Kinetics of spin trapping superoxide, hydroxyl, and aliphatic radicals by cyclic nitrones

Article abstract of DOI:10.1021/jp048441i

Spin trapping coupled with electron paramagnetic resonance (EPR) spectroscopy has surfaced as one of the most specific and reliable methods for identifying free radicals in biological systems. Despite extensive studies focused on the kinetics of radical trapping by cyclic nitrones, the mechanism has not been fully elucidated. Moreover, major controversies still persist even regarding the efficiency and the rate constants of the trapping reaction. The present research used pulse radiolysis for studying the reaction of 5,5-dimethyl-1-pyrroline N-oxide 1 (DMPO) and of the ester-containing derivative, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide 4, with OH, O₂^-, CO₂ ^-, C(OH)(CH₃)₂, CH₂OH, and CH₃. The results reveal that radical trapping is far more complex then previously realized. Radiation chemical experiments combined with EPR demonstrate that about 30% of OH add to nitrones 1 and 4 at position 2, yielding the corresponding persistent aminoxyls. The remaining OH radicals form transient intermediates that rapidly decay bimolecularly. These transient intermediates react with oxygen with rate constants that are significantly lower than those generally reported for alkyl radicals, which suggests that they are not simple carbon-centered radicals generated as a result of H-abstraction from the methyl or methylene groups of the nitrones. It is also shown that the addition of O₂^- and various aliphatic radicals to the nitrones is an equilibrium process. The upper limit for the rate constant of the reaction of nitrone 4 with O₂ ^- was 3 M^-1 s^-1. The rate constant for the reaction of nitrone 1 with O₂^- was determined to be 170 ± 40 M^-1 s^-1. This value is significantly higher than those previously determined by following the formation of the corresponding aminoxyl by EPR, which indicates that the yield of the aminoxyl is only a small fraction of the reacting O₂^-.

Full text of DOI:10.1021/jp048441i

J. Phys. Chem. A 2004, 108, 6679-6685
6679
Kinetics of Spin Trapping Superoxide, Hydroxyl, and Aliphatic Radicals by Cyclic Nitrones
Sara Goldstein,*,† Gerald M. Rosen,‡ Angelo Russo,§ and Amram Samuni
Department of Physical Chemistry, the Hebrew UniVersity of Jerusalem, Jerusalem 91904, Israel,
Department of Pharmaceutical Sciences, School of Pharmacy, UniVersity of Maryland, Medical Biotechnology
Center, UniVersity of Maryland Biotechnology Institute, and Center for Low Frequency EPR Imaging for
In ViVo Physiology, the UniVersity of Maryland, Baltimore, Maryland 21201, Radiation Biology Branch,
National Cancer Institute, NIH, Bethesda, Maryland 20892, and Department of Molecular Biology,
the Hebrew UniVersity of Jerusalem - Hadassah Medical School, Jerusalem 91120, Israel
ReceiVed: April 8, 2004; In Final Form: June 1, 2004
Spin trapping coupled with electron paramagnetic resonance (EPR) spectroscopy has surfaced as one of the
most specific and reliable methods for identifying free radicals in biological systems. Despite extensive studies
focused on the kinetics of radical trapping by cyclic nitrones, the mechanism has not been fully elucidated.
Moreover, major controversies still persist even regarding the efficiency and the rate constants of the trapping
reaction. The present research used pulse radiolysis for studying the reaction of 5,5-dimethyl-1-pyrroline
N-oxide 1 (DMPO) and of the ester-containing derivative, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide
4, with OH, O2•-, CO2•-, C(OH)(CH3)2, CH2OH, and CH3. The results reveal that radical trapping is far
more complex then previously realized. Radiation chemical experiments combined with EPR demonstrate
that about 30% of •OH add to nitrones 1 and 4 at position 2, yielding the corresponding persistent aminoxyls.
•
•
•
•
•
The remaining OH radicals form transient intermediates that rapidly decay bimolecularly. These transient
intermediates react with oxygen with rate constants that are significantly lower than those generally reported
for alkyl radicals, which suggests that they are not simple carbon-centered radicals generated as a result of
•-
H-abstraction from the methyl or methylene groups of the nitrones. It is also shown that the addition of O2
and various aliphatic radicals to the nitrones is an equilibrium process. The upper limit for the rate constant
•-
of the reaction of nitrone 4 with O2•- was 3 M-1 s-1. The rate constant for the reaction of nitrone 1 with O2
was determined to be 170 ( 40 M-1 s-1. This value is significantly higher than those previously determined
by following the formation of the corresponding aminoxyl by EPR, which indicates that the yield of the
•-
aminoxyl is only a small fraction of the reacting O2
.
Introduction
SCHEME 1
Given the theory that free radicals participate in cell signaling
pathways,1-5 the detection and characterization of these reactive
intermediates is critical to understanding the mechanism of these
physiological processes. Of the available methods for identifying
free radicals in a myriad of biological systems, spin trapping/
EPR spectroscopy has surfaced as one of the most specific and
reliable techniques.6 For instance, spin trapping was essential
in the discovery that nitric oxide synthase (NOS; EC 1.14.13.39)7
generates O2•- 8-16 along with •NO and H2O217-19 during NOS
reduction of O2.
Since the mid-1970s, 5,5-dimethyl-1-pyrroline N-oxide 1
(DMPO) has been the primary spin trap for O2•- and •OH, owing
this standing to the ease of synthesis and to the unique EPR
spectrum of aminoxyl 2 and aminoxyl 3, derived from the reac-
tion of nitrone 1 with O2 and OH, respectively (Scheme 1).
Nitrone 1 is the most frequently used trap; yet, despite
extensive studies focused on the kinetics of radical trapping,
the detailed mechanism has not been fully elucidated. Moreover,
although many potential artifacts associated with spin trapping
have been previously recognized, major controversies still persist
even regarding the efficiency and the rate constants of the
trapping reaction. Concomitantly, new spin traps have been
synthesized in an attempt to answer fundamental questions
pertinent to free radicals in biology. For example, the spin trap
5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide 4, whose
reaction with O2•- affords aminoxyl 5 yields a product having
a more robust EPR spectrum than that of aminoxyl 2,20-22
was critical for understanding how (6R)-5,6,7,8-tetrahydro-L-
•-
•
* To whom all correspondence should addressed.
† Department of Physical Chemistry, the Hebrew University of Jerusalem.
‡ Department of Pharmaceutical Sciences, School of Pharmacy, Univer-
sity of Maryland, Medical Biotechnology Center, University of Maryland
Biotechnology Institute, and Center for Low-Frequency EPR Imaging for
In Vivo Physiology, the University of Maryland.
biopterin (H4B) regulated NOS-generated O2 and H2O2.19
•-
In the present study we have used pulse- and γ-radiolysis
and EPR spectroscopy to study the mechanism of the reactions
of nitrones 1 and 4 with •OH, O2•-, CO2•-, and several aliphatic
radicals.
§ Radiation Biology Branch, National Cancer Institute, NIH.
Department of Molecular Biology, the Hebrew University of Jerusalem
- Hadassah Medical School.
10.1021/jp048441i CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/09/2004

6680 J. Phys. Chem. A, Vol. 108, No. 32, 2004
Goldstein et al.
Experimental Section
Chemicals. All chemicals were of analytical grade and were
used as received. Solutions were prepared with distilled water,
which was further purified using a Milli-Q water purification
system. 5,5-Dimethyl-1-pyroline N-oxide 1 was purchased from
Aldrich. The colored impurity in this commercial product was
removed with charcoal followed by filtration. This preparation
did not demonstrate any EPR signal before the trapping of the
radicals. Moreover, 1H NMR scanning did not detect any
contaminant either. The concentration of nitrone 1 was deter-
mined using ꢀ228 ) 8000 M-1 cm-1. 5-tert-Butoxycarbonyl-5-
methyl-1-pyroline N-oxide 4 was synthesized and purified as
previously described21,22 and showed no spurious EPR spectra.
Trace contaminants can lead to side reactions and erroneous
conclusions. This is true in the common spin-trapping experi-
ments where relatively high concentrations of nitrones are
applied (at the tens and hundreds millimolar range). Moreover,
since in the present study the concentration of the nitrones did
not exceed 4 mM, potential residual contaminants are not
anticipated to affect the reaction kinetics.
Figure 1. Competition kinetics using phenylalanine as a reference
solute. The yield of phenylalanine-OH adduct was measured upon
pulse irradiation of N2O-saturated solution containing nitrone 1 and
100 µM (b) or 300 µM (9) phenylalanine at pH 7.8 or nitrone 4 and
300 µM phenylalanine (4) at pH 7.4. All solutions contained 4 mM
PB, and the absorption was measured at 325 nm in the absence (Ao)
and presence (A) of various concentrations of the nitrone.
Pulse Radiolysis. Pulse radiolysis experiments were carried
out with a Varian 7715 linear accelerator delivering 5-MeV
electron pulses of 0.2-1.5 µs and 200 mA. All measurements
were made at ambient temperature using a 4-cm Spectrosil cell
applying three light passes (optical path length 12.1 cm). Each
measurement was repeated at least five times. Appropriate cutoff
filters were used to eliminate photolysis. Dosimetry was
performed with an oxygenated solution containing 5 mM
ferrocyanide using G(Fe(CN)63-)) ) 2.7 × 10-7 M Gy-1 and
Nitrone 1 or nitrone 4 (e2 mM) efficiently scavenges
•OH23-28 and H• radicals.26,27
nitrone + •OH f HO-aminoxyl• + products
nitrone + H• f H-aminoxyl• + products
(3)
(4)
The published values for k3 varied between 1.9 × 109 and 4.3
× 109 M-1 s-1 23-28
,
whereas the reports concerning the yield
of aminoxyl 3 are more controversial, namely 33-35%29,30 in
ꢀ420(Fe(CN)63-) ) 1000 M-1 cm-1
.
contrast to 94%.27,31
Steady-State γ-Irradiation. γ-Irradiation was carried out at
room temperature using either a 137Cs source with a dose rate
of 9 Gy min-1 or a 60Co source with a dose rate of 37 Gy min-1
as determined by Fricke dosimetry.
We determined the rate constant of reaction 3 using competi-
tion kinetics against the reaction of •OH with phenylalanine (PA)
as a reference solute.
Electron Paramagnetic Resonance (EPR) Measurements.
After irradiation, the samples were immediately frozen in liquid
nitrogen to prevent spin adduct decay. The frozen samples were
thawed just before recording their EPR spectra. To determine
aminoxyl concentration, samples were injected through a narrow
Teflon capillary inserted into a quartz tube that is placed within
the EPR spectrometer cavity. The EPR spectra were recorded
using JEOL JES-RE3X ESR spectrometer operating at X band
with center field set at 3290 G, 100 kHz modulation frequency,
1 G modulation amplitude, and 4 mW incident microwave
power. The concentration of the nitrone spin adduct was
calculated from the EPR signal using a standard solution of
2,2,6,6-tetramethyl-1-piperidinyloxyl and 4-hydroxy-2,2,6,6-
tetramethyl-1- piperidinyloxyl.
•OH + PA f HO-PA• k5 ) 6.5 × 109 M-1 s-1 32 (5)
The yield of HO-PA• was determined by monitoring the
absorbance at 325 nm immediately after pulse irradiation of
N2O-saturated solutions containing 100 or 300 µM PA and 4
mM phosphate buffer (PB), pH 7.4-7.8, in the absence (Ao)
and in the presence (A) of various nitrone concentrations. When
only reactions 3 and 5 take place, eq 6 describes the dependence
of the yield of HO-PA• on [nitrone]o and [PA]o:
A ) Aok5[PA]o/(k3[nitrone]o + k5[PA]o)
(6)
The plot of (Ao/A - 1) vs [nitrone]o/[PA]o was linear (Figure
1), and from the slope of the lines we calculated k3 ) (3.2 (
0.1) × 109 and (2.5 ( 0.1) × 109 M-1 s-1 for nitrone 1 and
nitrone 4, respectively. The value of k3 determined for nitrone
1 closely agrees with some of the literature values;23,25,27,33
whereas, k3 of for nitrone 4 is about half of that previously
Results and Discussion
•
Reaction of Nitrones with OH. Irradiation of aqueous
solutions at pH > 3 saturated with N2O (25 mM) produces ca.
•
90% OH and 10% H•.
•
determined using radiolytically formed OH.28
For a direct investigation of the kinetics of •OH reaction with
nitrones 1 and 4, we pulse irradiated solutions containing 0.15-2
mM nitrone and 4 mM PB (pH 7.5-7.8), which were saturated
either with N2O or with various mixtures of N2O and O2. A
rapid first-order formation of a transient with a maximum near
270 nm was observed for both nitrones in the absence and
presence of O2. In the absence of O2, this transient absorption
decayed via a second-order reaction, leaving a residual persistent
absorption as demonstrated for nitrone 1 in Figure 2. The
observed second-order rate constant and the extent of the
residual persistent absorption did not depend on the initial
H2O
9
γ8 e-aq (2.6), •OH (2.7), H• (0.6), H2 (0.45),
H2O2 (0.7), H3O+ (2.6) (1)
eaq- + N2O + H2O f •OH + N2 + OH-
k2 ) 9.1 × 109 M-1 s-1 (2)
The numbers in parentheses are G-values, which represent the
yields of the various species in 10-7 M Gy-1, and are about
7% higher in the presence of high solute concentrations.

Kinetics of Spin Trapping Radicals by Cyclic Nitrones
J. Phys. Chem. A, Vol. 108, No. 32, 2004 6681
Figure 2. Reaction of •OH with nitrone 1 in the absence and presence
of O2. Typical kinetic traces obtained upon pulse irradiation (13.4 Gy)
of solutions containing 300 µM nitrone 1 and 4mM PB at pH 7.5
saturated with N2O (A) or a mixture of 80% N2O and 20% O2 (B).
The apparent optical path length was 12.1 cm.
Figure 4. Dependence of the observed pseudo-first-order rate constant
of decay of the transient absorption on [O2]. The solutions containing
(9) 300 µM nitrone 1 or (O) nitrone 4 in 4 mM PB at pH 7.5 and
saturated with various mixtures of N2O and O2 were pulse irradiated
•
applying a dose of 14.5 Gy/pulse (7.7 µM OH).
TABLE 1: Summary of All the Rate Constants Determined
•
in the OH System
reaction
nitrone 1
nitrone 4
k3, M-1 s-1
k7, M-1 s-1
k8, M-1 s-1
(3.3 ( 0.1) × 109
(7.5 ( 0.4) × 107
(6.1 ( 0.5) × 106
(2.5 ( 0.1) × 109
(1.2 ( 0.1) × 108
(1.9 ( 0.6) × 106
and 6.24 Considering the total [•OH] formed by the radiation
and the concentration of the accumulated aminoxyls 3 and 6,
we calculated their yields as 26 ( 6% and 33 ( 5%,
respectively. These results suggest that about 30% of •OH add
to nitrones 1 or 4 at position 2, yielding the persistent aminoxyls
3 or 6, respectively. From the knowledge of the yields of
nitrone•, i.e., ca. 70%, we calculated ∆ꢀ270, which allowed us
to evaluate the second-order rate constant for reaction 7 as k7
) (7.5 ( 0.4) × 107 and (1.2 ( 0.1) × 108 M-1 s-1 for nitrone
1 and nitrone 4, respectively. Assuming that the products of
reaction 7 have no appreciable absorption at λ > 250 nm, the
residual absorption spectra shown in Figure 3 are attributable
to aminoxyls 3 and 6.
Figure 3. Difference absorption spectra between irradiated and
nonirradiated anoxic solutions containing 300 µM nitrone 4 (dashed
line) or nitrone 1 (solid line) in 4 mM PB at pH 7.5. The spectra of the
irradiated samples were recorded within 1 min after the pulse. The dose
•
was 14.5 Gy (7.7 µM OH), and the optical path length was 4 cm.
concentration of the nitrone between 0.15 and 2 mM. These
observations indicate, contrary to the conclusion of Neta et al.,24
who attributed the absorption with the maximum at 270 nm to
the formation of the persistent aminoxyl 3 alone, that the reaction
In the presence of oxygen the rate of the decay of the transient
absorption was enhanced and turned from second- to first-order
kinetics as demonstrated for nitrone 1 in Figure 2.
•
of OH with the nitrones yields at least two distinct species.
nitrone• + O2 f products
(8)
The transient absorption reflects the formation of some short-
lived nitrone radical (denoted nitrone•) via reaction 3, whereas
the accumulated aminoxyl 3 (or aminoxyl 6) is responsible for
the residual persistent absorption.
The observed first-order rate constant increased upon increasing
[O2] (Figure 4), and from the slope of the linear plots we
determined k8 ) (6.1 ( 0.5) × 106 and (1.9 ( 0.7) × 106 M-1
s-1 for nitrone 1 and nitrone 4, respectively.
nitrone• + nitrone• f products
(7)
All rate constants determined in the present study are
summarized in Table 1. The values of k7 and k8 are significantly
lower than those generally reported for alkyl radicals. These
observations suggest that the relatively short-lived nitrone• is
not a simple carbon-centered radical. This conclusion does not
agree with that of Carmichael et al.29,30 who assumed that •OH
radicals that do not add to the carbon of the NdC bond react
with the nitrone by H-abstraction from its methyl or methylene
group. Instead, •OH might add to the nitrogen of the NdC bond,
and nitrone• might represent a delocalized radical, e.g., an allylic
radical, which is generally less reactive than an alkyl radical
toward O2.
The analysis of the kinetics of the second-order decay yields
k7/∆ꢀ270l ) (3.6 ( 0.3) × 103 and (5.6 ( 0.5) × 103 s-1 for
nitrone 1 and nitrone 4, respectively.
The UV-vis spectrum of the residual persistent absorption
of the pulse-irradiated anoxic solution, which was monitored
using a diode array spectrophotometer within less than 45 s after
the pulse, was stable for at least 15 min. This spectrum was
compared with that of the nonirradiated sample, and the
difference absorption spectra monitored for nitrones 1 and 4
are shown in Figure 3.
The EPR spectra were recorded following γ-irradiation (4.5-
37 Gy) or pulse-irradiation (14.5 Gy/pulse) of 2 mM nitrone in
N2O-saturated solution at pH 7.5. The observed EPR signal
exhibited the well-known characteristic signals of aminoxyls 3
•
•
Reactions of Nitrones with CO2•-, CH3, CH2OH, and
•C(OH)(CH3)2. Employing pulse irradiation of N2O-saturated
solutions (4 mM PB at pH 7.4-7.8) containing 0.1 M formate,

6682 J. Phys. Chem. A, Vol. 108, No. 32, 2004
Goldstein et al.
The results are summarized in Table 2, and demonstrate that
in the case of nitrone 4, though not nitrone 1, most or even all
R• add at position 2 of the nitrone, yielding R-aminoxyl•
(reaction 9).
nitrone + R• h R-aminoxyl•
(9)
The reaction of the nitrone with R• forms also other species
(reaction 10). In the case of nitrone 1, though not nitrone 4, the
contribution of reaction 10 is quite significant.
nitrone + R• f products
(10)
Since both reactions 9 and 10 contribute to the loss of R•, the
rate constant that characterizes its decay as well as the formation
of the persistent R-aminoxyl• equals (k9 + k10), which were
evaluated from the slopes of the lines displayed in Figures 5
and 6. The results are summarized in Table 2 and compared to
Figure 5. Reaction of nitrone 1 with R•. The observed first-order rate
constant of the decay of the absorption formed following pulse
irradiation of N2O-saturated solutions containing 0.1 M formate (b),
0.2 M ethanol (9), or 0.2 M 2-propanol (2). All solutions contained 4
mM PB at pH 7.5-7.9, the dose was 9-16 Gy, and the decay of the
absorption was followed at 260-280 nm.
the values previously reported.
•-
Reaction of Nitrones with Superoxide Radicals (O2
/
•
HO2 ). The reaction of superoxide radicals with cyclic nitrones
has been extensively studied in the past. There are a few cases,
e.g., nitrones 1 and 4, where it was shown that the efficiency
•
of spin trapping of HO2 is far greater than that of O2•-, i.e.,
k11 . k12.22,23,34
nitrone + HO2 f HOO-aminoxyl•
(11)
(12)
•
nitrone + O2•- + H+ f HOO-aminoxyl•
Finkelstein et al.23 have studied the reaction of superoxide
radicals with nitrone 1 at pH 5-9 using competition kinetics
against superoxide dismutase (SOD), and a reasonable good fit
to the experimental data was obtained for k11 ) 6.6 × 103 M-1
s-1 and k12 ) 10 M-1 s-1. Nevertheless, major disagreements
still exist regarding the kinetics of this reaction. Obviously the
apparent rate constant, kapp, greatly depends on the pH; yet, the
values measured at pH 7.2-7.8 vary between 1.2 and 50 M-1
Figure 6. Reaction of nitrone 4 with R•. The observed first-order rate
constant of the decay of the absorption formed following pulse
irradiation of N2O-saturated solutions containing 0.1 M formate (b),
0.1 M DMSO (1), 0.2 M ethanol (9), or 0.1 M 2-propanol (2). All
solutions contained 4 mM PB at pH 7.4-7.5, the dose was 17 Gy, and
the decay of the absorption was followed at 290-310 nm.
s-1 23,35-40
In the case of nitrone 4, kapp was determined to be
.
239 M-1 s-1 at pH 5,21,22 0.24-77 M-1 s-1 at pH 7.0-7.422,38
and ca. 5 M-1 s-1 at pH 10 (competition kinetics against Cu/
Zn-SOD, unpublished results).
Pulse radiolysis enables the generation of superoxide radicals
within less than 1 µs, and hence allows direct study of the effect
of a tested molecule on their decay. This method has not yet
been employed for studying the reaction of nitrones with
superoxide radicals, which decay according to the well-
established mechanism described by reactions 13-15.
0.1 M DMSO, and 0.2 M ethanol or 0.1-0.2 M 2-propanol,
•
•
the reactions of nitrones 1 and 4 with CO2•-, CH3, CH2OH,
•
or C(OH)(CH3)2 (denoted as R•) were studied. Under such
experimental conditions all the primary radicals formed by the
radiation were converted into R•, which absorbs at 260-290
nm. In the absence of the nitrones, R• disappears by a second-
order kinetics,32 whereas in their presence the rate of its
decay increased and changed from second- to first-order kinetics.
The transient absorption resulting from R• did not decay to
zero but rather left a residual persistent absorption with a
maximum near 260 nm. The observed pseudo-first-order rate
constant was linearly dependent on [nitrone]o, demonstrating,
particularly in the case of nitrone 4, relatively high intercepts
(Figures 5 and 6). This suggests that the reaction of R• with
the nitrone is an equilibrium process. The knowledge of the
radiation dose, i.e., the yield of R•, combined with the
determination by EPR of the accumulation of R-aminoxyl•
allowed us to calculate the fractional yield of R-aminoxyl•.
Nitrone at 2 mM was used to minimize the competition between
the reaction of R• with the nitrones and the self-decomposition
of R•. In the case of nitrone 1 we were unable to study
•
•
HO2 + HO2 f O2 + H2O2
k13 ) 8.3 × 105 M-1 s-1 41 (13)
O2•- + HO2 + H+ f O2 + H2O2
•
k14 ) 9.7 × 107 M-1 s-1 41 (14)
HO2 h H+ + O2
pKa ) 4.841
(15)
•
•-
Because the dismutation of superoxide at physiological pH is
relatively fast, its slow reaction with nitrones can be studied
only at pH range where k12[nitrone]o . (k14/Ka) [H+][O2•-]o,
i.e., alkaline solutions.
In the present study, superoxide radicals were generated by
pulse-irradiation of solutions saturated with 50% N2O (12.5 mM)
and 50% O2 (0.6 mM), which contained 0.5 M 2-propanol, 25
µM DTPA, and 5 mM carbonate at pH 9.6-12.1. Under these
•
spectrophotometrically its reaction with CH3, which absorbs
too little at λ > 260 nm.

Kinetics of Spin Trapping Radicals by Cyclic Nitrones
J. Phys. Chem. A, Vol. 108, No. 32, 2004 6683
nitrone 4
TABLE 2: Summary of All the Rate Constants and Yields
nitrone 1
k, M-1 s-1
yield, %
k, M-1 s-1
yield, %
•OH
(3.3 ( 0.1) × 109 a
3.2 × 109 23
2.8 × 109 27
2.6 × 109 25
1.9 × 109 28
4.3 × 109 26
n.d.
26 ( 6a
(2.5 ( 0.1) × 109 a
33 ( 5a
9427
(4.5 ( 0.3) × 109 28
3529
3330
•CH3
63 ( 7a
86 ( 10a
73 ( 7a
(2.8 ( 0.2) × 107 a
(5.4 ( 0.2) × 107 a
(1.4 ( 0.1) × 108 a
n.d.
1.4 × 107 33
(4.4 ( 0.4) × 107 a
2.2 × 107 33
(6.6 ( 0.3) × 107 a
6.8 × 107 33
1.5 × 108 26
(1.15 ( 0.05) × 108 a
6.6 × 107 33
7.5 × 108 26
170 ( 40a
•CH2OH
•C(OH)(CH3)2
86 ( 7a
96 ( 5a
•-
CO2
55 ( 10a
(1.8 ( 0.1) × 108 a
95 ( 5a
•-
O2
n.d.
<3a
n.d.
5038
75 ( 1022
738
1023
2.439
0.5339
2.0 ( 0.240
1.337
a This study. The yields were determined upon γ-irradiation of N2O-saturated solutions containing 2 mM nitrone and 4 mM PB (pH 7.4) in the
absence and presence of 0.1 formate or 0.1 M DMSO or 0.5 M 2-propanol or 0.5 M methanol. The dose was 4.5-9 Gy.
conditions all the primary free radicals formed by the radiation
•-
are converted into O2 through reactions 1, 2, 16-19.
•OH + (CH3)2CHOH f •C(OH)(CH3)2 + H2O
k16 ) 1.9 × 109 M-1 s-1 32 (16)
H• + (CH3)2CHOH f •C(OH)(CH3)2 + H2
k17 ) 7.4 × 107 M-1 s-1 32 (17)
•C(OH)(CH3)2 + O2 f (CH2)2C(OH)OO•
k18 ) 4.2 × 109 M-1 s-1 32 (18)
•-
(CH2)2C(OH)OO•+OH- f (CH3)2CdO + O2•- + H2O
Figure 7. Reaction of O2 with nitrone 1. Typical kinetic traces
obtained upon pulse-irradiation of solutions saturated with 50% N2O
and 50% O2 that contained 0.5 M 2-propanol, 25 µM DTPA, and 5
mM carbonate at pH 10.6 without (trace A) and with 4 mM nitrone 1
k19 ) 5 × 109 M-1 s-1 32 (19)
H• + O2 f O2•- + H+ k20 ) 1.2 × 1010 M-1 s-1 32 (20)
(trace B). The dose was 11 Gy, and the apparent optical path length
was 12.1 cm.
Although the maximum absorption of O2•- is at 250 nm,32 the
absorption changes associated with its formation and decay were
followed at λ ) 270-290 nm due to the relatively high
order kinetics, and kobs increased with increasing [nitrone 1] as
demonstrated in Figure 8. The relatively high intercept seen in
•-
Figure 8 suggests that the addition of O2 to nitrone 1 is an
absorption of the nitrones at lower wavelengths. The decay of
equilibrium process (eq 21).
ca. 4 µM O2•- at pH 10 obeyed second-order kinetics with k )
(1.9 ( 0.2) × 103 M-1 s-1, and was hardly affected by the
presence of 0.5 mM nitrone 4. Under our experimental condi-
tions •C(OH)(CH3)2 reacts with O2 ca. 36-times faster than with
nitrone 4 (Table 2), and about 97% of all the radiolytically
formed primary radicals are converted into O2•-. Taking into
account that 20% acceleration of the rate of O2•- decay would
be detectable, the failure of nitrone 4 at 0.5 mM to affect the
decay kinetics of O2•- implies an upper limit of 3 M-1 s-1 for
k12.
nitrone 1 + O2•- h -OO-aminoxyl•
(21)
-OO-aminoxyl• + H+ h HOO-aminoxyl• (aminoxyl 2) (22)
Hence, kobs ) k21[nitrone 1] + k-21, and from the slope of the
line in Figure 8, we obtained k21 ) 170 ( 40 M-1 s-1
.
The pKa of aminoxyl 2 has not yet been determined but is
expected to be close to that of HOOH, namely 11.9. Therefore,
drawing the values of kobs obtained at pH > pKa as a function
of [nitrone 1] should yield a straight line with an intercept equal
to k-21. This intercept is expected to decrease at pH close to
and below the pKa of aminoxyl 2. The effect of the pH on the
rate of the faster decay was studied in the presence of 1.8 mM
nitrone 1. We measured kobs ) 1.3 ( 0.1 s-1 at pH 12.1 and
11.1, 0.9 ( 0.1 s-1 at pH 10.1 and 0.6 ( 0.1 s-1 at pH 9.6.
These results suggest that the pKa of aminoxyl 2 is close to 10.
In the presence of nitrone 1 at pH 9.6-12.1, the absorption
decayed via two sequential processes. Typical kinetic traces at
pH 10.6 in the absence and presence of 4 mM nitrone 1 are
shown in Figure 7. Under these experimental conditions ca. 90%
of all the primary radicals formed by the radiation are converted
into O2•-. The contribution of the first process increased with
increasing nitrone 1 concentration. This process obeyed first-

6684 J. Phys. Chem. A, Vol. 108, No. 32, 2004
Goldstein et al.
is equal to that of the formation of aminoxyl 2 or X, i.e., the
rate constant of the formation of aminoxyl 2 is the same as
•-
that of O2 decay. In the present study, we followed the
•-
absorption changes associated with O2 disappearance and
aminoxyl 2 formation and obtained (k21a + k21b). However, in
previous studies the differential rate equation (eq 24) for
aminoxyl 2 served for kinetics analysis using the concentration
of the nitrone and the initial rate of aminoxyl 2 accumulation,
which was followed by EPR. Such an analysis does not yield
(k21a + k21b) but rather k21a. Hence, the yield of aminoxyl 2
•-
does not exceed 30% of O2 reacting with nitrone 1, i.e., 50/
170. Such an analysis could be further complicated if O2•- reacts
with the persistent aminoxyl 2 and affect its accumulation. This
reaction is also supported by our recent observations where we
Figure 8. Dependence of kobs of the fast first-order decay of the
absorption at 270 nm on [nitrone 1]o (trace B, Figure 7). All solutions
contained 0.5 M 2-propanol, 25 µM DTPA, and 5 mM carbonate at
pH 10.6 and were saturated with N2O/O2 (1:1). The dose was 9 Gy,
and the optical path length was 12.1 cm.
•
showed that HO2 reacts with various aminoxyls much faster
than with nitrones.44 Consequently the analysis of the kinetics
of the accumulation of aminoxyls becomes even more complex,
which leaves pulse radiolysis solely as a valid tool for
determination of accurate rate constants.
The half-life of the slower decay decreased upon increasing
[nitrone 1], and at [nitrone 1] > 1 mM the decay obeyed first-
order kinetics. The observed rate constant increased somewhat
Conclusions
•
The rate constants for the reaction of nitrone 1 with OH,
upon increasing [nitrone 1] and decreasing the pH, e.g., kobs
)
•
•
CO2•-, C(OH)(CH3)2, and CH2OH were determined directly
to be (3.3 ( 0.1) × 109, (1.15 ( 0.05) × 108, (6.6 ( 0.3) ×
107, and (4.4 ( 0.4) × 107 M-1 s-1, respectively. The rate
constants for the reaction of nitrone 4 with •OH, CO2•-, •C(OH)-
0.018 ( 0.002 s-1 and 0.011 ( 0.001 s-1 in the presence of 4
mM nitrone 1 at pH 10 and 10.6, respectively, and 0.006 (
0.001 s-1 in the presence of 1.8 mM nitrone 1 at pH 11.1. The
analysis of these data is complex because the UV-vis spectra
of aminoxyl 2 and its anion and the half-life of the anion are
not known. Nevertheless, the measured value in the presence
of 4 mM nitrone 1 at pH 10 should approximate the reported
values for the half-life of aminoxyl 2 in neutral pH, i.e., k )
•
•
(CH3)2, CH2OH, and CH3 were determined to be (2.5 ( 0.1)
× 109, (1.8 ( 0.1) × 108, (1.4 ( 0.1) × 108, (5.4 ( 0.2) ×
107, and (2.8 ( 0.2) × 107 M-1 s-1, respectively. About 30%
•
of OH radicals add to nitrones 1 and 4 at position 2, yielding
•
the corresponding persistent aminoxyls. The remaining OH
0.014 s-1 42,43
9, i.e., k ) 0.026 s-1 42
,
and even more that previously measured at pH
radicals form transient intermediates that decay bimolecularly.
These transient intermediates react with oxygen with rate
constants that are significantly lower than those generally
reported for alkyl radicals. This suggests that these transient
intermediates are not simple carbon-centered radicals resulting
upon H-abstraction from the methyl or methylene groups of
the nitrones, but more likely delocalized radicals resulting from
.
Under our experimental conditions in the absence of radio-
lytically formed •OH radicals, we never detected the EPR signal
characteristic of aminoxyl 3 or 6 following the reaction of
superoxide radicals with the nitrones. This was true whether
superoxide radicals were produced by pulse- or γ-irradiation.
Hence, our results do not support the assumption frequently
made that the decomposition of aminoxyl 2 yields aminoxyl
3.23,35
•
the addition of OH to the nitrogen of the NdC bond that
subsequently yields an allylic radical. The failure of nitrone 4
at 0.5 mM to affect the decay kinetics of O2•- at pH 10 implies
an upper limit of 3 M-1 s-1 for this reaction. The rate constant
Assuming, as our results suggest, that the pKa of aminoxyl 2
is close to 10, the measured rate constant for the reaction of
•-
for the reaction of nitrone 1 with O2 has been determined to
•-
O2 with nitrone 1 at pH 7.4-7.8 should be close to k21
)
be 170 ( 40 M-1 s-1. This value is significantly higher than
those previously determined by following the formation of
aminoxyl 2 by EPR, which indicates that the yield of aminoxyl
2 is only a small fraction of O2•- radicals that react with nitrone
1.
170 ( 40 M-1 s-1. The latter value is considerably higher than
those previously reported, i.e., 1.2 and 50 M-1 s-1 23,35-40
.
This
apparent discrepancy can be explained as follows: As presented
•
above for the reaction of nitrone 1 with OH or R•, only a
fraction of the radicals yields the corresponding aminoxyls. The
situation is, most likely, the same for O2•-, where only part of
the radicals yield aminoxyl 2 and the rest give rise to different
species (denoted X):
Acknowledgment. This research was supported by grants
from the U.S.-Israel Binational Science Foundation (No.
2002013) and from the National Institutes of Health, EB-2034
(G.M.R.).
nitrone 1 + O2•- f aminoxyl 2
nitrone 1 + O2•- f X
k21a
k21b
(21a)
(21b)
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