is restored when the
nitroxide moiety is trapped by a reducing agent, such as a
hydrogen donor, to produce the diamagnetic hydroxylamine
(QTH).19 This methodology has been successfully used to
(
)
xe - x
(2[A0] - xe)
where A0 represents the initial nitroxide and xe is the
concentration of the corresponding diamagnetic N-hydroxyl-
amine in the equilibrium, since eq 3 allows for possible
reversibility in reaction 2.
In fact, similar considerations allow the evaluation of the
equilibrium constant from the fluorescence plateau intensity
reached at long times at different concentrations; a value of
8 × 10-3 is obtained for TROLOX. Despite the apparently
small equilibrium constant, the equilibrium should be largely
(14) Aliaga, C.; Lissi, E. A. Can. J. Chem. 2000, 78, 1052
(15) Fuchs, J.; Groth, N.; Herrling, T.; Zimmer, G. Free Rad. Biol. Med.
1997, 22, 967.
(16) Hiramoto, K.; Ojima, N.; Kikurawa, K. Free Rad. Biol. Med. 1997,
27, 45.
(17) Aliaga, C., Lissi, E. A.; Augusto, O.; Linares, E. Free Rad. Biol.
Med. 2003, 37, 225.
(18) The use of fluorescence to sense a variety of properties is well
established. See, for examples: Czarnik, A. W., Desvergne, J.-P., Eds.
Chemosensors of Ion and Molecule Recognition; Kluwer: Dordrecht, 1997.
Aspe´e, A.; Garcia, O.; Maretti, L.; Sastre, R.; Scaiano, J. C. Macromolecules
2003, 36, 4550.
(19) Blough, N. V.; Simpson, D. J. J. Am. Chem. Soc. 1988, 110, 1915.
Green, S. A.; Simpson, D. J.; Zhou, G.; Ho, P. S.; Blough, N. V. J. Am.
Chem. Soc. 1990, 112, 7337.
(20) Moad, G.; Shipp, D.; Smith, T. A.; Solomon, D. H. J. Phys. Chem.
1999, 103, 6580.
(21) Ahrens, B.; Davidson, M. G.; Forsyth, T.; Mahon, M. F.; Johnson,
A. L.; Mason, S. A.; Price, R. D.; Raithby, P. R. J. Am. Chem. Soc. 2001,
123, 9164.
(22) Lozinsky, E.; Martin, V. V.; Berezina, T. A.; Shames, A. I.; Weis,
A. L.; Likhtenshtein, G. I. J. Biophys. Methods 1999, 38, 29.
4146
Org. Lett., Vol. 5, No. 22, 2003

Table 1. Experimental Rate Constants of Hydrogen Transfer
from Phenolic Compounds to QT Probe
substrate
TROLOX
solvent
k, M-1 s-1
buffer pH 7
methanol
0.2
0.19
methanol-O-d
buffer pH 7
H2O metal free
methanol
methanol-O-d
D2O
0.018
gallic acid
8.8 × 10-3
5.5 × 10-3
4.6 × 10-3
1.3 × 10-3
1.4 × 10-3
3.3 × 10-3
1.3 × 10-3
BHT
methanol
methanol-O-d
could not be evaluated kinetically. Further, we assume that
reactions occur predominantly at the most reactive hydrogen
atom from every structure.
In principle, at least three mechanisms can be proposed
to rationalize the reaction of QT with phenolic hydrogen
donors, i.e., (a) hydrogen abstraction, (b) electron transfer
followed by proton transfer, and (c) substrate ionization
followed by electron transfer from the phenoxide to QT, as
recently demonstrated by Litwinienko and Ingold for reac-
tions of DPPH in hydroxylic solvents.27 The nitro substitution
in DPPH clearly favors this type of behavior. To address
this question, we examined the reactivity of TROLOX in
methanol and methanol-O-d, since in the latter solvent the
phenolic position in TROLOX should be fully deuterated.
The results are shown in Figure 2. From these data we obtain
Figure 1. (A) QT/QTH Fluorescence profile evaluated at 430 nm
after TROLOX addition (b) and in the absence of TROLOX (O)
with time (λexc360 nm). (B) Fluorescence kinetic adjusted. Experi-
mental conditions: 10 µM QT, 10 mM TROLOX, 50 mM
phosphate buffer, pH 7.0.
on the product side, since the concentration of phenolic
compound is typically 3 orders of magnitude larger than that
of QT. We anticipate that phenoxyl radicals, while long-
lived, will ultimately yield nonradical products23 by either
self-reaction or cross-reactions with QT. It is worth noting
that the calculation of the equilibrium constant for BHT could
be affected due to the fact that sterically hindered phenols
are efficient light quenchers.24 However, a rate constant
evaluated at a large concentration is a reliable value, since
the methodology is based on relative fluorescence intensities
(eq 1).
The data for the rate constants for the different phenolic
compounds are collected in Table 1. Note that the slow
reaction between the probe and the phenolic compound
makes it possible to evaluate substrates with high reactivity
using conventional spectroscopic instrumentation, in contrast
with the case of ABTS25,26 where the most reactive substrates
Figure 2. Relative fluorescence increase observed in methanol (b)
and methyl alcohol-d (O). Experimental conditions: 10 µM QT,
10 mM TROLOX (λexc 360 nm, λem 400 nm).
a deuterium isotope effect of 10.5, whereas a factor of ∼4
is observed for gallic acid. While large, values of this
magnitude for reactions of phenols have been known for over
40 years.28
(23) Bowry, V. W.; Ingold, K. U. J. Org. Chem. 1995, 60, 5456.
(24) Brede, O.; Naumov, S.; Hermann, R. Chem. Phys. Lett. 2002, 355,
1-7.
(25) Aliaga, C.; Lissi, E. A. Int. J. Chem. Kinet. 1998, 30, 565.
(26) Lissi, E. A.; Modak, B.; Torres, R.; Escobar, J.; Urzua, A. Free.
Rad. Res. 1999, 30, 471.
(27) Litwinienko, G.; Ingold, K. U. J. Org. Chem. 2003, 68, 3433
Org. Lett., Vol. 5, No. 22, 2003
4147

Interestingly, the rate constants for TROLOX are es-
sentially the same in water and in methanol; product
stabilization in the former probably compensates for reactiv-
ity reductions due to stronger hydrogen bonding. However,
the rate constant observed for gallic acid (a polyphenol) in
buffer is larger than in methanol. Further, the fact that the
less reactive substrate (gallic acid) shows a smaller isotope
effect suggests that some degree of charge transfer may be
involved in this example, probably mediated by the depro-
tonation of the acidic group. Additional studies of poly-
phenols could clarify this point.
The use of DPPH as a probe for radical reactions with
phenols has close to a half-century history,29 and in a recent
publication Ingold reports that publications on DPPH and
antioxidants approach 150 per year.27 Despite this, concerns
still remain, particularly in relation to the value of kinetic
parameters determined with DPPH in hydrogen-bonding
solvents. This is in part related to the strongly electron-
deficient radical center in DPPH. Nitroxide radicals, while
electrophilic (most oxygen centered radicals are), are not as
electron deficient as DPPH, and in this sense are better model
systems.
In summary, we report here a convenient approach for
the determination of the hydrogen-donor ability of phenols
which is very sensitive and appears less prone to suffer from
the problems of highly electron-deficient probes such as
DPPH. Prefluorescent probes offer a combination of high
sensitivity, reaction selectivity, and in many cases simple
synthetic routes; further, the concept can be easily exploited
while optimizing required properties such as hydrophobicity/
hydrophilicity, excitation wavelength, etc.
Acknowledgment. We thank L. Maretti for providing the
initial QT samples and acknowledge the financial support
from the Natural Sciences and Engineering Research Council
of Canada (J.C.S.) and the Fundacio´n Andes (Chile) for a
postdoctoral fellowship.
(28) Howard, J. A.; Ingold, K. U. Can. J. Chem. 1962, 40, 1851
(29) Bickel, A. F.; Kooyman, E. C. J. J. Chem. Soc. 1957, 2415.
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