Spin trapping of superoxide, alkyl- and lipid-derived radicals with derivatives of the spin trap EPPN

Article abstract of DOI:10.1016/S0006-2952(03)00479-9

The N-t-butyl-α-phenylnitrone derivative N-2-(2-ethoxycarbonyl-propyl)-α-phenylnitrone (EPPN) has recently been reported to form a superoxide spin adduct (t_1/2=5.25min at pH 7.0), which is considerably more stable than the respective N-t-butyl-α -phenylnitrone or 5,5-dimethylpyrroline N-oxide adducts (t_1/2 ～10 and 45s, respectively). In continuation of our previous studies on structure optimization of 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide derivatives, a series of six different EPPN derivatives was synthesized and characterized by ¹H NMR, ¹³C NMR and IR spectroscopy. The ethoxy group of EPPN was replaced by a propoxy, iso-propoxy, n-butoxy, sec-butoxy, and tert-butoxy moiety, as well as the phenyl by a pyridyl ring. Electron spin resonance spectra and stabilities of the superoxide adducts of the propoxy derivatives were found to be similar to those of the respective EPPN adduct, whereas the electron spin resonance spectra of the superoxide adducts of N-2-(2-ethoxycarbonyl-propyl)-α-(4-pyridyl) nitrone and the butoxy derivatives were accompanied by decomposition products. In contrast to the 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide series, no significant improvement of the superoxide adduct stability could be obtained when the ethoxy group was replaced by other substituents. Carbon centered radical adducts derived from methanol, ethanol, formic acid and linoleic acid hydroperoxide were more stable than those of 5,5-dimethylpyrroline N-oxide, whereas among the alkoxyl radicals only the methoxyl radical adduct could be detected.

Full text of DOI:10.1016/S0006-2952(03)00479-9

Biochemical Pharmacology 66 (2003) 1717–1726
Spin trapping of superoxide, alkyl- and lipid-derived radicals
with derivatives of the spin trap EPPN
Klaus Stolzea, Natascha Udilovaa, Thomas Rosenaub,
Andreas Hofingerb, Hans Nohla,*
aBasic Pharmacology and Toxicology, Institute of Applied Botany, University of Veterinary Medicine Vienna,
¨
Veterinarplatz 1, A-1210 Vienna, Austria
bInstitute of Chemistry, University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria
Received 10 February 2003; accepted 28 June 2003
Abstract
The N-t-butyl-a-phenylnitrone derivative N-2-(2-ethoxycarbonyl-propyl)-a-phenylnitrone (EPPN) has recently been reported to form a
superoxide spin adduct (t1=2 ¼ 5:25 min at pH 7.0), which is considerably more stable than the respective N-t-butyl-a-phenylnitrone or
5,5-dimethylpyrroline N-oxide adducts (t1/2 ꢀ10 and 45 s, respectively). In continuation of our previous studies on structure optimization
of 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide derivatives, a series of six different EPPN derivatives was synthesized and
1
characterized by H NMR, 13C NMR and IR spectroscopy. The ethoxy group of EPPN was replaced by a propoxy, iso-propoxy, n-
butoxy, sec-butoxy, and tert-butoxy moiety, as well as the phenyl by a pyridyl ring. Electron spin resonance spectra and stabilities of the
superoxide adducts of the propoxy derivatives were found to be similar to those of the respective EPPN adduct, whereas the electron spin
resonance spectra of the superoxide adducts of N-2-(2-ethoxycarbonyl-propyl)-a-(4-pyridyl) nitrone and the butoxy derivatives were
accompanied by decomposition products. In contrast to the 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide series, no significant
improvement of the superoxide adduct stability could be obtained when the ethoxy group was replaced by other substituents. Carbon
centered radical adducts derived from methanol, ethanol, formic acid and linoleic acid hydroperoxide were more stable than those of 5,5-
dimethylpyrroline N-oxide, whereas among the alkoxyl radicals only the methoxyl radical adduct could be detected.
# 2003 Elsevier Inc. All rights reserved.
Keywords: Spin traps; ESR; EPPN derivatives; Superoxide; Linoleic acid hydroperoxide; Free radicals
1. Introduction
rally related spin traps N-t-butyl-a-phenylnitrone (PBN) or
a-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN), the super-
oxide spin adducts of which are practically undetectable
due to their instability [2], the EPPN superoxide spin
adduct has a half-life of about 5 min at pH 7 [1]. This
value is comparable to the recently reported data of the spin
traps 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide
(EMPO) (t1=2 ¼ 8:6 min) [3–5] or 1,3,3-trimethyl-6-aza-
bicyclo [3.2.1]oct-6-ene-N-oxide (Trazon, t1=2 ¼ 3:6 min)
[6,7], even though lower than the respective value of
5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide
(DEPMPO) (t1=2 ¼ 14:8 min) [8,9]. Recent studies with
EMPO derivatives demonstrated that the stability of the
superoxide adducts was significantly increased when the
ethoxy group was replaced by bulky substituents such as
a sec-butyl or tert-butyl group [10]. Furthermore, due to
their higher lipophilicity, these compounds were expected
to be suitable for the investigation of radicals in lipid
The synthesis of the spin trap EPPN from benzaldoxime
and ethyl 2-bromo-2-methylpropionate has recently been
described by Roubaud et al. [1]. In contrast to the structu-
* Corresponding author. Tel.: þ43-1-25077-4400;
fax: þ43-1-25077-4490.
E-mail address: Hans.Nohl@vu-wien.ac.at (H. Nohl).
Abbreviations: BPPN, N-2-(2-butoxycarbonyl-propyl)-a-phenylni-
trone; DEPMPO, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide;
DMPO, 5,5-dimethylpyrroline N-oxide; DTPA, diethylenetriaminepentaa-
cetic acid; EMPO, 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide;
EPPN, N-2-(2-ethoxycarbonyl-propyl)-a-phenylnitrone; EPPyN, N-2-(2-
ethoxycarbonyl-propyl)-a-(4-pyridyl) nitrone; ESR, electron spin reso-
nance; HFS, hyperfine ꢁsplitting; LOꢁ, lipoxyl radical; NMR, nuclear
magnetic resonance; O2 À, superoxide anion radical; PBN, N-t-butyl-a-
phenylnitrone; POBN, a-(4-pyridyl-1-oxide)-N-t-butylnitrone; PPPN, N-2-
(2-propoxycarbonyl-propyl)-a-phenylnitrone; SOD, superoxide dismutase;
Trazon, 1,3,3-trimethyl-6-azabicyclo [3.2.1]oct-6-ene-N-oxide.
0006-2952/$ – see front matter # 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S0006-2952(03)00479-9

1718
K. Stolze et al. / Biochemical Pharmacology 66 (2003) 1717–1726
membranes. As a possible application, the detection of
lipid peroxidation-derived free radicals has recently been
investigated using different spin traps, such as 5,5-dimethyl-
pyrroline N-oxide (DMPO) [11], DEPMPO [9,12], EMPO
[5] or Trazon [7].
It was the aim of the present study to evaluate the effect
of substituent modification in the ester moiety of EPPN on
the stability of the radical adducts, especially with respect
to the stability of oxygen centered radicals such as super-
oxide, hydroxyl or alkoxyl radicals. For this purpose, a
modified synthetic approach was developed in accordance
with a recently published synthetic procedure for the
synthesis of the spin trap EMPO [3–5].
2.2.3. Synthesis of the N-oxides
To a concentrated solution of the respective alkyl
2-methyl-2-nitropropionate (25 mmol) in H2O/CH3OH
(v/v ¼ 6:4), benzaldehyde or pyridine-4-aldehyde
(30 mmol) and aqueous ammonium chloride solution
(1.87 g in 8 mL water) were added. The mixture was care-
fully kept at room temperature, while 3.27 g (50 mmol) zinc
dust was slowly added within 30 min. After stirring for
4.5 hr at room temperature, the white precipitate and the
remaining zinc powder was removed by filtration and the
solidresiduewaswashedfivetimeswith30 mLofmethanol.
The combined liquid phases were concentrated to a volume
of about 10 mL, saturated with borax and extracted four
times with 60 mL of dichloromethane. The combined
extracts were dried over sodium sulfate, filtered and con-
centrated. Column chromatography on silica gel (petroleum
ether/ethanol (1–5%) with gradient elution) yielded
approximately 20–30% of a light-brown product, which
2. Materials and methods
2.1. Chemicals
1
was recrystallized from pentane and characterized by H
NMR, 13C NMR, and IR spectroscopy. The purity of the
products was assessed by HPLC and UV spectroscopy.
2-Bromo-2-methylpropionyl bromide, linoleic acid,
superoxide dismutase (SOD) and xanthine oxidase were
commercially available from Sigma-Aldrich. Petroleum
ether (high boiling, 50–708) was obtained from Fluka,
all other chemicals from Merck.
2.2.4. Preparation of lipid hydroperoxides
Linoleic acid hydroperoxide was synthesized according
to O’Brien [13]. Briefly, linoleic acid was air-oxidized for
72 hr in the dark at room temperature. The oxidation
mixture was dissolved in petroleum ether (boiling range
60–908) and extracted four times with water/methanol
(v/v ¼ 1:3). The obtained methanolic phase was coun-
ter-extracted four times with petroleum ether (boiling
range 60–908) and evaporated under reduced pressure.
The obtained hydroperoxide was dissolved in ethanol
and stored in liquid nitrogen. The concentration of hydro-
peroxide was determined based on an extinction coefficient
of e233 nm ¼ 25,250 MÀ1 cmÀ1 in ethanol [13].
2.2. Syntheses
Synthesis and characterization of the compounds were
performed according to the synthesis of EMPO and its
derivatives [3–5] with minor adaptations.
2.2.1. Alkyl 2-bromo-2-methylpropionate
2-Bromo-2-methylpropionyl bromide (70 mmol) was
slowly added to a solution of the respective alcohol
(100 mmol) and pyridine (70 mmol) in chloroform at 08
(ice bath). After stirring for 1 hr, the reaction mixture was
successively washed with water (50 mL), sulfuric acid
(10%, 50 mL) and concentrated aqueous sodium bicarbo-
nate (50 mL), and dried over Na2SO4 overnight. Solvent and
excess alcohol were removed under reduced pressure. The
crude, almost colorless product was used without further
purification.
2.3. Instruments
UV-Vis spectra were recorded on Hitachi 150-20 and
U-3300 spectrophotometers in the double beam mode
against a reference sample of the respective solvent. Deter-
mination of the concentrations was done measuring the
absorption maxima in the range between 200 and 350 nm.
IR spectra were recorded as film spectra on a ATI
Mattson Genesis Series FTIR spectrometer.
For electron spin resonance (ESR) experiments the
Bruker spectrometers ER 200 D-SRC 9/2.7 with the data
system ESP1600 and the ESP300E were used, operating at
9.6 GHz with 100 kHz modulation frequency, equipped
with a rectangular TE102 or a TM110 microwave cavity.
1H NMR spectra were recorded at 300 MHz, 13C NMR
spectra at 75.47 MHz on a Bruker Avance. CDCl3 contain-
ing tetramethylsilane (TMS) as the internal standard was
used as the solvent throughout. 13C peaks were assigned by
means of APT (attached proton test), HMQC (1H-detected
heteronuclear multiple-quantum coherence) and HMBC
2.2.2. Alkyl 2-methyl-2-nitropropionate
The respective alkyl 2-bromo-2-methylpropionate
(60 mmol) was added under stirring to a solution of sodium
nitrite (7.2 g, 104 mmol) and phloroglucinol dihydrate
(8.5 g, 52 mmol) in dry dimethylformamide (120 mL) at
room temperature. The solution was stirred for 3 days,
poured into ice water (240 mL) and extracted four times
with ethyl acetate (100 mL). The combined extracts were
treated twice with 100 mL of saturated sodium bicarbonate
solution and dried over Na2SO4. After removal of the
solids by filtration, the solvent was evaporated in vacuo.
The obtained colorless or pale yellow products were used
without further purification.

K. Stolze et al. / Biochemical Pharmacology 66 (2003) 1717–1726
1719
(heteronuclear multiple bond connectivity) spectra. A com-
1
3.1. Spin trapping of superoxide radicals
plete set of H, H–H correlated, 13C, HMQC and HMBC
spectra was recorded for each compound. All chemical shift
data are given in ppm units.
Figure 1 shows the general structure of the spin traps
used. Figure 2a represents the ESR spectrum of the
superoxide adduct of EPPN (20 mM) generated in the
xanthine/xanthine oxidase system at pH 5.8, the optimal
value for its detection [1]. Five percent DMSO was
included to allow comparison with the less water-soluble
derivatives N-2-(2-propoxycarbonyl-propyl)-a-phenylni-
trone (PPPN) (Fig. 2b) and iPPPN (Fig. 2c), from which
ESR spectra of similar resolution and intensity could be
obtained. From the highly lipophilic derivatives BPPN,
sBPPN, and tBPPN only composite ESR spectra could
be obtained, with a high contribution of degradation pro-
ducts, most probably the bis(2-alkoxycarbonyl-propyl)
nitroxide, giving a triplet comparable to di-tert-butyl
nitroxide (spectrum not shown). The ESR spectrum
obtained from the N-2-(2-ethoxycarbonyl-propyl)-a-(4-
Computations, as implemented through Spartan Pro 02
by Wavefunction, Inc., Irvine, CA, USA, were carried out
on the superoxide spin adducts of EPPN, N-2-(2-butox-
ycarbonyl-propyl)-a-phenylnitrone (BPPN) and tBPPN.1
Starting geometries were obtained by conformational
search, followed by pre-optimization of the minimum
conformers by the semi-empirical PM3 method. For full
geometry optimization of each structure, the widely
employed hybrid method denoted by B3LYP [14], which
includes a mixture of HF and DFT exchange terms and the
gradient-corrected correlation functional of Lee et al.
[15,16], as proposed and parametrized by Becke [17,18],
was used, along with the double-zeta split valence basis
sets 6-31 þ G* [19], which includes diffuse functions.
pyridyl) nitrone (EPPyN) superoxide adduct (aN
13:67 G; aH ¼ 1:60 G) was accompanied by the six line
spectrum of an unknown degradation product (aN
¼
3. Results
¼
15:25 G; aH ¼ 2:6 G). The spectral parameters of the
latter compounds (see Table 5) had therefore to be
calculated from the difference spectra obtained by sub-
traction of the secondary product spectra. For this reason,
hyperfine splitting (HFS) values and half-life of the
superoxide adducts of EPPyN, BPPN, sBPPN, and tBPPN
could only be approximated (Tables 4 and 5). For the
determination of t1/2 of the superoxide adducts the best
results were obtained when the respective spin traps
(20 mM, final concentration) were incubated in water/
DMSO in the presence of approximately 0.5 mg solid
KO2 for 15 s, after which phosphate buffer (300 mM, final
pH 7.0, containing 20 mM DTPA, SOD (100 U/mL),
catalase (250 U/mL) and 5% DMSO) was added. Several
consecutive spectra were recorded until the superoxide-
related lines disappeared. Several accompanying lines of
low intensity, probably from a carbon-centered degrada-
tion product, were subtracted from the individual ESR
spectra before calculating the t1/2 values from the corrected
intensity decrease of the first two lines as a first order
exponential fit. The respective values are listed in Table 4.
The low stability of the superoxide adducts as compared
to those of the EMPO series can be correlated with the
molecular structure as obtained from DFT computations.
For EMPO derivatives it was found that the stability of the
superoxide adducts was largely governed by the steric
environment of the nitroxyl group, with bulky alkoxy
substituents shielding the nitroxyl group, thus increasing
the stability of the superoxide adducts, while the spin
density distribution was nearly identical for all EMPO
derivatives [10]. As in the case of EMPO derivatives, also
in EPPN derivatives the spin density distribution was not
influenced by alteration of the alkoxy substituent. How-
ever, while in the EMPO series the alkoxy substituents are
always held in close proximity to the nitroxyl group due to
The identity of the synthesized spin traps was unam-
1
biguously proven by NMR. In the H NMR spectra, the
benzylidenic proton appeared throughout as a singlet with-
out long-range couplings, usually at 7.49 ppm. The signal
with the highest down-field shift originates from the mag-
netically equivalent protons at C-2 and C-6 of the phenyl
ring. The resonances of the protons at C-3, C-5 and C-4 of
the phenyl ring generally overlapped, in some derivatives
also with the benzylidenic proton. The inductive effect of
secondary and tertiary alkoxy substituents in the ester
group was noticeable, but caused a small up-field shift
(0.03–0.07 ppm) only.
The four 13C NMR resonances (C-1, C-2/6, C-3/5, C-4)
of the phenyl substituent appeared at averaged values of
130.4, 129.0, 128.5 and 130.6 ppm, respectively. The car-
bonyl propyl moiety gave signals at 24.5 ppm (methyl),
77.1 ppm (quaternary carbon) and 170 ppm (carboxyl
group). The 13C NMR data were very consistent, without
significant influences of the ester moieties on the resonances
of the nitrone being noticeable. In N–O derivatives with a
C=N double bond, such as oximes, nitroxides or nitrones,
the Z-configured a-C atoms are characteristically shifted
upfield by 6–10 ppm [20]. With an expected resonance at
about 135–140 ppm, the chemical shift of the benzylidenic
carbon at 130 ppm is indicative of its Z-arrangement with
regard to the oxygen. Thus, only the isomers with the C=N
double bond in E-configuration were formed in the synth-
esis, so that the sterically demanding substituents, i.e. the
phenyl ring and the alkylester moiety, are placed trans. The
respective NMR and IR data are summarized in Tables 1–3.
1 The complete computational output can be obtained from the authors
upon request. Computational data of the spin trap starting materials are
available as well.

1720
K. Stolze et al. / Biochemical Pharmacology 66 (2003) 1717–1726
Table 1
13C NMR data (ppm) of the spin traps
0
0
100
200
300
400
1C
2;6C
3;5C
4C
CH
1 C
2 C
COO
C
C
C
C
EPPN
PPPN
130.4
130.4
130.56
130.4
130.52
130.7
137.3a
129.1
129.0
129.0
129.0
128.9
129.0
121.8a
128.6
128.5
128.5
128.5
128.5
128.6
149.7a
130.7
130.6
130.54
130.6
130.47
130.5
–
131.3
131.2
131.2
131.2
131.1
131.1
129.0a
77.1
77.1
77.1
77.2
77.2
77.2
78.3a
24.5
24.5
24.5
24.5
24.4
24.6
24.4a
170.6
170.6
170.1
170.6
170.2
169.8
169.9a
62.1
67.6
69.8
65.9
14.0
21.8
21.5
30.4
–
–
10.3
–
–
–
iPPPN
BPPN
sBPPN
tBPPN
EPPyN
19.0
28.6
–
13.7
9.6
–
00
00
74.2(2 )
19.2(1 )
82.5
62.3a
27.8
13.9a
a Positions in analogy to EPPN.
the geometry requirements of the heterocyclic ring system,
the aliphatic chain structure of EPPN derivatives renders
these compounds much more flexible. Thus, the alkoxy
substituent in the ester group loses its influence as the
spatial distance to the spin-bearing nitroxyl group is much
larger. DFT computations confirmed that the distance
between the nitrogen and carbon C-1 of the alkoxy group
in superoxide adducts of EMPO derivatives ranged around
not result in the detection of the respective hydroxyl
adducts. Instead, a carbon-centered radical adduct was
formed in the case of incubation times less than 30 s
(Table 5, spectrum not shown). After prolonged incubation,
the respective (2-alkoxycarbonyl-propyl)-2-aminoxyl [1]
was observed as the predominant species (shown below,
Fig. 4b).
In the presence of methanol and iron, two different
radical adducts could be detected. A Fenton system in
the presence of 5% methanol according to Roubaud et al.
[1] resulted in the formation of the respective hydroxy-
methyl radical adduct, as shown in Fig. 3a for the EPPN/
ꢁCH2OH species. In Fig. 3b the ESR spectrum of the
methoxyl radical adduct of EPPN is shown (marked
‘‘x’’), which was obtained after nucleophilic addition of
methanol to EPPN in the presence of Fe3þ using experi-
mental conditions reported by Dikalov and Mason [11],
except that the incubation time was reduced to 10 s in order
to suppress the formation of the (2-ethoxycarbonyl-pro-
pyl)-2-aminoxyl radical (marked ‘‘o’’).
˚
4.2 A, whereas that in EPPN derivatives is approximately
˚
0.5 A larger. The same applies to the distance between the
˚
nitroxyl oxygen and C-1, which is on average 4.3 A in
˚
EMPO superoxide spin adducts, but 4.8 A in those of
EPPN. The low steric shielding of the nitroxyl group by
the alkoxycarbonyl moiety in the spin adducts of EPPN
derivatives must thus be assumed to be the major cause of
their observed lower stability.
3.2. Spin adduct formation with oxygen-containing
radicals
Spin adduct formation in the presence of any EPPN
derivative with hydroxyl radicals using a Fenton system did
The observed HFS values (aN ¼ 14:56 G; aH ¼ 4:16 G)
are clearly different from the hydroxymethyl radical
Table 2
1H NMR data (ppm) of the spin traps
0
200
300
400
2;6CH
3;5CH
4CH
HC=N
2 CH3
OCHx
CHx
CHx
CH3
EPPN
8.28m
8.28m
8.27m
8.27m
8.27m
8.25m
8.09da
7.42m
7.42m
7.41m
7.42m
7.41m
7.42m
8.69da
7.42m
7.42m
7.41m
7.42m
7.41m
7.42m
–
7.49s
7.49s
7.45s
7.49s
7.46s
7.42s
7.55sa
1.82s
1.82s
1.80s
1.82s
1.81s
1.77s
1.84sa
4.25q
4.19t
1.27t
–
–
–
–
PPPN
1.66m
1.25d
1.63qi
1.22d(1 )
0.91t
–
iPPPN
BPPN
sBPPN
tBPPN
EPPyNa
5.10sp
4.20t
4.93sx(2 )
1.35m
1.59m
–
0.89t
0.87t
–
00
00
–
1.48s
1.28ta
4.26qa
a Positions in analogy to EPPN.

Table 3
IR data (cmÀ1) of the spin traps
EPPN 3091
PPPN 3080
iPPPN 3079
BPPN 3090
sBPPN 3085
tBPPN 3078
EPPyN 3085
3058
3057
3063
3057
3058
3053
3035
3054
3035
2985
2966
2983
2959
2973
2984
2983
2975
2982
2939
2939
2938
2936
2938
2928
2939
2934
2946
1742 1580 1563 1469 1446 1413 1387 1364 1280 1177 1154 1119
–
–
1025
1054 963
–
906
900
898
900
909
904
910
907
912
–
859
856
839
–
808
813
817
805
805
–
755 693
756 693
754 692
754 693
753 693
752 690
762 695
752 693
1743 1581 1563 1469 1447 1414 1390 1364 1283 1162
1742 1582 1567 1468 1447 1418 1386 1374 1278 1169
1745 1579 1555 1467 1446 1413 1390 1364 1281 1164
1740 1581 1564 1468 1447 1415 1385 1365 1280 1169
1738 1581 1566 1468 1445 1416 1386 1364 1288 1144
–
–
–
–
–
1112
–
1112 1101
1121
1113 1090 1028 969
–
957
–
–
1020
–
–
882
–
866
844
839
802
841
1112
–
–
–
954/927
1742 1593 1571 1469 1445 1416 1389 1365 1281 1177 1156 1133
1024 989
–
794
–
PBN 3092
POBN 3119
–
–
1577 1557 1479 1445 1409 1362
1555 1466 1436 1409 1361
–
–
– 1193
1265 1239
–
–
1125
–
–
–
–
–
–
1124 1095
–
–
–
–
Intensities: strong (1742), medium (1580), weak (1563).

1722
K. Stolze et al. / Biochemical Pharmacology 66 (2003) 1717–1726
EPPN: R = C2H5
PPPN: R = C3H7
CH3
C
iPPPN: R = iso-C3H7
BPPN: R = n-C4H9
sBPPN: R = sec-C4H9
tBPPN: R = tert-C4H9
CH
CH
N
COOR
CH3
O
CH3
C
N
COOC2H5
N
EPPyN:
CH3
O
Fig. 1. General structure of the spin traps.
adduct obtained in the above mentioned Fenton system
containing 5% methanol (aN ¼ 15:29 G; aH ¼ 3:80 G).
When 13C-labeled methanol was used, an additional
splitting was observed in the case of the carbon-centered
hydroxymethyl radical adduct (e.g. for EPPN/ꢁCH2OH:
radical adduct of EPPN was obtained after nucleophilic
addition of 2-propanol to EPPN in the presence of Fe3þ
using the same conditions as with methanol. Instead, a four-
line ESR spectrum from a decomposition product appeared
(Fig. 4b), which can be assigned to the (2-ethoxycarbonyl-
propyl)-2-aminoxyl (aN ¼ 13:5 G; aH ¼ 13:4 G) [1].
a
¼ 3:60 G), whereas the spectrum of the oxygen cen-
13C
tered methoxyl radical adduct (EPPN/ꢁOCH3) remained
unchanged (Table 5, spectra not shown).
3.3. Spin trapping of lipid-derived free radicals
With other alcohols only the respective hydroxyalkyl
radical adducts were observed, e.g. the EPPN/
ꢁC(CH3)2(OH) adduct shown in Fig. 4a, obtained in a
Fenton system containing 5% 2-propanol. No 2-propoxyl
For the detection of lipid-derived free radicals two dif-
ferent radical generating systems were selected. The first
system was anaerobic (flushed with nitrogen for several
minutes) and consisted of peroxidized linoleic acid and the
spin trap dissolved in 20 mM phosphate buffer, pH 7.4,
containing 1.5% acetonitrile. To this solution Fe2þ, dis-
solved in nitrogen-purged water, was added in order to start
the formation of free radicals in a Fenton-type reaction, as
recently tested with different DEPMPO derivatives in both
aerobic and anaerobic environment [3,4] using a stationary
system in combination with the rapid sampling technique.
In Fig. 5a, the EPPN adduct of the radical derived from
linoleic acid hydroperoxide generated in the anaerobic
Fenton is shown, its spectral parameters as obtained by
computer simulation (Fig. 5b) being typical of a partially
immobilized carbon centered radical adduct (see also
Table 5). No additional radical adducts could be detected
even when the concentration of EPPN was increased to
(a)
(b)
Table 4
(c)
Half-lives of the superoxide adducts and n-octanol/water partition
coefficients of the spin traps
Acronym
Apparent
Partition coefficient
n-octanol/phosphate
buffer (100 mM, pH 7.4)
t1/2 (min)
20 G
Fig. 2. Formation of the superoxide adducts of the spin traps EPPN, PPPN
and iPPPN using the xanthine/xanthine oxidase system. (a) EPPN
(20 mM), catalase (250 U/mL), xanthine (0.2 mM) and xanthine oxidase
(50 mU/mL) in oxygenated phosphate buffered saline (20 mM, pH 5.8,
containing 0.4 mM DTPA and 5% DMSO) were aspirated into the flat cell
using the rapid sampling technique and measured using the following ESR
parameters: sweep width, 60 G; modulation amplitude, 1.50 G; microwave
power, 50 mW; time constant, 0.16 s; receiver gain, 2 ꢂ 105; scan rate,
43.9 G/min. (b) Same as in (a), except that PPPN (20 mM) was used. (c)
Same as in (a), except that iPPPN (20 mM) was used.
EPPN
PPPN
5.90
3.45
5.37
<1
29.8a
57
iPPPN
BPPN
sBPPN
tBPPN
EPPyN
60
219
207
145
3.7
2.51
ꢀ1b
7.28
a Data from Roubaud et al. [1].
b Approximated value due to formation of decomposition products.

K. Stolze et al. / Biochemical Pharmacology 66 (2003) 1717–1726
1723
Table 5
Comparison of the ESR parameters of different radical adducts of various EPPN derivatives
Radical HFS (G)
ꢁOOH
EPPN
PPPN
iPPPN
BPPN
sBPPN
tBPPN
EPPyN
aN
aH
14.18
2.47
14.2
2.5
14.10
2.49
14.2
2.5
14.12
2.48
14.1
2.5
13.67
1.60
ꢁC (‘‘ꢁOH’’)
ꢁH
aN
aH
15.07
4.88
15.11
4.93
15.05
4.88
15.11
4.87
15.07
4.77
15.07
4.72
14.67
3.43
aN
aH
16.05
10.50
16.02
10.46
16.01
10.64
16.02
10.46
15.98
10.64
16.05
10.80
15.65
9.99
ꢁCH3
aN
aH
15.72
3.89
15.71
3.96
15.67
3.97
15.67
3.95
15.65
4.03
15.69
4.00
14.66
3.43
ꢁOCH3
ꢁCH2OH
aN
aH
14.56
4.16
14.55
4.15
14.54
4.19
14.50
4.10
14.47
4.15
14.53
4.26
14.05
3.03
aN
aH
15.29
3.80
3.60
15.29
3.87
3.57
15.24
3.85
3.85
15.29
3.80
3.64
15.19
3.95
3.67
15.24
4.02
3.71
14.92
3.09
3.99
a
13C
ꢁCH(OH)CH3
ꢁC(CH3)2OH
aN
aH
15.42
3.20
15.40
3.23
15.33
3.25
15.41
3.26
15.29
3.33
15.35
3.42
14.95
2.65
aN
aH
15.43
3.47
15.42
3.47
15.35
3.57
15.41
3.40
15.35
3.60
15.43
3.64
14.97
2.63
À
ꢁCO2
aN
aH
15.19
4.49
15.16
4.60
15.14
4.75
15.13
4.64
15.10
4.99
15.16
4.90
14.95
3.49
ꢁOOLa
ꢁOOLb
aN
aH
15.53
2.42
15.53
2.40
15.54
2.42
15.55
2.28
15.56
2.40
15.60
2.42
15.23
2.67
aN
aH
15.44
3.37
15.53
3.24
15.55
3.39
15.59
3.22
15.43
3.64
15.57
3.60
15.26
2.69
a LOOH/Fe3þ
.
b LOOH/Fe2þ
.
(a)
(a)
(b)
x
x
x
x
x
x
o
o
o
o
(b)
20 G
20 G
Fig. 4. Iron-dependent formation of two different EPPN spin adducts from
2-propanol. (a) EPPN (10 mM) was incubated with a Fenton system
containing 10% 2-propanol, FeSO4 (1 mM), EDTA (2 mM), H2O2 (0.2%)
and the reaction was stopped after 10 s by 1:1 dilution with phosphate
buffer (0.3 M, pH 7.4, containing 20 mM DTPA) and the solution
immediately aspirated into the flat cell. The spectrum was recorded with
the following spectrometer settings: sweep width, 60 G; modulation
amplitude, 0.24 G; microwave power, 2 mW; time constant, 0.164 s;
receiver gain, 2 ꢂ 105; scan rate, 21.5 G/min. (b) After a 10 s incubation of
EPPN (100 mM in 2-propanol) with FeCl3 (10 mM), the reaction was
stopped by 1:20 dilution with phosphate buffer (0.15 M, pH 7.4, containing
10 mM DTPA), and the solution aspirated into the flat cell using a rapid
sampler. The spectrum was recorded with the following spectrometer
settings: sweep width, 60 G; modulation amplitude, 0.67 G; microwave
power, 20 mW; time constant, 0.02 s; receiver gain, 2 ꢂ 105; scan rate,
171.7 G/min.
Fig. 3. Iron-dependent formation of two different EPPN spin adducts from
methanol. (a) EPPN (10 mM) was incubated with a Fenton system
containing 10% methanol, FeSO4 (1 mM), EDTA (2 mM), H2O2 (0.2%)
and the reaction was stopped after 10 s by 1:1 dilution with phosphate buffer
(0.3 M, pH 7.4, containing 20 mM DTPA) and the solution immediately
aspirated into the flat cell. The spectrum was recorded with the following
spectrometer settings: sweep width, 60 G; modulation amplitude, 0.24 G;
microwave power, 2 mW; time constant, 0.164 s; receiver gain, 2 ꢂ 105;
scan rate, 21.5 G/min. (b) After a 10 s incubation of EPPN (100 mM in
methanol) with FeCl3 (10 mM), the reaction was stopped by 1:20 dilution
with phosphate buffer (0.15 M, pH 7.4, containing 10 mM DTPA), and the
solution aspirated into the flat cell using a rapid sampler. The spectrum was
recorded with the following spectrometer settings: sweep width, 60 G;
modulation amplitude, 0.67 G; microwave power, 20 mW; time constant,
0.02 s; receiver gain, 2 ꢂ 105; scan rate, 171.7 G/min.

1724
K. Stolze et al. / Biochemical Pharmacology 66 (2003) 1717–1726
(a)
(a)
(b)
(c)
(b)
(c)
(d)
(d)
20 G
Fig. 6. Detection of lipid-derived radical adducts from preoxidized
linoleic acid after Fe3þ catalyzed addition in the presence of the spin
traps EPPN and iPPPN. (a) A mixture of peroxidized linoleic acid
(5.5 mM), FeCl3 (5 mM) and EPPN (20 mM), previously incubated for
5 min and then 1:30 diluted in with phosphate buffer (300 mM, pH 7.4,
containing 20 mM DTPA), was aspirated using a rapid sampler and
measured under the following conditions: sweep width, 60 G; modulation
amplitude, 1.5 G; microwave power, 2 mW; time constant, 0.04 s; receiver
gain, 1 ꢂ 105; scan rate, 171.7 G/min. (b) Computer simulation, using the
following parameters: aN ¼ 15:53 G; aH ¼ 2:42 G; DH ¼ 1:70, 1.60,
1.85 G (1st, 2nd and 3rd doublet, respectively); 0.4 Lorentz/Gauss. (c)
Same as in (a), except that iPPPN (10 mM) was used. (d) Computer
simulation, using the following parameters: aN ¼ 15:54 G; aH ¼ 2:42 G;
DH ¼ 1:75, 1.70, 1.95 G (1st, 2nd and 3rd doublet, respectively); 0.4
Lorentz/Gauss.
20 G
Fig. 5. Detection of a lipid-derived radical adduct from preoxidized
linoleic acid using a Fenton-type incubation system in the presence of the
spin traps EPPN and iPPPN. (a) To a nitrogen-bubbled solution of
peroxidized linoleic acid (2.5 mM) and EPPN (20 mM) in phosphate
buffer (20 mM, pH 7.4, containing 1.5% acetonitrile) FeSO4 (0.1 mM) was
added and the solution was aspirated under nitrogen into the flat cell using
a rapid sampler. The spectrum was recorded with the following spectro-
meter settings: sweep width, 60 G; modulation amplitude, 1 G; microwave
power, 2 mW; time constant, 0.08 s; receiver gain, 1 ꢂ 105; scan rate,
42.9 G/min. (b) Computer simulation using the following parameters:
aN ¼ 15:44 G; aH ¼ 3:37 G; DH ¼ 1:40, 1.00, 1.55 G (1st, 2nd and 3rd
doublet, respectively); 0.4 Lorentz/Gauss. (c) Same as in (a), except that
iPPPN (20 mM) was used. (d) Computer simulation, using the following
parameters: aN ¼ 15:55 G; aH ¼ 3:39 G; DH ¼ 1:30, 1.05, 1.55 G (1st, 2nd
and 3rd doublet, respectively; 0.4 Lorentz/Gauss.
exceptionally small. From iPPPN similar experimental
(Fig. 6c) and simulated (Fig. 6d) spectra were obtained.
The data of the other investigated spin traps are summar-
ized in Table 5.
100 mM, which was possible in the presence of 20%
acetonitrile or DMSO (spectra not shown).
Similarly, the respective iPPPN adduct was obtained
(Fig. 5c) and characterized by computer simulation
(Fig. 5d). As compared to the respective spin adducts with
DMPO or DEPMPO, the adducts of the EPPN derivatives
were considerably more stable (ca. 10–30 min).
The second system used aerobic incubation for 5 min in
the presence of Fe3þ [11], followed by addition of the iron
chelator DTPA in phosphate buffer.
The ESR spectrum obtained from EPPN and peroxi-
dized linoleic acid is shown in Fig. 6a. The spectral
parameters, obtained by computer simulation of the spec-
trum (Fig. 6b) can be interpreted in terms of a partially
immobilized ESR spectrum different from the species
obtained in the Fenton system. Although the nitrogen
coupling constant suggests the formation of a carbon-
centered radical, the value of the hydrogen splitting is
4. Discussion
Six novel EPPN derivatives were synthesized and char-
acterized in this study. EPPN and its derivatives form only
moderately stable superoxide adducts (t1=2 ¼ 1–7 min),
which are, however, more stable than the adducts with
DMPO (t1=2 ¼ 45 s [21]) or PBN [2]. In contrast, the
superoxide adducts of some derivatives of EMPO
(t1=2 > 20 min [10]) or DEPMPO (t1=2 ¼ꢀ 7–15 min
[8,9,12]) are considerably more stable.
Although in our experiments no alkoxyl radical spin
adducts could be detected, no definitive statement about
their stability can be made. Alkoxyl radicals undergo rapid
b-scission with a rate constant of around 106, which

K. Stolze et al. / Biochemical Pharmacology 66 (2003) 1717–1726
1725
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However, carbon centered radical adducts formed
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In conclusion, EPPN and its derivatives can be recom-
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since the stability of their carbon-centered radical spin
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The authors wish to thank G. Marik and R. Stadtmu¨ller
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and characterization of the spin traps. The present inves-
¨
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