Spin-trapping properties of 5-(Diphenylphosphinoyl)-5-methyl-4,5-dihydro- 3H-pyrrole N-Oxide (DPPMDPO)

Article abstract of DOI:10.1246/bcsj.80.495

Spin-trapping properties of a novel spin-trapping reagent, 5-(diphenylphosphinoyl)-5-methyl-4,5-dihydro-3H-pyrrole N-oxide (DPPMDPO), were investigated by ESR spectroscopy. DPPMDPO had larger rate constants than DMPO and DEPMPO. DPPMDPO should be better t

Full text of DOI:10.1246/bcsj.80.495

Short Articles
Bull. Chem. Soc. Jpn. Vol. 80, No. 3, 495–497 (2007)
495
Cl
O
Ph
H O
O
NH
Spin-Trapping Properties of
5-(Diphenylphosphinoyl)-
5-methyl-4,5-dihydro-3H-pyrrole
N-Oxide (DPPMDPO)
2
Ph
Ph
3
P
P
Cl
H
EtOH
CH CN
Ph
3
O
P
O
NaHCO aq.
3
Ph
Ph
Ph
Ph
P
N
O
N
H
Acetone
DPPMDPO
ꢀ1
Masahiro Nishizawa, Kosei Shioji,2
Yoshimitsu Kurauchi,2 Kentaro Okuma,2
and Masahiro Kohno1
Scheme 1.
phosphinoyl)-4,5-dihydro-3H-pyrrole N-oxide (MPPMDPO).7
MPPMDPO has acceptable solubility and half-life as a super-
oxide adduct. Unfortunately, MPPMDPO had disadvantage.
MPPMDPO exists as diastereomers that are difficult to sepa-
rate and that affect ESR measurements of the adducts. There-
fore, for good ESR measurements, a spin-trapping reagent
not have a diastereomer. In this paper, we introduce 5-(di-
phenylphosphinoyl)-5-methyl-4,5-dihydro-3H-pyrrole N-oxide
(DPPMDPO) as a new phosphinoyl derivative.
1New Industry Creation Hatchery Center,
Tohoku University, Aoba-ku, Sendai 980-8579
2Department of Chemistry, Faculty of Science,
Fukuoka University, Jonan-ku, Fukuoka 814-0180
Received January 30, 2006; E-mail: nisizawa@niche.tohoku.
ac.jp
DPPMDPO was synthesized as shown in Scheme 1. Di-
phenylphosphine oxide was prepared in 97% yield from chloro-
diphenylphosphine. Cyclization of 5-chloropentan-2-one with
ammonia and chlorodiphenylphosphine gave a pyrrolidine in
63% yield. OxoneÔ oxidation of diphenylphosphine in ace-
tone gave DPPMDPO in 47% yield. Using the OxoneÔ in-
stead of MCPBA enables large scale nitrone production in suf-
ficient yield. The purification of DPPMDPO was performed by
using recrystallization instead of column chromatography,
since pure DPPMDPO easily formed colorless crystals at room
temperature. DPPMDPO did not decompose in aqueous solu-
tion at room temperature for several months.
The partition coefficient is important parameter for a spin-
trapping reagent since a high lipophilicity can improve its dis-
tribution in cell membrane. Therefore, we measured the parti-
tion coefficient of DPPMDPO in 1-octanol/aqueous solution
by using the method described by Konorev et al.8 The partition
coefficient was calculated as the ratio between the DPPMDPO
concentration in 1-octanol solution and that in aqueous solu-
tion. The concentrations were acquired from optical absorption
at 227 nm for an 1-octanol solution and at 224 nm for an aque-
ous solutions. The observed partition coefficient value was
4:3 ꢁ 0:2 as summarized in Table 1. As shown in Table 1, the
partition coefficient of DPPMDPO was larger than that of
DMPO,9 DEPMPO,2 and MPPMDPO.7 This result showed that
two phenyl groups on phosphinoyl group improved lipophilic-
ity and that one of the drawbacks of DEPMPO was overcome.
Spin-trapping properties of
a novel spin-trapping
reagent, 5-(diphenylphosphinoyl)-5-methyl-4,5-dihydro-3H-
pyrrole N-oxide (DPPMDPO), were investigated by ESR
spectroscopy. DPPMDPO had larger rate constants than
DMPO and DEPMPO. DPPMDPO should be better than
DEPMPO as a spin-trapping reagent for superoxide detection.
Superoxide and hydroxyl radical have been widely re-
searched, because superoxide is the primary upstream radical
of the radical reaction chain that induces oxidative stress and
the hydroxyl radical is the most reactive radical species. De-
tection and measurement of reactive oxygen species (ROS)
in vivo and in vitro have been performed by spin-trapping
methods with spin-trapping reagents. Electron spin resonance
(ESR) spectroscopy is a popular method for detection and
identification of adducts since it is possible to detect specifi-
cally adducts with relatively stable radicals. Popular spin-trap-
ping reagents for ROS detection are 5,5-dimethyl-4,5-dihydro-
3H-pyrrole N-oxide (DMPO) and 5-(diethoxyphosphinoyl)-5-
methyl-4,5-dihydro-3H-pyrrole N-oxide (DEPMPO). DMPO
is most frequently used since the kinetic parameters of the
trapping reactions and ESR parameters of its adducts are well
documented.1 DEPMPO is used for most effective detection
and measurement of superoxide since DEPMPO has a larger
rate constant for trapping and a longer adduct lifetime in com-
parison with DMPO.2 However, the lifetime of DEPMPO–
OOH is not sufficient in vivo. Besides, DEPMPO has poor
distribution in cell membrane. Therefore, in order to improve
detection, several alkoxy derivatives of DEPMPO have been
developed.3 There are a few reasons for studying DEPMPO
analogues. The diethoxyphosphinoyl group increases the reac-
tivity forward ROS,4 and DEPMPO has sufficient amount of
application data for in vivo and in vitro ROS trapping.5,6
In our previous study, we have described the synthesis
and chemical properties of methyl- and phenyl-substituted
phosphinoyl derivatives, such as 5-methyl-5-(methylphenyl-
Table 1. Partition Coefficients, Rate Constants, and Half-
Lives of DMPO, DEPMPO, and DPPMDPO
DMPO8,11 DEPMPO2 DPPMDPO
Partition coefficient
Rate constant for
0.1
0.06
4:3 ꢁ 0:2
Superoxide/Mꢂ1 sꢂ1
Hydroxyl radical/109 Mꢂ1 sꢂ1
Half-life of adduct
Superoxide/min
15.7
3.4
23.5
7.1
39:5 ꢁ 0:2
8:50 ꢁ 0:01
1
60
14.8
57
8:3 ꢁ 0:2
Hydroxyl radical/min
13:2 ꢁ 1:0
Published on the web March 13, 2007; doi:10.1246/bcsj.80.495

496 Bull. Chem. Soc. Jpn. Vol. 80, No. 3 (2007)
Ó 2007 The Chemical Society of Japan
(a)
(a)
(b)
(c)
(b)
(c)
330
332
334
336
338
340
330
332
334
336
338
340
Magnetic Field / mT
Magnetic Field / mT
Fig. 1. ESR spectra of (a) DMPO–OOH, (b) DEPMPO–
OOH, and (c) DPPMDPO–OOH. All measurements and
trapping reaction were performed under the same condi-
tion.
Fig. 3. ESR spectra of (a) DMPO–R, (b) DEPMPO–R, and
(c) DPPMDPO–R. All trapping reactions were performed
under the same condition.
Table 2. Hyperfine Coupling Constants and g-Values of
DPPMDPO–OOH, –OH, and –R
Hyperfine coupling constant g-Value
(a)
aP/mT
aN/mT
aH/mT
DPPMDPO–OOH
Diastereomer A 65%
Diastereomer B 35%
DPPMDPO–OH
3.90
3.95
3.55
3.75
1.26
1.22
1.37
1.42
1.10
1.18
1.37
2.16
2.0069
2.0072
2.0068
2.0067
(b)
(c)
DPPMDPO–R
pH 7.4, and hydroxyl radical trapping was performed in the
Fenton reaction system at pH 7.4. Observed rate constants
for DPPMDPO and reported values for DMPO and DEPMPO
are summarized in Table 1. Unfortunately, the rate constants
of several DMPO analogues were not obtained from the com-
petitive reaction with DMPO but from inhibitory reaction of
cytochrome c reduction, because in order to compare the rate
constants the reactions must be done under the same condi-
tions.13 Furthermore, some of the rate constants of the other
analogues have been calculated by using the inaccurate value
for DEPMPO.14 Consequently, we performed the competitive
reaction with DMPO and ESR spin-trapping method the same
each time. Therefore, the diphenylphosphinoyl group increas-
ed the rate constants of the ROS trapping reactions more than
the diethoxyphosphinoyl group.
Monitoring of time dependent decay of relative ESR signal
intensities gave half-lives of DPPMDPO–OOH and –OH. The
half-lives were calculated from the first-order kinetic decay
date. We applied the HPX–XOD superoxide generation system
to produce DPPMDPO–OOH since it has been reported that
other systems, such as the light-riboflavin superoxide genera-
tion system, simultaneously generate persistent carbon-cen-
tered radicals, which leads to an overestimation of the mea-
sured half-life.15
330
332
334
336
338
340
Magnetic Field / mT
Fig. 2. ESR spectra of (a) DMPO–OH, (b) DEPMPO–OH,
and (c) DPPMDPO–OH. All measurements and trapping
reaction were performed under the same condition.
Figure 1 shows ESR spectra of DMPO–, DEPMPO–, and
DPPMDPO–OOH. Superoxide generation was performed by
using the hypoxanthine–xanthine oxidase (HPX–XOD) reac-
tion system at pH 7.4.10 The hyperfine splitting pattern of
ESR spectrum of DPPMDPO–OOH was similar to that of
DEPMPO–OOH. However, the signal of DPPMDPO–OOH
did not overlap that of Mn2þ as an external standard. Figure 2
shows ESR spectra of DMPO–, DEPMPO–, and DPPMDPO–
OH. Hydroxyl radical generation was performed by using
the Fenton reaction system at pH 7.4.11 Hyperfine splitting
pattern of the ESR spectrum of DPPMDPO–OH was also
similar to that of DEPMPO–OH. Figure 3 shows ESR spectra
of DMPO–, DEPMPO–, and DPPMDPO–R. Carbon-centered
radical generation was performed by using the Fenton reaction
system containing MeOH at pH 7.4. Computer simulations
of the ESR spectra of DPPMDPO–OOH, –OH, and –R gave
ESR parameters and showed that DPPMDPO–OOH has a dia-
stereomer. Calculated ESR parameters of DPPMDPO–OOH,
–OH, and –R are summarized in Table 2.
The signal intensity of DPPMDPO–OOH in the HPX–XOD
reaction system reached a maximum around 5 min from the
reaction initiation and decreased exponentially as shown in
Fig. 4a. In the exponentially decaying region, time dependency
completely obeyed first-order kinetic decay. In case of SOD ad-
dition to HPX–XOD reaction system to stop the superoxide
trapping reaction, signal intensity decayed exponentially. The
In many cases, the rate constant of a ROS trapping reaction
is obtained from competitive reaction by using the method de-
scribed by Finkelstein et al.12 For our study, DMPO was used
in the competitive reaction of DPPMDPO. Superoxide trap-
ping was performed in the HPX–XOD reaction system at

M. Nishizawa et al.
Bull. Chem. Soc. Jpn. Vol. 80, No. 3 (2007)
497
the diethoxyphosphinoyl group enhanced stability of nitrone
and increased the rate constants for superoxide and hydroxyl
radical trapping reactions. Furthermore, DPPMDPO did not
degrade in aqueous solution for several months. DPPMDPO
appears to be a better spin-trapping reagent for superoxide
detection.
200
(a)
150
100
50
0
0
5
10
15
20
25
Time / min
References
200
1
2
G. R. Buettner, Free Radical Biol. Med. 1987, 3, 259.
C. Frejaville, H. Karoui, B. Tuccio, F. Moigne, M. Culcasi,
(b)
150
100
50
S. Pietri, R. Lauricella, P. Tordo, J. Med. Chem. 1995, 38, 258.
3
2110.
4
F. Chalier, P. Tordo, J. Chem. Soc., Perkin Trans. 2 2002,
0
0
J. Katzhendler, I. Ringel, R. Karaman, H. Zaher, E. Breuer,
5
10
15
20
25
Time / min
J. Chem. Soc., Perkin Trans. 2 1997, 341.
K. J. Liu, M. Miyake, T. Panz, H. Swartz, Free Radical
Biol. Med. 1999, 26, 714.
6 S. Dikalov, P. Tordo, A. Motten, R. P. Mason, Free
Radical Res. 2003, 37, 705.
7 K. Shioji, S. Tsukumoto, H. Tanaka, K. Okuma, Chem.
Lett. 2003, 32, 604.
8 E. A. Konorev, J. E. Baker, B. Kalyanaraman, Free
Radical Biol. Med. 1993, 14, 127.
5
Fig. 4. Time-dependent decay of ESR signal intensities for
DPPMDPO–OOH (a) and DPPMDPO–OH (b) at pH 7.4.
time dependency also obeyed first-order kinetic decay. The time
dependence of the signal decay was the same regardless of the
presence of the SOD. Calculated half-life of DPPMDPO–OOH
was 8:3 ꢁ 0:2 min as summarized in Table 1.
Signal intensity of DPPMDPO–OH in Fenton reaction
system decayed exponentially after 3 min from reaction initia-
tion. In the exponentially decaying region, the time depen-
dency completely obeyed first-order kinetic decay. In addition,
the signal decay of DPPMDPO–OH in the H2O2-UV radiation
hydroxyl radical generation system showed also the same
time dependency. The calculated half-life of DPPMDPO–
OH was 13:2 ꢁ 1:0 min as summarized in Table 1. Half-
life of DPPMDPO–OH was shorter than those of DMPO–
and DEPMPO–OH. Furthermore, the signal intensity of
DPPMDPO–OH decayed rapidly immediately after initiation
of the Fenton reaction as shown in Fig. 4b. These results
showed that DPPMDPO–OH was not stable.
9
Biochem. Biophys. 1982, 215, 367.
10 K. Mitsuta, Y. Mizuta, M. Kohno, M. Hiramatsu, A. Mori,
Bull. Chem. Soc. Jpn. 1990, 63, 187.
11 M. Kohno, M. Yamada, K. Mitsuta, Y. Mizuta, T.
Yoshikawa, Bull. Chem. Soc. Jpn. 1991, 64, 1447.
12 E. Finkelstein, G. M. Rosen, E. J. Rauckman, J. Am. Chem.
Soc. 1980, 102, 4994.
13 P. Tsai, K. Ichikawa, C. Mailer, S. Pou, H. J. Halpern,
B. H. Robinson, R. Nielsen, G. M. Rosen, J. Org. Chem. 2003,
68, 7811.
G. M. Rosen, E. Finkelstein, E. J. Rauckman, Arch.
14 F. A. Villamena, J. L. Zweier, J. Chem. Soc., Perkin Trans.
2 2002, 1340.
15 G. R. Buettner, Free Radical Res. Commun. 1990, 10, 11.
Introduction of the diphenylphosphinoyl group instead of