Spin-trapping reactions of a novel gauchetype radical trapper G-CYPMPO

Article abstract of DOI:10.1021/ac2023926

Chemical reactions of a novel gauchetype spin trap, G-CYPMPO (sc-5-(5,5-dimethyl-2-oxo-1,3,2-dioxaphosphinan-2-yl)-5-methy-1-pyrroline N-oxide, O1-P1-C6-N1 torsion angle = 52.8°), with reactive oxygen species were examined by pulse radiolysis technique with 35 MeV electron beam and by electron spin resonance spectroscopy after ⁶⁰Co γ-ray irradiation. The spin-trapping reaction rate constants of G-CYPMPO toward the hydroxyl radical and the hydrated electron were estimated to be (4.2 ± 0.1) × 10⁹ and (11.8 ± 0.2) × 10⁹ M^-1s^-1, respectively. Half-lives of the spin adducts, hydroxyl radical, and perhydroxyl radical adducted G-CYPMPO were estimated to be ～35 and ～90 min, respectively. A comparison of the results with earlier reports using different radical sources suggests that the purity of the solution and/or the radical generation technique may influence the stability of the spin adducts.

Full text of DOI:10.1021/ac2023926

ARTICLE
pubs.acs.org/ac
Spin-Trapping Reactions of a Novel Gauchetype Radical Trapper
G-CYPMPO
Toshitaka Oka,*,† Shinichi Yamashita,‡ Masamichi Midorikawa,z Seiichi Saiki,‡ Yusa Muroya,§
Masato Kamibayashi,|| Masayuki Yamashita,|| Kazunori Anzai,^ and Yosuke Katsumura*,z
†Advanced Science Research Center, Japan Atomic Energy Agency (JAEA), Tokai-mura, Ibaraki 319ꢀ1195, Japan
‡Quantum Beam Science Directorate, Japan Atomic Energy Agency (JAEA), Takasaki, Gunma 370ꢀ1292, Japan
zSchool of Engineering, The University of Tokyo, Bunkyo, Tokyo 113ꢀ8656, Japan
§School of Engineering, The University of Tokyo, Tokai-mura, Ibaraki 319ꢀ1188, Japan
Pharmaceutical Manufacturing Chemistry, Kyoto Pharmaceutical University, Kyoto, Kyoto 607ꢀ8414, Japan
^School of Pharmaceutical Sciences, Nihon Pharmaceutical University, Kitaadachi, Saitama 362ꢀ0806, Japan
ABSTRACT: Chemical reactions of a novel gauchetype
spin trap, G-CYPMPO (sc-5-(5,5-dimethyl-2-oxo-1,3,2-dioxapho-
sphinan-2-yl)-5-methy-1-pyrroline N-oxide, O1ꢀP1ꢀC6ꢀN1
torsion angle = 52.8ꢀ), with reactive oxygen species were
examined by pulse radiolysis technique with 35 MeV electron
beam and by electron spin resonance spectroscopy after 60Co
γ-ray irradiation. The spin-trapping reaction rate constants of
G-CYPMPO toward the hydroxyl radical and the hydrated
electron were estimated to be (4.2 ( 0.1) ꢁ 109 and (11.8 (
0.2) ꢁ 109 Mꢀ1sꢀ1, respectively. Half-lives of the spin adducts, hydroxyl radical, and perhydroxyl radical adducted G-CYPMPO
were estimated to be ∼35 and ∼90 min, respectively. A comparison of the results with earlier reports using different radical sources
suggests that the purity of the solution and/or the radical generation technique may influence the stability of the spin adducts.
eactive oxygen species (ROS), such as the hydroxyl radical
(•OH), the superoxide anion radical (O•2ꢀ), and the perhy-
development of free-radical impurities under ambient condi-
tions, and short lifetime of O•2ꢀ adduct.14
R
droxyl radical (HO•2), are well-known to play a crucial role in
biological damages such as DNA strand breaks, heart attack,
cancer, ischemia-reperfusion injury, and so on.1ꢀ4 The detection
and quantification of these oxygen-derived free radicals in
biological systems is very important for an accurate evaluation
and understanding of these diseases.5ꢀ7
To overcome these drawbacks, a cyclic DEPMPO (5-diethox-
yphosphoryl-5-methyl-1-pyrroline N-oxide)-type novel spin
trap, CYPMPO (CAS No. 934182-09-9) was obtained as a
slightly hydroscopic colorless crystalline compound (mp 126 ꢀC)
that was determined to be an antitype conformation14 and was
evaluated for spin-trapping capabilities.14,15 The conformation
of CYPMPO was determined to be an antitype, in which the
O1ꢀP1ꢀC6ꢀN1 torsion angle of CYPMPO was 164.31ꢀ.
Recently, one of the authors, Kamibayashi, successfully synthe-
sized a nonhygroscopic new spin trap, G-CYPMPO (sc-5-(5,5-
dimethyl-2-oxo-1,3,2-dioxaphosphinan-2-yl)-5-methy-1-pyrroline
N-oxide) which has a gauche conformation as shown in
Figure 1a. Kitamura et al. reported that G-CYPMPO attenuates
the neuro-degeneration in a rat Parkinson’s disease model.16
Moreover, G-CYPMPO aqueous solution can be stored under
ambient conditions for at least one month without any develop-
ment of free-radical impurities. These results suggest that
G-CYPMPO is a useful tool in biochemical research, better than
either DMPO or DEPMPO, and widespread research using G-CY-
PMPO is expected. However, details of the chemical reaction
These radicals can be produced not only by an enzymatic
reaction in a biological system, but also in water radiolysis. The
estimation of these radical productions in water radiolysis is also
important, because it can offer useful information for ion-beam
radiation therapy. Baldacchino et al. determined the yield of •OH
using a fluorescent probe,8 and LaVerne et al. estimated the yield
of HO•2 from water radiolysis by heavy ion-beams.9 Meesung-
noen et al. calculated the yields and concentrations of O2 in ion-
beam tracks by Monte Carlo simulation.10
One of the most sensitive and definitive methods to detect
these free radicals is a spin-trapping method utilizing electron
spin resonance (ESR) spectroscopy.11 With this technique, very
short-lived oxygen derived radicals (•OH, O2•ꢀ, HO2•, etc.) react
with a spin trap and form rather long-lived radicals. DMPO (5,5-
dimethyl-1-pyrroline N-oxide) has been most widely used as a
spin trap to identify and quantify the oxygen-derived free
radicals,12,13 but it has several drawbacks, such as handling
difficulties due to its low melting point (35 ꢀC), ready
Received: September 9, 2011
Accepted: October 19, 2011
Published: October 19, 2011
r
2011 American Chemical Society
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|

Analytical Chemistry
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Table 1. O1ꢀP1ꢀC6ꢀN1 Torsion Angle and Wavenumber
of Specific IR Peak for A-CYPMPO and G-CYPMPO
spin trap
torsion angle/ꢀ
IR (KBr)/cmꢀ1
G-CYPMPO
A-CYPMPO
52.8
1579.6
1573.8
164.57
Figure 1. X-ray structure analysis of (a) a novel gauchetype spin-trap
G-CYPMPO and (b) antitype A-CYPMPO. The torsion angle was
determined from the O1ꢀP1ꢀC6ꢀN1 angle.
Figure 3. Transient absorption spectra of G-CYPMPOꢀOH. ΔAbs.
increases with increasing time from irradiation. Inset shows the chemical
structure of G-CYPMPOꢀOH.
•
scavenger for OH19 in pulse radiolysis experiment and ESR
Figure 2. Chemical structures of (a) G-CYPMPO and A-CYPMPO
and (b) DMPO.
measurement, respectively.
Electron Pulse Radiolysis. Pulse radiolysis experiments were
conducted at LINAC, the electron linear accelerator at the
Nuclear Professional School, University of Tokyo. The energy
and pulse duration of the electron beam were 35 MeV and 10 ns,
respectively. The average dose per pulse was 10ꢀ45 Gy, which
was measured with an N2O-saturated 10 mM KSCN aqueous
solution.18ꢀ20 The apparatus has been described in a previous
article.21 G-CYPMPO aqueous solution in a quartz cell with a 20-
mm optical path was irradiated at room temperature (ca. 20 ꢀC).
γ-ray Irradiation and an ESR Measurement. G-CYPMPO
aqueous solutions were irradiated with γ-rays from the 60Co
γ-ray source at the University of Tokyo. Dosimetry was done
preliminarily with the Fricke dosimeter; the dose rate was ca. 7
Gy/min, and the dose was set to 200 Gy. After irradiation, the
solutions were immediately transferred from a glass tube irradia-
tion cell to a quartz ESR flat cell and placed into the ESR cavity.
ESR measurements were performed utilizing a Model JES-RE2X
ESR spectrometer (JEOL, Tokyo, Japan) with a microwave
power of 1 mW, a modulation width of 0.1 mT, a time constant
of 0.1 s, a sweep time of 2ꢀ15 min, and the peak-to-peak
intensities of the spin adducts were observed. All the irradia-
tion and measurement were carried out at room temperature
(ca. 25 ꢀC).
between G-CYPMPO and the oxygen-derived free radicals are less
understood. In the present paper, we report rate constants of
G-CYPMPO toward reactive species and lifetime of spin-adducꢀted
G-CYPMPO. The reactive species such as hydrated electrons (eaq),
•OH and HO•2 were generated by water radiolysis. The rate constants
were studied by pulse radiolysis method, whereas identification and
lifetime estimation of G-CYPMPO adducts were followed by ESR
spectroscopy.
’ MATERIALS AND METHODS
Chemicals. A colorless needle-shaped spin trap, G-CYPMPO,
anti-CYPMPO (A-CYPMPO, ap-5-(5,5-dimethyl-2-oxo-1,3,2-dioxa-
phosphinan-2-yl)-5-methy-1-pyrroline N-oxide), and one of the most
popular spin traps (DMPO, the chemical structures of which are
shown in Figure 2) were used. G-CYPMPO was synthesized by
oxidizing CYPMPH (ap-2-(5,5-dimethyl-2-oxo-1,3,2-dioxaphosphi-
nan-2-yl)-2-methylpyrrolidine, CAS No. 902503-51-9) in diluted
hydrogen peroxide in the presence of a catalytic amount of sodium
tungstate and purified by silica gel column chromatography.16 X-ray
structure analysis of G-CYPMPO (mp 127.8ꢀ128.6 ꢀC) and highly
purified A-CYPMPO (mp 130 ꢀC) were performed utilizing a
R-AXIS RAPID II X-ray diffractometer (Rigaku, Japan).
All aqueous solutions were prepared with ultrapure water from
a Milli-Q system (Millipore, Bedford, MA), and saturated with
highly pure N2O, Ar, or O2 gases by bubbling for ∼30 min before
irradiation. N2O and Ar gases were used to enable the conversion
of eꢀaq to •OH, via the reaction17,18
’ RESULTS AND DISCUSSION
X-ray Structure Analysis. Figure 1 shows the results of the
X-ray structure analysis for G-CYPMPO and A-CYPMPO, and
the O1ꢀP1ꢀC6ꢀN1 torsion angles are listed in Table 1. A
torsion angle of 52.8ꢀ for G-CYPMPO indicates that the con-
formation of G-CYPMPO is gauche, whereas the torsion angle of
A-CYPMPO is 164.57ꢀ.
•
eꢀaq þ N2O þ H2O f N2 þ OH þ OHꢀ
Rate Constant Determination. Since eꢀaq is scavenged by
dissolved N2O and converted to •OH in N2O-saturated aqueous
or to remove dissolved O2, respectively. Tert-butyl alcohol
(t-BuOH) and formic acid (HCOOH) were added as a
•
solution, only the behavior of OH can be observed. Figure 3
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Table 2. Rate Constants (109 Mꢀ1sꢀ1) for G-CYPMPO,
A-CYPMPO, and DMPO toward •OH Observed by a Com-
petition Method and eꢀaq Measured at 715 nm
spin trap
•OH/109 Mꢀ1sꢀ1
eꢀaq/109 Mꢀ1sꢀ1
G-CYPMPO
A-CYPMPO
this work
reported15
4.2 ( 0.1
11.8 ( 0.2
>1
DMPO
this work
reported17
4.0 ( 0.1
8.3 ( 0.1
4.0
shows transient absorption spectra observed in pulse radiolysis of
an N2O-saturated 0.3-mM G-CYPMPO aqueous solution at
different elapsed times from the electron beam irradiation. Each
spectrum shows a strong absorption below 300 nm and a weak
broad peak at ∼400 nm, whereas unirradiated CYPMPO has an
absorption peak at λ = 233 nm but not at λ > 270 nm. These
•
Figure 4. ESR spectra of G-CYPMPOꢀOH and G-CYPMPOꢀOOH.
For G-CYPMPOꢀOH: (a) experimental spectrum generated with γ-ray
irradiation of a G-CYPMPO aqueous solution bubbled with N2O, and
(b) corresponding computer-simulated spectrum considering two dia-
stereomers. For G-CYPMPOꢀOOH: (c) experimental spectrum gen-
erated by γ-ray irradiation of a G-CYPMPO aqueous solution with
HCOOH bubbled with O2 gas, and (d) corresponding computer-
simulated spectrum considering two diastereomers.
peaks can be attributed to an OH adduct of G-CYPMPO,
G-CYPMPOꢀOH, the chemical structure of which is shown
in the inset of Figure 3. •OH itself also has an absorption band
below 300 nm and it survives ∼2 μs after electron beam
irradiation in pure water,22,23 so that both the production of
•
G-CYPMPOꢀOH and the consumption of OH appear in the
time region of Figure 3. However, it is confirmed by a competi-
tion method with a thiocyanate anion as described hereinafter
•
that G-CYPMPO reacts with OH at a diffusion-controlled
Table 3. Hyperfine Coupling Constants (hfcc) of G-CY-
PMPOꢀOH and G-CYPMPOꢀOOH Used for the Simula-
tion Spectra in Figure 4
rate constant, leading to identification of the observed peak
as absorption of G-CYPMPOꢀOH. Similar transient spectra
were obtained for DMPOꢀOH, suggesting that the reaction
•
spin adduct
diastereomer
AN/mT
AH/mT
AP/mT
sites of these spin traps for OH scavenging are same. By spin
•
trapping, OH adducts to the carbon of the CdN bond in a
G-CYPMPOꢀOH
A
B
A
B
1.40
1.40
1.32
1.30
1.40
1.22
1.04
1.15
5.04
4.90
5.11
5.24
pyrroline-ring (Figure 2), and the spin trap itself becomes a
radical.
G-CYPMPOꢀOOH
On the other hand, reaction with eꢀaq was observed by
•
removing OH via the addition of 100 mM t-BuOH. Sample
solutions were saturated with argon to purge O2, because O2 scavenges
eꢀaq rapidly with a reaction rate constant of 1.9 ꢁ 1010 Mꢀ1sꢀ1.17 The
transient absorption spectra of eꢀaq were observed, and the well-known
absorption peak of eaꢀq was confirmed at ∼715 nm.23,24 This peak
decays with first-order kinetics in the presence of G-CYPMPO, while
the decay without G-CYPMPO is known to be second-order kinetics.
The first-order reaction rate increꢀases as the G-CYPMPO con-
centration increases, showing that eaq is scavenged by G-CYPMPO.
Generally, the decay time of eꢀaq in pure water is longer than 1.5 μs,25,26
such eꢀaq decrement is due to the reaction of that with G-CYPMPO.
No other species were observed by this reaction in the UVꢀvis
domain (λ = 265ꢀ800 nꢀm). A similar tendency was observed from
the transient spectra for eaq in the DMPO.
was (11.8 ( 0.2) ꢁ 109 Mꢀ1 sꢀ1, determined from the time profiles
recorded at 715 nm with different G-CYPMPO concentrations.
The rate constants obtained in this work are listed in Table 2,
along with those for DMPO. The rate constants of G-CYPMPO/
DMPO toward •OH arꢀe comparable, whereas the rate constant
for G-CYPMPO with eaq is notably higher than that for DMPO
with regard to eꢀaq, suggesting that reaction sites of eaꢀq scavenging
might be different for DMPO and G-CYPMPO.
Selective Detection of Spin Adducts. An ESR spectrum for
G-CYPMPOꢀOH of a 1.0 mM G-CYPMPO aqueous solution
saturated with N2O gas irradiated with γ-rays and the corre-
sponding computer simulated spectrum are shown in Figures 4a
and 4b, respectively. The simulated spectrum was drawn using
two sets of hyperfine coupling constants (hfcc), representing two
diastereomers15 of G-CYPMPOꢀOH, summarized in Table 3.
The experimental spectrum consists of two identical groups of
lines which are split by a large hyperfine coupling with phos-
Because of the overlap of the production of G-CYPMPOꢀ
OH and the consumption of •OH (Figure 3), the rate constant
•
of G-CYPMPO toward OH was obtained via a competition
method, using KSCN as a reference, not by build-up kinetics
•
phorus, and is similar to the ESR spectra of the OH and HO2•
analysis. The reference reaction rate constant for SCNꢀ + OH
was 1.1 ꢁ 1010 Mꢀ1sꢀ1 17
.
By varying the ratio of the concen-
adducts trapped by DEPMPO and A-CYPMPO. On the other
hand, Figures 4c and 4d represent an experimental ESR spectrum
for G-CYPMPO-OOH and the corresponding computer simu-
lated spectrum, respectively. A 1.0 mM G-CYPMPO/200 mM
HCOOH aqueous solution under acidic conditions was bubbled
with O2 gas and then irradiated with γ-ray. The hfccs used in the
simulation of Figure 4d are also listed in Table 3. Compared to
tration of G-CYPMPO to that of KSCN, the rate constant was
estimated to be (4.2 ( 0.1) ꢁ 109 Mꢀ1sꢀ1. The reported rate
constant of A-CYPMPO, estimated using a competitive-trap-
ping method with ESR, was larger than 109 Mꢀ1sꢀ1 15
Our
.
result is consistent with the previous data, but more precise. Oꢀn
the other hand, the rate constant of G-CYPMPO toward eaq
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ARTICLE
trapping of oxygen-derived radicals with G-CYPMPO, and their
decay was investigated by ESR spectroscopy, which are useful for the
accurate evaluation of the function of ROS in the living body. From
the X-ray structure analysis, we determined the O1ꢀP1ꢀC6ꢀN1
torsion angle of 52.8ꢀ. The trapping reactions are all diffusion-
controlled and we found that the rate constant for G-CYPMPO with
eꢀaq is larger than that for DMPO. The hyperfine coupling constants
for G-CYPMPOꢀOH and G-CYPMPO-OOH are relatively higher
than those for CYPMPOꢀOH and CYPMPOꢀOOH. Half-lives of
G-CYPMPOꢀOH and G-CYPMPOꢀOOH are estimated to be
35 and 90 min, respectively. The half-life of G-CYPMPOꢀOOH
generated by a “pure” method becomes longer than the half-life of
CYPMPOꢀOOH generated by an enzymatic reaction, indicating
that the additive plays an important role in the stability of the
G-CYPMPO adduct.
Figure 5. Decay of the ESR signal for G-CYPMPOꢀOH and G-CY-
PMPOꢀOOH after γ-ray irradiation. The peak-to-peak intensity of the
fourth peak from the left in the ESR spectra was plotted versus time elapsed
after irradiation. The decay curves are fitted by double exponential.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail addresses: oka.toshitaka@jaea.go.jp (T.O.), katsu@
n.t.u-tokyo.ac.jp (Y.K.).
the hfcc of CYPMPOꢀOH and CYPMPOꢀOOH,15 the rela-
tively wide hfccs of phosphorus AP (2.0%ꢀ4.3% wider) may be
due to the difference of the conformation observed for G-CY-
PMPOꢀOH and G-CYPMPOꢀOOH.
Figure 5 displays the variations of the peak-to-peak intensities
of the G-CYPMPOꢀOH and G-CYPMPOꢀOOH as a function
of elapsed time from γ-ray irradiation. All the data are normalized
at the first-derivative intensities of each spin adduct. The decay
rate of G-CYPMPOꢀOH is faster than that of G-CYPMPO-
OOH. The decay curves are fitted by double exponential and the
half-lives of the G-CYPMPOꢀOH and G-CYPMPOꢀOOH are
estimated to be ∼35 and ∼90 min, respectively, using the faster
components. Previously, Saito et al. reported that the half-lives of
CYPMPOꢀOH and CYPMPOꢀOOH were 44.8 and 30.4 min,
respectively.27 In that report, •OH was generated by irradiation
with 1.0 MHz ultrasound without any additives. In our case, •OH
was generated by γ-ray irradiation without any additives. Both
’ ACKNOWLEDGMENT
Authors thank Mr. D. Hiroishi, Mr. T. Ueda, and Prof. M.
Uesaka (University of Tokyo) for their technical assistance in
γ-ray irradiation and pulse radiolysis experiments.
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’ CONCLUSION
The X-ray structure analysis of G-CYPMPO and determination
•
of the rate constants of G-CYPMPO toward OH and eꢀaq were
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