Hydrogen-transfer reactions from phenols to TEMPO prefluorescent probes in micellar systems

Article abstract of DOI:10.1021/ol800446c

(Chemical Equation Presented) A nitroxide prefluorescent probe has been used to evaluate local reactivity of antioxidants in micellar systems. An apparent rate constant that directly reflects the relevance of antioxidant hydrophobicity on the reaction tow

Full text of DOI:10.1021/ol800446c

ORGANIC
LETTERS
2008
Vol. 10, No. 11
2147-2150
Hydrogen-Transfer Reactions from
Phenols to TEMPO Prefluorescent
Probes in Micellar Systems
Carolina Aliaga,*,† Juan M. Jua´rez-Ruiz,‡ J. C. Scaiano,‡ and Alexis Aspe´e*,†
Facultad de Qu´ımica y Biolog´ıa, UniVersidad de Santiago de Chile,
Casilla 40 Correo 33, Santiago de Chile, Department of Chemistry, UniVersity of
Ottawa, Ottawa, Canada K1N 6N5
aaspee@usach.cl; caliaga@usach.cl
Received March 10, 2008
ABSTRACT
A nitroxide prefluorescent probe has been used to evaluate local reactivity of antioxidants in micellar systems. An apparent rate constant that
directly reflects the relevance of antioxidant hydrophobicity on the reaction toward nitroxide radicals has been defined. Dramatic increases in
this parameter for quercetin are shown on moving from methanol to micellar media: 90 and 230 fold enhancements for SDS and Triton X100
micelles, respectively. This is a clear consequence of the favorable partition of reactants in the micelles.
The antioxidant properties of phenols and polyphenols have
been mostly associated to the trapping of radicals involved
in the oxidative chain by efficient hydrogen transfer reactions
toward free radicals.1–4 Different methodologies have been
developed to evaluate the rate constant for hydrogen transfer
from phenols to peroxyl, alkoxyl, nitrogen and carbon-
centered radicals. These studies have allowed correlating the
reactivity of polyphenols in aqueous and organic solvents
with their chemical structure.5 However, there is limited
information related to the participation of free radical
processes and the antioxidant mechanism involved in bio-
logical membranes. We anticipate that the hydrophobicity
of flavonoids will be reflected in different antioxidant activity
due to the partition and preferential location of the antioxidant
in the membrane.
We have recently proposed the use of nitroxide prefluo-
rescent probes to evaluate hydrogen transfer rate constants
from highly reactive polyphenols such as flavonoids in
homogeneous media.6 The methodology reported is based
on the intramolecular quenching of the fluorescent chro-
mophore moiety by the tethered TEMPO moiety. The
abstraction of a hydrogen atom from the polyphenol by the
nitroxide produces the diamagnetic hydroxylamine thereby
restoring the fluorescence of the chromophore.7,8
† Facultad de Qu´ımica y Biolog´ıa, Universidad de Santiago de Chile.
‡ Department of Chemistry, University of Ottawa, Canada.
(1) Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and
Medicine; Oxford University Press: Oxford, 1999
(2) Valgimigli, L.; Banks, J. T.; Ingold, K. U.; Lusztyk, J. J. Am. Chem.
Soc. 1995, 117, 9966–9971
.
.
(3) Burton, G. W.; Ingold, K. U. J. Am. Chem. Soc. 1981, 103, 6472–
6477
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(6) Aliaga, C.; Aspe´e, A.; Scaiano, J. C. Org. Lett. 2003, 5, 4145–4148.
(7) Blough, N. V.; Simpson, D. J J. Am. Chem. Soc. 1988, 110, 1915–
1917.
(4) Litwinienko, G.; Ingold, K. U. J. Org. Chem. 2004, 69, 5888–5896
(5) Burton, G. W.; Ingold, K. U. Acc. Chem. Res. 1996, 19, 194–201.
.
10.1021/ol800446c CCC: $40.75
Published on Web 05/09/2008
2008 American Chemical Society

In the present work, we extend the potential use of these
nitroxide prefluorescent probes to evaluate local reactivity of
antioxidants in micellar systems. In this context, it is important
to note that not only the polyphenol could be distributed in the
micelle but also the probe. As the prefluorescent probe, we
employed C343T (see Figure 1 and Supporting Information) an
almost completely displaced toward the products allowing
an accurate determination of the rate constants.6 On the other
hand, in the case of polyphenol radicals that react rapidly
with oxygen in air-saturated solutions in competition with
fast self-reaction,10,11 the initial hydrogen abstraction by the
nitroxide is the limiting reaction evaluated in this kinetic
treatment. It is important to mention that the rate constant
evaluated from fluorescence measurements could be affected
at most by a factor of 2, if the antioxidant carbon centered
radical is exclusively trapped by the remaining nitroxide
probe to produce the fluorescent diamagnetic alcoxyamine,
as shown in 2.
The hydrogen abstraction rate constants toward the ni-
troxide unit evaluated under these conditions for R-tocopherol
in different solvents are shown in Table 1.
Figure 1. Structures of polyphenols employed and C343T.
Table 1. Hydrogen Transfer Rate Constants from R-Tocopherol
to C343T in Different Solvents at Room Temperature
solvent
k, M-1 s-1
appropriate probe for the study of hydrogen transfer reactions
in these systems due to its high hydrophobicity and absorption
at long wavelengths, therefore not interfering with the absor-
bance of the antioxidants.9
hexane
benzene
acetonitrile
water or methanol
0.33
0.13
0.06
0.21a
The hydrogen transfer rate reactions were evaluated
employing different phenol antioxidants, including R-toco-
pherol and Trolox, as models in different solvents and
microheterogeneous media (Figure 1). The micellar reactions
were carried out in phosphate buffer (20 mM, pH 7.0)
pretreated with Chelex100 in order to minimize the concen-
tration of 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; initial and at
time “t”, respectively.
a Evaluated using Trolox.
These hydrogen-transfer rate constants present a similar
solvent dependence to that observed for phenols toward
highly reactive free radicals. This solvent effect has been
attributed to hydrogen bond formation between the phenols
and the solvent. Ingold et al. have discussed in detail how
this effect depends on the hydrogen bond accepting (HBA)
properties of the solvent and is independent of the nature of
the attacking free radical.12
Interestingly, the hydrogen-transfer reactions from R-to-
copherol to nitroxides in hydroxylic solvents are considerably
faster than what could be expected according to the HBA
properties of the solvent. This behavior has been attributed
to a specific formation of hydrogen bonds between the
solvent hydroxyl group and the lone pair on the nitroxide’s
oxygen. This interaction increases the reactivity of the
nitroxide as it has been previously observed in quenching
of carbon-centered radicals by TEMPO.13
I∞ - I0
ln
) kobs
t
(1)
(
)
I∞ - It
The reactions in aqueous or organic solvens were evaluated
under pseudo-first-order reaction conditions employing an
excess of the phenols (0.5-20 mM) in relation to the
nitroxide probe (2.5 µM). Kinetic analysis leads to the rate
constant for hydrogen abstraction directly from the slope of
kobs vs the intial antioxidant concentration plot.
We have previously demonstrated that for antioxidants
such as Trolox in water or methanol, the reaction with
nitroxide shows reversibility in nitrogen-purged solutions.
However, at high Trolox concentration the equilibrium is
(10) Burton, G. W.; Doba, T.; Gabe, E.; Hughes, L.; Lee, F. L.; Pasad,
L.; Ingold, K. U. J. Am. Chem. Soc. 1985, 107, 7053–7065
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.
78–84
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J. Am. Chem. Soc. 1990, 112, 7337–7346.
(12) Valgimigli, L.; Banks, J. T.; Ingold, K. U.; Lusztyk, J. J. Am. Chem.
Soc. 1995, 117, 9966–9971.
(9) Coenjarts, C.; Garc´ıa, O.; Llauger, L.; Palfreyman, J.; Vinette, A. L.;
Scaiano, J. C. J. Am. Chem. Soc. 2003, 125, 620–621.
(13) Beckwith, A. L. J.; Bowry, V. W.; Ingold, K. U. J. Am. Chem.
Soc. 1992, 114, 4983–4992.
2148
Org. Lett., Vol. 10, No. 11, 2008

Table 2. Rate Constants for Hydrogen Abstraction from Phenolic Compounds toward C343T Probe
antioxidant
kH, M-1 s-1
kD, M-1 s-1
TARd
k′ SDSe
k′ Tritone
b,c
Trolox
BHT
caffeic acid
gallic acid
quercetin
rutin
0.19a,c
0.5 × 10-3
1.3 × 10-3
0.5 × 10-3
4.6 × 10-3
0.6 × 10-3
unreactive
1.0
0.83
0.15
0.03
0.02
0.35
0.52
0.39
0.09
0.01
0.93
b,c
b,c
b
3.3 × 10-3
a,f
b
4.0 × 10-2 2.2 × 10-3
1.0
2.0
3.3
1.0
b,c
b,c
b
8.8 × 10-3
a
b
22.7 × 10-3 * 4.0 × 10-3
*
unreactive
a Phosphate buffer, Pi (10 mM, pH 7.0) or in D2O pD 7.0. b MeOH or MeOD. c Reference 6. d TAR (total antioxidant reactivity) evaluated from competitive
experiments of c-phycocyanin protection against peroxyl radicals in Pi (10 mM pH 7.0)21 e Experimental conditions for micelles: 2.5 µM C343T, 20 mM
SDS, 15 mM Triton X100, 20 mM phosphate buffer pH 7.0, phenol concentrations from 100 µM to 5 mM. f Pi (100 mM, pH 7.0). * Data obtained by EPR
using TEMPO.
In order to make a systematic comparison between the
two systems we have defined an apparent rate constant in
micelles as
The micellar reaction with Trolox, a predominantly water-
soluble antioxidant, showed pseudo-first-order kinetics, but
the reaction was faster than in aqueous media (Figure 2).
kobs
k′ )
(3)
[phenol]0
where [phenol]0 is the initial concentration of the phenol
added. This value allows us to have a parameter that directly
reflects the importance of the distribution of the antioxidant
between the micelle and the dispersing media reflecting the
antioxidant hydrophobicity. It is worth mentioning that this
apparent rate constant k′ depends on the micellar concentra-
tion. In the present study, we decided to employ 20 mM
SDS and 15 mM Triton X100 after taking into account the
critical micelle concentration (CMC) values of the surfac-
tants.14
The nitroxide probe reaction with R-tocopherol solubilized
in micelles of SDS shows an increase in the apparent rate
constant value (17 M-1s-1) relative to the rate constant in
organic solvents (Table 1). It has been reported that the
tocopheryl radical self-reaction rate constant decreases in
micelles (showing even longer lifetimes than the millisecond
values that are common in SDS micelles);15 in principle, this
could increase the proportion of its cross-reaction with
nitroxide prefluorescent probes, however, the reaction with
oxygen is still considerably faster (and thus favored), given
the very low concentration of probe employed (2.5 µM).
On the other hand, the reaction of ascorbic acid with the
nitroxide probe in SDS micelles was much slower (0.5 M-1
s-1) than in aqueous media at pH 7.0 (13.1 M-1 s-1).16 These
results suggest a very efficient micellar compartmentalization
of the antioxidants depending on their hydrophobicity;
further, it also suggests a preferred location of the nitroxide
probe near the micellar interface. The latter is supported by
the high partition constant determined for C343T (KSDS ) 500
M-1) and a high polarity microenvironment sensed by the
fluorescence emission maximum of the chromophore unit
in SDS micelles (489 nm) relative to water or methanol (492
and 481 nm, respectively).
Figure 2. (A) Fluorescence-time profile associated to the reduction
of C343T to the N-hydroxylamine by hydrogen transfer from 10
mM (9) and 5 mM (O) Trolox, in 20 mM SDS-phosphate buffer
(20 mM, pH 7). (B) Experimental rate constant evaluated from eq
1. Experimental conditions: 2.5 µM C343T, 20 mM phosphate buffer,
pH 7.0. λexc 420nm, λem 470nm.
The same tendency is observed when Triton X100 micelles
are employed.
Detailed analysis of the data in Table 2 reveals a
remarkable increase of the rate constant for quercetin as we
move from water to micelles: 15 and 41 times faster in SDS
and Triton, respectively. These dramatic changes can be
attributed to the favorable partition of quercetin into micelles
due to its hydrophobic character. In fact, the difference is
even larger between methanol and micelles (increases of 88-
and 230-fold for SDS and Triton, respectively). This is a
consequence of the lower rate constant of quercetin in
methanol compared with water at pH 7, where the molecule
is partially deprotonated (pKa 7.03),17,18 thus increasing the
reaction rate by an electron transfer mechanism. This
argument is supported on the reduction potential of quercetin
(14) Fuguet, E.; Ra`fols, C.; Rose´s, M.; Bosch, E. Anal. Chim. Acta 2005,
548, 95–100.
(15) Patist, A.; Chhabra, V.; Pagidipati, R.; Shah, R.; Shah, D. O.
Langmuir 1997, 13, 432–434.
(17) Jovanovic, S. V.; Steenken, S.; Tosic, M.; Marjanovic, B.; Simic,
M. G. J. Am. Chem. Soc. 1994, 116, 4846–4851
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(16) Aspe´e, A.; Aliaga, C.; Scaiano, J. C. Photochem. Photobiol. 2007,
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Org. Lett., Vol. 10, No. 11, 2008
2149

and Tempol evaluated at pH 7.0,19,20 and the secondary
reactions of quercetin radicals, such as crossed reaction with
unreacted nitroxides. The difference between the two micelles
is due to a less efficient incorporation of this antioxidant in
the anionic micelle compared to the neutral one. The negative
headgroup of the SDS molecule and the enolate form present
at high pH are the subject of repulsive interactions.15 In fact,
in CTAC micelles the kinetics appear almost instantaneous;
here the enolate form acts as a micellar counterion, com-
plicating the determination of reliable kinetic parameters
(data not shown).
In the case of BHT, the relative rate constants are 1:45:
120 for methanol/SDS micelles/Triton micelles, respectively.
This is a clear consequence of BHT partition in the micelles
while gallic acid shows almost the same reactivity as in
methanol.
In methanol, no reaction between the probe and rutin was
observed while for quercetin a rate constant of 4.0 × 10-3
M-1 s-1 was obtained. This is a remarkable result in terms
of the selectivity of the nitroxyl moiety of the prefluorescent
probe, revealing that the more reactive center in quercetin
(among the 5 OH groups) is the 3′-hydroxyl group, which
in rutin is protected by a sugar residue. This result is in
agreement with theoretical results where Fukui indexes of
reactivity were employed.22 Further, the hydrogen transfer
selectivity shown by C343T is higher than that of related
species, such as nitronyl nitroxide radicals.23 Nitroxide
radicals exhibit a ratio between the rate of hydrogen transfer
for ascorbic acid (13.1 M-1 s-1)16 and quercetin (22.7 ×
10-3 M-1 s-1) of 570, whereas for nitronyl nitroxide radicals
the ratio is only 57, with rate constant values of 43 and 0.76
M-1 s-1 for ascorbic acid and quercetin, respectively.
It is worth mentioning that other methodologies involving
peroxyl radicals in competitive experiments in homogeneous
systems are not able to discriminate well between two highly
reactive phenols. A typical example is the TAR index, which
is based on the relative antioxidant protection of the target
molecule by the decrease on the stationary concentration of
peroxyl radicals (derived from 2,2′-azobis(2-amidinopropane)
dihydrochloride, AAPH) caused by the antioxidant. However,
as shown in Table 2, with this methodology, antioxidants
such as caffeic acid are not distinguishable from the reference
compound, Trolox. Whereas the analysis of the rate hydrogen
transfer toward the nitroxide probe shows a noticeable higher
reactivity for Trolox than caffeic acid (5 times) in phosphate
buffer, pH 7. Moreover, there is a clear tendency for higher
TAR values with the increase on number of phenol groups
in the molecules without relation to their actual reactivity.21
In fact, we have recently shown that most of the reactivity
parameters obtained in competitive experiments are strongly
influenced of stoichiometric factors and also by the reactivity
of the reference compound employed.24,25
In summary, we propose the use of persistent substituted
nitroxide radicals in micellar systems as a new and simple
tool to evaluate antioxidant efficiency; this approach takes
into account both the hydrophobicity of the antioxidant and
also the high selectivity of the nitroxide radicals toward very
reactive phenols such as flavonoids. Further, the use of
different chromophore units tethered to nitroxide moieties,
of different hydrophobicity,26–29 can be employed to control
the partition constant, and also the location of the nitroxide
prefluorescent probe within the micelles.
Acknowledgment. C.A. acknowledges the World Bank
CONICYT: Programa Bicentenario de Ciencia y Tecnolog´ıa
and DICYT-USACH. A.A. thanks FONDECYT (project
1050137), and J.C.S. and A.A. thank the Natural Sciences
and Engineering Research Council of Canada for financial
support.
Supporting Information Available: Synthetic procedures
and full spectroscopy data for all new compounds, an
example of kinetic determination using fluorescence spec-
troscopy, and procedures for the determination of partition
constants. This material is available free of charge via the
Internet at http://pubs.acs.org.
OL800446C
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