Very stable superoxide radical adducts of 5-ethoxycarbonyl-3,5-dimethyl- pyrroline N-oxide (3,5-EDPO) and its derivatives

Article abstract of DOI:10.1016/j.bcp.2005.01.019

Oxygen radicals are involved in the onset of many diseases. Adequate spin traps are required for identification and localisation of free radical formation in biological systems. Superoxide spin adducts with half-lives up to 20 min at physiological pH have recently been reported to be formed from derivatives of the spin trap 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO). This is a major improvement over DMPO (t_1/2 ca. 45 s), and even DEPMPO (t _1/2 ca. 14 min). In this study, an additional methyl group was introduced into position 3 or 4 of the pyrroline ring which greatly increases the stability of the respective superoxide spin adducts. In addition, the ethoxy group of EMPO was exchanged by either a propoxy- or an iso-propoxy group in order to test the influence of increasing lipophilic properties of the investigated spin traps. The structure of all compounds was confirmed by ¹H and ¹³C-NMR with full signal assignment. In comparison with EMPO (t_1/2 ca. 8 min) or DEPMPO (t_1/2 ca. 14 min), the superoxide adducts of all novel spin traps were considerably higher (t _1/2 ca. 12-55 min). In addition, various other spin adducts obtained from oxygen-centered as well as carbon-centered radicals (e.g. derived from methanol or linoleic acid hydroperoxide) were also detected.

Full text of DOI:10.1016/j.bcp.2005.01.019

Biochemical Pharmacology 69 (2005) 1351–1361
www.elsevier.com/locate/biochempharm
Very stable superoxide radical adducts of 5-ethoxycarbonyl-
,5-dimethyl-pyrroline N-oxide (3,5-EDPO) and its derivatives
3
a
Klaus Stolze , Natascha Rohr-Udilova , Thomas Rosenau ,
a
b
a
Roswitha Stadtm u¨ ller , Hans Nohl
a,
*
a
Research Institute of Biochemical Pharmacology and Toxicology, University of Veterinary Medicine Vienna,
Veterin a¨ rplatz 1, A-1210 Vienna, Austria
b
Division of Organic Chemistry, University of Natural Resources and Applied Life Sciences (BOKU),
Department of Chemistry, Muthgasse 18, A-1190 Vienna, Austria
Received 14 December 2004; accepted 31 January 2005
Abstract
Oxygen radicals are involved in the onset of many diseases. Adequate spin traps are required for identification and localisation of free
radical formation in biological systems. Superoxide spin adducts with half-lives up to 20 min at physiological pH have recently been
reported to be formed from derivatives of the spin trap 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO). This is a major
improvement over DMPO (t1/2 ca. 45 s), and even DEPMPO (t1/2 ca. 14 min). In this study, an additional methyl group was introduced into
position 3 or 4 of the pyrroline ring which greatly increases the stability of the respective superoxide spin adducts. In addition, the ethoxy
group of EMPO was exchanged by either a propoxy- or an iso-propoxy group in order to test the influence of increasing lipophilic
1
13
properties of the investigated spin traps. The structure of all compounds was confirmed by H and C-NMR with full signal assignment. In
comparison with EMPO (t1/2 ca. 8 min) or DEPMPO (t1/2 ca. 14 min), the superoxide adducts of all novel spin traps were considerably
higher (t1/2 ca. 12–55 min). In addition, various other spin adducts obtained from oxygen-centered as well as carbon-centered radicals (e.g.
derived from methanol or linoleic acid hydroperoxide) were also detected.
#
2005 Elsevier Inc. All rights reserved.
Keywords: Spin traps; EPR; EMPO derivatives; Superoxide; Linoleic acid hydroperoxide; Free radicals
1
. Introduction
adducts (t1/2 = 15–25 min [1,3]) as compared to the struc-
turally related spin traps DMPO (t1/2 < 1 min [4]), EMPO
Derivatives of the spin trap EMPO (5-ethoxycarbonyl-5-
(
t1/2 ca. 8 min [5–7]), or even DEPMPO (t1/2 ca. 14 min [8–
0]). Since the observed spin adduct stabilisation was
methyl-1-pyrroline N-oxide) have recently been described
by several authors to be superior to the parent compound
itself [1–3]. Compounds having bulky alkoxycarbonyl
substituents formed considerably more stable superoxide
1
obviously not only due to the electron-withdrawing effect,
but also due to steric influences of the alkoxycarbonyl
group, it was expected that the incorporation of an addi-
tional methyl group at position 3 or 4 of the pyrroline ring
of EMPO derivatives might cause increased steric shield-
ing and thus lead to an even higher stability of the super-
oxide adducts.
Abbreviations: DEPMPO, 5-(diethoxyphosphoryl)-5-methyl-1-pyrro-
line N-oxide; DMPO, 5,5-dimethylpyrroline N-oxide; DTPA, diethylene-
triaminepentaacetic acid; 3,5-EDPO, 5-(ethoxycarbonyl)-3,5-dimethyl-1-
pyrroline N-oxide; 4,5-EDPO, 5-(ethoxycarbonyl)-4,5-dimethyl-1-pyrro-
line N-oxide; 3,5-DIPPO, 3,5-dimethyl-5-(iso-propoxycarbonyl)-1-pyrro-
line N-oxide; 4,5-DIPPO, 4,5-dimethyl-5-(iso-propoxycarbonyl)-1-
pyrroline N-oxide; 3,5-DPPO, 3,5-dimethyl-5-(propoxycarbonyl)-1-pyrro-
line N-oxide; 4,5-DPPO, 4,5-dimethyl-5-(propoxycarbonyl)-1-pyrroline N-
oxide; EMPO, 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide; EPR,
Preliminary experiments on trapping of radicals derived
from peroxidized linoleic acid using different spin traps,
such as DMPO [11], DEPMPO [9,10], EMPO [7] or
Trazon [12,13], have recently been reported, although an
optimal spin trap for the detection of all types of oxygen-
and carbon-centered radicals has not yet been found.
Aim of the present work was the synthesis of a series of
3,5- or 4,5-dimethylated pyrroline derivatives with differ-
ꢀ
electron paramagnetic resonance; HFS, hyperfine splitting; LO , lipoxyl
ꢀꢁ
radical; NMR, nuclear magnetic resonance; O
SOD, superoxide dismutase
2
, superoxide anion radical;
*
Corresponding author. Tel.: +43 1 25077 4400; fax: +43 1 25077 4490.
E-mail address: hans.nohl@vu-wien.ac.at (H. Nohl).
0006-2952/$ – see front matter # 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2005.01.019

1352
K. Stolze et al. / Biochemical Pharmacology 69 (2005) 1351–1361
ent lipophilic properties for the detection of superoxide and
other radicals in aqueous solution as well as within lipid
membranes.
104 mmol) and phloroglucinol dihydrate (8.5 g, 52 mmol)
in dry dimethylformamide (120 mL) at room temperature.
The solution was stirred overnight, poured into ice water
(
240 mL), and extracted four times with ethyl acetate/
petroleum ether (v/v = 4:1, 100 mL). The combined
extracts were treated twice with 100 mL of saturated
Na CO solution and dried over Na SO . After removal
2
. Materials and methods
2
3
2
4
2
.1. Chemicals
of the solids by filtration, the solvent was evaporated in
vacuo. The obtained colorless or pale yellow products were
used further without purification.
2-Bromopropionyl bromide, crotonaldehyde, linoleic
acid, methacrolein, superoxide dismutase and xanthine
oxidase were commercially available from Sigma–Aldrich.
Petroleum ether (high boiling, 50–70 8C) was obtained
from Fluka, all other chemicals from Merck.
2.2.3. Alkyl 2,3-dimethyl-4-formyl-2-nitro-butanoate and
alkyl 2,4-dimethyl-4-formyl-2-nitro-butanoate
23 mmol of the respective alkyl 2-nitropropionate was
dissolved in a mixture of acetonitrile (10 g, 244 mmol) and
triethylamine (0.2 g, 2 mmol). Crotonaldehyde (2.5 g,
2
.2. Syntheses
38 mmol: for the 4-methyl derivatives) or methacrolein
Synthesis and characterization of the compounds were
(2.5 g, 38 mmol: for the 3-methyl derivatives) was slowly
added at 0 8C. The solution was stirred at room tempera-
ture overnight and then poured into a solution of ice-cold
HCl (5 mL concentrated HCl in 150 mL water). The
solution was extracted three times with CH Cl and dried
performed as reported earlier [1,7], in analogy to the
synthesis of EMPO and its derivatives [5,6] with minor
adaptations as given below.
2
2
2
.2.1. Alkyl 2-bromopropionate
-Bromopropionyl bromide (70 mmol) was slowly
added to a solution of the respective alcohol (100 mmol)
over anhydrous Na SO . After filtration the mixture was
2 4
2
distilled under reduced pressure, and the purity of the
obtained product was assessed by thin layer chromatogra-
phy and IR spectroscopy. If necessary, the product was re-
purified by column chromatography.
and pyridine (70 mmol) in CHCl at 0 8C (ice bath). After
3
stirring for 1 h, the reaction mixture was successively
washed with water (50 mL), sulfuric acid (10%, 50 mL)
and concentrated aqueous Na CO (50 mL), and dried over
2.2.4. Synthesis of the N-oxides
2
3
Na SO overnight. Solvent and excess alcohol were
4
Synthesis of the nitrones was performed according to the
procedure described previously for the synthesis of EMPO
derivatives [1,7]. To a solution of the respective alkyl
2
removed under reduced pressure. The crude, nearly color-
less product was used without further purification.
dimethyl-4-formyl-2-nitrobutanoate (25 mmol) in H O/
2
2
.2.2. Alkyl 2-nitropropionate
The respective alkyl 2-bromopropionate (60 mmol) was
added under stirring to a solution of sodium nitrite (7.2 g,
CH OH (v/v = 3:2, ca. 250 mL) an aqueous NH Cl solu-
3
4
tion (1.87 g in 8 mL water) was added. The mixture was
carefully kept at room temperature while 8.5 g (130 mmol)
Table 1
13
C NMR data (ppm) of the spin traps
0
0
0
3
2
C
3
3a
4
4a
5
C
1
2
5a
CH
3
N
C
C
C
C
COO
C
C
C
EMPO*
134.9
138.8
139.8
135.6
133.8
139.7
135.9
134.1
139.5
139.8
25.4
33.3
33.3
33.8
34.4
33.2
33.9
34.5
33.3
33.2
–
31.9
40.1
41.5
40.7
36.8
41.5
40.8
36.9
40.1
41.5
–
–
–
78.5
79.6
79.6
82.0
82.0
79.6
82.2
82.1
79.6
79.6
169.3
170.1
169.8
168.1
170.0
169.8
168.2
170.0
169.5
169.2
61.7
62.2
62.2
61.9
62.0
67.5
67.5
67.6
69.9
69.8
13.4
13.9
13.9
14.0
13.9
21.68
21.7
21.7
21.2
21.3
–
–
–
–
–
20.3
21.3
21.7
14.7
14.7
c-3,5EDPO
t-3,5EDPO
c-4,5EDPO
t-4,5EDPO
t-3,5DPPO
c-4,5DPPO
t-4,5DPPO
c-3,5DIPPO
t-3,5DIPPO
18.6
18.6
–
19.9
15.3
–
–
18.5
–
10.1
10.3
10.2
–
21.66
14.7
14.7
21.3
21.7
19.9
15.4
–
–
18.5
18.5
–
–
2
1.4
c-4,5DIPPO
t-4,5DIPPO
135.7
134.2
34.0
34.6
–
–
41.0
37.0
20.0
15.5
82.1
82.1
168.3
169.5
70.1
70.0
21.7
21.7
–
–
14.8
14.8

K. Stolze et al. / Biochemical Pharmacology 69 (2005) 1351–1361
1353
zinc dust was slowly added within 30 min. The mixture
was stirred for several hours at room temperature, the white
precipitate and the remaining zinc powder were removed
by filtration, and the residue was washed five times with
methanol (30 mL). The liquid phase was concentrated to
about 10 mL and extracted four times with CH Cl
cis-forms. The purity of the obtained products was assessed
by TLC and UV spectroscopy. Final identification of the
1
13
purified products was performed by H NMR, C NMR,
and IR spectroscopy (see Tables 1–3).
2.2.6. Preparation of lipid hydroperoxides
Linoleic acid hydroperoxide was synthesized according
to O’Brien [14]. Briefly, linoleic acid was air-oxidized for
2
2
(60 mL). The organic phase was dried over Na SO ,
filtered and concentrated.
2
4
72 h in the dark at room temperature. The oxidation
2.2.5. Purification and separation of the different
stereoisomers
mixture was dissolved in petroleum ether (boiling range
60–90 8C) and extracted four times with water/methanol
(v/v = 1:3). The obtained methanolic phase was counter-
extracted four times with petroleum ether (boiling range
60–90 8C) and evaporated under reduced pressure. The
obtained hydroperoxide was dissolved in ethanol and
stored in liquid nitrogen. The concentration of hydroper-
oxide was determined by UV spectroscopy based on an
The crude products were purified chromatographically
in two steps, first on silica gel using a petroleum
ether/ethanol gradient, followed by a final purification
immediately before the EPR experiments on 1 mL solid
phase extraction columns using a Chromabond C-18
1
00 mg column obtained from Macherey-Nagel (D u¨ ren,
Germany).
ꢁ1
ꢁ1
extinction coefficient of e233 nm = 25,250 M cm
ethanol [14].
in
In this way pure colourless products were obtained from
all investigated spin traps. In the case of 3,5-EDPO the two
possible diastereomeric products could be separated into
the cis- and trans-forms. From 3,5-DPPO and 3,5-DIPPO
the pure trans-form and very small amounts of the cis-form
2.3. Instruments
UV–vis spectra were recorded on Hitachi 150–20 and
U-3300 spectrophotometers in double-beam mode against
a blank of the respective solvent. Determination of the
(ca. 95% pure) were obtained. 4,5-EDPO, -DPPO and -
DIPPO could, however, not be separated into the trans- and
Table 2
1
H NMR data (ppm) of the spin traps
0
0
0
3
2
3
3a
4
4a
1
2
5a
CH
N
CH
x
CH
3
CH
2.16m
.60m
x
CH
3
CH
x
CH
x
CH
x
CH
3
EMPO*
6.97t
6.85d
6.91d
7.03t
6.92t
6.91d
7.06t
6.95t
6.92d
6.90d
7.06t
6.92t
2.75m
3.10m
3.15m
2.40m
–
–
4.26m
4.27m
4.25m
4.26m
4.27m
4.15m
4.16m
4.16m
5.09sp
5.06sp
5.10sp
5.10sp
1.31t
–
–
–
–
–
1.72s
1.71s
1.73s
1.67s
1.58s
1.73s
1.68s
1.58s
1.69s
1.71s
1.66s
1.56s
2
c-3,5EDPO
t-3,5EDPO
c-4,5EDPO
t-4,5EDPO
t-3,5DPPO
c-4,5DPPO
t-4,5DPPO
c-3,5DIPPO
t-3,5DIPPO
c-4,5DIPPO
t-4,5DIPPO
1.25d
ci2.17dd
tr2.40dd
ci2.76dd
tr1.70dd
2.48m
–
–
1.32t
1.20d
1.29t
–
1.11d
1.12d
–
1.31t
2
.72m
2.26m
.94m
–
2.87m
1.31t
2
3.15dsx
1.21d
ci2.77dd
tr1.71m
2.47m
1.71sx
1.70sx
1.70sx
1.29d
1.25d
1.29d
1.28d
0.95t
2.40m
–
1.11d
1.12d
–
0.96t
2
.72m
2.26m
.95m
–
2.89m
0.95t
2
3.09m
3.14dsx
2.37m
1.25d
1.20d
–
ci2.13dd
tr2.40dd
ci2.77dd
tr1.69dd
2.45m
–
–
–
–
–
1.12d
1.11d
2
.70m
2.24m
.93m
–
2.87m
2
Abbreviations: s, singlet; d, doublet; t, triplet; sx, sextet; sp, septet; m, multiplet; dd, doublet of doublets; dsx, doublet of sextets; ci, cis to ester; tr, trans to ester.

1354
K. Stolze et al. / Biochemical Pharmacology 69 (2005) 1351–1361
concentrations was done measuring the absorption maxima
in the range between 200 and 350 nm. IR spectra were
recorded as film on an ATI Mattson Genesis Series FTIR
spectrometer.
For EPR experiments Bruker spectrometers (ESP300E
and EMX) were used, operating at 9.7 GHz with 100 kHz
modulation frequency, equipped with a rectangular TE102
or a TM microwave cavity.
1
10
1
13
H NMR spectra were recorded at 300.13 MHz,
C
NMR spectra at 75.47 MHz on a Bruker Avance. CDCl3
containing tetramethylsilane (TMS) as the internal stan-
13
dard was used as the solvent throughout. C peaks were
1
assigned by means of APT (attached proton test), H-
detected heteronuclear multiple-quantum coherence
(
(
HMQC) and heteronuclear multiple bond connectivity
1
HMBC) spectra. A complete set of H, H–H correlated,
1
3
C, HMQC and HMBC spectra was recorded for each
compound. All chemical shift data are given in ppm units.
3. Results
3.1. Structure of the spin traps
The identity of the synthesized spin traps was estab-
lished unambiguously by NMR. In the current compound
series, an additional substituent at either C-3 or C-4 was
present, which could be configured either trans (E) or cis
(
Z) with regard to the bulky alkoxycarbonyl substituent at
C-5, so that two diastereomers of each spin trap were
obtained, a cis-compound and the respective trans-isomer.
The carbon NMR data of the spin traps clearly reflected
the substitution pattern at the pyrroline system. The four
signals of the carbons C-2 to C-5 in the heterocyclic ring
appeared at averaged values of 139, 33, 41 and 80 ppm for
the 3,5,5-trisubstituted derivatives, and at 135, 34, 37
(
trans)/41 (cis) and 82 ppm, for the 4,5,5-trisubstituted
derivatives, respectively. Substitution site and configura-
tion had no influence on the shifts of the carboxyl carbon
and the alkoxy moieties. In contrast, the 5a-methyl group
gave a resonance at about 21.5 ppm for 3,5,5-trisubstituted
and at 14.7 ppm for 4,5,5-trisubstituted compounds. Due to
this pronounced shift difference, the 5a-CH signal can
3
serve as a quick indicator as to the presence of a 3,5,5-
substituted or 4,5,5-substituted compound.
In the 3,5,5-trisubstituted derivatives, the cis/trans-con-
figuration had no influence on C-3, and only a small effect
on C-4: the C-4 in the trans-compounds showed a weak
downfield shift of about 1.4 ppm relative to the cis-counter-
part. In contrast, in the 4,5,5-trisubstituted derivatives the
difference between the two diastereomers was much larger.
C-4 in the trans-compounds experienced a strong highfield
shift by 4 ppm relative to the cis-compound, and C-3 a
0.6 ppm downfield shift. These data reflect that 3- and 5-
substituents in the pyrrolidine are positioned relatively
remote from each other, whereas 4- and 5-substituents

K. Stolze et al. / Biochemical Pharmacology 69 (2005) 1351–1361
1355
are placed in close proximity showing strong interactions.
This was also seen by the shift values of the 3a- and 4a-
methyl groups; 3a-CH appeared at 18.5 ppm independent
ester). In the cis-4,5-derivative, the highfield proton
(2.40 ppm) is now configured trans to 4-CH , (cis to the
3
ester), the downfield proton (2.72 ppm) consequently cis to
4-CH and trans to the ester. The resonances of the alkoxy
3
of the configuration, whereas the 4a-CH moiety gave
3
3
0
resonances at 20 ppm in cis-compounds and at 15.4 ppm
in trans-derivatives.
moieties fell in the expected ranges, the 1 -methylene
groups in the ethoxy and propoxy residues showed pro-
nounced diastereotopic splitting. The respective NMR data
are summarized in Tables 1 and 2.
1
Also the H NMR data fully agreed with the expecta-
tions. The H-2 appeared as doublet in the case of 3-
substituted derivatives. In 4-substituted compounds, H-2
showed a triplet – not a doublet of doublets, indicating
similar coupling constants to the geminal H-3 protons. 3a-
IR spectra of the different compounds show character-
istic differences especially in the so-called fingerprint
ꢁ
1
range around 1000 cm . In this way the different fractions
obtained from the chromatography columns could be pre-
selected before NMR characterization.
CH substituents were found at 1.25 ppm for cis-deriva-
3
tives and at 1.20 ppm for the trans-compounds, 4a-CH3
groups resonated more up-field at about 1.12 ppm inde-
pendent of the configuration. The 5a-CH group was found
The respective IR data are summarized in Table 3.
3
at approx. 1.70 ppm in 3,5,5-trisubstituted pyrrolines with
almost no difference between cis- and trans-isomers,
whereas in the 4,5,5-trisubstituted counterparts there was
an obvious influence of the configuration, with the 5a-CH3-
resonance of the cis-isomer (1.67 ppm) being shifted
3.2. Spin trapping of superoxide radicals
The general structure of the spin traps is shown in Fig. 1.
In Fig. 2(a), the EPR spectrum of the superoxide adduct of
trans-3,5-EDPO is given as an example. The adduct was
generated in the xanthine/xanthine oxidase system at pH
7.4. No EPR spectrum was observed in the presence of
SOD (Fig. 2(b)). Under these conditions similar EPR
spectra were obtained from trans-3,5-DPPO and trans-
3,5-DIPPO (not shown). The superoxide adduct of the
diastereomeric cis-3,5-EDPO, on the other hand, could
be readily distinguished due to its characteristically dif-
ferent spectral parameters (Fig. 2(c)) and considerably
lower stability (t1/2 = 11.5 min) as compared to the
trans-compound (t1/2 = 44.2 min). The remarkable line-
width difference between the cis- and the trans-form is
probably due to stereochemically sensitive contributions of
the unresolved methyl protons. The respective cis-isomers
0
.1 ppm downfield relative to the trans-isomer (1.57 ppm).
1
An interesting feature of the H spectra were the signal
patterns of the H-3 and H-4 protons, which showed sig-
nificantly different patterns according to both substitution
site and configuration. In 3,5,5-derivatives, the proton at
C-3 appeared at around 3.10 ppm, and the shift difference
between cis- and trans-isomers was rather small
(
0.05 ppm). The two geminal H-4 protons appeared at
about 2.15 ppm/2.40 ppm for the cis-compound and
.70 ppm/2.76 ppm for the trans-compound. Thus, the
1
shift difference between these two protons was much more
pronounced for the trans-isomers – more than 1 ppm. In
the trans-3,5-derivative, the highfield proton (1.70 ppm) is
configured cis to both methyl groups at C-3 and C-4 and
trans to the alkoxycarbonyl moiety at C-5, the downfield
proton (2.76 ppm) is consequently placed trans to both
methyl groups and cis to the ester. In the cis-3,5-derivative,
the situation is reversed: the highfield proton (2.15 ppm) is
now configured trans to the two vicinal methyl groups
at C-3 and C-4 and cis to the ester, the downfield proton
(2.40 ppm) is in cis-arrangement to the methyl groups and
trans to the ester.
In the 4,5,5-trisubstituted pyrroline derivatives, the reso-
nance of H-4 was found at 2.46 ppm for the cis-isomers and
at 2.88 ppm for the trans-isomers. H-4 was thus found
more upfield than H-3 in the 3,5,5-compounds, and there
was also a clear difference between the cis- and trans-
derivatives. The two geminal H-3 protons appeared at
2
2
.40 ppm/2.72 ppm for the cis-compound and at
.26 ppm/2.94 ppm for the trans-compound. In analogy
to the 3,5,5-derivatives, the shift differences between the
two geminal ring protons was more pronounced for the
trans-isomer. In the trans-4,5-compounds, the highfield
proton (2.26 ppm) is placed cis to the methyl at C-4 (trans
to the ester), the downfield proton (2.94 ppm) is conse-
quently in trans-configuration relative to 4a-CH (cis to the
3
Fig. 1. General structure of the spin traps.

1356
K. Stolze et al. / Biochemical Pharmacology 69 (2005) 1351–1361
Table 4
Half-life of the superoxide adducts and n-octanol/buffer partition coeffi-
cients of the spin traps
compound
Apparent
Partition coefficient
n-octanol/phosphate
buffer (100 mM, pH 7)
t
1/2 (min)
a
EMPO
8.6
0.15
0.45
0.46
0.44
1.66
1.36
cis-3,5EDPO
11.5
44.2
44.6
47.3
47.0
trans-3,5EDPO
b
,5EDPO
4
trans-3,5DPPO
c
4
,5DPPO
cis-3,5DIPPO
trans-3,5DIPPO
d
d
–
d
0.90
55.0
42.8
1.12
1.03
e
4
,5DIPPO
a
Data from Stolze et al. [7].
b
c
d
e
cis/trans = 21/79.
cis/trans = 29.6/70.4.
ca. 95% cis form.
cis/trans = 24/76.
pH 7.0) containing 20 mM DTPA, SOD (100 U/mL), and
catalase (250 U/mL). The final pH was 7.4. The decay of
the EPR signal was recorded as a series of consecutive
spectra until the superoxide-related lines gradually disap-
peared. After prolonged incubation an increasing amount
of the hydroxyl radical adduct was observed (e.g. for trans-
Fig. 2. Formation of the superoxide adducts of the spin traps trans-3,5-
EDPO, cis-3,5-EDPO, and cis/trans-4,5-EDPO; (a) Trans-3,5-EDPO
3,5-EDPO: ca. 15 % after 30 min). The spectral contribu-
(
(
20 mM), catalase (250 U/mL), xanthine (0.2 mM) and xanthine oxidase
50 mU/mL) in oxygenated phosphate buffer (20 mM, pH 7.4, containing
tion of this secondary spin adduct was subtracted from each
individual EPR spectrum before calculating the respective
half-life. For all spin traps the resulting intensity decrease
of the first line (or set of lines) was approximated by a first-
order exponential decay, which was in good agreement
with the experimental data. The Pearson correlation coef-
ficient, which characterizes the quality of approximation,
was higher than 0.99 for all spin traps investigated. The
respective values of apparent superoxide half-lives are
listed in Table 4.
0.4 mM DTPA) were incubated and measured using the following EPR
parameters: sweep width, 60 G; modulation amplitude, 0.7 G; microwave
4
power, 20 mW; time constant, 0.16 s; receiver gain, 6.3 ꢂ 10 ; scan rate,
21.46 G/min. The bars represent 10,000 arbitrary units; (b) Same as in (a),
except that SOD (100 U/mL) was added; (c) Same as in (a), except that cis-
,5-EDPO (20 mM) was used. EPR parameters: sweep width, 60 G; mod-
3
ulation amplitude, 0.7 G; microwave power, 20 mW; time constant, 0.08 s;
5
receiver gain, 1 ꢂ 10 ; scan rate, 42.9 G/min; (d) same as in (a), except that
cis/trans-4,5-EDPO (20 mM) was used. EPR parameters: sweep width,
6
0 G; modulation amplitude, 0.7 G; microwave power, 20 mW; time con-
5
stant, 0.16 s; receiver gain, 1 ꢂ 10 ; scan rate, 21.46 G/min. The species
marked ‘‘*’’ was not detected when KO was used instead of Xa/XOD.
2
Preliminary tests regarding the overall efficiency of the
spin traps (combination of rate constant, line width and
half-life) revealed that the obtainable spectral intensity
(measured after 3 and 15 min incubation with the
xanthine/xanthine oxidase system) was higher than with
DEPMPO.
of 3,5-DPPO and 3,5-DIPPO could, however, only be
isolated in minor amounts. 4,5-EDPO consisted of 21%
cis- and 79% trans-isomer (NMR), which could not be
separated by conventional chromatographic procedures. In
Fig. 2(d) the spectrum obtained from 4,5-EDPO is shown,
recorded under conditions identical to those in Fig. 2(a).
3.3. Spin adduct formation with other
oxygen-containing radicals
N
H
One species (a = 13.48 G; a = 15.97 G; marked ‘‘*’’)
was not detected in the KO system and is therefore not
listed in Table 5.
2
Using a Fenton system in the presence of the spin traps,
EPR spectra of the respective hydroxyl radical adducts
could be obtained from all investigated spin traps, e.g. from
trans-3,5-EDPO (Fig. 3(a)), cis-3,5-EDPO (Fig. 3(b)), or
4,5-EDPO (Fig. 3(c)). The hydroxyl radical adducts were
rather stable, and only minor changes were observed in the
EPR spectra during the first 20 min of incubation.
As already shown above for 3,5-EDPO, no signals were
detected in control experiments in which SOD had been
added to the incubation mixture prior to xanthine/xanthine
oxidase (data not shown).
Half-lives of the respective superoxide adducts were
determined as follows: the respective spin traps (40 mM,
final concentration) were incubated in water in the pre-
In order to discriminate between the formation of oxy-
gen- and carbon-centered radical adducts from alcohols
such as methanol, two simple model systems were chosen:
sence of ca. 0.5 mg solid KO for 15 s, after which the
2
solution was diluted 1:1 with phosphate buffer (300 mM,

K. Stolze et al. / Biochemical Pharmacology 69 (2005) 1351–1361
1357
Fig. 4. Methanol-derivedoxygen- and carbon-centered spin adducts from the
Fig. 3. Formation of the hydroxyl radical adducts of the spin traps trans-
,5-EDPO, cis-3,5-EDPO, and cis/trans-4,5-EDPO; (a) trans-3,5-EDPO
20 mM) was incubated with a Fenton system containing FeSO (0.8 mM),
(0.2%) and the reaction was stopped after 10 s by 1:1
spin traps trans- and cis-3,5-EDPO; (a) after a 2 min incubation of trans-3,5-
3
3
EDPO (1 M in methanol) with FeCl (10 mM), the reaction was stopped by
(
4
1:20 dilution with phosphate buffer (0.15 M, pH 7.4, containing 10 mM
DTPA), and the spectrum was recorded with the following spectrometer
settings: sweep width, 80 G; modulation amplitude, 0.1 G; microwave power,
2 2
EDTA (2 mM), H O
dilution with phosphate buffer (300 mM, pH 7.4, containing 20 mM DTPA)
and the spectrum was recorded using the following spectrometer settings:
5
20 mW;timeconstant,0.08 s;receivergain, 1 ꢂ 10 ;scanrate,57 G/min.The
sweep width, 60 G; modulation amplitude, 0.1 G; microwave power,
4
bars represent 10,000 arbitrary units; (b) difference spectrum between two
succeeding scans, clearly showing the minor species (marked ‘‘*’’ in (a)); (c)
same conditions as in (a), except that cis-3,5-EDPO was used; (d) trans-3,5-
2
0 mW; time constant, 0.81 s; receiver gain, 5 ꢂ 10 ; scan rate, 42.9 G/
min. The bars represent 10,000 arbitrary units; (b) same conditions, except
that cis-3,5-EDPO was used. EPR parameters: sweep width, 60 G; mod-
ulation amplitude, 0.1 G; microwave power, 2 mW; time constant, 0.82 s;
EDPO (20 mM) was incubated with a Fenton system containing FeSO
(1 mM), methanol (10%), EDTA (2 mM), H (0.2%) and the reaction
4
2 2
O
4
receiver gain, 5 ꢂ 10 ; scan rate, 42.9 G/min. The bars represent 10,000
was stopped after 10 s by 1:1 dilution with phosphate buffer (300 mM, pH
7.4, containing 20 mM DTPA). The spectrum was recorded using the follow-
ing spectrometer settings: sweep width, 80 G; modulation amplitude, 0.24 G;
arbitrary units; (c) same conditions, except that cis/trans-4,5-EDPO was
used. EPR parameters: sweep width, 60 G; modulation amplitude, 0.1 G;
4
4
microwave power, 0.2 mW; time constant, 0.82 s; receiver gain, 5 ꢂ 10 ;
microwave power, 20 mW; time constant, 0.08 s; receiver gain, 1 ꢂ 10 ; scan
scan rate, 42.9 G/min. The bars represent 10,000 arbitrary units.
rate, 57 G/min. The bars represent 10,000 arbitrary units.
3
a) the Fe -catalyzed formation of alkoxyl radical adducts
+
(
clearly different from those of the methoxyl radical
N
adduct mentioned above (a = 13.55 G; a = 6.77 G;
H
according to Dikalov and Mason [11] and (b) the a-
hydroxymethyl radical generating Fenton system in the
presence of 5% methanol according to Roubaud et al. [15].
H
a = 0.99 G; 61% and a = 13.95 G; a = 16.10 G;
N
H
H
a = 0.95 G; 39%). This can be seen in Fig. 4(d), where
3
+
The first system (Fe /methanol [11]) lead to a mixture
ꢀ
a Fenton system in the presence of methanol [15] was used
to generate an almost pure EPR spectrum of the two
of two diastereomeric forms of trans-3,5-EDPO/ OCH
3
ꢀ
(Fig. 4(a)), from which the spectrum of the less stable
species (Fig. 4(b), marked ‘‘*’’) was obtained by mathe-
matical subtraction of the succeeding scan (recorded after
diastereomeric forms of the trans-3,5-EDPO/ CH OH
2
adduct. The spectral contribution of the different species
(see Table 5) was assessed by computer simulation (Bruker
WINEPR SimFonia).
6
the EPR spectrum obtained from the two diastereomeric
min) from the first scan shown in Fig. 4(a). In Fig. 4(c),
ꢀ
forms of cis-3,5-EDPO/ OCH are shown, recorded under
3
3.4. Spin trapping of lipid-derived free radicals
otherwise identical conditions. Upon prolonged incubation
the methoxyl radical adduct gradually disappeared and
increasing amounts of the more stable carbon-centered
hydroxymethyl radical adduct were detected, leading to
very complex spectra (not shown). The HFS values of the
Radicals derived from linoleic acid by lipid peroxida-
tion-type processes were also investigated. Radicals were
formed in a Fenton-type reaction from peroxidized linoleic
2+
acid in the presence of Fe [9,10]. An anaerobic setup,
obtained by flushing with nitrogen for several minutes, was
chosen. The reaction system consisted of peroxidized
ꢀ
N
H
trans-3,5-EDPO/ CH OH (a = 14.73 G; a = 17.10 G;
2
N
4% and a = 14.90 G; a = 24.33 G; 36%) are, however,
H
6

Table 5
Comparison of the EPR parameters of different radical adducts of various EMPO derivatives
Radical HFS (G) EMPO
c-3,5EDPO
t-3,5EDPO
4,5EDPO
t-3,5DPPO
4,5DPPO
c-3,5DIPPO
t-3,5DIPPO
4,5DIPPO
ꢀ
a
(36%) (35%) (29%) (100%)
OOH
OH
(57%)
13.28
11.89
–
(43%) (69%) (31%) (100%)
13.28 13.50 13.55 13.13
–
–
–
–
(44%) (35%) (21%) (76%) (34%) (100%)
13.49 13.49 13.49 13.15 13.45 13.22
(35%) (33%) (32%)
13.48 13.48 13.43
N
H
H
a
a
a
13.48 13.48 13.48
13.15
8.48
9.67
15.40 5.98
8.40
0.90
9.10
11.60 8.55
1.90
9.25
11.40 6.75
8.18
15.36 8.48
9.70
11.73 7.45
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
ꢀ
ꢀ
(50%)
14.00
15.00
0.90
(50%) (67%) (33%) (67%)
14.00 14.50 13.90 13.85
(33%) (75%) (25%)
(86%)
13.85
9.05
(14%) (80%) (20%)
(62%) (38%) (85%) (15%) (66%) (34%)
14.53 13.92 13.85 14.30 13.98 14.47
N
H
H
a
a
a
14.30
19.30
0.95
14.00 14.45
10.00 18.60
14.30
19.30
0.95
13.96 14.45
12.58 19.67 8.57
9.00
9.92
18.58
19.57 8.80
9.08
19.55 10.07 18.52
–
–
0.75
0.75
1.05
–
0.75
1.11
–
–
0.75
0.75
0.95
1.11
–
(100%)
(100%)
15.52
25.96
16.04
0.57
(100%)
15.45
25.87
17.50
–
(100%)
15.57
25.31
18.03
–
(100%)
15.42
25.84
17.53
–
(100%)
15.51
25.16
17.82
–
(100%)
15.61
25.90
16.20
0.50
(100%)
15.46
25.79
17.65
–
(100%)
15.59
25.12
17.96
–
N
H
H
H
H
a
a
a
a
15.52
22.21
20.82
-
ꢀ
ꢀ
CH
3
(100%)
15.42
22.30
(50%)
13.74
10.87
–
(52%) (48%) (89%)
15.45 15.35 15.32
27.00 16.60 16.74
(11%) (98%) (2%)
(89%)
15.31
16.69
(11%) (98%) (2%)
(52%) (48%) (88%) (12%) (95%) (5%)
15.40 15.50 15.35 15.35 15.37 15.48
16.45 27.08 16.82 26.25 18.61 26.52
N
H
a
a
15.35
26.20
15.40 15.50
18.43 26.73
15.30
26.22
15.35 15.35
18.26 26.70
b
OCH
3
(50%) (60%) (40%) (61%)
13.74 13.62 14.07 13.55
(39%) (56%) (24%) (20%) (56%)
(44%) (47%) (29%) (24%) (72%) (28%) (50%) (50%) (44%) (30%) (26%)
N
H
H
a
a
a
13.95
16.10
0.95
13.57 13.52 14.13
13.55
6.70
0.99
(65%)
14.78
17.07
–
13.95
16.01
0.95
13.65 13.57 14.08 13.68 13.99 13.62 13.95 13.57 13.57 14.10
7.81
4.60
16.06 6.77
0.99
8.55
0.70
4.50
16.43
8.30
0.70
4.30
16.37 4.93
15.73 6.65
0.90
15.90 8.25
0.90 0.70
4.10
16.25
–
–
–
–
–
–
–
–
–
–
–
ꢀ
ꢀ
CH OH
2
(100%)
14.95
21.25
–
(79%) (21%) (64%)
15.03 15.00 14.73
25.41 16.00 17.10
(36%) (98%) (2%)
(35%) (98%) (2%)
(76%) (24%) (72%) (28%) (96%) (4%)
15.08 14.95 14.84 14.97 15.00 15.02
25.31 16.04 17.18 24.58 18.05 25.26
N
H
a
a
14.90
24.33
–
15.00 15.00
18.20 25.30
14.94
24.53
–
15.00 15.02
18.02 25.10
3
3
3
3
0.50
0.50
–
–
–
–
–
0.51
0.50
–
–
–
–
c
C (LOOH)
(62%)
13.45
11.45
–
(38%) (80%) (10%) (50%)
13.45 14.45 15.30 14.40
(45%) (68%) (32%)
(45%)
13.79
8.82
–
(45%) (63%) (37%) (38%) (28%) (64%) (24%) (12%) (58%) (42%)
N
H
H
a
a
a
13.75
8.83
13.85 14.60
14.20
18.90
–
13.85 14.58 14.45 15.35 14.30 13.64 14.40 13.85 14.65
8.55
19.67 24.20 18.90
9.90
1.05
18.30
9.94
1.10
18.18 19.60 24.30 19.55 8.82
15.50 10.20 18.20
–
–
–
–
–
–
–
–
–
0.95
–
–
1.10
–
d
d
(
16.70/8.95)
(15.30/23.00)
(15.70/13.70)
N
(15.40/11.55)
(12.90/10.50)
d
d
d
–
–
(12.90/10.45)
–
–
–
–
–
–
–
–
–
–
–
–
a
H
Fourth species only detected in the Xa/XOD system (a = 13.48 G; a = 15.97 G).
b
c
d
Data from difference spectrum.
ꢀ
Rest mainly OH-adduct.
Approximate values of secondary products.

K. Stolze et al. / Biochemical Pharmacology 69 (2005) 1351–1361
1359
be seen in Fig. 5(b). The respective experiments performed
with cis-3,5-EDPO can be seen in Fig. 5(c) (cis-3,5-EDPO/
2
+
ꢀ
LOOH/Fe ) and Fig. 5d (cis-3,5-EDPO/ CH ). Compar-
3
ison with data from Table 5 makes the contribution of the
N
H
hydroxyl radical adduct (a = 14.45 G; a = 19.67 G;
0%, see also above, Fig. 3(b)) as well as a carbon-centered
8
N
H
radical adduct (a = 15.30 G; a = 24.20 G; 10%) likely.
The spectral parameters of lipid-derived adducts of the
other spin traps are listed in Table 5.
4. Discussion
Several novel compounds derived from the spin trap
EMPO with an extra methyl group in position 3 or 4 of the
pyrroline ring were synthesized and fully characterized in
1
13
this study. Complete H and C signal assignment was
performed.
The substances form very stable superoxide adducts (t1/
=
ca. 12–55 min), which are at least 15 times more stable
2
than the respective adducts with DMPO (t1/2 = 45 s) [4].
The stabilities of the superoxide adducts of trans-3,5-
EDPO, trans-DPPO and trans-DIPPO even exceed those
of the EMPO derivatives iPrMPO or sBuMPO (t1/
>
20 min [1]) or DEPMPO and its derivatives (t1/
2
2
= ca. 7–15 min [8–10]).
The half-lives of the superoxide adducts were deter-
Fig. 5. Lipid-derived radical adducts from peroxidized linoleic acid using a
Fenton-type incubation system in the presence of the spin traps cis- and
trans-3,5-EDPO. Comparison with a methyl radical generating model
system; (a) to a nitrogen-bubbled solution of peroxidized linoleic acid
mined according to a first-order exponential decay approx-
2
imation (Pearson correlation coefficient r > 0.99). Under
the experimental conditions used a second order decay of
the superoxide adducts was negligible. An important
aspect to consider is the fact that the observed spectral
intensity does not only depend on the half-life of the spin
adducts, but also on the rate constant of the spin trapping
reaction, the spectral line width, and the total number of
lines, and additional factors, such as enzyme binding or the
solubility (aggregate formation) of the spin trap. Prelimin-
ary tests, based on the spectral intensity of the respective
superoxide spin adducts after 3 and 15 min, have shown
that the performance of the investigated spin traps is almost
two-fold higher than with DEPMPO, increasing with
incubation time due to its higher stability.
(2 mM) and trans-3,5-EDPO (20 mM) in phosphate buffer (20 mM, pH 7.4,
containing 1.5% acetonitrile) FeSO (0.2 mM) was added and the spectrum
4
was recorded with the following spectrometer settings: sweep width, 80 G;
modulation amplitude, 1.42 G; microwave power, 20 mW; time constant,
6
0
.16 s; receiver gain, 1 ꢂ 10 ; scan rate, 57.27 G/min. 5 Scans accumulated.
The bars represent 10,000 arbitrary units; (b) trans-3,5-EDPO (20 mM) was
incubated with a Fenton system containing FeSO (1 mM), DMSO (10%),
EDTA (2 mM), H (0.2%) and the reaction was stopped after 10 s by 1:1
4
2 2
O
dilution with phosphate buffer (300 mM, pH 7.4, containing 20 mM
DTPA). The spectrum was recorded using the following spectrometer
settings: sweep width, 70 G; modulation amplitude, 0.1 G; microwave
5
power, 20 mW; time constant, 0.16 s; receiver gain, 5 ꢂ 10 ; scan rate,
25.03 G/min. The bars represent 10,000 arbitrary units; (c) same conditions
as in a), except that cis-3,5-EDPO (20 mM) was used; (d) same conditions
as in (b), except that cis-3,5-EDPO (20 mM) was used.
All novel compounds formed hydroxyl and methoxyl
radical adducts. Whereas hydroxyl radical adducts were
quite stable, the respective methoxyl radical adducts gra-
dually disappeared and the more stable hydroxymethyl
radical adducts became the predominant species. Several
radicals adducts were formed from peroxidized linoleic
acid, the major component being the hydroxyl radical
adduct possibly formed according to secondary reactions
or iron-catalyzed hydrolysis of the spin trap. Secondary
linoleic acid and the respective spin trap dissolved in
2
0 mM phosphate buffer, pH 7.4, containing 1.5% acet-
2
+
onitrile. To this solution Fe , dissolved in nitrogen-purged
water, was added in order to start the formation of free
radicals, as recently tested with different derivatives of
DEPMPO [9,10], EMPO [7], Trazon [12] or EPPN [16,17].
In Fig. 5(a), the EPR spectrum obtained from trans-3,5-
EDPO is shown, consisting of four different species, one of
which was identified as the hydroxyl radical adduct (see
ꢀ
ꢀ
product formation from LOO and LO has also been
reported by other authors [18–24]. The structure of various
secondary radical adducts could not be determined, but the
oxygen in the initially formed alkoxyl radical formed from
peroxidized linoleic acid is most likely situated at position
above, Fig. 3(a)). For comparison, a typical carbon-cen-
ꢀ
tered radical adduct (trans-3,5-EDPO/ CH ), generated in
3
a Fenton-type model system in the presence of DMSO can

1360
K. Stolze et al. / Biochemical Pharmacology 69 (2005) 1351–1361
[
3] Tsai P, Ichikawa K, Mailer C, Pou S, Halpern HJ, Robinson BH, et al.
9
or 13. Before trapping these very short-lived species
Esters of 5-carboxyl-5-methyl-1-pyrroline N-oxide: A family of spin
traps for superoxide. J Org Chem 2003;68:7811–7.
undergo rapid b-scission [25,26] leading to secondary
carbon-centered radical species such as the pentyl radical
or the octanoic acid radical, from which stable spin adducts
can be expected.
[
4] Buettner GR, Oberley LW. Considerations in the spin trapping of
superoxide and hydroxyl radical in aqueous systems using 5,5-
dimethyl-1-pyrroline-1-oxide. Biochem Biophys Res Commun
1
978;83:69–74.
[
5] Olive G, Mercier A, LeMoigne F, Rockenbauer A, Tordo P. 2-Ethoxy-
carbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide: Evaluation of the
spin trapping properties. Free Radic Biol Med 2000;28:403–8.
6] Zhang H, Joseph J, Vasquez-Vivar J, Karoui H, Nsanzumuhire C,
Mart a´ sek P, et al. Detection of superoxide anion using an isotopically
labeled nitrone spin trap: Potential biological applications. FEBS Lett
The investigated spin traps posses two chiral centers
within the molecule (located at the positions of the methyl
groups (3 and 5 or 4 and 5). Only 3,5-EDPO could be fully
separated into two pure diasteromers by column chroma-
tography. Upon reaction with the attacking radical a third
chiral center is formed, as the radical can attack the
pyrroline ring from two sides:
[
2
000;473:58–62.
[
7] Stolze K, Udilova N, Nohl H. Spin adducts of superoxide, alkoxyl, and
lipid-derived radicals with EMPO and its derivatives. Biol Chem
2
002;383:813–20.
[
8] Fr e´ javille C, Karoui H, Tuccio B, Le Moigne F, Culcasi M, Pietri S, et
al. 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide: A new
efficient phosphorylated nitrone for the in vitro and in vivo spin
trapping of oxygen-centered radicals. J Med Chem 1995;38:258–65.
9] Stolze K, Udilova N, Nohl H. Spin trapping of lipid radicals with
DEPMPO-derived spin traps: Detection of superoxide, alkyl and
alkoxyl radicals in aqueous and lipid phase. Free Radic Biol Med
[
In some cases only one product was detected in the EPR
spectrum, which indicates significant differences in reac-
tion rate and adduct stability. Otherwise, a superimposed
spectrum was observed which had to be deconvoluted
mathematically. In the case of the respective trans-3,5-
isomers, exceptionally stable superoxide adducts were
observed with half-lives ranging from about 44–55 min.
These spin traps can therefore be recommended for super-
oxide trapping. Additional studies in biological systems
including cytotoxicity tests with various cell lines will
follow in the near future.
2
000;29:1005–14.
[10] Stolze K, Udilova N, Nohl H. Lipid radicals: Properties and detection
by spin trapping. Acta Biochim Polonica 2000;47:923–30.
[
[
[
[
[
[
[
11] Dikalov SI, Mason RP. Spin trapping of polyunsaturated fatty acid-
derived peroxyl radicals: Reassignment to alkoxyl radical adducts.
Free Radic Biol Med 2001;30:187–97.
12] Stolze K, Udilova N, Nohl H. ESR analysis of spin adducts of alkoxyl
and lipid-derived radicals with the spin trap Trazon. Biochem Phar-
macol 2002;63:1465–70.
13] Sankuratri N, Janzen EG. Synthesis and spin trapping chemistry of a
novel bicyclic nitrone: 13,3-trimethyl-6-azabicyclo[3.2.1]oct-6-ene-
N-oxide (Trazon). Tetrahedron Lett 1996;37:5313–6.
14] O’Brien PJ. Intracellular mechanisms for the decomposition of a lipid
peroxide. I. Decomposition of a lipid peroxide by metal ions, heme
compounds, and nucleophiles. Can J Biochem 1969;47:485–92.
15] Roubaud V, Lauricella R, Bouteiller JC, Tuccio B. N-2-(2-Ethoxy-
carbonyl-propyl)-a-phenylnitrone: An efficacious lipophilic spin trap
for superoxide detection. Arch Biochem Biophys 2002;397:51–6.
16] Stolze K, Udilova N, Rosenau T, Hofinger A, Nohl H. Spin trapping of
superoxide, alkyl- and lipid-derived radicals with derivatives of the
spin trap EPPN. Biochem Pharmacol 2003;66:1717–26.
Acknowledgements
The authors would like to thank R. Stadtm u¨ ller and A.
Hofinger for skilful technical assistance in synthesis, pur-
ification, and characterization of the spin traps. The present
study was supported by the Austrian Fonds zur F o¨ rderung
der wissenschaftlichen Forschung.
17] Stolze K, Udilova N, Rosenau T, Hofinger A, Nohl H. Spin adducts of
several N-2-(2-alkoxycarbonyl-propyl)-a-pyridylnitrone derivatives
with superoxide, alkyl and lipid-derived radicals. Biochem Pharmacol
2
004;68:185–93.
References
[18] Rota C, Barr DP, Martin MV, Guengerich FP, Tomasi A, Mason RP.
Detection of free radicals produced from the reaction of cytochrome P-
4
50 with linoleic acid hydroperoxide. Biochem J 1997;328:565–71.
[
1] Stolze K, Udilova N, Rosenau T, Hofinger A, Nohl H. Synthesis
and characterization of EMPO-derived 5,5-disubstituted 1-pyrroline
N-oxides as spin traps forming exceptionally stable superoxide spin
adducts. Biol Chem 2003;384:493–500.
[
[
19] Van der Zee J, Barr DP, Mason RP. ESR spin trapping investigation of
radical formation from the reaction between hematin and tert-butyl
hydroperoxide. Free Radic Biol Med 1996;20:199–206.
20] Akaike T, Sato K, Ijiri S, Miyamoto Y, Kohno M, Ando M, et al.
Bactericidal activity of alkyl peroxyl radicals generated by heme-iron-
catalyzed decomposition of organic peroxides. Arch Biochem Bio-
phys 1992;294:55–63.
[
2] Zhao H, Joseph J, Zhang H, Karoui H, Kalyanaraman B. Synthesis and
biochemical applications of a solid cyclic nitrone spin trap: A rela-
tively superior trap for detecting superoxide anions and glutathiyl
radicals. Free Rad Biol Med 2001;31:599–606.

K. Stolze et al. / Biochemical Pharmacology 69 (2005) 1351–1361
1361
[
21] Kalyanaraman B, Mottley C, Mason RP. A direct ESR and
[24] Davies MJ. Detection of peroxyl and alkoxyl radicals produced by
reaction of hydroperoxides with rat liver microsomal fractions. Bio-
chem J 1989;257:603–6.
spin-trapping investigation of peroxyl free radical formation
by hematin/hydroperoxide systems.
855–8.
J Biol Chem 1983;258:
3
[25] Iwahashi H, Deterding LJ, Parker CE, Mason RP, Tomer KB. Identi-
fication of radical adducts formed in the reactions of unsaturated fatty
acids with soybean lipoxygenase using continuous flow fast atom
bombardment with tandem mass spectrometry. Free Rad Res
1996;25:255–74.
[
[
22] Chamulitrat W, Takahashi N, Mason RP. Peroxyl, alkoxyl, and carbon-
centered radical formation from organic hydroperoxides by chloro-
peroxidase. J Biol Chem 1989;264:7889–99.
23] Davies MJ. Detection of peroxyl and alkoxyl radicals produced by
reaction of hydroperoxides with heme-proteins by ESR spectroscopy.
Biochim Biophys Acta 1988;964:28–35.
[26] Dikalov SI, Mason RP. Reassignment of organic peroxyl radical
adducts. Free Radic Biol Med 1999;27:864–72.