Synthesis of a new spin trap: 2-(diethoxyphosphoryl)-2-phenyl-3,4-dihydro-2H-pyrrole 1-oxide

Article abstract of DOI:10.1021/jo981875n

Recently, the synthesis of a new phosphorylated nitrone, 2-(diethoxyphosphoryl)-2-methyl-3,4-dihydro-2H-pyrrole 1-oxide (DEPMPO) (2) was described. The presence of the phosphorylated group strongly stabilized the DEPMPO-superoxide spin adduct. To understand the role of the diethoxy-phosphoryl group in this stabilization, a new phosphorylated nitrone, 2-(diethoxyphosphoryl)-2-phenyl-3,4-dihydro-2H-pyrrole 1-oxide (DEPPPO) (7), was prepared through a four-step synthetic pathway, and its ability to trap free radicals was investigated. Data obtained from spin trapping experiments of a wide variety of free radicals generated in situ showed the formation of two diastereoisomers spin adducts with different phosphorus and hydrogen coupling constants. Superoxide trapping by DEPPPO gave a persistent nitroxide spin adduct, and its half-time life was measured and compared to that of the DEPMPO analogue.

Full text of DOI:10.1021/jo981875n

J . Org. Chem. 1999, 64, 1471-1477
1471
Syn th esis of a New Sp in Tr a p :
2-(Dieth oxyp h osp h or yl)-2-p h en yl-3,4-d ih yd r o-2H-p yr r ole 1-Oxid e†
Hakim Karoui, Ce´line Nsanzumuhire, Franc¸ois Le Moigne, and Paul Tordo*
Laboratoire Structure et Re´activite´ des Espe`ces Paramagne´tiques, case 521, CNRS UMR 6517 “Chimie,
Biologie et Radicaux Libres”, Universite´s d’Aix-Marseille I et III, Centre de Saint J e´roˆme, Av. Escadrille
Normandie Niemen, 13397 Marseille Cedex 20, France
Received September 15, 1998
Recently, the synthesis of a new phosphorylated nitrone, 2-(diethoxyphosphoryl)-2-methyl-3,4-
dihydro-2H-pyrrole 1-oxide (DEPMPO) (2) was described. The presence of the phosphorylated group
strongly stabilized the DEPMPO-superoxide spin adduct. To understand the role of the diethoxy-
phosphoryl group in this stabilization, a new phosphorylated nitrone, 2-(diethoxyphosphoryl)-2-
phenyl-3,4-dihydro-2H-pyrrole 1-oxide (DEPPPO) (7), was prepared through a four-step synthetic
pathway, and its ability to trap free radicals was investigated. Data obtained from spin trapping
experiments of a wide variety of free radicals generated in situ showed the formation of two
diastereoisomers spin adducts with different phosphorus and hydrogen coupling constants.
Superoxide trapping by DEPPPO gave a persistent nitroxide spin adduct, and its half-time life
was measured and compared to that of the DEPMPO analogue.
Sch em e 1
In tr od u ction
Superoxide anion radical (O2•-) is the most important
radical formed in aerobic organisms. During the mito-
chondrial respiratory chain, 1-4% of the metabolized
oxygen escape the normal four-electron reduction process
after accepting the first electron. Superoxide is also
generated in vivo from various enzymatic processes and
by the oxidation of a number of biologically significant
compounds. Superoxide and reactive oxygen species
derived therefrom (HOO•, HO•, and H2O2) are considered
to be involved in a host of pathological processes includ-
ing simple inflammation,1 ischemia-reperfusion syn-
drome,2 DNA damage,3 and neuron death.4
ESR-spin trapping is a useful method to detect free
radicals and has been widely used to trap oxygen-
centered radicals in biological milieu.5 2,2-Dimethyl-3,4-
dihydro-2H-pyrrole 1-oxide (DMPO) (1, Scheme 1) is the
most popular spin trap, and ESR features of its resulting
nitroxide spin adducts are well-known.6 However, a major
limitation with superoxide trapping is the low rate
constant for the reaction between O2•- and nitrones and
also the poor stability of the resulting nitroxide spin
adduct.7,8 DMPO-superoxide spin adduct has a half-life
time estimated to be 1 min at physiological pH,7 and a
small percentage (3-5%) decomposes to DMPO-hydroxyl
adduct,8 thus, in some extent, limiting the use of DMPO
to trap superoxide.
Studies of â-phosphorylated five-membered ring ni-
troxides reported previously showed that their ESR
phosphorus coupling was very sensitive to the ring
conformation and represents a valuable structural probe.9
We thus developed the synthesis of a new class of
phosphorylated nitrones which would give â-phosphory-
lated five-membered ring nitroxide spin adducts by
trapping free radicals (Scheme 2).
†Abbr evia tion s: DEPPPO, 2-(diethoxyphosphoryl)-2-phenyl-3,4-
dihydro-2H-pyrrole 1-oxide; DEPMPO, 2-(diethoxyphosphoryl)-2-meth-
yl-3,4-dihydro-2H-pyrrole 1-oxide; DEP-DMPO, 5-(diethoxyphosphoryl)-
2,2-dimethyl-3,4-dihydro-2H-pyrrole 1-oxide; DMPO, 2,2-dimethyl-3,4-
dihydro-2H-pyrrole 1-oxide; O2•-, superoxide anion radical.
(1) Halliwell, B.; Gutteridge, J . M. C. In Free Radicals in Biology
and Medicine, 2nd ed.; Oxford: Clarendon Press: 1989; p 422.
(2) Korthuis, D. L.; Carden, D. L.; Granger, D. N. In Biological
Consequences of Oxidative Stress; Spatz, L., Bloom, D., Eds.; Oxford
Press: London, 1992; p 50.
(3) (a) Wiseman, H.; Halliwell, B. Biochem. J . 1996, 313, 17. (b)
Keyer, K.; Imlay, J . A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13635.
(4) Schulz, J . B.; Henshaw, D. R.; Siwek, D.; J enkins, B. G.;
Ferrante, R. J .; Cipolloni, P. B.; Kowall, N. W.; Rosen, B. R.; Beal, M.
F. J . Neurochem. 1995, 64, 2239.
(8) (a) Finkelstein, E.; Rosen, G. M.; Rauckman, E. J .; Paxton, J .
Mol. Pharmacol. 1979, 16, 676. (b) Finkelstein, E.; Rosen, G. M.;
Rauckman, E. J . Mol. Pharmacol. 1982, 21, 262. (c) Finkelstein, E.;
Rosen, G. M.; Rauckman, E. J . J . Am. Chem. Soc. 1980, 102, 4994. (d)
Finkelstein, E.; Rosen, G. M.; Rauckman, E. J . Arch. Biochem. Biophys.
1980, 200, 1. (e) Buettner, G. R. Free Rad. Res. Commun. 1993, 19,
S79.
(9) (a) Tordo, P.; Boyer, M.; Santero, O.; Pujol, L. J . Phys. Chem.
1978, 82, 1742. (b) Mercier, A.; Berchadsky, Y.; Badrudin, Y.; Pie´tri,
S.; Tordo, P. Tetrahedron Lett. 1991, 32, 2125. (c) Le Moigne, F.;
Mercier, A.; Tordo, P. Tetrahedron Lett. 1991, 32, 3841. (d) Dem-
browski, L.; Finet, J . P.; Fre´javille, C.; Le Moigne, F.; Mercier, A.;
Pages, P.; Tordo, P. Free Rad. Res. Commun. 1993, 19, S23. (e) Stipa,
P.; Finet, J . P.; Le Moigne, F.; Tordo, P. J . Org. Chem. 1993, 58, 4465.
(5) Degray, J . A.; Mason, R. P. In Electron Spin Resonance; Atherton,
N. M., Davies, M. J ., Gilbert, B. C., Eds.; Athenæum Press Ltd.:
Cambridge, 1994; Vol. 14, p 246.
(6) Buettner, G. R. Free Radical Biol. Med. 1987, 3, 259.
(7) Buettner, G. R.; Oberley, L. W. Biochim. Biophys. Res. Commun.
1978, 83, 69.
10.1021/jo981875n CCC: $18.00 © 1999 American Chemical Society
Published on Web 02/10/1999

1472 J . Org. Chem., Vol. 64, No. 5, 1999
Karoui et al.
Sch em e 2
Sch em e 3a
The first phosphorylated five-membered ring nitrone
synthesized, (2-diethoxyphosphoryl)-2-methyl-3,4-dihy-
dro-2H-pyrrole 1-oxide (DEPMPO) (2, Scheme 1), exhib-
ited interesting spin trapping properties and yielded long-
lived superoxide spin adduct (half-life time estimated to
15 min at physiological pH).10 Zeghdaoui et al. reported
the synthesis of PBN-type phosphorylated nitrones,11a
and Roubaud et al. showed that for these nitrones also
the phosphorylated group significantly stabilized the
superoxide spin adducts.11b In the case of diethyl (1-N-
oxy-N-[4-(N′-oxypyridinyl)formylene]amino-1-methyleth-
yl)phosphonate (4-PyOPN) (4), the phosphorylated ana-
logue of commercially available R-(4-pyridyl-1-oxide)-N-
tert-butylnitrone (4-PyOBN) (3), the superoxide spin
adduct is 22 times more persistent in 0.1 M phosphate
buffer at pH 5.8.11b Recently, Roubaud et al. showed by
synthesizing the 2-(diethoxyphosphorylmethyl)-2-methyl-
3,4-dihydro-2H-pyrrole 1-oxide (5) that the lifetime of the
superoxide spin adduct depended on the phosphorylated
group position in the five-membered ring nitrone.12 The
same observations were reported by J anzen et al. who
used 5-(diethoxyphosphoryl)-2,2-dimethyl-3,4-dihydro-
2H-pyrrole 1-oxide (DEP-DMPO) (6).13 The half-life time
of DEP-DMPO-superoxide spin adduct was only 3.6 s.13
Although the presence of a phosphorylated group on the
double bond will facilitate a spectral pattern (6-line
spectra), it decreased dramatically the superoxide adduct
persistency.
a(i) NaN3, DME, 75 °C, 16 h (86%, crude product); (ii) PPh3,
Et2O (70%); (iii) HP(O)(OEt)2, cat. BF3(Et2O) (68%); (iv) m-CPBA,
CHCl3, -5 °C (30%).
ethoxyphosphoryl)-2-phenyl-3,4-dihydro-2H-pyrrole 1-ox-
ide (DEPPPO) (7), and then investigating their ESR
features.
Resu lts
Syn th esis. According to a method described by Vault-
ier,15 we obtained the γ-azidobutyrophenone (86%,
crude product) by adding sodium azide to a solution of
commercially available γ-chlorobutyrophenone in 1,2-
dimethoxyethane. The reaction mixture was stirred at
75 °C during 16 h. The 2-phenyl-1-pyrroline 7a (70%,
after crystallization in pentane) was obtained in two steps
from the γ-azidobutyrophenone via cyclization of the
iminophosphorane (not isolated). Reaction of diethyl
phosphite with 7a in the presence of a catalytic amount
of boron trifluoride diethyl etherate (Et2O‚BF3) at room
temperature led to 68% of diethyl (2-phenylpyrrolidin-
2-yl)phosphonate 7b (Scheme 3) obtained as a colorless
oil after column chromatography. Oxidation of 7b was
the most sensitive step of the synthetic pathway and was
carried out with a solution of 70% m-chloroperbenzoic
acid in chloroform at -5 °C to yield 7 as a yellow oil (30%)
after column chromatography (CH2Cl2/EtOH 90/10).
E SR St u d ies. (a ) Sp in Tr a p p in g of Hyd r oxyl
Ra d ica l. The hydroxyl radical was obtained by incubat-
ing DEPPPO (50 mM) with H2O2 (1 mM) and FeSO4 (0.3
mM) in 0.1 M phosphate buffer at pH 7.0. We obtained
an ESR signal simulated as a superimposition of spectra
corresponding respectively to two diastereoisomers (aN
) 1.35 mT, aH ) 1.05 mT (1H), aP ) 3.06 mT (48%); aN
) 1.37 mT, aH ) 1.49 mT (1H), aP ) 3.18 mT, aHγ ) 0.043
mT (1H) (52%)) (Figure 1a). This signal was inhibited
by the presence of catalase (50 U mL-1) in the incubation
mixture. We observed the same signal by nucleophilic
addition of water to 7 in the presence of Fe3+ (data not
shown).16 In this case, the percentage contribution for
each diastereoisomer determined by computer simulation
was 54/46% showing that we were getting a different
ratio by radical addition. Due to the presence of two
diastereoisomers, the DEPPPO/•OH signal is relatively
For the DEPMPO-superoxide spin adduct, the average
value of the phosphorus coupling is rather important (aP
≈ 5.29 mT at pH 7.4 in phosphate buffer). The large
magnitude of the phosphorus coupling shows a significant
hyperconjugative interaction between the carbon-phos-
phorus bond and the nitroxyl moiety. The existence of
this interaction shows that in the DEPMPO-superoxide
conformers the carbon-phosphorus bond adopts an axial
or pseudoaxial orientation. To appreciate if stereoelec-
tronic effects could influence the persistency of the
DEPMPO-superoxide spin adduct, we sought to generate
spin adducts in which the hyperconjugative interaction
between the carbon-phosphorus bond and the nitroxyl
moiety would be weakened. Rockenbauer has shown that
for pyrrolidinoxyl radicals the phenyl group adopts an
axial position.14 We thus decided to generate spin adducts
of the pyrrolidinoxyl series bearing a diethoxyphosphoryl
and a phenyl group on the same carbon (Scheme 2, R )
Ph) by trapping free radicals with a new nitrone, 2-(di-
(10) (a) Fre´javille, C.; Karoui, H.; Le Moigne, F.; Culcasi, M.; Pie´tri,
S.; Lauricella, R.; Tuccio, B.; Tordo, P. J . Chem. Soc., Chem. Commun.
1994, 1793. (b) Fre´javille, C.; Karoui, H.; Le Moigne, F.; Culcasi, M.;
Pie´tri, S.; Lauricella, R.; Tuccio, B.; Tordo, P. J . Med. Chem. 1995, 38,
258.
(11) (a) Zeghdaoui, A.; Tuccio, B.; Finet, J . P.; Cerri, V.; Tordo, P.
J . Chem. Soc., Perkin Trans. 2 1995, 12, 2087. (b) Roubaud, V.;
Lauricella, R.; Tuccio, B.; Bouteiller, J . C.; Tordo, P. Res. Chem.
Intermed. 1996, 22 (4), 405.
(12) Roubaud, V.; Mercier, A.; Olive, G.; LeMoigne, F.; Tordo, P. J .
Chem. Soc., Perkin Trans. 2 1997, 9, 1827.
(13) J anzen, E. G.; Zhang, Y.-K. J . Org. Chem. 1995, 60, 5441.
(14) Rockenbauer, A.; Korecz, L.; Hideg, K. J . Chem. Soc., Perkin
Trans. 2 1993, 2149.
(15) Vaultier, M.; Lambert, P. H.; Carrie´, R. Bull. Soc. Chim. Fr.
1986, 1.
(16) (a) Makino, K.; Hagiwara, T.; Hagi, A.; Nishi, M.; Murakami,
A. Biochem. Biophys. Res. Commun. 1990, 172, 1073. (b) Hanna, P.
M.; Chamulitrat, W.; Mason, R. P. Arch. Biochem. Biophys. 1992, 296,
640.

Synthesis of a New Spin Trap
J . Org. Chem., Vol. 64, No. 5, 1999 1473
F igu r e 1. Spin trapping of hydroxyl radical. (a) Spectrum
obtained upon incubating H2O2 (1 mM), FeSO4 (0.3 mM), and
DEPPPO (50 mM) at room temperature in phosphate buffer
(0.1 M, pH 7.4). (b) As in a but in the presence of DEPMPO
(50 mM). The dotted lines in a and b represent computer
simulation of the respective spectrum. Open and closed circles
indicate the low-field line of DEPPPO/•OH diastereoisomers.
Spectrometer settings: microwave power, 10 mW; modulation
amplitude, 0.05 mT; time constant, 0.128 s; gain, 2.5 × 104;
scan range, 20 mT; and scan time, 120 s.
F igu r e 2. Spin trapping of superoxide radical anion. (a)
Incubation mixture contained hypoxanthine (0.4 mM), xan-
thine oxidase (0.4 U mL-1), DTPA (1 mM), and DEPPPO (100
mM) in phosphate buffer (0.1 M, pH 7.4). (b) As in a but in
the presence of SOD (0.5 mg.mL-1). (c) As in a but in the
presence of Gpx (10 U mL-1) and GSH (0.3 mM). The dotted
lines in
a and c represents computer simulation of the
respective spectrum and the closed circles (b) indicate the lines
corresponding to the diastereoisomer A. Spectrometer set-
tings: microwave power, 10 mW; modulation amplitude, 0.1
mT; time constant, 0.128 s; gain, 5 × 104; scan range, 20 mT;
and scan time, 120 s.
complicated compared to its DEPMPO analogue (Figure
1b). Monitoring of the ESR signal showed that the two
diastereoisomers decayed with a different rate (data not
shown).
mers with only one diastereoisomer involved in a chemi-
cal exchange (Figure 3, Table 1).
(b) Sp in Tr a p p in g of Su p er oxid e An ion Ra d ica l.
Spin trapping of superoxide with DEPPPO was per-
formed at pH 7.4 by using two different superoxide-
generating systems. The same signal was observed by
using either hypoxanthine (0.4 mM) in the presence of
xanthine oxidase (0.4 U mL-1) or a light-riboflavin-
DTPA combination (Figure 2a). With both superoxide
generating systems, the ESR signal was inhibited by
addition of SOD (0.5 mg mL-1) proving that the spectra
(c) Kin etic Deca y of th e Su p er oxid e Ad d u ct. The
use of the light-riboflavin-DTPA superoxide generating
system was not possible in this study because a residual
carbon-centered radical coming from the electron donor
arises.8c Carbon-centered radical spin adducts are very
persistent and overlapping of lines can occur, leading to
an overestimation of the measured half-life times. So we
used the xanthine-xanthine oxidase system to generate
superoxide in 0.1 M phosphate buffer, pH 7.4 and 8.2.
After incubating the spin trap (17 mM) with hypoxan-
thine (0.4 mM), xanthine oxidase (0.04 U mL-1) in the
presence of DTPA (300 µM) for 10 min to reach the
steady-state concentration, we added superoxide dismu-
tase (0.5 mg mL-1) to dismutate superoxide and thus to
stop the formation of the DEPPPO-superoxide spin
adduct. Decay of the superoxide spin adduct was then
monitored by measuring the decrease of the ESR spec-
trum low-field line of the diastereoisomer with the most
intense signal considered as the major diastereoisomer.
The kinetic was simulated as a pseudo-first-order process.
Calculated half-life times for DEPPPO-superoxide ad-
duct were (13.1 ( 0.1) min at pH 7.4 and (8.5 ( 0.5) min
at pH 8.2. Under the same conditions, the DEPMPO
superoxide spin adduct had a similar persistency: (14.8
•-
were due to the trapping of O2 (Figure 2b). In the
presence of a glutathione (0.3 mM)-glutathione peroxi-
dase (10 U mL-1) system with both superoxide-generating
systems, we only observed the hydroxyl radical spin
adduct with a percentage contribution for each diaste-
reoisomer of 33/67% determined by computer simulation
of the experimental spectrum (Figure 2c). 7-superoxide
spin adduct was detected as a complicated signal which
was computer-simulated considering two diastereoiso-
mers A and B, both involved in a chemical exchange
(Table 1). The existence of a chemical exchange between
two conformers inducing an important alternate line
width in the ESR spectrum has been reported for the
DEPMPO-superoxide spin adduct.10b Trapping of tert-
butylperoxyl radical (t-BuOO•) gave also a complicated
signal pattern, computer simulated as two diastereoiso-

1474 J . Org. Chem., Vol. 64, No. 5, 1999
Ta ble 1: ESR Hyp er fin e Sp littin g Con sta n ts for P er oxyl Ad d u cts of Nitr on e 7
Karoui et al.
spin adduct
O2•-/HOO•
source
HY/XO
solvent
waterb
%
AN/mT
AP/mT
A
Hâ/mT
A
Hγ/mT
ka/s-1 × 10-6
(A): 51c
40d
60d
28d
72d
47d
53d
17c
1.17
1.35
1.25
1.31
1.25
1.24
1.30
3.72
1.88
4.64
3.32
4.20
3.99
2.50
0.83
1.19
1.16
1.02
0.95
0.71
0.95
23
(B): 49c
83c
2
t-BuOO•
t-BuOOH/hν
benzene
0.16
0.18
0.10
2.5
17c
a
b
d
Exchange rate. 0.1 M phosphate buffer, pH 7.4. c Percentage contribution of diastereoisomer. Percentage contribution of conformer.
Different results were obtained during the generation
of the DEPPPO-OBut spin adduct depending on the
nature of the generating system. By UV photolysis of a
solution containing DEPPPO and di-tert-butyl peroxide
in benzene, two diastereoisomers were observed (Table
2). Surprisingly, only one species (24-line spectra) was
observed when t-BuO• was tentatively generated by
addition of a small amount of Pb(OAc)4 to a solution of
the tert-butyl alcohol in benzene. The same results were
obtained with CH3OH, CH3CH2OH, and i-PrOH. On the
other hand, in the presence of Pb(OAc)4, we always
observed the same paramagnetic byproducts. Our results
suggest that a nucleophilic addition of the alcohol to the
nitrone assisted by Pb(OAc)4 could occur followed by a
rapid oxidation of the hydroxylamine to the correspond-
ing aminoxyl.
F igu r e 3. tert-Butyl peroxyl spin adduct of DEPPPO and its
Only a weak signal was observed with other carbon-
computer simulation. Spectrum obtained upon UV photolysis
of a solution containing tert-butyl hydroperoxide (1.5 M) and
DEPPPO (50 mM) at room temperature in benzene. Spectrom-
eter settings: microwave power, 10 mW; modulation ampli-
tude, 0.032 mT; time constant, 0.128 s; gain, 2.5 × 104; scan
range, 20 mT; and scan time, 480 s.
•
•
centered radicals such as CH3 and CH(CH3)OH gener-
ated via a Fenton reaction (H2O2, Fe2+) in the presence
of dimethyl sulfoxide (DMSO) and ethanol (EtOH),
respectively (data not shown). UV photolysis of CH3I gave
no detectable signals.
(e) P a r tition Coefficien t (Kp ). In a previous paper,
we reported the value of the DEPMPO partition coef-
ficient to be between those of 1-octanol and water (Kp )
0.06).10b Using a method described by Konorev et al.,17
we evaluated the DEPPPO partition coefficient (Kp ) 2.4).
It is interesting to note that the presence of a phenyl
group greatly enhanced lipophilicity of the spin trap;
5-phenyl-2,2-methyl-3,4-dihydro-2H-pyrrole 1-oxide (8),18
a phenylated analogue of DMPO, exhibited a partition
coefficient 100 times greater than that of DMPO (Kp-
(DMPO) ) 0.1).19
( 1.4) min at pH 7.4 and (9.9 ( 0.2) min at pH 8.2. A
comparison with the commonly used spin trap, DMPO,
was impossible to perform under the same conditions
because of the short half-life time of the corresponding
superoxide spin adduct (∼50 s at pH 7.0). However, it is
important to recall that DEPMPO-superoxide spin ad-
duct is 15 times more persistent than DMPO-superoxide
spin adduct.
(d ) Sp in Tr a p p in g of Oth er Ra d ica ls. tert-Butoxyl
radical (t-BuO•) was produced by UV photolysis of a
solution of di-tert-butyl peroxide in benzene. In the
presence of DEPPPO, we observed a spectrum, computer-
simulated as a superimposition of two diastereoisomers
Discu ssion
Con for m a tion of th e Dieth oxyp h osp h or yl Gr ou p .
We have already shown in a previous paper that for
DEPMPO the position of the diethoxyphosphoryl group
is very important in the stabilization of its superoxide
spin adduct.12 Thus if the diethoxyphosphoryl group is
moved away from the C2 position as in the 2-(diethoxy-
phosphorylmethyl)-2-methyl-3,4-dihydro-2H-pyrrole 1-ox-
ide (5), the corresponding superoxide spin adduct has a
half-life time comparable to that of its DMPO analogue.12
Removing the diethoxyphosphoryl group from the C2
position can modify both the steric and electronic influ-
ences on the stabilization of the resulting superoxide spin
adduct.
•
(Table 2). Spectra obtained by trapping CO2-, Ph•, and
thiyl radicals showed the presence of two species, prob-
ably diastereoisomers, with slightly different hyperfine
coupling constants (Table 2). For most of the radicals
generated and trapped by DEPPPO, we obtained a
similar percentage contribution for the two diastereoi-
somers (approximately 70% of the diastereoisomer with
the largest phosphorus coupling value and 30% of the
other). Trapping of the glutathionyl radical (GS•) gener-
ated by UV photolysis of GSNO in water gave a broad
12-line spectrum with alternating line width (Table 2).
We simulated this signal by assuming the existence of
two diastereoisomers (data not shown). In the case of the
DEPPPO/•SG spin adduct, the major diastereoisomer was
the one with the lowest phosphorus coupling value. Spin
adducts obtained by trapping of thiyl radicals were not
persistent, and continuous photolysis was needed to
observe them.
(17) Konorev, E. G.; Baker, J . E.; J oseph, J .; Kalyanaraman, B. Free
Rad. Biol. Med. 1993, 14, 127.
(18) J anzen, E. G.; Zhang, Y-K.; Haire, D. L. J . Am. Chem. Soc.
1994, 116, 3738.
(19) J anzen, E. G.; Poyer, J . L.; Schaefer, C. F.; Downs, P. E.;
Dubose, C. M. J . Biochem. Biophys. Methods 1995, 30, 239.

Synthesis of a New Spin Trap
J . Org. Chem., Vol. 64, No. 5, 1999 1475
Ta ble 2: ESR Hyp er fin e Sp littin g Con sta n ts for Sp in Ad d u cts of Nitr on e 7
a
adduct
HO•
source
H2O2/Fe2+
solvent
waterb
%
AN/mT
AP/mT
A
Hâ/mT
A
Hγ/mT
other
52
48
46
54
100
100
100
100
1.37
1.35
1.37
1.35
1.28
1.28
1.29
1.31
3.18
3.06
3.18
3.06
3.81
3.84
3.80
3.70
1.49
1.05
1.49
1.05
0.71
0.72
0.73
0.94
0.043
0.043
H2O/Fe3+
waterc
CH3Od
EtOd
CH3OH/Pb(OAc)4
EtOH/Pb(OAc)4
iPrOH/Pb(OAc)4
tBuOH/Pb(OAc)4
benzene
benzene
benzene
benzene
0.18
0.18
0.18
0.17
i-PrOd
t-BuOd
0.02
0.07
tBuO•
(t-BuO)2/hν
benzene
benzene
watera
67.5
1.29
1.24
1.37
1.33
1.44
1.42
1.31
1.28
1.34
1.29
1.29
1.25
1.39
1.41
1.40
3.82
3.02
3.41
3.26
3.45
2.60
3.62
3.01
3.49
3.18
3.50
3.10
3.09
3.25
3.42
0.80
0.92
2.0
0.19
0.11
32.5
63
Ph•
(PhCOO)2/hν
37
1.92
1.94
1.80
1.36
1.36
1.37
1.42
1.43
1.42
1.62
1.54
1.63
-
•CO2
H2O2/Fe2+
/HCO2Na
(CH3S)2/hν
67.5
32.5
68
CH3S•
t-BuS•
PhS•
GS•
benzene
benzene
benzene
watera
32
(tBuS)2/hν
(PhS)2/hν
GSNO/hν
2/DMDe
67
33
68
32
64
36
H•
watera
100
AHâ2
2.02
a
b
d
Percentage contribution of diastereoisomer. 0.1 M phosphate buffer, pH 7.4. c Doubly distilled water, pH 7.0. The spin adducts are
formed via nucleophilic attack. e DMD = dimethyldioxirane.
If we could maintain the diethoxyphosphoryl group in
the C2 position and change the resulting spin adducts
conformation, we should observe the influence of the
conformation on the spin adduct persistency. Rocken-
bauer et al. have reported in pyrrolidinoxyl radicals that
a phenyl group will adopt preferentially a pseudoaxial
position.14 To study the influence of the conformation of
the phosphorylated group, we prepared DEPPPO, a
phenylated analogue of DEPMPO bearing a phenyl and
a diethoxyphosphoryl group on the C2 position (see
Scheme 2).
that the steric hindrance of the phenyl group in the
pseudoaxial position is more important than that of the
pseudoequatorial diethoxyphosphoryl group. By trapping
free radicals with DEPPPO, we obtained two diastere-
oisomers with different percentage contributions, and the
major diastereoisomer should be the one resulting from
the radical addition on the opposite face of the phenyl
group. Half-life times for the diastereoisomer A of the
DEPPPO-superoxide spin adduct exhibiting the smaller
average phosphorus coupling (Table 1, Figure 2) were
measured at two different pHs and compared to those
obtained under the same conditions for the DEPMPO
analogue. Our results suggest that the diethoxyphospho-
ryl group conformation does not have a major influence
on superoxide spin adduct persistency.
Trapping of the hydroxyl radical with DEPPPO yielded
an intense and persistent ESR signal corresponding to
two diastereoisomer-spin adducts that decayed at dif-
ferent rates. It is interesting to mention that, keeping
all the EPR parameters (coupling constants, line widths)
equal, the calculated diastereoisomer ratio was different
when the DEPPPO-OH spin adducts were generated via
nucleophilic addition of water (Table 2). This observation
could be used to differentiate the two modes of formation
of DEPPPO-OH. However, it is necessary to mention
that a good fit between experimental and calculated
spectra could also be obtained using the same diastere-
oisomer ratio but with a different set of parameters.
For DEPMPO, 13C NMR data showed a 7.35 Hz 3J P-C4
coupling; however, for DEPPPO this coupling was not
3
resolved. J P-C4 couplings have an angular dependence,
and by using the Karplus relation proposed by Thiem and
Meyer we found dihedral angle (PC2C3C4) values equal
to 131° and 98° for DEPPPO and DEPMPO, respec-
tively.20 Thus, the preferred geometry of the diethoxy-
phosphoryl group is pseudoaxial for DEPMPO and pseu-
doequatorial for DEPPPO.
Radical additions on DEPMPO are stereoselective. The
bulkiness of the diethoxyphosphoryl group in the pseudo-
axial position hinders one nitronyl face and favors radical
addition on the opposite face. As a consequence, in most
cases, only one diastereoisomer was observed (RS•, HO•,
R3C•, ...).21 Although two diastereoisomers were obtained
in some cases, the second diastereoisomer had a very low
percentage contribution.21 For DEPMPO spin adducts,
the average phosphorus coupling value is 5 mT, indicat-
ing that in the most populated conformers the diethoxy-
phosphoryl group adopts a pseudoaxial position. Con-
cerning the DEPPPO spin adducts, the phosphorus
coupling values are smaller than 4 mT, which is in a good
agreement with a phosphorus mainly in the pseudoequa-
torial position. Furthermore, the selectivity observed for
DEPPPO is poor, indicating that both faces of the
nitronyl moiety are hindered. We can reasonably assume
Con clu sion
2-(Diethoxyphosphoryl)-2-phenyl-3,4-dihydro-2H-pyr-
role 1-oxide (DEPPPO) was prepared according to an
original four-step synthesis, and the ability of DEPPPO
to trap free radicals, especially hydroxyl and superoxide,
was investigated. For DEPPPO and its spin adducts the
preferred geometry of the diethoxyphosphoryl group is
pseudoequatorial while a pseudoaxial geometry is favored
for DEPMPO and its spin adducts. We found that the
DEPMPO- and DEPPPO-superoxide spin adducts have
almost the same half-life time, thus indicating that the
(20) Thiem, T.; Meyer, B. Org. Magn. Reson. 1978, 11(1), 50.
(21) Barbati, Ste´phane. Ph.D. Thesis, 1997, University of Aix-
Marseille I, France.

1476 J . Org. Chem., Vol. 64, No. 5, 1999
Karoui et al.
layer was dried over magnesium sulfate. The solvent was dried
under vacuum. Column chromatography of the residue (silica,
THF/EtOH 9:1 and CH2Cl2/EtOH 9:1) afforded 7 as a hygro-
scopic yellow oil (0.5 g, 25%) which solidified at low temper-
conformational geometry of the diethoxyphosphoryl group
does not influence the decay of these phosphorylated spin
adducts. The two faces of DEPPPO are hindered, and the
free radical additions on the nitronyl moiety were not
stereoselective. Although persistent spin adducts were
ature. NMR 31P (40.53 MHz): δ 18.22 ppm (CDCl3), 18.87 ppm
3
(C6D6). 1H (400 MHz, C6D6): 0.96 (3H, t, OCH2CH3, J HH
)
•
-
observed by trapping CO2 and Ph•, the observation of
an unstable signal during methyl and hydroxyethyl
radical trapping remains unexplainable. Concerning the
trapping of O2•- and HO•, DEPPPO appears as a promis-
ing lipophilic analogue of DEPMPO.
3
7.1 Hz), 1.02 (3H, t, OCH2CH3, J HH ) 7.1 Hz, J PH ) 0.3 Hz),
1.7-1.8 (1H, m, CH), 1.91-2.0 (1H, m, CH), 2.20-2.28 (1H,
m, CH), 2.94-3.06 (1H, m, CH), 3.87-3.95 (2H, m, OCH2CH3),
4.31-4.4 (2H, m, OCH2CH3), 6.57 (1H, dt, dCH, J HH ) 2.8
Hz, J PH ) 1.5 Hz), 7.1 (1H, m, CHar), 7.2 (2H, m, CHar), and
3
5
8.0 (2H, m, CHar). 13C (100.06 MHz, C6D6): 16.70 and 16.76
(OCH2CH3, J ) 11.1 and 11.0 Hz, respectively), 26.33 (CH2-
(3), J ) 7.2 Hz), 33.77 (CH2(4)), 63.10 (OCH2CH3, J ) 7.0 Hz),
64.78 (OCH2CH3, J ) 6.0 Hz), 82.97 (Ph-C2-P, J ) 158 Hz),
127.9 (CH(9), J ) 4 Hz), 128.45 (CH(7), J ) 3 Hz), 128.77 (CH-
(8), J ) 2 Hz), 134.41 (CH(5), J ) 8 Hz), 137.75 (C(6)) (Calcd
for C14H20NO4P (297.29): C, 56.56; H, 6.78; N, 4.71 O, 21.53;
P, 10.42. Found: C, 56.36; H, 6.83; N, 4.34).
Exp er im en ta l Section
Syn th esis a n d Ch a r a cter iza tion s. (a ) Gen er a l. 1H NMR
spectra were recorded at 100 and 400 MHz in CDCl3 and C6D6
using TMS as internal references. 31P NMR (40.53 MHz) was
taken in CDCl3 and C6D6 using 85% H3PO4 as an internal
standard with broad-band 1H decoupling. 13C NMR spectral
measurements were performed at 100.6 MHz using CDCl3 and
C6D6 as internal standards. δ values are given in ppm and J
values in hertz. Elemental analyses were determined at the
University of Aix-Marseille III.
Sp in Tr a p p in g Stu d ies. (a ) Gen er a l. Xanthine oxidase
(XOD), bovine erythrocyte superoxide dismutase (SOD), and
catalase were purchased from Boehringer Mannheim Bio-
chemical Co.; glutathione (GSH), glutathione peroxidase (Gpx),
diethylenetriaminepentaacetic acid (DTPA), di-tert-butyl per-
oxide, dibenzoyl peroxide, tert-butyl hydroperoxide, riboflavin,
and other chemicals were from Sigma Chemical Co. S-
Nitrosoglutathione (GSNO) was kindly provided by Pr. B.
Kalyanaraman from the Biophysics Research Institute in
Milwaukee, USA.
(b ) E SR Mea su r em en t s. ESR spectra were recorded at
room temperature using a computer-controlled Varian E-3 or
a Bruker ESP 300 ESR 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 7.4
and 8.2). Standard ESR spectra were simulated using ESR
software developed by D. Duling from the Laboratory of
Molecular Biophysics, NIEHS22 (this software is available via
the World Wide Web at http://www.epr.niehs.nih.gov/PEST/)
and with the ESR software developed by A. Rockenbauer from
the Central Research Institute of Chemistry, Hungary.23 The
UV photolysis was performed by a 1000 W xenon-mercury
Oriel lamp.
(c) Su p er oxid e Tr a p p in g. Hyp oxa n th in e-Xa n th in e
Oxid a se System . Xanthine oxidase (0.4 U mL-1) was added
to a solution of DEPPPO (0.1 M), DTPA (1 mM), and hypox-
anthine (0.4 mM) in phosphate buffer (0.1 M, pH 7.4). The
ESR spectrum was recorded 60 s after the addition of XOD.
(d ) Su p er oxid e Tr a p p in g. Ribofla vin -DTP A-Ligh t
System . This superoxide generating system contained DEP-
PPO (0.1 M), DTPA (4.5 mM), and riboflavin (0.1 mM) in
phosphate buffer (0.1 M, pH 7.4). The ESR spectrum was
recorded 2 min after irradiating the ESR cell inside the
spectrometer cavity (blue light λ ) 430 nm, 600 mCd).
With both superoxide generating systems, the ESR signal
was inhibited by addition of SOD (0.5 mg mL-1). In the
presence of GSH (0.3 mM) and Gpx (10 U mL-1), only the
hydroxyl adduct was observed.
(b) 2-P h en yl-1-p yr r olin e (7a ). Sodium azide (7.6 g, 117
mmol) was added to a solution of γ-chlorobutyrophenone (14
g, 78 mmol) and tetrabutylammonium chloride (0.5 g, 1.8
mmol) in 1,2-dimethoxyethane (60 mL). After stirring for 16
h at 75 °C, the solution was filtered on Celite 545 and washed
with ether, and the solvent was evaporated under reduced
pressure to yield a brown oil. To this oil diluted in dry ether
(200 mL) was added triphenylphosphine (20.4 g, 777 mmol).
When the nitrogen release was over, we added 100 mL of
pentane, and the mixture was stirred at room temperature
for 12 h. The solution was then filtered and the solvent
evaporated under reduced pressure to yield a yellow oil which
was purified by crystallization in pentane (7.7 g, 68%). NMR
1H (100 MHz, CDCl3, TMS): 1.9-2.1 (m, 2H), 2.8-2.9 (tt, 2H),
4-4.1 (tt, 2H), 7.3-7.4 (m, 3H), 7.8-7.9 (m, 2H) (Calcd for
C
10H11N (145.20): C, 82.72; H, 7.64; N, 9.65. Found: C, 82.81;
H, 7.62; N, 9.58).
(c) Dieth yl (2-P h en ylp yr r olid in -2-yl)p h osp h on a te (7b).
To a solution of 7a (12.5 g, 86 mmol) was added diethyl
phosphite (14.2 g, 100 mmol) in the presence of boron trifluo-
ride ethyl etherate (1 mL, 8 mmol). The mixture was stirred
at room temperature under nitrogen for 24 h. A 2 N HCl
solution (40 mL) was added in the mixture up to pH 2. After
having been extracted with dichloromethane (2 × 30 mL), the
aqueous layer was treated first with sodium hydroxide and
then with sodium bicarbonate to reach pH 10. After extraction
with dichloromethane (4 × 30 mL), the organic layer was dried
over magnesium sulfate, filtered, and evaporated under re-
duced pressure to yield a yellow oil which was chromato-
graphed on a silica column (acetone/pentane 1:3) and evapo-
rated as a colorless oil (6.6 g, 60%). NMR 31P (40.53 MHz): δ
26.9 ppm (C6D6), 26.4 ppm (CDCl3). 1H (400 MHz, C6D6): 0.92
(3H, t, OCH2CH3, 3J HH ) 7.07 Hz), 0.97 (3H, t, OCH2CH3, 3J HH
) 7.07 Hz), 1.34-1.45 (1H, m, CH), 1.58-1.69 (1H, m, CH),
2,15-2,24 (1H, m, CH), 2.49 (1H, s large, NH), 2.51-2.63 (1H,
m, CH), 2.79 (1H, dd, CH), 2.94-3.0 (1H, m, CH), 3.67-3.85
(2H, m, OCH2CH3), 3.87-3.94 (2H, m, OCH2CH3), 7.06-7.11
(1H, m, Har), 7.20-7.24 (2H, m, Har), 7.79-7.83 (2H, m, Har).
13C (100.6 MHz, C6D6): 16.78-16.83 (OCH2CH3), 25.94 (CH2-
(4), J ) 9.05 Hz), 37.33 (CH2(3)); 47.22 (CH2(5), J ) 9.05 Hz),
62.66 et 63.04 (OCH2CH3, J ) 7.0 Hz), 67.7 (Ph-C2-P, J )
149.9 Hz), 127.41 (CH(9), J ) 3.0 Hz), 128.36 (CH(7), J ) 3.0
(e) HO• Tr a p p in g. F en ton System . The hydroxyl radical
was generated by adding FeSO4 (0.5 mM) to a solution of
DEPPPO (50 mM) and H2O2 (1 mM) in phosphate buffer (0.1
M, pH 7.4). The ESR spectrum of the hydroxyl adduct was
recorded 60 s after addition of ferrous sulfate. No ESR signal
was observed in the presence of catalase (50 U mL-1) in the
incubation mixture.
(f) Nu cleop h ilic Ad d ition of Wa ter . The hydroxyl spin
adduct was obtained by adding FeCl3 (1 mM) to a solution of
DEPPPO (50 mM) in doubly distilled water.
Hz), 128.49 (CH(8), J ) 4.01 Hz), 142,7 (C(6)) (Calcd for C14H22
-
NO3P: 283.31 C, 59.35; H, 7.83; N, 4.94; O, 16.94; P, 10.93.
Found: C, 59.37; H, 7.82; N, 4.94).
(g) •CO2 (•CO2H) Tr a p p in g. A Fenton system in the
-
presence of NaHCO2 was used to generate •CO2 (•CO2H).
-
(d ) 2-(Dieth oxyp h osp h or yl)-2-p h en yl-3,4-d ih yd r o-2H-
p yr r ole 1-Oxid e (DEP P P O) (7). A solution of 70% m-
chloroperbenzoic acid (3.4 g, 14 mmol) in chloroform (30 mL)
was added over a period of 1 h to a stirred solution of 7b (2 g,
7 mmol) in choloroform (20 mL) at -5 °C. The reaction mixture
was then washed with saturated aqueous sodium bicarbonate
(20 mL) and sodium chloride (20 mL) solutions. The organic
FeSO4 (0.5 mM) was added to a solution of DEPPPO (50 mM),
H2O2 (1 mM), and NaHCO2 (0.1 M) in phosphate buffer (0.1
(22) Duling, D. R.; Motten, A. G.; Mason, R. P. J . Magn. Reson. 1988,
77, 504.
(23) Rockenbauer, A.; Korecz, L. Appl. Magn. Reson. 1996, 10, 29.

Synthesis of a New Spin Trap
J . Org. Chem., Vol. 64, No. 5, 1999 1477
M, pH 7.4). An ESR spectrum of the hydroxyl adduct was
recorded 60 s after addition of ferrous sulfate.
amplitude at time tn was calculated from the signal amplitude
at time tn-1 using the chosen rate equation. The standard least-
squares method was then applied to fit the calculated curves
with the experimental ones. In these calculations, the moni-
tored ESR peak intensity is related to the actual radical
concentration [SA] by a scale factor. The first-order constant
ka and the product kb[SA]0 are independent of this scale factor.
Half-life times are given as means ( SD (n g 3).
(h ) t-Bu OO• Tr a p p in g. t-BuOO• was produced by UV
photolysis of a solution of tert-butyl hydroperoxide (1.5 M) and
DEPPPO (50 mM) in benzene.
(i) P h • Tr a p p in g. Ph• was generated by UV photolysis of a
solution of dibenzoyl peroxide (1 M) in the presence of
DEPPPO (50 mM) in benzene.
(j) Th iyl Ra d ica ls Tr a p p in g. CH3S•, PhS•, and t-BuS• were
produced by UV photolysis of the respective dialkyl sulfide (1.5
M) and DEPPPO (50 mM) in benzene. Glutathion thiyl radical
(GS•) was produced by UV photolysis of GSNO (2 mM) and
DEPPPO (50 mM) in phosphate buffer (0.1 M, pH 7.4).
(k ) Alk oxyl Ra d ica ls Tr a p p in g. DEPPPO/•OCH3, DEP-
PPO/•OCH2CH3, and DEPPPO/ •O-i-Pr were produced by
addition of a small amount of Pb(OAc)4 in a benzenic solution
containing DEPPPO (50 mM) and 1.5 M of the desired alcohol.
t-BuO• was generated by UV photolysis of a solution of di-tert-
butyl peroxide (0.15 M) and DEPPPO (50 mM) in benzene.
(l) Kin etics of Deca y of Su p er oxid e Sp in Ad d u cts. We
used the hypoxanthine-xanthine oxidase system described
previously to generate superoxide in phosphate buffer (0.1 M,
pH 7.4 and 8.2). The spin trap concentration was 0.017 M for
DEPPPO and DEPMPO. The superoxide generation was
initiated by incubating xanthine oxidase in the reaction
mixture for 10 min and suppressed by adding SOD (0.5 mg
mL-1). The spin adduct decay was followed by monitoring the
decrease of an appropriate line of the spin adduct. Computer
simulations were performed using the DAPHNIS program
developed by R. Lauricella at Laboratoire SREP: the signal
(m ) P a r tition Coefficien t (Kp ) Deter m in a tion . The
lipophilicity of DEPPPO was evaluated from its 1-octanol-
water partition coefficient as it was previously described by
Konorev et al.17 A solution of DEPPPO was prepared in
1-octanol at a concentration of 0.25 mM. The concentration of
spin trap was measured at its optical absorption maximum
(λmax ) 245 nm in 1-octanol and λmax ) 238 nm in water) using
a computer-controlled UNICAM UV/visible UV4 spectrometer.
Equal volumes (5 mL) of freshly prepared 1-octanol solution
of DEPPPO and of doubly distilled water were vigorously
mixed at 37 °C during 1 h, and the two phases were separated
by brief centrifugation (3500g, 2 min). Kp was measured as
the ratio between the absorbency of the spin trap in 1-octanol
to that in water.
Ack n ow led gm en t. We thank Professor A. Rocken-
bauer from the Central Research Institute for Chemis-
try in Budapest, Hungary, for providing the ESR
simulation program and for his help on spectra simula-
tion.
J O981875N