Spin adduct formation from lipophilic EMPO-derived spin traps with various oxygen- and carbon-centered radicals

Article abstract of DOI:10.1016/j.bcp.2004.09.021

Free radicals are involved in the onset of many diseases, therefore the availability of adequate spin traps is crucial to the identification and localization of free radical formation in biological systems. In recent studies several hydrophilic compounds of 2-ethoxycarbonyl-2-methyl-pyrroline-N-oxide (EMPO) have been found to form rather stable Superoxide spin adducts with half-lives up to twenty minutes at physiological pH. This is a major improvement over DMPO (t_1/2 = ca. 45 s), and even over DEPMPO (t_1/2 = ca. 14 min), the best commercially available spin trap for the unambiguous detection of superoxide radicals. In order to allow the detection of superoxide and also other radicals in lipid environment a series of more lipophilic derivatives of EMPO was synthesized and their structure unambiguously characterized by ¹H and ¹³C NMR spectroscopy. In this way, six different compounds with a n-butyl group in position 5 and either an ethoxy- (EBPO), propoxy- (PBPO), iso-propoxy- (iPBPO), butoxy- (BBPO), sec-butoxy- (sBBPO) or tert-butoxycarbonyl group (tBBPO) in position 5 of the pyrroline ring were obtained and fully analytically characterized (NMR, IR). The stability of the superoxide adducts of all investigated spin traps were comparable with EMPO (t1/2 = ca. 8 min), except for the two compounds bearing an additional methyl group in position 3 or 4 of the pyrroline ring, 5-butyl-5-ethoxycarbonyl-3-methyl-pyrroline-N-oxide (BEMPO-3) and 5-butyl-5-ethoxycarbonyl-4-methyl-pyrroline-N-oxide (BEMPO-4), of which the superoxide adducts were stable for more than 30 min. Spin adducts of other carbon- and oxygen-centered radicals were also investigated.

Full text of DOI:10.1016/j.bcp.2004.09.021

Biochemical Pharmacology 69 (2005) 297–305
www.elsevier.com/locate/biochempharm
Spin adduct formation from lipophilic EMPO-derived spin traps with
various oxygen- and carbon-centered radicals
Klaus Stolzea, Natascha Udilovaa, Thomas Rosenaub, Andreas Hofingerb, Hans Nohla,
*
a
¨
Research Institute of Pharmacology and Toxicology, University of Veterinary Medicine Vienna, Veterinarplatz 1, A-1210 Vienna, Austria
bDepartment of Chemistry, University of Natural Resources and Applied Life Sciences (BOKU), Muthgasse 18, A-1190 Vienna, Austria
Received 20 August 2004; accepted 28 September 2004
Abstract
Free radicals are involved in the onset of many diseases, therefore the availability of adequate spin traps is crucial to the identification
and localization of free radical formation in biological systems. In recent studies several hydrophilic compounds of 2-ethoxycarbonyl-2-
methyl-pyrroline-N-oxide (EMPO) have been found to form rather stable superoxide spin adducts with half-lives up to twenty minutes at
physiological pH. This is a major improvement over DMPO (t1/2 = ca. 45 s), and even over DEPMPO (t1/2 = ca. 14 min), the best
commercially available spin trap for the unambiguous detection of superoxide radicals. In order to allow the detection of superoxide and
also other radicals in lipid environment a series of more lipophilic derivatives of EMPO was synthesized and their structure
unambiguously characterized by 1H and 13C NMR spectroscopy. In this way, six different compounds with a n-butyl group in position
5 and either an ethoxy- (EBPO), propoxy- (PBPO), iso-propoxy- (iPBPO), butoxy- (BBPO), sec-butoxy- (sBBPO) or tert-butoxycarbonyl
group (tBBPO) in position 5 of the pyrroline ring were obtained and fully analytically characterized (NMR, IR). The stability of the
superoxide adducts of all investigated spin traps were comparable with EMPO (t1/2 = ca. 8 min), except for the two compounds bearing an
additional methyl group in position 3 or 4 of the pyrroline ring, 5-butyl-5-ethoxycarbonyl-3-methyl-pyrroline-N-oxide (BEMPO-3) and 5-
butyl-5-ethoxycarbonyl-4-methyl-pyrroline-N-oxide (BEMPO-4), of which the superoxide adducts were stable for more than 30 min.
Spin adducts of other carbon- and oxygen-centered radicals were also investigated.
# 2004 Elsevier Inc. All rights reserved.
Keywords: Spin traps; EPR; Lipophilic EMPO derivatives; Superoxide; Linoleic acid hydroperoxide; Free radicals
1. Introduction
(t1/2 < 1min [4]) or EMPO (t1/2 = ca. 8 min [5–7]) the
compounds bearing bulky substituents on the alkoxy-
carbonyl moiety formed considerably more stable
superoxide adducts (t1/2 = 15–25 min [1,3]) even when
compared to the spin trap DEPMPO (t1/2 = ca. 14 min
[8–10]).
The spin trap EMPO (5-ethoxycarbonyl-5-methyl-1-
pyrroline-N-oxide) and its derivatives have recently been
described by several authors [1–3] to be excellent spin
traps for the detection of superoxide radicals. When
compared to the structurally related spin traps DMPO
In addition to spin adduct stabilization due to electron-
withdrawing as well as steric effects, variation of the 5-
alkyl substituent and the 5-alkoxycarbonyl substituents
also allows tuning the lipophilic properties of the spin
trap. In this regard, the 5-butyl substituent will increase the
lipophilicity of the trap. This aspect is of great importance
if higher concentrations of the spin trap in a particular
biological environment is needed, e.g. within or close to
the surface of mitochondrial or other membranes. The two
novel compounds BEMPO-3 and BEMPO-4 bear an addi-
tional methyl group at position 3 or 4 of the pyrroline ring,
thus providing an increased steric shielding effect leading
to an even higher stability of the respective superoxide
Abbreviations: BBPO,
5-(butoxycarbonyl)-5-butyl-1-pyrroline-N-
oxide; DEPMPO, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide;
DMPO, 5,5-dimethylpyrroline-N-oxide; DTPA, diethylenetriaminepentaa-
cetic acid; EBPO, 5-(ethoxycarbonyl)-5-butyl-1-pyrroline-N-oxide;
EMPO, 5-(ethoxycarbonyl)-5-methyl-1-pyrroline-N-oxide; BEMPO-3, 5-
butyl-5-(ethoxycarbonyl)-3-methyl-1-pyrroline-N-oxide; BEMPO-4, 5-
butyl-5-(ethoxycarbonyl)-4-methyl-1-pyrroline-N-oxide; EPR, electron
paramagnetic resonance; HFS, hyperfine splitting; LOꢀ, lipoxyl radical;
NMR, nuclear magnetic resonance; O2ꢀꢁ, superoxide anion radical; PBN,
N-tert-butyl-a-phenylnitrone; PBPO, 5-(propoxycarbonyl)-5-butyl-1-pyr-
roline-N-oxide; SOD, superoxide dismutase
* 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 # 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2004.09.021

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K. Stolze et al. / Biochemical Pharmacology 69 (2005) 297–305
adducts. In the present paper we wish to communicate
synthesis, analytical properties and spin traping behavior
of a series of substituted pyrroline derivatives with increas-
ing lipophilicity, suitable for the detection of superoxide
radicals as well as other oxygen- or carbon-centered
radicals in analogy to recently published experiments using
peroxidized linoleic acid with the spin traps DMPO [11],
DEPMPO [9,10], EMPO [7] and Trazon [12,13].
added at 0 8C. The solution was kept at 10 8C for 1.5 h
and then poured into a solution of ice-cold HCl (5 ml of
concentrated HCl in 150 ml of water). The solution was
extracted three times with CH2Cl2 and dried over Na2SO4.
After filtration, the mixture was distilled under reduced
pressure, and the purity of the remaining product was
assessed by thin layer chromatography and IR spectro-
scopy.
2.2.4. Ethyl 2-butyl-4-formyl-2-nitropentanoate
2. Materials and methods
Same conditions as above, except that methacrolein
(2.5 g, 38 mmol) was used instead of acrolein. After slow
addition of methacrolein at 0 8C, the solution had to be
stirred for several days at room temperature.
2.1. Chemicals
Acrolein, croton aldehyde, 2-bromohexanoyl bromide,
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.
2.2.5. Ethyl 2-butyl-4-formyl-3-methyl-2-nitrobutanoate
Same conditions as above, except that croton aldehyde
(2.5 g, 38 mmol) was used instead of acrolein. After slow
addition of methacrolein at 0 8C, the solution had to be
stirred for several days at room temperature.
2.2. Syntheses
2.2.6. Synthesis of the N-oxides
Synthesis and characterization of the compounds were
performed as reported previously [1,7], in analogy to the
synthesis of EMPO and its derivatives [5,6] with minor
adaptations as given below.
Synthesis of the nitrones was performed according to the
procedure described recently for the synthesis of EMPO
derivatives [1,7]. To a concentrated solution of 25 mmol of
the respective alkyl 2-butyl-4-formyl-2-nitrobutanoate
(ethyl 2-butyl-4-formyl-2-nitropentanoate for BEMPO-3;
ethyl 2-butyl-4-formyl-3-methyl-2-nitrobutanoate for
BEMPO-4) in H2O/CH3OH (v/v = 6:4) an aqueous solu-
tion of ammonium chloride (1.87 g in 8 ml of water) was
added. While zinc dust (8.5 g, 130 mmol) was slowly
added within 30 min, the mixture was carefully kept at
room temperature. The mixture was stirred for 4.5 h 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, saturated with
borax and extracted four times with 60 ml CH2Cl2. The
organic phase was dried with Na2SO4, filtered and con-
centrated. Careful purification by column chromatography
on silica gel with a petroleum ether/ethanol gradient
allowed the separation from the majority of side products
and provided the product as a yellow oil or yellow needles.
Additional purification was done immediately before the
EPR experiments on a 1-ml solid phase extraction column
using a Chromabond C-18 100-mg column obtained from
Macherey-Nagel (Du¨ren, Germany). The purity of the
obtained products was assessed by TLC and UV spectro-
scopy. Final identification of the purified products was
2.2.1. Alkyl 2-bromohexanoate
2-Bromohexanoyl bromide (70 mmol) was slowly
added to a solution of the respective alcohol (100 mmol)
and pyridine (70 mmol) in CHCl3 at 0 8C (ice bath). After
stirring for 1 h, the reaction mixture was successively
washed with water (50 ml), sulfuric acid (10 %, 50 ml)
and concentrated aqueous Na2CO3 (50 ml), and dried over
Na2SO4 overnight. Solvent and excess alcohol were
removed under reduced pressure. The crude, nearly color-
less product was used without further purification.
2.2.2. Alkyl 2-nitrohexanoate
The respective alkyl 2-bromohexanoate (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 N,N-dimethylformamide (120 ml) at room tempera-
ture. The solution was stirred overnight, 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 Na2CO3 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 further without purifi-
cation.
1
performed by H NMR, 13C NMR, and IR spectroscopy
(see Tables 1–3).
2.2.3. Alkyl 2-butyl-4-formyl-2-nitrobutanoate
2.2.7. Preparation of lipid hydroperoxides
Alkyl 2-nitrohexanoate (23 mmol) was dissolved in a
mixture of acetonitrile (10 g, 244 mmol) and triethylamine
(0.2 g, 2 mmol). Acrolein (2 g, 38 mmol) was slowly
Linoleic acid hydroperoxide was synthesized according
to O’Brien [14]. Briefly, linoleic acid was air-oxidized for
72 h in the dark at room temperature. The oxidation

K. Stolze et al. / Biochemical Pharmacology 69 (2005) 297–305
299
Table 1
13C NMR data (ppm) of the spin traps
0
0
0
0
100
200
300
400
2C
3C
3aCH3 4C
4aCH3 5C
COO
1 C
2 C
3 C
4 C
C
C
C
C
EMPOa
EBPO
134.9 25.4
135.2 26.0
135.3 26.1
135.1 26.1
135.4 26.1
135.3 26.1
135.3 26.1
–
–
–
–
–
–
–
31.9
–
–
–
–
–
–
–
–
–
78.5 169.3 61.7
81.8 169.8 61.9
82.0 169.9 67.5
82.0 169.3 69.8
82.0 170.0 65.9
13.4
–
–
–
20.3
–
–
–
28.3
28.4
28.4
28.4
28.4
28.4
37.2
36.8
13.7
–
32.2 24.8 22.5 13.8
32.3 24.9 22.6 13.8
32.1 24.9 22.7 13.8
32.4 25.0 22.7 13.9
PBPO
21.8
10.3
–
–
iPBPO
BBPO
21.5, 21.6
30.4
–
19.0
13.6
sBBPO
tBBPO
t-BEMPO-3
82.0 169.5 18.9, 19.1 74.2, 74.3 28.4, 28.5
9.4, 9.5 32.3 24.9 22.6 13.8
82.6 168.6 82.4
82.7 169.9 62.1
82.6 170.2 62.1
84.4 169.9 62.0
85.1 168.5 61.8
27.7
14.0
14.0
14.0
14.1
–
–
–
–
–
–
–
–
–
–
32.2 25.0 22.7 13.9
32.2 25.1 22.7 13.8
32.7 24.8 22.6 13.8
29.3 26.1 23.0 13.8
30.8 24.8 22.8 13.9
140.2 33.2 18.4
c-BEMPO-3 139.7 33.6 19.1
t-BEMPO-4
134.5 35.0
–
–
37.2 14.5
37.2 15.4
c-BEMPO-4 136.4 34.2
a
Data from Stolze et al. [1].
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
extinction coefficient of e233 nm = 25,250 Mꢁ1 cmꢁ1 in
ethanol [14].
blank of the respective solvent. Determination of the
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 (see also Table 3).
For 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 TM110 microwave cavity.
NMR spectra were recorded on a Bruker Avance at
1
300.13 MHz for H, and 75.47 MHz for 13C. CDCl3 was
used as the solvent throughout, TMS (tetramethylsilane) as
the internal standard. 13C peaks were assigned by means of
APT (attached proton test), HMQC (1H-detected hetero-
nuclear multiple-quantum coherence) and HMBC (hetero-
2.3. Instruments
UV–vis spectra were recorded on Hitachi 150-20 and U-
3300 spectrophotometers in double-beam mode against a
Table 2
1H NMR data (ppm) of the spin traps
0
0
0
0
100
200
300
400
2CH=N
3CHx
3aCH3 4CHx
4aCH3 1 CHx 2 CHx 3 CHx 4 CH3
CHx
CHx
CHx
CH3
EMPOa
EBPO
PBPO
6.97t
6.98t
6.99t
2.75m
–
–
–
–
–
–
–
2.16m, 2.60m
–
–
–
–
–
–
–
–
–
4.26m 1.31t
4.26m 1.30t
–
–
–
–
–
–
1.72s
–
–
–
2.64m, 2.78m
2.63m, 2.78m
2.27m, 2.50m
2.27m, 2.50m
2.27m, 2.46m
2.26m, 2.49m
2.26m, 2.47m
2.23m, 2.44m
2.03m, 2.17m 1.31m 1.40m 0.93t
2.05m, 2.16m 1.30m 1.41m 0.93t
2.05m, 2.15m 1.31m 1.41m 0.93t
4.16m 1.69sx 0.95t
5.08sp 1.28d
iPBPO
BBPO
sBBPO
tBBPO
6.91t, 2.76m 2.62m
–
7.00t
7.00t
6.98t
2.63m, 2.76m
4.19m 1.66qi 1.39m 0.93t 2.05m, 2.16m 1.30m 1.41m 0.93t
1.25d 4.92sx 1.61m 0.91t 2.03m, 2.14m 1.30m 1.40m 0.93t
2.62m, 2.77m
2.61m, 2.75m
3.15m
–
1.49s
–
–
–
–
–
–
–
–
–
–
1.97m, 2.13m 1.26m 1.37m 0.93t
1.95m, 2.21m 1.30m 1.40m 0.93t
1.95m, 2.21m 1.30m 1.40m 0.92t
1.96m, 2.23m 1.32m 1.38m 0.93t
1.96m, 2.23m 1.32m 1.38m 0.93t
t-BEMPO-3 6.88t
c-BEMPO-3 6.88t
t-BEMPO-4 6.96t
c-BEMPO-4 7.00t
1.19d 1.76dd, 2.64dd
1.25d 2.10dd, 2.50dd
4.24m 1.29t
4.24m 1.30t
3.02m
2.32m, 2.82m
2.38m, 2.71m
–
–
2.96m
2.71m
1.17d 4.26m 1.30t
1.09d 4.26m 1.30t
Abbreviations: s: singlet; d: doublet; t: triplet; qi: quintuplett; sx: sextet; sp: septet; m: multiplet; dd: doublet of doublets.
a
Data from Stolze et al. [1].

300
K. Stolze et al. / Biochemical Pharmacology 69 (2005) 297–305
nuclear multiple bond connectivity) spectra. All chemical
shift data are given in ppm units, coupling constants in Hz.
3. Results
3.1. Structure of the spin traps
The identity of the novel spin traps was confirmed by
NMR spectroscopy. A complete set of 1H, H–H correlated,
13C, HMQC and HMBC spectra was recorded for each
1
compound, and a full signal assignment for both H and
13C was performed. In the current compound series, all
derivatives carry a butyl group as 5-substituent, along with
a 5-alkoxycarbonyl moiety with varying alkoxy groups. In
two compounds, BEMPO-3 and BEMPO-4, an additional
methyl substituent was present at C-3 and C-4, respec-
tively. This caused the presence of configurational isomers
in these compounds, since the CH3 group could be
arranged either trans (E) or cis (Z) to the bulky alkox-
ycarbonyl substituent at C-5. Thus, synthesis provided two
diastereomers of each spin trap, a trans-compound and the
respective cis-isomer, so that BEMPO-3 and BEMPO-4
were obtained as a mixture of cis- and trans-isomers in the
ratio 45:55 and 35:65, respectively.
Also in the case of sBBPO, two diastereomers were
obtained which were not separable by column chromato-
graphy, as the C-20 in the 2-butoxy group introduces an
additional chiral center into the molecule.
The carbon NMR data of the spin traps confirmed the
presence of the pyrroline system, the four signals of the
carbons C-2 to C-5 in the heterocyclic ring appearing at
averaged values of 135, 26, 28 and 82 ppm. In comparison
to EMPO with its 5-methyl substituents, the butyl group in
the present series causes a slight downfield shift at C-3
(0.5 ppm), a highfiled shift at C-4 (3.5 ppm), and a down-
field shift at C-5 (3.5 ppm), cf. Table 1. The resonances of
the butyl substituent (C-100 to C-400) were found at 32.2,
24.9, 22.6 and 13.9 ppm, respectively. The nature of the
alkoxy substituent had no significant effect on the carbon
resonances at the pyrroline ring or the butyl group. The
effects of additional methyl substituents at either C-3 or C-
4, as well as the shifts of the respective cis- and trans-
isomers will not be addressed here, as they are compre-
hensively covered in a discussion of EDPO derivatives,
which will follow this study in due course [15].
1
Also the H NMR data showed the expected patterns.
The geminal protons at H-3, H-4 and H-100 are diaster-
eotopic and thus magnetically inequivalent caused by the
stereogenic center C-5, with shift differences of about
0.14 ppm for H-3, 0.22 ppm for C-4 and 0.12 ppm for
H-100. Similarly, two geminal C-10 protons in alkoxycar-
bonyl group at C-5 are diasterotopic, giving a multiplet. As
already previously noted [1], the magnetic inequivalence
of the two H-3 protons increases with increasing bulkiness
of substituents at C-5. Their shift difference is small for 5-

K. Stolze et al. / Biochemical Pharmacology 69 (2005) 297–305
301
methyl substituents, i.e. in EMPO derivatives, becomes
larger (0.04–0.12 ppm) for ethyl and propyl, and reaches
approx. 0.14 ppm in the present EBPO derivatives. sBBPO
was obtained as a mixture of two diastereomers due to the
chiral 2-butoxy substituents, which was introduced as
racemic 2-butanol. Only the resonances of the butoxy
group showed different 13C resonances, the resonances
of the respective other signals of the two isomers were
indistinguishable.
As in the case of the 13C resonances, the effect of methyl
1
substituents at C-3 or C-4 on the H spectra shall not be
discussed here, as it will be addressed in detail in a
following paper [15]. It should just be mentioned that the
signal patterns of the H-3 and H-4 protons in the BEMPO-3
and BEMPO-4 derivative showed the expected significant
differences in dependence of both substitution site (C-3 or
C-4) and configuration (cis or trans). The cis/trans-ratio of
BEMPO-3 and BEMPO-4 was easily determined from the
integrals in the 1H spectrum. In BEMPO-3, the proton at C-
3 appeared at around 3.10 ppm, and the shift difference
between cis- and trans-isomers was rather small
(0.10 ppm). The two geminal H-4 protons appeared at about
2.10/2.50 ppm for the cis-compound and 1.76/2.64 ppm for
the trans-compound. In BEMPO-4, the resonance of H-4
was found at 2.71 ppm for the cis-isomers and at 2.96 ppm
for the trans-isomers. H-4 was thus found more upfield than
H-3 in BEMPO-3, and there was a clear difference between
cis- and trans-derivatives in addition. The two geminal H-3
protons appeared at 2.38/2.71 ppm for the cis-compound
and at 2.32/2.82 ppm for the trans-compound. In analogy to
the EDPO derivatives, the shift differences of the geminal
protons (H-4 in BEMPO-3 and H-3 in BEMPO-4) was
much more pronounced for the respective trans-isomers.
The NMR data are given in Table 1 (13C) and 2 (1H).
Fig. 1. General structure of the spin traps.
the EPR signal was recorded as a series of consecutive
spectra until the superoxide-related lines disappeared and
additional lines of lower intensity, consisting mainly of
the hydroxyl radical adduct and in some cases a small
contribution of a carbon-centered adduct, became predo-
3.2. Spin trapping of superoxide radicals
The general structure of the spin traps is depicted in Fig.
1. In Fig. 2a, the EPR spectrum of the superoxide adduct of
EBPO 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. 2b).
Under these conditions similar spectra were obtained from
most of the other spin traps, except for BEMPO-3 (Fig. 2c)
and BEMPO-4 (Fig. 2d), where additional diastereomeric
forms contributed to the observed EPR spectra, some of
which were stable for about 30 min. As already shown
above for EBPO, no signals were detected in control
experiments in which SOD had been added to the incuba-
tion mixture prior to xanthine/xanthine oxidase (experi-
ments not shown). Determination of the half-life of
superoxide adducts was performed as follows: With the
exception of PBPO, the respective spin traps (40 mM in
oxygenated phosphate buffer pH 7.4) were pre-incubated
for 10 min in the above-mentioned xanthine/xanthine oxi-
dase system, SOD (100 U/ml) was added and the decay of
Fig. 2. Formation of the superoxide adducts of the spin traps EBPO,
BEMPO-3 and BEMPO-4. (a) EBPO (40 mM), catalase (250 U/ml),
xanthine (0.2 mM) and xanthine oxidase (50 mU/ml) in oxygenated phos-
phate buffer (20 mM, pH 7.4, containing 0.4 mM DTPA) were incubated and
measured using the following EPR parameters: sweep width, 60 G; mod-
ulation amplitude, 1.0 G; microwave power, 20 mW; time constant, 0.16 s;
receiver gain, 2 ꢂ 105; scan rate, 42.9 G/min. The bars represent 10,000
arbitrary units. (b) Same as in panel a, except that SOD (100 U/ml) was
added. (c) Same as in panel a, except that BEMPO-3 (40 mM) was used. (d)
Same as in panel a, except that BEMPO-4 (40 mM) was used. EPR
parameters: sweep width, 50 G; modulation amplitude, 1.0 G; microwave
power, 20 mW; time constant, 0.16 s; receiver gain, 5 ꢂ 104; scan rate,
35.8 G/min.

302
K. Stolze et al. / Biochemical Pharmacology 69 (2005) 297–305
Table 4
(100 U/ml) and catalase (250 U/ml) in phosphate buffer
(150 mM, final pH 7.4). With all spin traps the resulting
intensity decrease of the first two lines was approximated
by a first-order exponential decay, which was in a good
agreement with the experimental data. The Pearson corre-
lation coefficient, which characterizes the quality of
approximation, was higher than 0.99 for all spin traps
investigated, except for EBPO (0.97) and PBPO (0.96).
The respective values of apparent superoxide half-lives are
listed in 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 7.0)
t1/2 (min)
EMPOa
EBPO
8.6
4.2
0.15
4.92
PBPO
0.36
15.8
6.4
14.48
11.79
55.62
39.03
22.75
10.84
10.33
iPBPO
BBPO
sBBPO
tBBPO
BEMPO-3
BEMPO-4
a
6.8
17.6
47.2
36.2
3.3. Spin adduct formation with oxygen-containing
radicals
Data from Stolze et al. [1].
EPR spectra of the respective hydroxyl radical adducts
were detected in a Fenton system in the presence of the
investigated spin traps. Typical EPR spectra are shown in
Fig. 3, e.g. from EBPO (Fig. 3a), tBBPO (Fig. 3b),
BEMPO-3 (Fig. 3c) or BEMPO-4 (Fig. 3d). The EPR
spectra of the hydroxyl radical adducts gradually disap-
minant. The contribution of these secondary lines was then
subtracted from each individual EPR spectrum before
calculating the respective half-life. Due to the short
half-life of its superoxide adduct, a system using solid
KO2 (ca. 0.5 mg added to 1 ml of an unbuffered aqueous
solution) had to be used in the experiments with the spin
trap PBPO, immediately followed by addition of SOD
Fig. 4. Iron-dependent formation of spin adducts from methanol and the
spin traps EBPO and BEMPO-3. (a) After a 10-s incubation of EBPO (1 M
in methanol) with FeCl3 (2 mM), the reaction was stopped by 1:20 dilution
with phosphate buffer (0.3 M, pH 7.4, containing 20 mM DTPA), and the
spectrum was recorded with the following spectrometer settings: sweep
width, 60 G; modulation amplitude, 0.7 G; microwave power, 20 mW; time
constant, 0.08 s; receiver gain, 4 ꢂ 105; scan rate, 42.9 G/min. The bars
represent 10,000 arbitrary units. (b) EBPO (40 mM) was incubated with a
Fenton system containing FeSO4 (1 mM), EDTA (2 mM), H2O2 (0.2%) in
10% methanol. The reaction was stopped after 10 s by 1:1 dilution with
phosphate buffer (300 mM, pH 7.4, containing 20 mM DTPA) and the
spectrum was recorded using the following spectrometer settings: sweep
width, 70 G; modulation amplitude, 0.1 G; microwave power, 20 mW; time
constant, 0.08 s; receiver gain, 1 ꢂ 105; scan rate, 50.1 G/min. (c) Same
conditions as in a), except that BEMPO-3 was used. Sweep width, 60 G;
modulation amplitude, 0.7 G; microwave power, 20 mW; time constant,
0.08 s; receiver gain, 8 ꢂ 104; scan rate, 171.8 G/min. (d) Same conditions
as in panel b, except that BEMPO-3 was used. Sweep width, 70 G;
modulation amplitude, 0.1 G; microwave power, 20 mW; time constant,
0.08 s; receiver gain, 1 ꢂ 105; scan rate, 50.1 G/min.
Fig. 3. Formation of hydroxyl radical adducts of the spin traps EBPO,
tBBPO, BEMPO-3, and BEMPO-4. (a) EBPO (20 mM) was incubated with
a Fenton system containing FeSO4 (1 mM), EDTA (2 mM), H2O2 (0.2%).
The reaction was stopped after 10 s by 1:1 dilution with phosphate buffer
(300 mM, pH 7.4, containing 20 mM DTPA) and the spectrum was recorded
using the following spectrometer settings: sweep width, 60.0 G; modulation
amplitude, 0.1 G; microwave power, 20 mW; time constant, 0.08 s; receiver
gain, 2 ꢂ 105; scan rate, 42.9 G/min. The bars represent 10,000 arbitrary
units. (b) Same conditions, except that tBBPO (20 mM) was used. EPR
parameters: sweep width, 60 G; modulation amplitude, 0.1 G; microwave
power, 20 mW; time constant, 0.08 s; receiver gain, 1 ꢂ 105; scan rate,
42.9 G/min. (c) Same conditions, except that BEMPO-3 (20 mM) was used.
EPR parameters: sweep width, 70 G; modulation amplitude, 0.1 G; micro-
wave power, 20 mW; time constant, 0.08 s; receiver gain, 1 ꢂ 105; scan rate,
50.1 G/min. (d) Same conditions, except that BEMPO-4 (20 mM) was used.
EPR parameters: sweep width, 70.0 G; modulation amplitude, 0.1 G; micro-
wave power, 20 mW; time constant, 0.08 s; receiver gain, 1 ꢂ 105; scan rate,
50.1 G/min.

K. Stolze et al. / Biochemical Pharmacology 69 (2005) 297–305
303
Table 5
Comparison of the EPR parameters of different radical adducts of various lipophilic EMPO derivatives
Radical
EMPOa
EBPO
PBPO
iPBPO
BBPO
sBBPO
tBBPO
BEMPO-3
BEMPO-4
HFS (G)
ꢀOOH
aN
aH
(43%) (57%) (37%) (63%) (42%) (58%) (37%) (63%) (37%) (63%) (37%) (63%) (37%) (63%) (63%) (25%) (12%) (60%) (40%)
13.28 13.28 13.20 13.18 13.21 13.21 13.20 13.22 13.22 13.24 13.25 13.20 13.28 13.28 12.95 13.40 13.40 13.45 13.45
9.67 11.89 9.12 11.60 9.35 11.54 9.20 11.80 9.14 11.70 9.20 11.75 9.12 11.79 7.50 14.50 17.00
10.80 7.90
ꢀOH
aN
aH
(50%) (50%) (45%) (55%) (45%) (55%) (45%) (55%) (45%) (55%) (42%) (58%) (38%) (62%) (71%) (29%)
14.00 14.00 14.05 13.93 14.12 13.93 14.07 13.90 14.01 13.86 14.05 13.87 14.10 13.93 13.75 14.30
15.00 12.58 15.89 12.21 16.02 12.21 15.93 12.15 15.80 12.08 15.89 12.09 16.00 12.20 8.70 19.20
(55%) (45%)
13.80 13.80
11.00 8.80
0.90
(0.65) (0.80) (0.65) (0.80) (0.65) (0.80) (0.65) (0.80) (0.65) (0.80) (0.65) (0.80) (0.85)
ꢀH
aN
aH
aH
(100%)
15.52
22.21
20.82
(100%)
15.34
22.94
19.92
(100%)
15.32
22.99
19.88
(100%)
15.37
22.92
19.93
(71%) (29%) (82%) (18%) (100%)
15.35 15.20 15.32 15.22 15.42
22.95 25.20 22.98 25.23 22.96
19.98 17.35 19.93 17.33 20.04
(70%) (30%)
15.20 15.40
16.45 17.45
26.36 24.90
(90%) (10%)
15.40 15.20
17.60 18.45
26.00 23.96
ꢀCH3
aN
aH
(100%)
15.42
(100%)
14.75
(40%) (60%) (36%) (64%) (26%) (74%) (29%) (71%) (18%) (82%) (46%) (42%) (12%) (59%) (41%)
15.20 15.12 15.25 15.25 15.10 15.13 15.21 15.22 15.21 15.32 15.25 15.10 15.35 15.20 15.00
21.10 23.80 24.30 21.65 24.25 21.34 24.10 21.35 24.38 21.38 26.60 15.75 18.40 17.75 19.30
22.30
21.48
ꢀOCH3
aN
aH
(50%) (50%) (56%) (44%) –
–
–
–
(47%) (53%) –
(47%) (53%) (60%) (21%) (19%) (70%) (23%) (7%)
13.50 13.50 13.45 13.80 13.90 13.38 14.71 13.87
13.74 13.74 13.45 13.45
10.87 7.81 10.42 7.62
–
–
13.45 13.45
10.56 7.62
–
–
10.90 8.10 5.70 15.10 17.50
7.33 18.89 17.97 1.45
ꢀCH2OH (100%)
(100%)
14.80
(100%)
14.83
(100%)
14.83
(100%)
14.84
(56%) (44%) (58%) (42%) (56%) (44%)
14.87 14.75 14.82 15.02 14.86 14.66
20.40 22.35 21.45 20.45 24.60 16.80
(100%)
14.76
aN
aH
14.95
21.25
21.44
21.31
21.39
21.28
18.79
ꢀC(LOOH) (62%) (38%) (60%)b
(53%)b
14.70
22.00
(58%)b
14.95
21.90
(45%)b
14.80
21.95
(46%)b
15.00
22.10
(62%)b
15.00
21.50
(30%)b (30%)b
15.00 14.10
24.20 19.20
(100%)
15.00
aN
aH
a
13.45 13.45 15.00
11.45 8.55 21.70
18.80
Data from Stolze et al. [7].
Rest mainly ꢀOH-adduct.
b
peared within about 15–20 min with concomitant forma-
tion of hitherto unidentified secondary products.
several radicals was observed with EBPO (Fig. 5a),
whereas in oxygenated buffer mainly the EBPO hydroxyl
radical adduct was observed (Fig. 5b). A rather complex
mixture of radicals was observed when BEMPO-3 was
used (Fig. 5c). With BEMPO-4 on the other hand, only one
species predominated the spectrum, most probably a lipid-
derived carbon-centered radical adduct (Fig. 5d). The
spectral parameters of lipid-derived adducts of the other
spin traps are listed in Table 5.
Two simple model systems were chosen to discriminate
between the formation of oxygen-centered and carbon-
centered radical adducts from alcohols such as methanol:
Fe3+-catalyzed formation of alkoxyl radical adducts
according to Dikalov and Mason [11], leading to the
short-lived EBPO/ꢀOCH3 adduct (Fig. 4a), and the Fenton
system containing 5% methanol described by Roubaud et
al. [16], giving rise to the more stable EBPO/ꢀCH2OH spin
adduct (Fig. 4b), of which the observed HFS values
(aN = 14.80 G; aH = 21.44 G) are clearly different from
those of the methoxyl radical adduct (aN = 13.45 G;
aH = 10.42 G; 56% and aN = 13.45 G; aH = 7.62 G;
44%). The compounds BEMPO-3 and BEMPO-4 possess
two asymmetric C-centers, therefore the formation of diffe-
rent diastereomers upon radical trapping can be expected.
In Fig. 4c, the EPR spectra of the two stereoisomeric forms
of BEMPO-3/ꢀOCH3 are shown, and the EPR spectrum of
BEMPO-3/ꢀCH2OH can be seen in Fig. 4d. The spectral
contribution of the different isomers was assessed by
computer simulation (see Table 5).
4. Discussion
Eight derivatives of the spin trap EMPO were synthe-
sized in this study, which possess increased lipophilicity
due to a butyl side chain at C-5. All compounds were
comprehensively analytically characterized, and a full
NMR assignment (1H, 13C) was performed. The two
substances with an extra methyl group in position 3
(BEMPO-3) and 4 (BEMPO-4) of the pyrroline ring form
very stable superoxide adducts (t1/2 = ca. 36–47 min), at
least 30 times more stable than the respective adducts
with DMPO (t1/2 = 45 s) [4] and approximately two times
more stable than superoxide adducts of DEPMPO deriva-
tives (t1/2 = ca. 7–15 min [8–10]). This stabilizing effect
is most probably due to steric shielding rather than elec-
tronic influences as has already been mentioned previously
[1].
3.4. Spin trapping of lipid-derived free radicals
We also investigated radicals formed in a Fenton-type
reaction from peroxidized linoleic acid in the presence of
Fe2+ [11,12]. Under anaerobic conditions a mixture of

304
K. Stolze et al. / Biochemical Pharmacology 69 (2005) 297–305
BEMPO-4, which gave the respective methoxyl radical
adducts that rearranged within several minutes into the
respective carbon-centered adducts. Furthermore, in the
experiments performed with peroxidized linoleic acid
carbon-centered radical adducts were also detected (under
anaerobic conditions). Whether originally trapped oxygen-
centered radical adducts rearranged rapidly into the respec-
tive carbon-centered form or whether the originally formed
alkoxyl radicals underwent rapid b-scission (rate constant
ca. 106) prior to radical trapping cannot be decided. A
variety of secondary reactions has been reported leading to
carbon-centered radicals according to different pathways
[17] involving intermediates such as LOOꢀ [18–23] or
LOꢀ. Most probably the oxygen in the primary alkoxyl
radical formed from peroxidized linoleic acid is located
either in position 9 or 13, as shown in the following figure.
Fig. 5. Detection of a lipid-derived radical adduct from peroxidized linoleic
acid using a Fenton-type incubation system in the presence of the spin traps
EBPO, BEMPO-3 or BEMPO-4. (a) To a nitrogen-bubbled solution of
peroxidized linoleic acid (1 mM) and EBPO (40 mM) in phosphate buffer
(10 mM, pH 7.4) FeSO4 (0.2 mM) was added and the spectrum was
recorded with the following spectrometer settings: sweep width, 80.0 G;
modulation amplitude, 1 G; microwave power, 20 mW; time constant,
0.08 s; receiver gain, 2 ꢂ 105; scan rate, 57.2 G/min, 6 scans accumulated.
The bar represents 10,000 arbitrary units. (b) Same conditions as in panel a,
except that the solution was oxygen bubbled. Sweep width, 80.0 G;
modulation amplitude, 1 G; microwave power, 20 mW; time constant,
0.08 s; receiver gain, 1 ꢂ 105; scan rate, 57.2 G/min, 6 scans accumulated.
The bar represents 10,000 arbitrary units. (c) Same conditions as in panel a,
except that BEMPO-3 (20 mM) was used. Sweep width, 100.0 G; modula-
tion amplitude, 1 G; microwave power, 20 mW; time constant, 0.16 s;
receiver gain, 5 ꢂ 105; scan rate, 71.5 G/min, 20 Scans accumulated.
The bar represents 10,000 arbitrary units. (d) Same conditions as in panel
a, except that BEMPO-4 (20 mM) was used. Sweep width, 70.0 G; mod-
ulation amplitude, 0.1 G; microwave power, 20 mW; time constant, 0.08 s;
receiver gain, 2.5 ꢂ 104; scan rate, 50.1 G/min. The bar represents 1000
arbitrary units.
In conclusion, the two ring-methylated EMPO deriva-
tives BEMPO-3 and BEMPO-4 can be recommended for
trapping of superoxide radicals, since the half-life of their
superoxide adducts is about 36–47 min. Moderately stable
hydroxyl radical adducts were detected from all spin traps
investigated, but the stability of alkoxyl radical adducts,
such as the methoxyl radical adduct, was rather low, and
most of these adducts rearranged into the more stable
hydroxymethyl radical adduct.
From these results it can be concluded that minor
changes of the geometry around the attack site of the
radical have a profound influence on the spin adduct
stability as well as the EPR spectral shape. More detailed
studies covering compounds bearing substituents in posi-
tions 3 or 4 of the pyrroline moiety will therefore be
conducted in the near future.
Determination of the half-lives of the superoxide adducts
was done using a first-order exponential decay approxima-
tion (Pearson correlation coefficient r2 > 0.99, except for
EBPO (0.97) and PBPO (0.96)). Under the experimental
conditions used a second order decay of the superoxide
adducts was insignificant. An important issue 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, the total number of lines, and additional factors, such
as enzyme binding or the solubility (aggregate formation) of
the spin trap, reduction by antioxidants or buffer constitu-
ents, and degradation by transition metal catalyzed reactions
(Fenton-type reactions, oxidation by Fe3+, etc.). In view of
these facts the half-life values given in Table 4 are therefore
to be interpreted only as preliminary, and further tests will be
necessary to test these compounds in different biological
systems.
Acknowledgements
The authors wish to thank R. Stadtmu¨ller for skilful
technical assistance in synthesis, purification, and charac-
terization of the spin traps. The present study was supported
¨
by the Austrian Fonds zur Forderung der wissenschaftli-
chen Forschung.
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