A simple method for the determination of the partitioning of nitroxide probes in microheterogeneous media

Article abstract of DOI:10.1002/mrc.3871

The use of the ESR g-factor of spin labels for the easy and clean assessment of their partitioning in microheterogeneous media is described. The method constitutes a valuable alternative in those cases where the classical Benesi-Hildebrand treatment, based on UV-visible measurements, is difficult or not feasible. Copyright

Full text of DOI:10.1002/mrc.3871

Research Article
Received: 3 November 2011
Revised: 4 July 2012
Accepted: 15 August 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/mrc.3871
A simple method for the determination of the
partitioning of nitroxide probes in
microheterogeneous media
Carolina Aliaga,a,b* Paulina Torresa and Fernanda Silvaa
The use of the ESR g-factor of spin labels for the easy and clean assessment of their partitioning in microheterogeneous media
is described. The method constitutes a valuable alternative in those cases where the classical Benesi–Hildebrand treatment,
based on UV–visible measurements, is difficult or not feasible. Copyright © 2012 John Wiley & Sons, Ltd.
Supporting Information maybe found in the online version of this article.
Keywords: NMR; g-factor; micelle; partition constant; nitroxide probe
set, arylsulfonamides 3 and 4. Ortep projections of their
structures, obtained by X-ray analysis, are given in the Supporting
Information. Compound 5 (QT), a quinoline fluorophore deriva-
Introduction
Nitroxides have been extensively used for the assesment of
antioxidants in homogeneous and microheterogeneous media.[1–3]
tized with a TEMPO fragment, has been used as a prefluorescent
probe in hydrogen abstractions reactions[1,15] (Scheme 1).
In the latter, knowledge of the partitioning of substrates and radical
labels between two different environments is an important factor
that is often neglected when evaluating antioxidant activities. This
aspect has been stressed by various groups when comparing the
Experimental
reactivity of substrates or radical probes of variable hydrophobicity
in the presence of micelles.[4–6] In particular, we have recently drawn
Equipments
attention to the importance of partitioning factors in the assessment
of antioxidants in microheterogeneous media, showing that their
Melting points were recorded with a Kruss Optronic apparatus
(Hamburg, Germany) and were not corrected. IR spectra were
observed activities reflect the solubilities of both the probe and the
obtained with a Bruker IFS 66v, FT-IR spectrometer (Billerica, MA,
antioxidant in the heterogeneous system.[6]
USA). ESR spectra were measured in a Bruker EMX-1572 series spec-
Nitroxide labels have been widely employed in membrane
trometer, operating at the X-band (Ettlingen, Germany) X-ray analyses
studies because of their versatility, stability and sensitivity toward
of compounds 3 and 4 were obtained with a Bruker AXS SMART single
their microenvironment.[7] This sensitivity is reflected in their ESR
crystal diffractometer (Madison, WI, USA). ESR spectra were measured
spectra.[8] The interaction of the probes with micellar aggregates
in a Bruker EMX-1572 series spectrometer, operating at the X-band.
X-ray analyses of compounds 3 and 4 were obtained with a Bruker
AXS SMART single crystal diffractometer. Details of data collection
is responsible for g-factor variations[7–11] and for line shifts and
line shape changes.[12–14]
In the present communication, we took advantage of the
and treatment, and the corresponding CIF files for the two
sensitivity of the g-factor parameter to the probe microenvironment,
compounds are given in the Supporting Information.
to develop an easy and clean method for the determination of its
partitionining in microheterogeneous media. Partitioning measure-
ments in these media are often carried out with the aid of UV–visible
spectroscopic techniques and the use of Benesi–Hildebrand plots.
When applied to biological fluids, food or plant extracts, the determi-
nation of the partitioning constants of spin labels is often difficult,
because of interferences from components of the complex sample
in the same region where the probe absorbs. In addition, its absor-
bance variations are often rather small, being masked by absorptions
from other species in solution and leading to less precise partition
constants. The present method constitutes a valuable alternative
for the partitioning of spin labels that obviates these spectral
interferences.
Reagents
All solvents were of spectroscopic grade from Sigma-Aldrich
(St Louis, MO, USA) and were used as received. RTX-100, the
4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (4-acetamidoTEMPO)
1 and the 2,2,6,6-tetramethylpiperidine–1-oxyl (TEMPO) 2 were all
*
Correspondence to: Carolina Aliaga, Facultad de Química y Biología, Universi-
dad de Santiago de Chile, Casilla 40 Correo 33, Santiago de Chile, Chile. E-mail:
carolina.aliaga@usach.cl
a Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40
Correo 33, Santiago de Chile, Chile
We illustrate the method with five nitroxide radicals 1–5 in
micellar solutions of reduced Triton-X 100 (RTX-100).
Nitroxide radicals 1 and 2 were commercially available TEMPO
derivatives. Two new TEMPO radicals were also included in the
b Centro para el Desarrollo de la Nanociencia y la Nanotecnología (CEDENNA),
Chile
Magn. Reson. Chem. (2012)
Copyright © 2012 John Wiley & Sons, Ltd.

C. Aliaga, P. Torres and F. Silva
ESR spectroscopy equation ΔE= hv =gbB0, where b is the Bohr
magneton, equivalent to 9.27400915 ꢃ 10ꢁ21 erg Gꢁ1, B0 represents
the magnetic field at the center of the spectrum in gauss, obtained
from the experimental spectrum, h represents Planck’s constant
equivalent to 6.6260755 ꢃ 10ꢁ27 erg s and v represents frequency
tuned and registered for each experiment in gigahertz.
Reliable g-factor values were obtained by employing DPPH as a
standard magnetic-field reference in each experiment, yielding a
microwave frequency that was stable (automatically controlled)
during acquisition of the probe samples. This ensured reproduc-
ible g-values that were not affected by the sample preparation.
In order to discard any effect by dissolved oxygen on the
sample signals, spectra were compared in the presence and
absence of oxygen. Nitrogen-purged and air-exposed samples
showed the same g-values, with no evidence of signal broaden-
ing in the latter.
Scheme 1. Structures of the employed spin probes.
purchased from Sigma-Aldrich and used as received. All reagents
employed in the preparation of new TEMPO derivatives 3 and 4
were purchased from Aldrich. The 4-(3-hydroxy-2-methyl-4-
quinolinoyloxy)-2,2,6,6 tetramethylpiperidine-1-oxyl radical (QT) 5
was prepared by the procedure described by Hassner.[16]
Preparation of N-(2,2,6,6- tetramethylpiperidine-1-oxyl)-4-arenesulfonamides 3
and 4 (general procedure)
Results and Discussion
A solution of 4-aminoTEMPO (0.25 g, 1.4 mmol), N,N-dimethylami-
nopyridine (0.18 g, 1.4 mmol), the corresponding arenesulfonyl
chloride (1.9 mmol) and pyridine (0.23 g, 2.9 mmol) in chloroform
(20 mL) was left standing at room temperature for 12 h. The solution
was washed with dilute HCl (0.1 M), with sodium hydroxide and then
with water, and the organic layer dried over anhydrous sodium
sulfate. The product crystallized by slow evaporation of the solvent
at room temperature.
The ESR spectrum of probe 5 in water is shown in Fig. 1(a),
together with its simulated spectrum. A good fitting was obtained,
In this way, the following sulfonamides were prepared:
•
N-(2,2,6,6-tetramethylpiperidine-1-oxyl)-4-(4-nitrobenzene)
sulfonamide (3): yield 0.17 g (33%), m.p. 174–176 ꢀC. Ir (KBr) nmax
(cmꢁ1) 3208, 3160, 3120, 1524, 1346, 1313, 1165, 860, 740.
1
Because of its paramagnetic nature, adequate H and 13C NMR
spectra of the TEMPO radical 3 are not reported.[17] Further
confirmation of its structure was obtained by X-ray analysis of
its crystals (see Supporting Information).
•
N-(2,2,6,6-tetramethylpiperidine-1-oxyl)-4-toluenesulfonamide
(4): yield 0,49 g (51%), m.p. 126–128 ꢀC. Ir (KBr) nmax (cmꢁ1
)
3192, 1464, 1328, 1159, 1075, 820, 660. Because of its
paramagnetic nature, adequate 1H and 13C NMR spectra of
the TEMPO radical 4 are not reported.[17] Further confirmation
of its structure was obtained by X-ray analysis of its crystals
(see Supporting Information).
Sample preparation and spectral measurements
ESR spectra of compounds 1–5 were recorded in a thermostated
cavity (25ꢂ 0.5 ꢀC) in absence and in presence of surfactant solutions
with different concentrations (20–250 mM) above the critical michelle
concentration (0.24 mM[18]). To a volume of 200 ml of a freshly
prepared buffered solution (pH 7) of reduced Triton-X 100, 10 ml of
an ethanolic solution of the radical probe was added, attaining a
probe concentration of 50 mM. The solution was stirred vigorously
and transferred to a quartz capillary tube placed in a 3-mm ESR
quartz tube before being placed into the spectrometer cavity. The
ESR spectrometer employed automatic frequency control that
ensured the stability of the frequency for each experiment, using a
power of 25 mW, 100 kHz modulation frequency, modulation
amplitude of one-third of the line-width of each nitroxide and a time
constant of 0.01 s. Final spectra were recorded after ten scans.
Reported g-values were obtained by processing the ESR spectra with
the WINEPR software version 2.11b of Bruker, where the g-factor
values for each sample were calculated from the fundamental
Figure 1. (a) Experimental (gray solid line) and simulated (black dashed line)
ESR spectra of probe 5 (QT) in a pH 7 buffer. The simulated spectrum had a
Lorentzian contribution of 0.7, g-factor = 2.00547, aN = 16.73 G. (b) Experimen-
tal (gray solid line) and simulated (black dashed line) spectra of probe 5 in a
pH 7 buffered solution of 20 mM of TXR 100. The simulated spectrum had a
Lorentzian contribution of 0, g-factor = 2.00547, aN = 16.38 G.
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Magn. Reson. Chem. (2012)

Partitioning of nitroxide probes in microheterogeneous media
with a high contribution (0.7) of the Lorentzian component. This
spectrum is compared with the spectrum of 5 incorporated into an
aqueous Triton-X 100 solution (Fig. 1(b)). The corresponding simu-
lated spectrum had no contribution of the Lorentzian component,
with an especially good fitting of the first two lines, reproducing
what had been observed in Fig. 1(a). The fact that a good fitting for
different lines was obtained with the same contribution of the
Lorentzian component was taken as an indication that the obtained
triplets were not superpositions of two signals from different
environments, but rather constituted time-averaged spectra.
Assuming therefore that the equilibrium between probes i in two
different microenvironments was fast enough in the ESR time scale,
the observed g-factor could be expressed as a linear combination
of the gi factors of each probe, affected by their pi fraction in the
system.[19] This allows us to express the observed g-factor of a spin
label in an aqueous micellar solution as the sum of the contributions
in the water and micellar phases according to Eqn (1):
aqueous medium according to the equilibrium (2), between the
probe in the water (w) and in the micelle (m).
Surfactant þ Probew⇌Probem
(2)
The partition constant in the presence of a large excess of
surfactant may then be defined by[20]
½Probemꢅ
K ¼
(3)
(4)
½Probewꢅ½Surfactantꢅ
or expressed in terms of a as
K ¼
a
ð1 ꢁ aÞ½Surfactantꢅ
Substitution of the value of a from Eqn (1) in (4) leads to the
expression (5), where Δg = g ꢁ gw.
ðgm ꢁ gwÞꢄKꢄ½Surfactantꢅ
Δg ¼
(5)
ð1 þ Kꢄ½Surfactantꢅ Þ
g ¼ gmꢄa þ gwꢄð1 ꢁ aÞ
(1)
Plots of Δg against the surfactant concentration [Surfactant],
for a constant concentration of the radical probe, yield curves
that rise with [Surfactant], as the label is partitioned between
water and the micelle, until a maximum value is reached, indicat-
ing its full incorporation into the more hydrophobic domain of
the micelle. Examples of such curves are shown in Fig. 2, for the
TEMPO derivatives 1–5. The partitioning constant K and the
probe g-factor in the surfactant domain (gm) may be obtained
by nonlinear fitting of the data to Eqn (5). Alternatively, if gm is
known, K may be obtained from K = 1/[Surfactant]1/2, where
[Surfactant]1/2 is the surfactant concentration for which
Δg = (gm ꢁ gw)/2. Values of K, gm, gw and Δg = gm ꢁ gw are given
in Table 1. The g-factor values were essentially independent of
the employed probe concentration.
where gw and gm are the g-factors of the radical in water and in
the surfactant domain, respectively, a is the fraction of the probe in
the surfactant domain and (1 ꢁ a) the fraction of the probe in the
In order to validate the method, the obtained value of one of
the K partitioning constants in Table 1 was compared with a
value obtained by a different methodology, from measurements
by fluorescence quenching.[20,21] In addition, comparison of the
method based on g-factor measurements was made with other
classical methods that employ ESR spectral parameters for
determining the partition of spin labels in microheterogeneous
media. Figure 3 reproduces the obtained spectra of QT (probe 5)
in the presence of increasing concentrations of surfactant.
Variations of line intensities and line-width with the surfactant
concentration are plotted in Fig. 4.
Figure 2. Variation of the probe g-factor in the presence of increasing
concentrations of RTX-100 in water, for the TEMPO derivatives 1–5. The
concentration of the radical probes was 50 mM in all cases. Data were fitted
to curves corresponding to Eqn (5).
Table 1. Partitioning constants K, estimated log P values, g values in water (gw) and inside the micelle (gm) for spin labels 1–5 in aqueous solutions
of RTX-100
ꢁ1
Spin label
K (M
)
log Pb
gw valuec
gm valuec
(gm ꢁ gw) 104
1
2
3
4
5
6.3 ꢂ 2.1a,d
15.7 ꢂ3.5a,e (9.9 ꢂ2,5)f
20.0 ꢂ 3.4a,d
0.81
1.78
2.21
3.02
2.92
2.00580
2.00584
2.00578
2.00604
2.00547
2.00589
2.00598
2.00606
2.00608
2.00598
0.9
1.4
2.8
3.4
5.1
28.0 ꢂ 1.8a, e
19.8 ꢂ 1.9a,e
aDetermined by the present methodology.
blog P values estimated with the aid of the ICM software based on fragment contributions.[22]
cRecorded for a constant probe concentration of 50 mM.
dRecorded for a maximum micellar concentration of 200 mM.
eRecorded for a maximum micellar concentration of 250 mM.
fValue between brackets obtained by other methodology, from measurements of fluorescence quenching.[20,21]
Magn. Reson. Chem. (2012)
Copyright © 2012 John Wiley & Sons, Ltd.
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C. Aliaga, P. Torres and F. Silva
Table 2. Values of the partitioning constant of QT (probe 5) in aque-
ous TXR-100 solutions, calculated with the aid of Eqn (6), where P, Pw
and Pm refer to an ESR spectral parameter P
ꢁ1
Parameter P
K (M
)
r2a
1st line intensity
3rd line intensity
3rd line line-width
g-factor
17.0 ꢂ3.0
13.7 ꢂ3.5
18.1 ꢂ9.9
19.8 ꢂ1.9
0.97
0.95
0.81
0.99
aCorrelation coefficient between experimental and fitted values
according to Eqn 6.
Figure 3. Variation of the ESR spectrum of probe 5 (QT, 50 mM) in the
presence of increasing concentrations of RTX-100 (0, 20, 40, 60, 80, 100,
120, 140, 160, 180 and 200 mM) buffered solutions.
very close, showing that the classical methods yield the same
partitioning constant obtained by measurements of g-factors.
Interestingly, the method based on g-factor measurements
yielded the smallest error among all the methods that employ
ESR-based parameters.
An interesting observation from the curves of Fig. 1 is the fact that
the g-factor variations of analogous compounds, when transferred
from water to the micellar core, are not uniform. Although the
variation of (gm ꢁ gw) correlates to a certain extent with K for
compounds 1, 2, 3 and 4, radical probe 5 departs from this trend,
with a partitioning constant reasonably close to that of TEMPO 2
and a g-factor variation three times larger.
The data were fitted to Eqn (6), where P stands for the
variable parameter employed and K is the corresponding
partition constant.
ðPm ꢁ PwÞꢄKꢄ½Surfactantꢅ
ΔP ¼
(6)
ð1 þ Kꢄ½Surfactantꢅ Þ
Partitioning coefficients between water and a more hydropho-
bic environment should in principle reflect differences in the
In Table 2, we list the values and errors obtained in each case
for this particular K constant. It is seen that all of them are
Figure 4. Plots of the variations of various spectral parameters P from ESR spectra of probe 5 (QT) in the presence of increasing concentrations of surfactant
RTX-100. Data were fitted to the general Eqn (6), where the subscritps w and m refer to water and micelle, respectively. The parameter P was (a) the intensity
of the first line of the nitroxide triplet, Pm ꢁ Pw =7.1ꢃ 106, (b) the third line intensity of the nitroxide triplet, Pm ꢁ Pw =2.0ꢃ 106 and (c) the line-width of the third
line of the nitroxide triplet, Pm ꢁ Pw = 1.252.
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Magn. Reson. Chem. (2012)

Partitioning of nitroxide probes in microheterogeneous media
lipophilicities of the probes. This was investigated by estimating
the octanol–water partitioning coefficients (log P) for the series
1–5 with an available software.[22] The results are given in Table 1.
It is seen that the estimated coefficients increase in the order 1
(0.81) < 2(1.78) < 3(2.21) < 4(3.02), following the same order of
the obtained K values. Although the absolute values of log P for
these compounds should be regarded with caution, since their
value is the result of an estimate and a particular method of cal-
culation, their relative order is a more reliable result, pointing to
an increase of their lipophilicity in going from 1 to 4. Compound
5 (2.92) constituted an exception to this, with a K value somewhat
smaller than what might be expected from its estimated lipophi-
licity. This may be due to the presence of acidic and basic groups
in the molecule and the possibility of equilibria in water with
ionic structures, making this TEMPO derivative more hydrophilic
than predicted by a crude log P estimation.
In conclusion, g-factor measurements of spin probes in micellar
solutions may be used to determine partitioning constants, using
a clean and easy alternative to the classical Benesi–Hildebrand
plots based on UV–visible spectroscopic measurements. The probe
g-factor in the presence of increasing micellar concentrations is a
sensitive parameter that is not disturbed by interferences from
other species in the mixture. The g-factor variations are also large
enough to allow a clean and reliable determination of the
corresponding K values. This simple method constitutes a valuable
alternative for the assessement of antioxidant capabilities of phenols
or polyphenols by spin labels.
University of Ottawa for the X-ray diffraction analyses of radicals
3 and 4. We thank Drs Gerald Zapata-Torres and Angélica Fierro
for calculating log P values.
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Acknowledgements
This work was financed by FONDECYT project 1110736. We are
grateful to Drs María Gonzalez-Bejar and Ilia Korobkov of the
Magn. Reson. Chem. (2012)
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