Diastereoselective synthesis and ESR study of 4-phenylDEPMPO spin traps

Article abstract of DOI:10.1021/jo0479516

(Chemical Equation Presented) The cis and trans diastereoisomers of 5-(diethoxyphosphoryl)-5-methyl-4-phenylpyrroline N-oxide (4-PhDEPMPOt 8 and 4-PhDEPMPOc 9) were prepared stereoselectively and used as spin traps for hydroxyl and superoxide radicals. Th

Full text of DOI:10.1021/jo0479516

Diastereoselective Synthesis and ESR Study of 4-PhenylDEPMPO
Spin Traps
Micae¨l Hardy,† Florence Chalier,† Jean-Pierre Finet,*,† Antal Rockenbauer,‡ and Paul Tordo*,†
Laboratoire SREP, UMR 6517 CNRS et Universite´s Aix-Marseille 1, 2 et 3, Centre de Saint Je´roˆme,
13397 Marseille Cedex 20, France, and Chemical Research Center, Institute for Structural Chemistry,
H-1525 Budapest, PO Box 17, Hungary
finet@srepir1.univ-mrs.fr
Received November 18, 2004
The cis and trans diastereoisomers of 5-(diethoxyphosphoryl)-5-methyl-4-phenylpyrroline N-oxide
(4-PhDEPMPOt 8 and 4-PhDEPMPOc 9) were prepared stereoselectively and used as spin traps
for hydroxyl and superoxide radicals. The spin adduct formed by reaction of the cis stereoisomer 9
with superoxide radical anion exhibited an 8-line ESR spectrum, showing only a reduced alternating
line width phenomenon. This spectrum is simpler than the 12-line spectrum of DEPMPO-OOH,
which exhibits a strong alternating line width phenomenon. The half-life times of the 4-PhDEP-
MPOc-OOH and DEPMPO-OOH adducts were of the same order: 14.5 and 15.5 min, respectively.
Introduction
the most popular to detect oxygen-centered radicals.13-15
However, detection of the superoxide radical is limited
by the instability of the DMPO-OOH adduct (half-life
time < 1 min).14 In the past 10 years, a new generation
of spin traps related to DMPO has been developed.16-18
Among them, DEPMPO 2 is becoming a widely used spin
trap for biological studies because of the considerably
better stability of the DEPMPO-OOH spin adduct (half-
life time ∼18 min at physiological pH).17,19 The related
diisopropyl ester (DIPPMPO) 3 is even slightly better,
with a half-life time of 21 min for the DIPPMPO-OOH
adduct at physiological pH.20 However, the dissymmetric
Reactive oxygen species (HOO•, HO•, H2O2) are con-
sidered to play an important role in many pathological
disorders, such as ischemia-reperfusion, heart attack,
stroke, Alzheimer’s disease, aging, DNA damage, cancer,1-5
and so on. To study the involvement of oxygen short-lived
free radical species, the technique of spin trapping is one
of the most valuable tools.6-13 Among the nitrones used
as spin traps, DMPO 1 has been for a long time one of
* To whom correspondence should be addressed. Phone: 33 4 91 28
85 62. Fax: 33 4 91 28 87 58.
† UMR 6517 CNRS et Universite´s Aix-Marseille 1, 2 et 3.
‡ Institute for Structural Chemistry.
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10.1021/jo0479516 CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/08/2005
J. Org. Chem. 2005, 70, 2135-2142
2135

Hardy et al.
nature of these nitrones, DEPMPO and DIPPMPO (Me-5
and phosphoryl-5 groups), leads to the formation of
complex spectra because of the superimposition of the
signals of two superoxide spin adducts. These signals are
due to the formation of the cis and trans adducts 4 and
5, respectively, in a ∼1:9 ratio, which result from attack
of the incipient free radical on both sides of the ni-
trone.17,20 Moreover, the 12-line spectrum of the major
signal, the trans spin adduct 5, exhibits an alternating
line width phenomenon because of the presence of two
quickly exchanging conformers.21,22 To limit these effects,
introduction of a phenyl substituent on the pyrroline ring
could be beneficial. Earlier studies showed that the
presence of a phenyl group slows down the pseudorota-
tion occurring within the ring of stable â-phosphorylated
five-membered ring nitroxides.23,24 Introduction of a
phenyl group on the DMPO spin trap induced a rigidi-
fication of the ring conformation, beneficial for superoxide
radical trapping.25 In the DEPMPO series, synthesis of
the cis-3-phenyl DEPMPO analogue 6 (DEPMPPOc) was
recently reported.26 The presence of a phenyl group led
to an important steric effect on the course of addition of
the superoxide radical. The DEPMPPOc-OOH adduct 7
showed an EPR spectrum with 12 separated lines with
the same intensity, indicative of the absence of an
alternating line width phenomenon. Unfortunately, the
half-life time of this adduct was only around 2 min,
compared to the 18 min of the DEPMPO-OOH adduct.
Thus, the presence of a phenyl group on the C-3 carbon
center has a largely positive effect on the pattern of the
spectrum, but a negative one on the stability of the
adducts. We considered that shifting the phenyl group
from the C-3 center to the C-4 center should result in
two beneficial effects: (a) the steric hindrance leading
to only one free radical adduct should be maintained and
(b) the absence of vicinal interactions destabilizing the
spin adducts should be observed. In this paper, we report
the stereoselective syntheses of the two diastereoisomers
of 4-phenylDEPMPO, the trans isomer 8 (4-PhDEPM-
POt) and the cis isomer 9 (4-PhDEPMPOc), and the study
of their behavior toward oxygen free radicals (Figure 1).
FIGURE 1. Chemical structures of DMPO, phosphorylated
analogues, and some of their superoxide adducts.
SCHEME 1. General Routes for the Synthesis of
DMPO Analogues
SCHEME 2. Synthesis of 4-PhDEPMPO
Derivatives
step.29,30 In the case of ring-substituted DEPMPO deriva-
tives, this reductive cyclization step leads to the forma-
tion of a pair of diastereoisomers.26 However, diastereo-
selective synthesis should be possible by the route
involving a pyrroline-type intermediate. Addition of a
phosphite to 3-phenyl-2-methylpyrroline (10, R1 ) Me)
should lead after oxidation to the trans-5-phosphoryl-4-
phenyl nitrone derivative 8 [Scheme 2, R1 ) Me, R2 )
P(O)(OEt)2], and addition of a methyl group to 3-phenyl-
2-phosphorylpyrroline [10, R1 ) P(O)(OEt)2] should lead
after oxidation to the cis-5-phosphoryl-4-phenyl nitrone
derivative 9 [Scheme 2, R1 ) P(O)(OEt)2, R2 ) Me].
The trans nitrone 8, 5-(diethoxyphosphoryl)-5-methyl-
4-phenylpyrroline N-oxide, was prepared in six steps
from cinnamonitrile by the sequence described in Scheme
3. 3-Phenyl-4-oxopentane-1-nitrile 11 was obtained by
treatment of cinnamonitrile with Mg turnings in the
presence of acetic anhydride/TMSCl in DMF according
to the method of Ohno et al.31 Protection of the carbonyl
group as a dioxolane was performed by reaction of 11
with ethylene glycol in the presence of a catalytic amount
of camphorsulfonic acid (CSA) in toluene under reflux,
giving compound 12 in 91% yield. Reduction of 12 with
Results and Discussion
DMPO-type nitrones can be prepared by three main
routes (Scheme 1).27,28 Two of them (a and b) require the
synthesis of an intermediate pyrrolidine, eventually
oxidized into the nitrone.28 The third method (c) involves
the reductive cyclization of a γ-nitroaldehyde in the last
(20) Chalier, F.; Tordo, P. J. Chem. Soc., Perkin Trans. 2 2002,
2110-2117.
(21) Krishna, M. C.; Grahame, D. A.; Samuni, A.; Mitchell, J. B.;
Russo, A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 5537-5541.
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H.; Samuni, A. J. Biol. Chem. 1996, 271, 26026-26031.
(23) Dembkowski, L.; Finet, J.-P.; Frejaville, C.; Le Moigne, F.;
Maurin, R.; Mercier, A.; Pages, P.; Stipa, P.; Tordo, P. Free Rad. Res.
Commun. 1993, 19, S23-S32.
(24) Chachaty, C.; Mathieu, C.; Mercier, A.; Tordo, P. Magn. Reson.
Chem. 1998, 36, 46-54.
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West, M.; Janzen, E. G. Magn. Reson. Chem. 1996, 34, 351-359.
(26) Nsanzumuhire, C.; Cle´ment, J.-L.; Ouari, O.; Karoui, H.; Finet,
J.-P.; Tordo, P. Tetrahedron Lett. 2004, 45, 6385-6389.
(27) Barbati, S.; Clement, J.-L.; Frejaville, C.; Bouteiller, J.-C.;
Tordo, P.; Michel, J.-C.; Yadan, J.-C. Synthesis 1999, 2036-2040.
(28) Cle´ment, J.-L.; Fre´javille, C.; Tordo, P. Res. Chem. Intermed.
2002, 28, 175-190.
(29) Rosen, G. M.; Turner, M. J. J. Med. Chem. 1988, 31, 428-432.
(30) Haire, D. L.; Hiborn, J. W. J. Org. Chem. 1986, 51, 4298-4300.
(31) Ohno, T.; Sakai, M.; Ishino, Y.; Shibata, T.; Maekawa, H.;
Nishiguchi, I. Org. Lett. 2001, 3, 3439-3442.
2136 J. Org. Chem., Vol. 70, No. 6, 2005

Synthesis and ESR Study of 4-PhenylDEPMPO Spin Traps
SCHEME 3. Synthesis of Nitrone 8a
SCHEME 4 . Synthesis of Nitrone 9a
a Reaction conditions: (i) Mg, Ac2O-TMSCl, DMF, 70%; (ii) CSA,
ethylene glycol, toluene, reflux, 8 h, 91%; (iii) LiAlH4, THF, 0 °C,
85%; (iv) 3 N HCl, THF/H2O, 4 h, then K2CO3, 1 h, 93%; (v)
HP(O)(OEt)2, cat. BF3‚OEt2, 7 d, 54%; (vi) H2O2, cat. Na2WO4,
EtOH/H2O, 0 °C, 72 h, 90%.
a Reaction conditions: (i) LDA, -78 °C, THF, then 1-azido-2-
iodoethane, 12 h, rt, 59%; (ii) NaOH 1 M, EtOH, 3 h, rt, 100%;
(iii) (COCl)2, CH2Cl2; (iv) P(OEt)3, CH2Cl2, 15 h, rt, 100% for the
two steps; (v) PPh3, Et2O, 16 h, rt, 95%; (vi) cat. BF3‚OEt2,
MeMgBr, 4 h, -78 °C, 76%; (vii) H2O2, cat. Na2WO4, EtOH/H2O,
0 °C, 50 h, 64%
LiAlH4 in THF at 0 °C led to 13 in 85% yield. The
3-phenyl-2-methylpyrroline32 14 was obtained in two
steps from 13 by deprotection of the carbonyl by treat-
ment with 3 N HCl followed by intramolecular cyclization
of the amine in the presence of K2CO3 in a mixture of
THF/H2O. Reaction of 14 with diethyl phosphite in the
presence of a catalytic amount of boron trifluoride diethyl
etherate (BF3‚OEt2) at room temperature led to 3-Ph-
DEPMP 15 in 54% yield. The addition of diethyl phos-
phite was stereoselective leading to the phosphorylated
group in a trans position in relation with the phenyl
group. Oxidation of 15 with H2O2 in the presence of a
catalytic amount of sodium tungstate in a mixture of
THF/H2O led finally to 4-PhDEPMPOt 8 in 90% yield.
The cis nitrone 9, 5-diethoxyphosphoryl-5-methyl-4-
phenylpyrroline N-oxide, was prepared by the route
described in Scheme 4. The azidoester 16 was obtained
in 59% yield by alkylation of ethyl phenylacetate with
1-azido-2-iodoethane33 by an adaptation of the procedure
of Flintoft et al.34 The azidoester 16 was easily saponified
by treatment with dilute sodium hydroxide in methanol/
water at room temperature leading to the acid 17 (100%).
Synthesis of the iminophosphonate 19 was performed by
the method of Vaultier et al.35,36 Treatment of the acid
17 with oxalyl chloride in CH2Cl2 gave the crude acid
chloride which was used without further purification.
Arbuzov reaction of the azidoacid chloride with triethyl
phosphite in CH2Cl2 led to the moisture-sensitive azido
acylphosphonate 18 in a quantitative yield, after distil-
lation under vacuum (10-3 Torr) of the volatile com-
pounds. The crude acylphosphonate 18 was treated
directly with 1 equiv of triphenylphosphane in anhydrous
ether to give the 3-phenyl-2-diethoxyphosphorylpyrroline
19 in 95% yield. Addition of methylmagnesium bromide
on the pyrroline 19 in the presence of BF3‚OEt2 at -78
°C in THF37-39 afforded the 3-PhDEPMPc 20 in 76%
yield. Attack of the nucleophile occurred anti to the
phenyl group. Thus, the 3-phenyl group is now in the cis
position in relation with the phosphoryl group. Oxidation
of the pyrrolidine 20 by H2O2 in the presence of a catalytic
amount of sodium tungstate in a mixture of THF/H2O
led to the nitrone 9, 4-PhDEPMPOc, in 64% yield.
ESR Studies. In the present work, the spin-trapping
capacities of the two nitrones 8 and 9 were tested only
toward two oxygen-centered radical species, the super-
oxide and hydroxyl radicals, that are of the largest
biological relevance.
(a) Spin Trapping of Superoxide Radical. Spin
trapping of superoxide radical with 4-PhDEPMPOc 9 (20
mM) was performed in a buffered aqueous solution at
pH 7.3, using two different superoxide generating sys-
tems. Similar signals were observed either by using
hypoxanthine with xanthine oxidase (HX/XO system) in
the presence of diethylenetriaminepentaacetic acid (DTPA)
or by reaction with KO2. When superoxide dismutase
(SOD) was added to the HX/XO generating system, the
ESR signal disappeared (Figure 2c) proving that spec-
trum (a) is due to the trapping of superoxide.
The two ESR spectra of the 4-PhDEPMPOc-OOH
adducts consisted of a doublet of quadruplet showing a
certain degree of dissymmetry together with a broaden-
ing of the lines (Figure 2a,b). The relative simplicity of
the spectra can be attributed to the stereoselective
(32) Stevens, R. V.; Ellis, M. C.; Wentland, M. P. J. Am. Chem. Soc.
1968, 90, 5576-5579.
(33) Khoukhi, M.; Vaultier, M.; Carrie´, R. Tetrahedron Lett. 1986,
27, 1031-1034.
(34) Flintoft, R. J.; Buzby, J. C.; Tucker, J. A. Tetrahedron Lett.
1999, 40, 4485-4488.
(37) Bloch, R. Chem. Rev. 1998, 98, 1407-1438.
(38) Enders, D.; Reinhold, U. Tetrahedron: Asymmetry 1997, 8,
1895-1946.
(39) Harwood, L. M.; Vines, K. J.; Drew, M. G. B. Synlett 1996,
1051-1053.
(35) Khoukhi, M.; Vaultier, M.; Carrie´, R. Tetrahedron 1987, 43,
1811-1822.
(36) Vaultier, M.; Lambert, P. H.; Carrie´, R. Bull. Soc. Chim. Fr.
1986, 83-92.
J. Org. Chem, Vol. 70, No. 6, 2005 2137

Hardy et al.
spectrum of 4-PhDEPMPOc-OOH is simpler (8 lines)
than the spectrum of DEPMPO-OOH showing 12 lines.19
Moreover, the dissymmetry of the signal (compared to
the baseline) and the alternate line width phenomenon
are much less important in the case of the 4-PhDEPM-
POc-OOH adduct than in the case of the corresponding
DEPMPO-OOH adduct, even under the low amplitude
modulation conditions that were used. Under these
conditions, the γAH hyperfine coupling constants were not
detected. The exchange rate constants between the
respective pairs of conformers were not very different
between 4-PhDEPMPOc-OOH and DEPMPO-OOH (0.34
× 108 s-1 for the system 9/O2•- and 0.67 × 108 s-1 for the
system 2/O2•-).41 The values of the coupling constants of
the 4-PhDEPMPOc-OOH and DEPMPO-OOH spin ad-
ducts are similar even if smaller differences between the
coupling constants of the two conformers of 4-PhDEPM-
POc-OOH compared to the corresponding values of the
DEPMPO-OOH conformers can be noted (Table 1). These
different facts can explain why, in the case of the
4-PhDEPMPO-OOH adduct, only a widening of the lines
but no significant alternating line width phenomenon are
observed.
(b) Spin Trapping of Hydroxyl Radical. The hy-
droxyl radical spin adduct, 4-PhDEPMPOc-OH, was
obtained by incubation of 4-PhDEPMPOc 9 (20 mM) with
a mixture of H2O2 (2 mM), FeSO4 (2 mM), and DTPA (1
mM) in 0.1 M phosphate buffer at pH 7.3. The ESR
spectrum of the 4-PhDEPMPOc-OH adduct consisted of
12 main lines (Figure 3), compared to the doublet of
quadruplet detected for the corresponding DEPMPO-OH
adduct.19 This signal disappeared upon addition of cata-
lase to the incubation mixture. Computer simulation
showed that spectrum (a) results from the superimposi-
tion of the signals of the adducts of OH (80.4%), alkyl
(14%) and one unidentified species. The parameters are
given in Table 1.
FIGURE 2. Spin trapping of superoxide radical by 4-PhDEP-
MPOc 9: (a) Signal obtained after 5 min incubation of a
mixture containing hypoxanthine (0.4 mM), xanthine oxidase
(0.04 U mL-1), DTPA (1 mM), and 4-PhDEPMPOc 9 (20 mM)
in phosphate buffer (0.1 M, pH 7.3). (b) Signal obtained after
1 min incubation of a mixture containing KO2 (10 mM) and
4-PhDEPMPOc 9 (20 mM) in phosphate buffer (0.1 M, pH 7.3).
(c) As in (a) but in the presence of SOD (606 U mL-1). The
dotted lines in (a) and (b) represent the computer simulation
of the spectra. Spectrometer settings: microwave power 10
mW; modulation amplitude, 0.702; time constant, 0.128 s; gain
105; sweep time, 83.89 s; conversion time, 0.819 s.
(c) Kinetics of Decay of the Superoxide Adduct.
The half-life time of the superoxide spin adducts depends
on a variety of parameters, such as nitrone concentration,
nature of the generating system, pH, etc.42,43 In the
classical method used for determination of the kinetics
of decay of the DEPMPO-OOH adduct, the HX/XO system
in the presence of DEPMPO in a phosphate buffer is used
to generate the DEPMPO-OOH adduct.41 Once the ad-
duct concentration has reached an optimum value, the
formation of superoxide is stopped by addition of a large
amount of SOD. As superimposition of the ESR spectra
of the DEPMPO-OOH and DEPMPO-OH adducts does
not lead to overlap of the two signals, the decay of
DEPMPO-OOH can be monitored by following the de-
crease of an appropriate line of the spectrum. In contrast,
in the case of the 4-PhDEPMPOc nitrone, superimposi-
tion of the ESR signals of the two adducts (OOH and OH)
leads to complete overlap of the two signals, and there-
fore, the decrease of one line cannot be used to determine
the kinetics of decay of the 4-PhDEPMPOc-OOH adduct.
addition of the superoxide species on the less hindered
face of 4-PhDEPMPOc giving only one adduct. The
difference between the steric bulk of the methyl and the
diethoxyphosphoryl groups present on the C-5 position
combined with the presence of a phenyl group on the C-4
position forces the addition to take place on the face
opposite to the phosphoryl group. The specific patterns
of the two spectra (a) and (b) (Figure 2) could be explained
either by an exchange model between two conformers in
chemical equilibrium or by a superimposition model of
two species. These species could be either two rigid
conformers of the superoxide adduct or the combination
of a hydroxyl adduct with one superoxide adduct con-
former. Computer simulation of the spectra were made
with the ROKI program40 (dotted lines in Figure 2a,b).
The best fit was obtained with the exchange model, with
a regression parameter of 0.98739, the respective pa-
rameters being given in Table 1. Thus, the geometry of
the 4-PhDEPMPOc-OOH adduct does not freeze the
chemical exchange between two conformers of the su-
peroxide spin adduct.
(41) Barbati, S.; Cle´ment, J.-L.; Olive, G.; Roubaud, V.; Tuccio, B.;
Tordo, P. Free Rad. Biol. Environ. 1997, 39-47.
(42) Keszler, A.; Kalyanaraman, B.; Hogg, N. Free Rad. Biol. Med.
2003, 35, 1149-1157.
(43) Lauricella, R.; Allouch, A.; Roubaud, V.; Bouteiller, J.-C.; Tuccio,
B. Org. Biomol. Chem. 2004, 2, 1304-1309.
If we compare the ESR signals of the adducts of
superoxide with 4-PhDEPMPOc and with DEPMPO, the
(40) Rockenbauer, A.; Korecz, L. Appl. Magn. Reson. 1996, 10, 29-
43.
2138 J. Org. Chem., Vol. 70, No. 6, 2005

Synthesis and ESR Study of 4-PhenylDEPMPO Spin Traps
TABLE 1. Simulated Coupling Constants for Superoxide and Hydroxyl Adducts of 4-PhDEPMPOc 9
nitrone/R•
generating system
diastereoisomer conformer Ap (mT) AN (mT) AHâ (mT) AHγ (mT)
•-
9/O2
HX/XO system or KO2/18-crown-6/DMSO
in 0.1 M phosphate buffer
trans (100%)
T1 (38%)a
5.458
1.284
1.316
0.074
T2 (62%)a
5.241
5.333
5.594
1.289
1.369
1.410
1.08
0.982
1.660
9/HO•
Fenton reaction in 0.1 M phosphate buffer
trans (80.4%)
alkyl (14%)
a Exchange rate constant between T1 and T2, K ) 0.34 × 108 s-1
.
FIGURE 4. Kinetics of decay of the superoxide spin adducts.
Signal obtained after incubation of a mixture containing
hypoxanthine (0.4 mM), xanthine oxidase (0.04 U mL-1), DTPA
(1 mM), and 4-PhDEPMPOc 9 (20 mM) in phosphate buffer
(0.1 M, pH 7.3). The formation of superoxide anion radicals is
stopped by addition of a large amount of SOD (606 U mL-1),
and the spectrum was recorded by slow scan (5368.71 s).
Spectrometer settings: microwave power 10 mW; modulation
amplitude, 0.625; time constant, 0.128 s; gain 105; sweep time,
5368.71 s; conversion time, 2621.44 s.
FIGURE 3. Spin trapping of hydroxyl radical by 4-PhDEP-
MPOc 9. (a) Signal obtained after 1 min incubation of a
mixture containing 4-PhDEPMPOc 9 (20 mM), H2O2 (2 mM),
FeSO4 (2 mM), DTPA (1 mM) in phosphate buffer (0.1 M, pH
7.3). (b) As in (a) but in the presence of catalase (600 U mL-1).
The dotted line in (a) represents the computer simulation of
the spectrum. Spectrometer settings: microwave power 10
mW; modulation amplitude, 0.497; time constant, 0.128 s; gain
105; sweep time, 83.89 s; conversion time, 0.819 s.
(half-life time of 2 min),30 the superoxide adducts ob-
tained with the two nitrones 2 and 9 showed quite similar
values. Thus, the steric hindrance induced by the pres-
ence of the 4-phenyl group does not lead to a destabiliza-
tion of the superoxide spin adduct. In fact, addition of
the superoxide radical on the face opposite to the phos-
phoryl group results in a conformation of the nitroxide
in which the HOO and phosphoryl groups are in a
pseudoaxial position, favorable for the stabilization by
anomeric or hyperconjugative effects.26,44 Moreover, the
relative cis position between the 5-phosphoryl and 4-phe-
nyl groups results, upon addition of the superoxide
radical, in a conformation of the spin adduct 21, in which
the 4-phenyl group is in an equatorial position (Figure
5). A similar conformation is observed in spin adduct 5,
which is also stabilized. In contrast, the phosphoryl and
OOH substituents occupy equatorial positions in spin
adduct 7, a fact which is not favorable for the stabilizing
anomeric or hyperconjugative effects.26,44
A different method was used, taking advantage of the
fact that the ROKI program is able to compute the
concomitant evolution of different species. To compare
the kinetics of decay of the 4-PhDEPMPOc-OOH and
DEPMPO-OOH adducts, the two trapping systems were
tested in parallel under the same set of conditions. A
mixture containing hypoxanthine (0.4 mM), xanthine
oxidase (0.04 U mL-1), DTPA (1 mM), and the nitrone,
either 4-PhDEPMPOc 9 or DEPMPO 2 (20 mM), in
phosphate buffer (0.1 M, pH 7.3) was incubated at room
temperature. Once the adduct concentration had reached
a significant value (approximately 7 min), the formation
of superoxide anion radicals was stopped by addition of
a large amount of SOD and the spectra were recorded.
Fast scan recording (30 s) gave the complete ESR
spectrum. Slow scan recording (5368.71 s) was used to
determine the decay rate of the superoxide spin adducts,
which was computed by comparing the variation of ESR
amplitudes recorded in the two different sweeping condi-
tions (Figure 4). The ROKI program was first used to
simulate the fast recording spectra in the traditional
manner. Then the slow recording spectra were simulated,
and the previous ESR amplitudes were modulated by a
kinetic function describing either first or second order
or complex decay process.
(d) Addition on 4-PhDEPMPOt, 8. When addition
of the two oxygen radical species, HOO• and HO•, was
performed with the trans diastereoisomer (4-PhDEPM-
POt, 8), mixtures of diastereoisomeric spin adducts were
observed, leading to complex ESR spectra. In the case of
nitrone 8, the steric bulk of the 5-phosphoryl and 4-phen-
yl groups is evenly distributed on the two faces of the
pyrroline ring and stereoselectivity in the addition step
cannot be observed. Therefore, the trans diastereoisomer
8 does not present the same interest as the cis nitrone 9
for spin-trapping experiments.
The computations showed that the half-life times were
14.5 min for the 4-PhDEPMPOc-OOH adduct and 15.5
min for the DEPMPO-OOH adduct and that the decay
of the 4-PhDEPMPOc-OOH has a predominant first-
order character. In contrast to the 3-PhDEPMPOc-OOH
(44) Cle´ment, J.-L.; Ferre´, N.; Siri, D.; Karoui, H.; Rockenbauer, A.;
Tordo, P. J. Org. Chem. 2005, 70, 1198-1203.
J. Org. Chem, Vol. 70, No. 6, 2005 2139

Hardy et al.
FIGURE 5. Conformations of the superoxide adducts 5, 7, and 21.
m), 3.26 (1H, dd, J ) 5.1, 9.8), 2.92 (1H, dd, J ) 16.8, 5.1),
2.72 (1H, dd, J ) 16.8, 9.8), 1.13 (3H, s); 13C NMR (50.32 MHz)
δ 137.8 (1CIV, s), 128.8 (2C, d), 128.5 (2C, s), 127.8 (1C, s),
119.1 (1C, s), 109.8 (1CIV, s), 65.3 (1C, s), 64.4 (1C, s), 50.7
(1C,s), 22.6 (1C, s), 18.8 (1C, s). Anal. Calcd for C13H15NO2:
217.26; C, 71.87; H, 6.96; N, 6.45. Found: C, 72.06; H, 6.85;
N, 6.41.
3-(2-Methyl-1,3-dioxolan-2-yl)-3-phenylpropylamine
(13). Lithium aluminum hydride (6 mL of a 1 M solution in
THF) was added dropwise to a solution of 12 (430 mg, 1.98
mmol) in 10 mL of THF at 0 °C under argon. The mixture
was stirred for 2 h at 0 °C then 7 h at room temperature.
Potassium hydroxide (1 mL of a 10% aqueous solution) was
added dropwise at 0 °C. The solution was filtered and diluted
with CH2Cl2 (50 mL). The organic layer was washed with
water (10 mL) and brine (10 mL). The organic layer was dried
over Na2SO4, filtered, and distilled under reduced pressure to
yield 13 as a pale yellow oil (370 mg, 85%): 1H NMR (200.13
MHz) δ 7.40-7.10 (5H, m), 4.25-3.82 (4H, m), 2.86 (1H, dd, J
) 10.9, 4.1), 2.44 (2H, td, J ) 8.5, 6.2), 2.10-1.73 (2H, m),
1.27 (2H, s), 1.13 (3H, s); 13C NMR (75.47 MHz) δ 140.4 (1CIV,
s), 129.2 (2C, d), 127.9 (2C, s), 126.5 (1C, s), 111.2 (1CIV, s),
65.0 (1C, s), 64.5 (1C, s), 52.1 (1C, s), 40.3 (1C, s), 33.2 (1C, s)
22.9 (1C, s).
2-Methyl-3-phenylpyrroline (14). A solution of 13 (3.7 g,
16.8 mmol) in a mixture of THF (75 mL) and aqueous HCl (50
mL of a 3 M solution) was stirred for 4 h. The mixture was
colored in red in a few moments. Solid K2CO3 was added to
the solution up to pH 9, and the mixture was stirred for 1 h.
A fluorescent orange color appeared. The aqueous layer was
then saturated with NaCl and extracted with CH2Cl2 (3 × 60
mL) and ethyl acetate (50 mL). The combined organic layers
were dried over Na2SO4 and distilled under reduced pressure
to afford 14 (2.5 g, 93%) as a yellow oil: lit.32 bp 58-60 °C/
0.25 mm); 1H NMR (300.13 MHz) δ 7.38-7.20 (3H, m), 7.15-
7.10 (2H, m), 3.85-3.75 (2H, m), 2.55-2.39 (1H, m), 1.98-
1.88 (2H, m), 1.85 (3H, s); 13C NMR (75.47 MHz) δ 176.3 (1CIV,
s), 141.7 (1CIV, s), 128.7 (2C, s), 127.5 (2C, s), 126.7 (1C, s),
59.5 (1C, s), 57.6 (1C, s), 33.5 (1C, s), 18.0 (1C, s).
2-(Diethoxyphosphoryl)-2-methyl-3-phenylpyrroli-
dine (15). A mixture of 14 (0.9 g, 5.65 mmol) and boron
trifluoride ethyl etherate (71 µL, 0.565 mmol) was stirred for
20 min. Diethyl phosphite (86.8 µL, 6.78 mmol) was then
added, and the solution was stirred at room temperature for
7 days. After dilution with water (2 mL), aqueous HCl (5%)
was added to the mixture to reach pH 2, and the mixture was
washed with CH2Cl2. The aqueous layer was treated with a
saturated NaHCO3 solution to reach pH 9. After extraction
with CH2Cl2 (3 × 10 mL), the organic layer was dried over
Na2SO4 and distilled under reduced pressure to yield the
pyrrolidine 15 (900 mg, 54%) as a brown oil: 31P NMR (81.01
MHz) δ 29.30; 1H NMR (200.13 MHz) δ 7.81-7.32 (5H, m),
4.59-4.18 (4H, m), 3.94 (1H, m), 3.54-3.24 (2H, m), 2.74-
2.23 (3H, m), 1.52 (6H, t, J ) 7.1), 1.14 (3H, d, J ) 16.3); 13C
NMR (50.32 MHz) δ 139.9 (1CIV, d, J ) 4.6), 129.0 (2C, s),
127.6 (2C, s), 126.4 (1C, s), 62.5 (1C, d, J ) 164.1), 62.3 (1C,
d, J ) 7.4), 62.1 (1C, d, J ) 8.0), 48.8 (1C, d, J ) 4.0), 45.6
(1C, d, J ) 6.3), 32.8 (1C, d, J ) 5.7), 20.5 (1C, d, J ) 5.7),
16.3 (2C, d, J ) 5.7).
Conclusion
From the two diastereoisomeric C(4)-phenyl analogues
of DEPMPO 8 and 9, only the cis isomer, 4-PhDEPMPOc
9, trapped hydroxyl and superoxide radicals in a stereo-
selective manner. The ESR spectrum of the 4-PhDEP-
MPOc-OOH spin adduct was more easily assignable than
that of DEPMPO-OOH since the alternating line width
phenomenon due to a chemical exchange has consider-
ably been reduced. Moreover the 4-PhDEPMPOc-super-
oxide spin adduct 21 has a persistency similar to the one
of DEPMPO-OOH (∼15 min) but a greater persistency
than the DEPMPPO-OOH adduct 7 (T1/2 DEPMPPO < 2
min). The conformational effect exerted by the presence
of a phenyl group on the pyrroline ring is strongly
dependent upon its position. When the phenyl is on the
C-3 position, a strong destabilization takes place.26 In
contrast, when the phenyl is on the C-4 position, a modest
stabilization of the superoxide adduct is observed. Thus,
the presence of a phenyl group on the pyrroline ring in
the most suitable position as in the 4-PhDEPMPO-c 9
does not freeze enough the conformational flexibility of
the ring to forbid the occurrence of the particular
conformation of the superoxide spin adduct that is prone
to dismutation of the nitroxide, leading to decrease of the
ESR signal.
Experimental Section
General Methods. The solvents were distilled under dry
argon atmosphere: THF, Et2O, and toluene in the presence
of sodium and benzophenone and CH2Cl2 in the presence of
P2O5. All chemicals and organic solvents were commercially
available and were used as supplied. The reactions were
monitored by CCM using silica gel and by 31P NMR. Crude
materials were purified by flash chromatography on silica gel
60 (0.040-0.063 mm). 31P NMR, 1H NMR, and 13C NMR
spectra were recorded at 121.49, 300.13, and 75.54 MHz,
respectively, or at 81.01, 200.13, and 50.32 MHz, respectively.
31P NMR was taken in CDCl3 using 85% H3PO4 as an internal
standard with broadband 1H decoupling. 1H NMR and 13C
NMR were taken in CDCl3 using TMS or CDCl3 as internal
reference, respectively. Chemical shifts (δ) are reported in ppm
and J values in hertz. The assignments of 1H NMR signals of
the compounds 20 and 9 were facilitated by use of the HMQC
(1H-13C) sequence. Melting points are uncorrected. Elemental
analyses and mass spectra were determined at the University
of Aix-Marseille III. HRMS was determined at the Service
Central d’Analyse du CNRS at Vernaison, France.
Synthesis of the Trans Nitrone (4-PhDEPMPOt) (8).
3-(2-Methyl-1,3-dioxolan-2-yl)-3-phenylpropionitrile (12).
In a Dean-Stark apparatus a mixture of 3-phenyl-4-oxopen-
tanenitrile31 (2.5 g, 14 mmol), ethylene glycol (6.5 mL, 0.11
mol), and camphorsulfonic acid (0.5 g, 2.1 mmol) in anhydrous
toluene (40 mL) was stirred under reflux for 8 h. The solvent
was distilled under reduced pressure. The oily residue was
purified by flash chromatography over silica gel (pentane/Et2O
3:1) to afford 12 as a white solid (2.82 g, 91%): mp 65.9 °C;
1H NMR (200.13 MHz) δ 7.35-7.25 (5H, m), 4.05-3.90 (4H,
2140 J. Org. Chem., Vol. 70, No. 6, 2005

Synthesis and ESR Study of 4-PhenylDEPMPO Spin Traps
trans-5-(Diethoxyphosphoryl)-5-methyl-4-phenylpyr-
roline N-Oxide (4-PhDEPMPOt) (8). A solution of the
pyrrolidine 15 (360 mg, 1.2 mmol) in ethyl alcohol (3 mL) was
added to a solution of sodium tungstate (40 mg, 0.12 mmol)
in demineralized water (6 mL). The mixture was cooled at -5
°C, and hydrogen peroxide (0.25 mL of a 30% aqueous solution,
2.54 mmol) was then added dropwise over a period of 1 h. The
mixture was then stirred at 3-4 °C for 48 h. The aqueous layer
was saturated with sodium chloride and extracted with CH2-
Cl2 (3 × 10 mL). The organic layer was dried over Na2SO4 and
distilled under reduced pressure. The residual oil was purified
by flash chromatography on silica gel (CH2Cl2/EtOH 9:1) to
crude oil that was dissolved in CH2Cl2 (20 mL). Triethyl
phosphite (3.38 mL, 0.019 mol) was then added dropwise over
a period of 2 h at 0 °C. The mixture was kept at room
temperature for 15 h. Distillation under reduced pressure (10-3
Torr) afforded 18 as a pale yellow oil (5.8 g, 100%): 31P NMR
(121.49 MHz) δ -2.69; 1H NMR (300.13 MHz) δ 7.41-7.18 (5H,
m), 4.45 (1H, t, J ) 7.6), 4.18-3.94 (2H, m), 3.87-3.68 (2H,
m), 3.27 (1H, ddt, J ) 12.5, 6.5, 6.6), 3.11 (1H, ddd, J ) 12.5,
6.5, 8.0), 2.31 (1H, m, J ) 8.0, 6.6, 14.2, 7.6), 1.98 (1H, m, J )
14.2, 7.6, 6.5, 6.5), 1.27 (3H, t, J ) 7.1), 1.10 (3H, t, J ) 7.2);
13C NMR (75.47 MHz) δ 209.3 (1CIV, d, J ) 166.3), 134.2 (1CIV,
s), 129.3 (2C, s), 129.0 (2C, s), 128.0 (1C, s), 63.7 (1C, d, J )
7.1), 63.2 (1C, d, J ) 7.1), 55.2 (1C, d, J ) 53.3), 48.7 (1C, s),
30.5 (1C, d, J ) 3.9), 16.1 (1C, d, J ) 5.5), 15.9 (1C, d, J )
6.0). Anal. Calcd for C14H20N3O4P: 325.30; C, 51.69; H, 6.20.
Found: C, 51.89; H, 6.17.
2-Diethoxyphosphoryl-3-phenylpyrroline (19). Under
argon, a solution of triphenylphosphane (4.3 g, 0.018 mol) in
Et2O (30 mL) was added dropwise to a solution of the
acylphosphonate 18 (5.8 g, 0.018 mol) in Et2O (20 mL) at 10
°C. The mixture was stirred at room temperature for 16 h.
The solution was filtered, and the solid was washed with
anhydrous Et2O. After distillation of the solvent under reduced
pressure, a mixture of anhydrous Et2O/petroleum ether (1/1)
was added. The precipitate was filtered and washed with
anhydrous Et2O at 0 °C. After distillation of the solvent under
reduced pressure, the procedure was repeated until complete
removal of triphenylphosphine oxide. The iminophosphonate
19 was obtained as a pale yellow oil (4.8 g, 95%): bp 140 °C/
10-3 Torr; 31P NMR (121.49 MHz) δ 7.94; 1H NMR (300.13
MHz) δ 7.36-7.27 (3H, m), 7.17-7.13 (2H, m), 4.32-4.70 (6H,
m), 2.56-2.39 (1H, m, J ) 13.2, 7.0), 2.03-1.88 (1H, m, J )
18.7, 13.2), 1.38-1.24 (1H, m, J ) 18.7, 7.0), 1.21 (3H, t, J )
6.9), 1.07 (3H, t, J ) 6.8); 13C NMR (75.47 MHz) δ 173.8 (1CIV,
d, J ) 207.9), 140.4 (1CIV, s), 128.5 (2C, s), 127.7 (2C, s), 126.9
(1C, s), 63.4 (1C, d, J ) 34.4), 62.8 (1C, d, J ) 6.2), 62.2 (1C,
d, J ) 6.2), 57.7 (1C, d, J ) 30.4), 32.6 (1C, d, J ) 5.1), 15.9
(1C, d, J ) 6.6), 15.8 (1C, d, J ) 6.6).
1
afford 8 (300 mg, 80%): 31P NMR (121.49 MHz) δ 21.81; H
NMR (300.13 MHz) δ 7.35-7.26 (3H, m), 7.18-7.09 (3H, m),
4.37-4.15 (4H, m), 3.42-3.28 (1H, m), 2.86-2.74 (1H, m),
2.65-2.10 (1H, m), 1.37 (3H, t, J ) 7.0), 1.35 (3H, t, J ) 7.2),
1.18 (3H, d, J ) 15.8); 13C NMR (75.47 MHz) δ 139.1 (1CIV, d,
J ) 9.7), 134.2 (1C, d, J ) 9.2), 128.6 (2C, d), 128.6 (2C, s),
127.8 (1C, s), 78.4 (1C, d, J ) 173.8), 64.2 (1C, d, J ) 6.3),
62.8 (1C, d, J ) 8.0), 44.9 (1C, d, J ) 1.8), 34.1 (1C, s), 16.4
(2C, d, J ) 6.3), 16.3 (1C, s); HRMS calcd for C15H22NO4P
[C15H22NO4P]+ + Na+ 334.1184, found 334.1172.
Synthesis of the Cis Nitrone (4-PhDEPMPOc) (9).
4-Azido-2-phenylbutyric Acid Ethyl Ester (16). Under
argon, a stirred solution of lithium diisopropylamide (36.5 mL
of a 2 M solution in hexanes) in THF (20 mL) was cooled to
-78 °C, and a solution of ethyl phenylacetate (8 g, 0.049 mol)
in THF (40 mL) was added dropwise. The mixture was stirred
at -78 °C for 1 h. A solution of 1-azido-2-iodoethane33 (12.42
g, 0.0633 mol) in THF (20 mL) was added over a period of 10
min. The solution was gradually warmed to room temperature
and stirred for 12 h. A saturated aqueous NH4Cl solution was
added to the mixture up to pH 8, and the aqueous layer was
extracted with CH2Cl2 (3 × 80 mL). The organic layers were
combined, washed with brine, and dried over Na2SO4. The
solvent was distilled under reduced pressure, and the residual
oil was purified by flash chromatography on silica gel (gradient
of pentane/CH2Cl2 4:1 to pure CH2Cl2) to afford 16 as a pale
yellow oil (6.73 g, 59%): 1H NMR (300.13 MHz) δ 7.39-7.20
(5H, m), 4.24-4.01 (2H, m), 3.68 (1H, t, J ) 7.7), 3.29 (1H,
ddd, J ) 12.3, 6.3, 6.6), 3.18 (1H, ddd, J ) 12.3, 6.3, 7.2), 2.33
(1H, m, J ) 14.1, 7.7, 7.2, 6.5), 2.01 (1H, m, J ) 14.1, 7.7, 6.3,
6.3), 1.20 (3H, t, J ) 7.1). 13C NMR (75.47 MHz) δ 173.1 (1CIV,
s), 138.0 (1CIV, s), 128.8 (2C, s), 127.9 (2C, s), 127.5 (1C, s),
60.9 (1C, s), 49.2 (1C, s), 48.5 (1C, s), 32.5 (1C, s), 14.0 (1C, s).
Anal. Calcd for C12H15N3O2: 233.27; C, 61.79; H, 6.48; N, 18.01.
Found: C, 61.91; H, 6.50; N, 18.01.
4-Azido-2-phenylbutyric Acid (17). A minimum of metha-
nol was added to a mixture of the azidoester 16 (6.62 g, 0.028
mol) and aqueous solution of NaOH (1 N, 0.035 mol) to make
the reaction mixture homogeneous. After the mixture was
stirred for 3 h at room temperature, the methanol was distilled
under reduced pressure. The aqueous solution was acidified
with HCl (10% aqueous solution) to reach pH 0. The aqueous
phase was extracted with Et2O (3 × 70 mL), and the organic
phase was dried over Na2SO4. The solvent was distilled under
reduced pressure to afford 17 as a white solid (5.8 g, 100%):
mp 58-59 °C; 1H NMR (300.13 MHz) δ 10.11-9.19 (1H, s,
OH), 7.30-7.13 (5H, m), 3.64 (1H, t, J ) 7.8), 3.22 (1H, ddt, J
) 12.7, 6.4, 6.4), 3.09 (1H, ddd, J ) 12.7, 6.4, 7.4), 2.25 (1H,
m, J ) 7.4, 6.4, 14.2, 7.8), 1.94 (1H, m, J ) 14.2, 7.8, 6.4, 6.4);
13C NMR (75.47 MHz) δ 179.2 (1CIV, s), 137.1 (1CIV, s), 128.9
(2C, s), 128.0 (2C, s), 127.8 (1C, s), 48.9 (1C, s), 48.3 (1C, s),
31.9 (1C, s). Anal. Calcd for C10H11N3O2: 205.21; C, 58.53; H,
5.40; N, 20.48. Found: C, 58.77; H, 5.49; N, 20.30.
2-(Diethoxyphosphoryl)-2-methyl-3-phenylpyrroli-
dine (20). Under argon, boron trifluoride diethyl etherate (1.97
mL, 7.46 mmol) was added dropwise to a solution of the
iminophosphonate 19 (1 g, 3.55 mmol) in THF (20 mL) at -78
°C. The mixture was stirred for 2 h at this temperature.
Methylmagnesium bromide (2.5 mL of a 3 M solution in THF)
was added dropwise over a period of 1 h at -78 °C. The
solution was stirred at this temperature for 4 h, and then a
saturated NaHCO3 aqueous solution was added to the mixture
kept at -78 °C. The aqueous layer was washed with CH2Cl2
(3 × 50 mL). The aqueous phase was treated with an aqueous
HCl (10%) solution to reach pH 1. The aqueous layer was
washed with CH2Cl2 and treated with a saturated NaHCO3
solution to reach pH 9. After extraction with CH2Cl2, the
organic layer was dried over Na2SO4 and the solvent distilled
under reduced pressure to give the pyrrolidine 20 as a pale
yellow oil (0.8 g, 76%): 31P NMR (121.49 MHz) δ 26.50; 1H
NMR (300.13 MHz) δ 7.46-7.43 (2H, d, J ) 7.37, Har), 7.33-
7.18 (3H, m, Har), 4.04-3.92 (2H, m, CH2OP), 3.74-3.62 (1H,
m, J ) 6.7, CHOP), 3.47-3.30 (2H, m, J ) 6.7, CHOP, Hd,
dtd, JHdHe ) 16.4, JHdHb ) 8.7, JHdHc ) 2.4), 3.17 (He, ddd, JHeHd
) 16.4, JHeHb ) 9.1, JHeP ) 1.1), 3.02 (Ha, ddd, JHaP ) 30.9,
JHaHb ) 12.1, JHaHc ) 7.4), 2.81 (Hb, m, JHbHc ) 17.6, JHbHa
)
12.1, JHbHe ) 9.1), 2.18-2.05 (Hc, m, JHcHb ) 17.6, JHcHa ) 7.4,
JHcHd ) 2.4), 1.91 (1H, s, NH), 1.47 (3H, d, JHP ) 13.6, CH3CIV),
1.16 (3H, t, J ) 7.2, CH3CH2OP), 0.88 (3H, t, J ) 7.2, CH3-
CH2OP); 13C NMR (75.47 MHz) δ 138.0 (1CIV, s, Car), 128.9
(2C, s, Car), 127.4 (2C, s, Car), 126.4 (1C, s, Car), 64.7 (1C, d,
JCP ) 158.6, CIVNH), 61.9 (1C, d, JCP ) 7.7, CH2OP), 60.6 (1C,
d, JCP ) 7.7, CH2OP), 55.5 (1C, d, JCP ) 5.5, CHPh), 44.1 (1C,
4-Azido-2-phenylbutyrylphosphonic Acid Diethyl Es-
ter (18). Under argon, oxalyl chloride (2.31 mL, 0.027 mol)
was added via a syringe to a solution of the acid 17 (3.68 g,
0.018 mol) in CH2Cl2 (11 mL) at 0 °C. The solution was stirred
at room temperature for 1 h and refluxed for 2 h. After the
solution was cooled to room temperature, distillation of the
volatiles under reduced pressure (10-3 Torr, 2 h) afforded a
d, JCP ) 2.7, CH2NH), 30.6 (1C, s, CH2CH), 24.0 (1C, d, JCP
)
6.0, CH3CIV), 16.2 (1C, d, JCP ) 5.5, CH3CH2OP), 15.6 (1C, d,
JCP ) 6.0, CH3CH2OP).
J. Org. Chem, Vol. 70, No. 6, 2005 2141

Hardy et al.
The pyrrolidine 20 was characterized as the picrate, after
recrystallization in ethyl alcohol. Anal. Calcd for
C21H27N4O10P: 526.43; C, 47.91; H, 5.17; N, 10.64. Found: C,
47.93; H, 5.31; N, 10.46.
Standard ESR spectra were simulated using the ESR software
developed by A. Rockenbauer from the Central Research
Institute of Chemistry, Hungary.40
(c) Superoxide Trapping: Hypoxanthine-Xanthine
Oxidase System. Xanthine oxidase (0.04 U mL-1) was added
to a solution of 4-PhDEPMPO (20 mM), DTPA (1 mM), and
hypoxanthine (0.4 U mL-1) in phosphate buffer (0.1 M, pH 7.3).
When SOD (606 U mL-1) was added to the HX/XO generat-
ing system, the ESR signal was not observed.
(d) Superoxide Trapping: KO2/18-Crown-6 System.
The ESR signal was observed upon incubating the reaction
mixture obtained after adding a DMSO solution of KO2 (10
mM final concentration) and 18-crown-6 ether (10 mM final
concentration) to a phosphate buffer solution (0.1 M) contain-
ing 4-PhDEPMPOc (20 mM final concentration).
cis-5-(Diethoxyphosphoryl)-5-methyl-4-phenylpyrro-
line N-Oxide (4-PhDEPMPOc) (9). A solution of the pyr-
rolidine 20 (300 mg, 1 mmol) in ethyl alcohol (3 mL) was added
to a solution of sodium tungstate (33 mg, 0.1 mmol) in
demineralized water (6 mL). The mixture was cooled at -5
°C, and hydrogen peroxide (0.21 mL of a 30% aqueous solution,
2.22 mmol) was then added dropwise over a period of 1 h. The
mixture was then stirred at 3-4 °C for 50 h. The aqueous layer
was saturated with sodium chloride and extracted with CH2-
Cl2 (3 × 10 mL). The organic layer was dried over Na2SO4 and
distilled under reduced pressure. The crude oil was purified
by flash chromatography on silica gel (CH2Cl2/EtOH 95:5) to
(e) Hydroxyl Trapping. The hydroxyl radical was gener-
ated by addition of FeSO4 (2 mM) to a solution of 4-PhDEP-
MPO (20 mM), DTPA (1 mM), and H2O2 (2 mM) in phosphate
buffer (0.1 M, pH 7.3).
1
afford 9 (200 mg, 64%): 31P NMR (121.49 MHz) δ 19.93; H
NMR (300.13 MHz) δ 7.55-7.25 (5H, m, Har), 7.19 (1H, q, J )
2.5, CHN), 4.24-4.04 (2H, m, J ) 7.0, CH2OP), 3.75-3.58 (1H,
m, J ) 15.5, CHOP), 3.67 (Ha, ddd, JHaP ) 20.5, JHaHc ) 11.7,
No ESR signal was observed when catalase (600 U mL-1
was added to the incubation mixture.
)
JHaHb ) 9.7), 3.51 (Hb, dtd, JHbHc ) 20.2, JHbHa ) 9.7, JHbP
)
2.4), 3.35 (1H, m, J ) 15.5, CHOP), 2.69 (Hc, dtd, JHcHb ) 20.2,
JHcHa ) 9.7, JHcP ) 2.5), 1.77 (3H, d, JHP ) 13.5, CH3CIV), 1.25
(3H, t, J ) 7.1, CH3CH2OP), 0.75 (3H, t, J ) 7.1, CH3CH2OP);
13C NMR (75.47 MHz) δ 135.7 (1C, d, JCP ) 6.0, HCN), 135.3
(1CIV, d, JCP ) 3.3, Car), 129.16 (2C, s, Car), 128.1 (2C, s, Car),
127.7 (1C, s, Car), 78.5 (1C, d, JCP ) 149.8, CIVNO), 64.1 (1C,
d, JCP ) 6.0, CH2OP), 61.0 (1C, d, JCP ) 7.7, CH2OP), 52.9
(1C, d, JCP ) 2.2, CHPh), 30.7 (1C, d, JCP ) 1.7, CH2CH), 19.6
(1C, s, CH3CIV), 16.3 (1C, d, JCP ) 6.0, CH3CH2OP), 15.4 (1C,
d, JCP ) 7.1, CH3CH2OP); ESI/MS/MS (20 eV) m/z 312.1 (M+
+ H), 294.3 (51.3), 266.1 (19.5), 238.0 (64.6), 219.9 (16.8), 174.1
(74.3).
(f) Kinetics of Decay of the Superoxide Spin Adducts.
The previously described HX/XO system was used to generate
superoxide in phosphate buffer (0.1 M, pH 7.3). The spin-trap
concentration was 20 mM for DEPMPO and 4-PhDEPMPOc.
Once the adduct concentration had reached a significant value
(approximately 7 min), the formation of superoxide anion
radicals was stopped by addition of a large amount of SOD
(606 U mL-1), and the spectrum was recorded by slow scan
(5368.71 s). Computer simulations were performed using the
ROKI40 program developed by A. Rockenbauer at the Central
Research Institute for Chemistry in Budapest. Spectrometer
settings: microwave power 10 mW; modulation amplitude,
0.625; time constant, 0.128 s; gain 105; sweep time, 5368.71 s;
conversion time, 2621.44 s.
Spin-Trapping Studies. (a) General Information. Xan-
thine oxidase (XO), bovine erythrocyte superoxide dismutase
(SOD), catalase, diethylenetriaminepentaacetic acid (DTPA),
and other chemicals were purchased from commercial suppli-
ers.
(b) ESR Measurements. ESR spectra were recorded at
room temperature using a spectrometer at 9.5 GHz (X-band)
employing 100 kHz field modulation. Reaction mixtures were
prepared in a chelex-treated phosphate buffer (0.1 M, pH 7.3).
Acknowledgment. A.R. thanks the Hungarian Sci-
ence Fund for partial funding of this work (Grant No.
OTKA T-046953).
JO0479516
2142 J. Org. Chem., Vol. 70, No. 6, 2005