Spin adducts of several N-2-(2-alkoxycarbonyl-propyl)-α- pyridylnitrone derivatives with superoxide, alkyl and lipid-derived radicals

Article abstract of DOI:10.1016/j.bcp.2004.03.017

Several derivatives of N-t-butyl-α-phenylnitrone (PBN) such as N-2-(2-ethoxycarbonyl-propyl)-α-phenylnitrone (EPPN) have recently been reported to form superoxide spin adducts (t_1/2 ca. 2-7 min at pH 7.0), which are considerably more stable than their respective PBN or DMPO adducts (t_1/2 ca. 10 and 45 s, respectively). In continuation of our studies on structure optimization of EPPN derivatives, a series of 12 novel spin traps with 2-, 3- and 4-pyridinyl substituents was synthesized and fully characterized by ¹H NMR, ¹³C NMR and IR spectroscopy. In addition to the replacement of the phenyl ring by a 2-, 3- or 4-pyridinyl substituent, the ethoxy group of the parent compound EPPN was replaced by either a propoxy, iso-propoxy, or cyclopropylmethoxy moiety. Superoxide adducts of all PPyN derivatives were considerably more stable than those of the respective EPPN derivatives with half-lives ranging from about 6 to 11 min. In addition, alkoxyl radical adducts were also considerably more stable than those of the EPPN series. Hydroxyl radical adducts were not detected, on the other hand, very stable spin adducts were formed from a series of carbon centered radicals, e.g. from the methyl or hydroxymethyl radical. The novel spin traps are offering an alternative to PBN or POBN, especially where the higher stability of oxygen-centered radical adducts is of major importance. All of them can easily be synthesized from commercially available compounds in two or three steps.

Full text of DOI:10.1016/j.bcp.2004.03.017

Biochemical Pharmacology 68 (2004) 185–193
Spin adducts of several N-2-(2-alkoxycarbonyl-propyl)-a-pyridylnitrone
derivatives with superoxide, alkyl and lipid-derived radicals
a
Klaus Stolze , Natascha Udilova , Thomas Rosenau , Andreas Hofinger , Hans Nohl
a
b
b
a,*
a
Research Institute for Pharmacology and Toxicology, University of Veterinary Medicine Vienna, Veterin a¨ rplatz 1, A-1210 Vienna, Austria
b
Institute of Chemistry, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria
Received 22 December 2003; accepted 16 March 2004
Abstract
Several derivatives of N-t-butyl-a-phenylnitrone (PBN) such as N-2-(2-ethoxycarbonyl-propyl)-a-phenylnitrone (EPPN) have recently
been reported to form superoxide spin adducts (t1/2 ca. 2–7 min at pH 7.0), which are considerably more stable than their respective PBN
or DMPO adducts (t1/2 ca. 10 and 45 s, respectively). In continuation of our studies on structure optimization of EPPN derivatives, a series
of 12 novel spin traps with 2-, 3- and 4-pyridinyl substituents was synthesized and fully characterized by H NMR, 13C NMR and IR
spectroscopy. In addition to the replacement of the phenyl ring by a 2-, 3- or 4-pyridinyl substituent, the ethoxy group of the parent
compound EPPN was replaced by either a propoxy, iso-propoxy, or cyclopropylmethoxy moiety. Superoxide adducts of all PPyN
derivatives were considerably more stable than those of the respective EPPN derivatives with half-lives ranging from about 6 to 11 min. In
addition, alkoxyl radical adducts were also considerably more stable than those of the EPPN series. Hydroxyl radical adducts were not
detected, on the other hand, very stable spin adducts were formed from a series of carbon centered radicals, e.g. from the methyl or
hydroxymethyl radical. The novel spin traps are offering an alternative to PBN or POBN, especially where the higher stability of oxygen-
centered radical adducts is of major importance. All of them can easily be synthesized from commercially available compounds in two or
three steps.
1
#
2004 Elsevier Inc. All rights reserved.
Keywords: Spin traps; EPR; PPyN derivatives; Superoxide; Linoleic acid hydroperoxide; Free radicals
1
. Introduction
[3]), the half-lives of the superoxide spin adducts of EPPN
derivatives (t1=2 ¼ 2–7 min [1,2]) are similar to the recently
The synthesis of N-2-(2-ethoxycarbonyl-propyl)-a-phe-
reported values for the spin traps EMPO (t1 ¼ 8:6 min
=2
nylnitrone (EPPN) and its derivatives has recently been
described by several authors [1,2]. When compared to the
structurally related spin traps PBN or POBN (t1=2 ! 1 min
[4–7]) or Trazon (t1=2 ¼ 3:6 min [8,9]), but lower than the
respective number for DEPMPO (t1 ¼ 14:8 min [10,11]).
=2
Since the latter compound bears strongly electron-with-
drawing groups, it was expected that the incorporation
of an additional electron acceptor substituent into the
molecule of EPPN derivatives might lead to an increased
stability of the superoxide adducts. Furthermore, the pos-
sibility to obtain a series of compounds with different
lipophilicity will facilitate the investigation of radicals in
lipid membranes. In this respect, the detection of free
radicals from lipid peroxidation has already been investi-
gated using different spin traps, such as DMPO [12],
DEPMPO [11,13], EMPO [6] or Trazon [9], but an optimal
spin trap for the detection of alkoxyl radicals has not yet
been found.
Abbreviations: DEPMPO, 5-(diethoxyphosphoryl)-5-methyl-1-pyrro-
line N-oxide; DMPO, 5,5-dimethylpyrroline N-oxide; DTPA, diethylene-
triaminepentaacetic acid; EMPO, 5-(ethoxycarbonyl)-5-methyl-1-
pyrroline N-oxide; EPPN, N-2-(2-ethoxycarbonyl-propyl)-a-phenylni-
trone; EPPyN-2, N-2-(2-ethoxycarbonyl-propyl)-a-(2-pyridinyl)nitrone;
EPPyN-3, N-2-(2-ethoxycarbonyl-propyl)-a-(3-pyridinyl)nitrone; EPPyN-
4, N-2-(2-ethoxycarbonyl-propyl)-a-(4-pyridinyl)nitrone; EPR, electron
ꢀ
paramagnetic resonance; HFS, hyperfine splitting; LO , lipoxyl radical;
À
ꢀ
NMR, nuclear magnetic resonance; O
2
, superoxide anion radical; PBN,
N-tert-butyl-a-phenylnitrone; POBN, a-(4-pyridinyl-1-oxide)-N-tert-bu-
tylnitrone; SOD, superoxide dismutase; Trazon, 1,3,3-trimethyl-6-azabi-
cyclo[3.2.1]oct-6-ene-N-oxide
*
Corresponding author. Tel.: þ43-1-25077-4400;
It was thus the aim of the present study to evaluate the
effect of the introduction of a pyridinyl ring (2-, 3- and
fax: þ43-1-25077-4490.
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.2004.03.017

186
K. Stolze et al. / Biochemical Pharmacology 68 (2004) 185–193
4
-pyridinyl derivatives) as well as the alteration of the
2.2.2. Alkyl 2-methyl-2-nitropropionate
alkoxy group in the ester moiety of the PPyN derivatives on
the stability of the radical adducts, especially with regard to
the stability of oxygen-centered radicals, such as super-
oxide, hydroxyl or alkoxyl radicals.
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 N,N-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 Na SO . After
2
. Materials and methods
2
.1. Chemicals
2
4
removal of the solids by filtration, the solvent was evapo-
rated in vacuo. The obtained colorless or pale yellow
products were used further without purification.
2-Bromo-2-methylpropionyl bromide, linoleic acid,
superoxide dismutase and xanthine oxidase were commer-
cially available from Sigma–Aldrich. Petroleum ether
(
high boiling, 50–70 8C) was obtained from Fluka, all
2.2.3. Synthesis of nitrones
To a concentrated solution of the respective alkyl
other chemicals from Merck.
2-methyl-2-nitropropionate (25 mmol) in H O/CH OH
2 3
2
.2. Syntheses
(v=v ¼ 6:4) the respective aldehyde (30 mmol of either
2
-, 3-, 4-pyridine carbaldehyde or benzaldehyde) and
Synthesis and characterization of the compounds were
aqueous ammonium chloride solution (1.87 g in 8 ml of
water) were added. The mixture was carefully kept at room
temperature, while 3.27 g (50 mmol) of zinc dust was
slowly added within 30 min. After stirring for 4.5 h at
room temperature, the white precipitate and the remaining
zinc powder was removed by filtration, and the solid
residue was washed five times with 30 ml of methanol.
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 Na SO , filtered, and concen-
performed as reported earlier [2], analogous to the syn-
thesis of EMPO and its derivatives [4–7] with minor
adaptations as given below.
2
.2.1. Alkyl 2-bromo-2-methylpropionate
-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
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 sodium bicarbo-
nate (50 ml), and dried over Na SO overnight. Solvent
2
(
0
2
4
(
trated. Column chromatography on silica gel (petroleum
ether/ethanol (1–5%) with gradient elution) afforded
20–30% (overall yield) of a light-brown product, which
2
4
and excess alcohol were removed under reduced pressure.
The crude, nearly colorless product was used without
further purification.
1
was recrystallized from pentane and characterized by H
NMR, 13C NMR, and IR spectroscopy (see Tables 1–3).
Table 1
13C NMR data (ppm) of the spin traps
0
0
00
00
00
3
1Ar
2Ar
3Ar
4Ar
5Ar
6Ar
CH
1
C
2
C
COO
1
C
2
C
C
EPPyN2
EPPyN3
EPPyN4
PPPyN2
PPPyN3
PPPyN4
iPPPyN2
iPPPyN3
iPPPyN4
CPPyN2
CPPyN3
CPPyN4
CPPN
(N)
(N)
(N)
(N)
(N)
(N)
(N)
(N)
(N)
(N)
(N)
(N)
130.4
130.4
149.87
150.6
149.7
149.47
150.6
150.8
149.57
150.0
149.4
149.45
150.0
150.3
128.4
129.1
124.8
127.3
121.8
124.3
127.4
122.1
124.3
126.9
120.7
124.4
127.0
121.6
128.9
128.6
137.3
135.1
137.3
136.9
135.3
137.2
136.8
134.8
135.9
136.8
134.9
136.8
130.5
130.7
124.4
123.9
121.8
123.9
123.9
122.1
123.9
123.4
120.7
124.0
123.5
121.6
128.9
128.6
149.92
151.1
149.7
149.42
151.0
150.8
149.41
150.5
149.4
149.38
150.5
150.3
128.4
129.1
133.1
128.7
129.0
132.6
128.7
129.6
132.6
128.2
128.2
132.7
128.4
129.3
131.2
131.3
78.2
77.9
78.3
77.8
78.0
78.7
77.7
77.4
77.2
77.8
77.6
78.3
77.1
77.1
24.8
24.8
24.4
24.4
24.8
24.8
24.3
24.3
23.6
24.4
24.4
24.4
24.5
24.5
170.5
170.5
169.9
170.1
170.6
170.4
169.6
169.6
169.0
170.2
170.3
170.1
170.6
170.6
62.5
62.6
62.3
67.5
68.1
68.2
69.8
69.8
69.1
70.7
70.8
70.8
70.6
62.1
14.3
14.3
13.9
21.7
22.2
22.2
21.1
21.4
20.4
9.5
–
–
–
10.1
10.6
10.6
–
–
–
3.1
3.1
3.1
3.1
–
9.5
9.5
9.6
EPPN
14.0

K. Stolze et al. / Biochemical Pharmacology 68 (2004) 185–193
187
Table 2
1H NMR data (ppm) of the spin traps
0
00
00
3
1ArH
2ArH
3ArH
4ArH
5ArH
6ArH
HC¼N
2
CH3
OCH
2
CHx
CHx
x
EPPyN2
EPPyN3
EPPyN4
PPPyN2
PPPyN3
PPPyN4
iPPPyN2
iPPPyN3
iPPPyN4
CPPyN2
CPPyN3
CPPyN4
CPPN
(N)
(N)
(N)
(N)
(N)
(N)
(N)
(N)
(N)
(N)
(N)
(N)
–
–
8.61d
–
7.28m
8.59d
–
7.76td
7.36t
9.15d
9.06d
8.69d
9.15d
9.05d
8.62d
9.16d
9.03d
8.68d
9.18d
9.08d
8.69d
8.27m
8.28m
7.82s
7.58s
7.55s
7.86s
7.55s
7.51s
7.82s
7.58s
7.52s
7.86s
7.56s
7.53s
7.48s
7.49s
1.84s
1.85s
1.84s
1.86s
1.81s
1.81s
1.82s
1.85s
1.84s
1.86s
1.85s
1.85s
1.84s
1.82s
4.25q
4.26q
4.26q
4.18t
1.26t
–
–
–
8.97s
8.69d
–
1.30t
8.09d
8.62d
–
8.09d
7.80td
7.35t
1.28t
7.29m
8.59d
–
1.68m
1.67m
1.68m
1.26d
1.23d
1.26d
1.14m
1.15m
1.14m
1.15m
1.27t
0.95t
0.92t
0.90t
–
8.98s
8.62d
–
4.16t
8.05d
8.63d
–
8.05d
7.78td
7.34t
4.14q
5.09spt
5.08spt
5.09spt
4.05d
4.26d
4.05d
4.05d
4.25q
7.29m
8.57d
–
8.97s
8.68d
–
–
8.04d
8.65d
–
8.04d
7.79td
7.38t
–
7.30m
8.61d
–
0.53m/0.29m
0.55m/0.30m
0.55m/0.29m
0.52m/0.27m
8.99s
8.69d
8.27m
8.28m
8.05d
7.42m
7.42m
8.05d
7.42m
7.42m
7.42m
7.42m
EPPN
–
s, singlet; d, doublet; t, triplet; q, quadruplet; spt, septet; m, multiplet; td, triplet of doublets.
The purity of the products was assessed by TLC and UV
spectroscopy.
HMBC spectra was recorded for each compound. All
chemical shift data are given in ppm units.
2
.2.4. Preparation of lipid hydroperoxides
Linoleic acid hydroperoxide was synthesized according
3. Results
to O’Brien [14]. Briefly, linoleic acid was air-oxidized for
2 h in the dark at room temperature. The oxidation
mixture was dissolved in petroleum ether (boiling range
0–90 8C) and extracted four times with water/methanol
v/v ¼ 1:3). The obtained methanolic phase was counter-
extracted four times with petroleum ether (boiling range
0–90 8C) and evaporated under reduced pressure. The
7
3.1. Structure of the spin traps
6
(
The identity of the synthesized spin traps was unam-
1
biguously proven by NMR. In the H NMR spectra, the
typical pattern of differently substituted pyridines was
produced. In general, the electron-deficient character of
the aromatic pyridine system became evident by a sig-
nificant down-field shift of the resonances: in comparison
to benzene as the standard aromatic system (7.28 ppm) the
pyridine resonances appear between 7.3 and 9.2 ppm
according to the respective substitution pattern. The signals
with the largest downfield shift originate from the protons 2
and 6 neighboring the electronegative pyridine nitrogen.
The 2-substituted aromatic gave a typical higher-order
pattern consisting of two duplets (H-3 and H-6) and two
multiplets (H-4 and H-5). The 3-substituted pyridine ring
showed a prominent singlet for H-2 and two duplets and a
triplet for H-4, H-6, and H-5, respectively. In the 4-sub-
stituted derivatives with their additional mirror symmetry,
H-2/H-6 and H-3/H-5 become magnetically equivalent
resulting in two duplets of double intensity.
The benzylidenic proton appeared throughout as a sing-
let without long-range couplings. Also here, the influence
of the electronegative nitrogen with its electron-acceptor
properties was noticeable. Increasing proximity of the
pyridine nitrogen causes a down-field shift: the resonances
of the PPyN-2 derivatives appear at about 7.82 ppm, those
of the PPyN-3 species at about 7.58 ppm, and those of the
PPyN-4 compounds at 7.52 ppm.
6
obtained hydroperoxide was dissolved in ethanol and stored
in liquid nitrogen. The concentration of hydroperoxide was
determined by UC spectroscopy based on an extinction
coefficient of e233nm ¼ 25,250 MÀ1 cm in ethanol [14].
À1
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
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 EPR experiments the Bruker spectrometer ESP300E
was used, operating at 9.7 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. CDCl contain-
3
ing tetramethylsilane (TMS) as the internal standard was
used as the solvent throughout. 13C peaks were assigned by
1
means of attached proton test (APT), H-detected hetero-
nuclear multiple-quantum coherence (HMQC) and hetero-
nuclear multiple bond connectivity (HMBC) spectra. A
complete set of H, H–H correlated, 13C, HMQC and
The resonance of the two magnetically equivalent
2 -methyl groups is found at about 1.85 ppm throughout.
In general, the influence of the alkoxy moiety on the NMR
0
1

188
K. Stolze et al. / Biochemical Pharmacology 68 (2004) 185–193
shifts of the acid part is generally rather low, so that—as
intended—the variation of the alkoxy part did indeed influ-
ence solubility and lipophilicity properties, but did not alter
the stability of the spin adducts. The cyclopropyl protons of
the cyclopropylmethoxy moiety showed the expected
upfield shift with resonances at 1.15, 0.50, and 0.30 ppm.
The 13C NMR data were very consistent. 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 [15]. With
an expected resonance at about 135–140 ppm, the chemi-
cal shift of the benzylidenic carbon at 128 ppm is indica-
tive of its Z-arrangement with regard to the oxygen. Thus,
only the isomers with the C=N double bond in E-config-
uration were formed in the synthesis, so that the sterically
demanding substituents, i.e. the pyridine ring and the alkyl
13
ester moiety, are placed trans. Also in the C NMR spectra
the electron-deficient nature of the pyridine moiety was
reflected. While the aromatic resonances in EPPN deriva-
tives were found between 128 and 130 ppm, the resonances
of the pyridine rings in the different PPyN derivatives
experienced significant changes: a very strong downfield
shift for C-2 and C-6, which neighbor the nitrogen
(
150 ppm), a weak downfield shift for C-3 and C-5 at
about 124 ppm and an upfield shift for C-4 at about
34 ppm. The nitrone substituent causes a slight downfield
1
shift in ipso position by 2–3 ppm and a very weak up-field
effect at the neighboring carbon atoms (<1 ppm).
In analogy to the H spectra, also the 13C results proved
that changes in the alkoxy part of the ester moieties
remained without significant influences on the resonances
1
0
of the nitrone. C-1 appeared generally at about 78 ppm,
0
C-2 at 24.5 ppm and the carboxyl carbon at 169–170 ppm.
The respective NMR data are summarized in Tables 1 and 2.
3.2. Spin trapping of superoxide radicals
In Fig. 1 thegeneral structure of the spin traps is shown. In
Fig. 2a, the EPR spectrum of the superoxide adduct of
iPPPyN-2 (20 mM) is given as an example. The adduct
wasgeneratedinthexanthine/xanthineoxidasesystematpH
7.4. No EPR spectrum was observed in the presence of SOD
(Fig. 2b). Under these conditions similar spectra were
obtained from EPPyN-4 in the absence (Fig. 2c) or presence
of SOD (Fig. 2d). From CPPN (Fig. 2e) however, an
intensive and well-resolved spectrum could only obtained
using an incubation system with solid KO . This system was
2
also used in order to determine the half-life of superoxide
adducts: the respective spin traps (20 mM, final concentra-
tion) were incubated in water/DMSO in the presence of ca.
0.5 mg solid KO for 15 s, after which phosphate buffer
2
(
(
300 mM, final pH 7.0) containing 20 mM DTPA, SOD
100 U/mL), catalase (250 U/mL) and 5% DMSO was
added. A series of consecutive spectra was recorded until
the superoxide-related lines gradually disappeared (after
about 15–20 min) and additional lines of lower intensity,

K. Stolze et al. / Biochemical Pharmacology 68 (2004) 185–193
189
CH
3
O
O
+
R
1
CH=N
C
C
(a)
(b)
(c )
-
R
2
O
CH
3
+
SOD
EPPyN2:
EPPyN3:
EPPyN4:
PPPyN2:
PPPyN3:
PPPyN4:
iPPPyN2:
iPPPyN3:
iPPPyN4:
CPPyN2:
CPPyN3:
CPPyN4:
CPPN:
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
= 2-Pyridinyl
= C
= 3-Pyridinyl
= C
= 4-Pyridinyl
= C
= 2-Pyridinyl
= C
= 3-Pyridinyl
= C
= 4-Pyridinyl
= C
= 2-Pyridinyl
= iso-C
= 3-Pyridinyl
= iso-C
= 4-Pyridinyl
= iso-C
= 2-Pyridinyl
= cyclo-C
= 3-Pyridinyl
= cyclo-C
= 4-Pyridinyl
2
H
5
2
H
5
2
H
5
+
SOD
3
H
7
(
d)
e)
3
H
7
3
H
7
(
3
H
7
3
H
7
20 G
3
H
7
Fig. 2. Formation of the superoxide adducts of the spin traps iPPPyN-2,
EPPyN-4 and CPPN. (a) iPPPyN-2 (20 mM), catalase (250 U/mL),
xanthine (0.2 mM) and xanthine oxidase (50 mU/mL) in oxygenated
phosphate buffered saline (20 mM, pH 7.4, containing 0.4 mM DTPA and
3
H
5
-CH
-CH
-CH
-CH
2
2
2
2
5
% DMSO) were incubated and measured using the following EPR
3
H
5
parameters: sweep width, 50 G; modulation amplitude, 1 G; microwave
5
power, 20 mW; time constant, 0.16 s; receiver gain, 2 ꢁ 10 ; scan rate,
= cyclo-C
= Phenyl
= cyclo-C
3
H
5
35.8 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
EPPyN-4 (20 mM) was used. EPR parameters: sweep width, 50 G;
3
H
5
modulation amplitude, 1 G; microwave power, 20 mW; time constant,
5
0
.16 s; receiver gain, 2 ꢁ 10 ; scan rate, 35.8 G/min. (d) Same as in (c),
Fig. 1. General structure of the spin traps.
except that SOD (100 U/ml) was added. (e) CPPN (20 mM in H
2
O
containing 10% DMSO) was incubated for 10 s with 0.5 mg solid KO and
2
coming from a carbon-centered degradation product,
became predominant. The contribution of these secondary
lines was then subtracted from each individual EPR spec-
trum before calculating the respective half-life. The result-
ingintensitydecreaseofthefirsttwolineswasapproximated
by a first-order exponential decay. This kind of approxima-
tion was in a good agreement with the experimental data.
The Pearson correlation coefficient, which characterizes the
certainty of approximation, was higher than 0.99 for all spin
traps investigated, except for CPPN and CPPyN-2, where
thesevalues were 0.97 and 0.98, respectively. The respective
values of apparent superoxide half-lives are listed in Table 4.
The obtainable spectral intensity (measured after 5 min
incubation with the xanthine/xanthine oxidase system)
was higher compared with DMPO, but lower than with
DEPMPO, decreasing from the hydrophilic to the lipophilic
compounds due to increasing steric hindrance.
then diluted 1:1 with phosphate buffer (300 mM, pH 7, containing 20 mM
DTPA). EPR parameters: sweep width, 50 G; modulation amplitude, 1 G;
5
microwave power, 20 mW; time constant, 0.04 s; receiver gain, 2 ꢁ 10 ;
scan rate, 143.1 G/min.
Table 4
Half-life of the superoxide adducts and n-octanol/buffer partition
coefficients of the spin traps
Compound
Apparent
Partition coefficient
n-octanol/phosphate
buffer (100 mM, pH 7.4)
t1/2 (min)
EPPyN-2
EPPyN-3
EPPyN-4
PPPyN-2
PPPyN-3
PPPyN-4
iPPPyN-2
iPPPyN-3
iPPPyN-4
CPPyN-2
CPPyN-3
CPPyN-4
CPPN
5.79
7.2
2.4
10.68
7.28
4.61
10.07
6.14
7.54
9.60
6.49
8.50
10.20
6.40
4.13
a
a
3.7
23.2
7.9
12.7
25.1
5.5
8.2
3.3. Spin adduct formation with oxygen-containing
radicals
19.4
10.0
11.9
72.6
No hydroxyl radical adducts were detected in a Fenton
system in the presence of any of the investigated spin traps.
a
Data from Stolze et al. [2].

190
K. Stolze et al. / Biochemical Pharmacology 68 (2004) 185–193
(a)
(a)
(b)
(b)
(c)
(c)
20 G
(d)
Fig. 3. Iron-dependent formation of two different EPPyN-4 spin adducts
from methanol (a) EPPyN-4 (20 mM) was incubated with a Fenton system
containing 10% methanol, FeSO
4
(1 mM), EDTA (2 mM), H
2
O
2
(0.2%)
20 G
and 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 G;
Fig. 4. Iron-dependent formation of spin adducts from n-octanol and the
spin traps iPPPyN-2 and iPPPyN-3. (a) After a 10 s incubation of iPPPyN-
modulation amplitude, 0.24 G; microwave power, 20 mW; time constant,
4
2
3
(1 M in n-octanol) with FeCl (10 mM), the reaction was stopped by 1:20
0
.08 s; receiver gain, 1 ꢁ 10 ; scan rate, 42.9 G/min. The bars represent
dilution with phosphate buffer (0.15 M, pH 7.4, containing 10 mM DTPA),
and the spectrum was recorded with the following spectrometer settings:
1
3
1
0,000 arbitrary units. (b) Same conditions, except that methanol- C-d
3
was used. (c) After a 10 s incubation of EPPyN-4 (1 M in methanol) with
FeCl (10 mM), the reaction was stopped by 1:20 dilution with phosphate
sweep width, 50 G; modulation amplitude, 1 G; microwave power,
5
3
2
0 mW; time constant, 0.33 s; receiver gain, 4 ꢁ 10 ; scan rate, 35.8 G/
buffer (0.15 M, pH 7.4, containing 10 mM DTPA), and the spectrum was
recorded with the following spectrometer settings: sweep width, 60 G;
min. The bars represent 10,000 arbitrary units. (b) Same conditions, except
that iPPPyN-3 was used. (c) Same as (a), secondary product formed after
modulation amplitude, 0.68 G; microwave power, 20 mW; time constant,
4
4
2 min. (d) Same as (b), secondary product formed after 33 min.
0.08 s; receiver gain, 1 ꢁ 10 ; scan rate, 42.9 G/min.
However, within a few minutes the EPR spectrum of a
secondary product gradually appeared, with parameters
being typical of a carbon-centered radical adduct (Table 5,
spectrum not shown). After prolonged incubation, other
secondary species, such as the respective (2-alkoxy-carbo-
nyl-propyl)-2-aminoxyl [1,2] were also detected.
In the presence of methanol and iron, two different
radical adducts were found. A Fenton system in the pre-
sence of 5% methanol according to Roubaud et al. [1]
resulted in the formation of the respective hydroxymethyl
13C-labeled methanol did not result in an additional split-
ting. The data of radical adducts obtained from the other
spin traps under study are listed in Table 5.
This set of experiments was repeated with higher alco-
hols such as ethanol or n-octanol. With increasing chain
length the formation of the respective oxygen centered
radical adducts became more and more difficult. The n-
octyloxyl adduct was only obtained as a rather short-lived
species from the spin traps iPPPyN-2 (Fig. 4a) and iPP-
PyN-3 (Fig. 4b). In both cases, the original EPR spectra
faded after several minutes, and signals of secondary
carbon-centered radical adducts from iPPPyN-2 and iPP-
PyN-3 appeared (Fig. 4c and d, respectively). The asym-
metric lines observed with adducts from asymmetric
radicals (e.g. from the a-hydroxyethyl radical) are most
probably due to the formation of different stereoisomers
(diastereomers and conformers) with HSF splitting differ-
ences being comparable to the line width. In those cases
where the line asymmetry was big enough, the spectral
contribution of the two individual stereoisomers was
assessed by computer simulation (see Table 5).
radical adduct, as shown in Fig. 3a for the EPPyN-4/
ꢀ
CH OH species. The identification as a carbon-centered
2
radical adduct was possible by using 13C-labeled methanol
(
Fig. 3b), where an additional carbon splitting became
visible.
In Fig. 3c the EPR spectrum of the methoxyl radical
adduct of EPPyN-4 is shown, which was obtained after
nucleophilic addition of methanol to EPPyN-4 in the
3
þ
presence of Fe
under the experimental conditions
reported by Dikalov and Mason [12], except that the
incubation time was reduced to 10 s in order to suppress
the formation of secondary products. The observed HFS
N
H
values (a ¼ 14:05 G; a ¼ 3:03 G) are clearly different
from those of the hydroxymethyl radical adduct obtained in
the above-mentioned Fenton system containing 5% metha-
3.4. Spin trapping of lipid-derived free radicals
We also investigated the detection of radicals derived
from linoleic acid by lipid peroxidation-type processes. An
N
H
nol (a ¼ 14:92 G; a ¼ 3:09 G). In addition, the use of

Table 5
Comparison of the EPR parameters of different radical adducts of various PPyN derivatives
a
a
a
Radical HFS (G)
EPPyN-2
EPPyN-3
EPPyN-4
PPPyN-2
PPPyN-3
PPPyN-4
iPPPyN-2
iPPPyN-3
iPPPyN-4
CPPyN-2
CPPyN-3
CPPyN-4
CPPN
ꢀ
N
H
b
OOH
a
13.85
2.03
13.85
1.68
13.67
13.83
2.07
13.85
1.65
13.69
1.66
13.77
2.05
13.83
1.67
13.67
1.64
13.81
2.12
13.82
1.67
13.67
1.67
14.16
2.45
a
1.60
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
N
H
b
C( OH)
a
a
15.05
3.51
15.26
2.81
14.67
3.43
15.04
3.51
15.23
2.83
15.03
3.08
15.00
3.52
15.17
2.77
15.02
2.92
15.06
3.59
15.30
2.98
15.05
3.15
15.44
3.84
N
b
H
a
a
15.78
10.30
15.90
10.56
15.65
15.75
10.28
15.90
10.56
15.60
9.93
15.72
10.33
15.86
10.60
15.63
9.99
15.73
10.33
15.89
10.73
15.64
10.04
16.05
10.60
H(2)
9.99
N
H
b
CH
3
a
a
15.41
3.91
15.53
2.95
14.66
15.39
3.99
15.51
3.00
15.35
3.27
15.35
3.88
15.50
2.98
15.33
3.20
15.38
3.90
15.52
2.95
15.34
3.20
15.69
3.92
3.43
N
H
b
OCH
3
a
a
14.30
3.60
14.31
3.20
14.05
3.03
14.34
3.61
14.30
3.15
14.16
3.23
14.20
3.58
14.27
3.16
14.12
3.16
14.25
3.80
14.26
3.35
14.10
3.17
14.56
4.33
N
H
b
CH
2
2
OH
a
a
14.98
3.58
15.11
2.83
14.92
14.95
3.59
15.10
2.83
14.94
3.08
14.87
3.52
15.04
2.81
14.89
3.06
14.93
3.57
15.07
2.85
14.90
3.09
15.26
3.86
3.09
N
H
OC
H
5
a
a
14.39
4.01
14.42
3.60
14.27
3.56
14.40
3.96
14.40
3.60
14.30
3.60
14.29
3.91
14.34
3.53
14.20
3.43
14.30
4.10
14.36
3.80
14.24
3.72
14.64
4.62
N
H
H
CH(OH)CH
3
a
a
a
15.08
15.21
14.99
15.04
15.17
15.01
14.96
15.13
14.97
15.03
15.16
14.99
15.38
3.81 (47%) 3.03 (50%) 3.08 (50%) 3.86 (47%) 3.03 (50%) 3.20 (50%) 3.85 (47%) 3.00 (50%) 3.17 (50%) 4.00 (47%) 3.10 (50%) 3.28 (50%) 3.71 (50%)
2.31 (53%) 2.01 (50%) 2.19 (50%) 2.36 (53%) 2.03 (50%) 2.20 (50%) 2.39 (53%) 2.00 (50%) 2.17 (50%) 2.52 (53%) 2.10 (50%) 2.28 (50%) 3.01 (50%)
ꢀ
ꢀ
N
H
OC
8
H
17
a
a
–
–
–
–
–
–
–
–
–
–
–
–
13.75
3.75
13.75
4.40
–
–
–
–
–
–
–
–
–
–
N
H
H
C(CH
3
)
2
OH
a
a
a
15.05 15.00
3.96 3.33
2.36 (50%) 2.04 (50%) 2.13 (50%) 2.28 (50%) 2.00 (50%) 3.17 (50%) 2.33 (50%) 2.03 (50%) 3.19 (50%) 2.46 (50%) 2.11 (50%) 2.23 (50%) 4.05 (50%)
15.19
3.14
14.97
3.13
15.04
3.92
15.17
3.16
15.01
2.17
14.95
3.93
15.11
3.13
14.98
2.19
15.02
4.06
15.15
3.21
15.37
3.05
ꢀ
ꢀ
2À
N
H
b
CO
a
a
15.01
3.30
15.08
3.64
14.95
3.49
14.98
3.31
15.05
3.65
14.90
3.44
14.92
3.23
15.04
3.66
14.89
3.45
14.97
3.33
15.04
3.82
14.90
3.54
15.16
4.62
N
H
N
H
C(LOOH)
a
a
a
a
14.81
14.94
15.02
14.86
15.17
14.73
14.60
14.88
14.71
14.66
15.03
14.74
15.48
3.18 (60%) 2.60 (40%) 2.75 (40%) 3.23 (60%) 2.49 (40%) 2.82 (40%) 3.11 (40%) 2.52 (40%) 2.78 (40%) 3.23 (40%) 2.63 (40%) 2.87 (40%) 2.95 (50%)
15.62 15.50 15.42 15.66 15.55 15.39 15.30 15.44 15.31 15.36 15.41 15.30 15.48
3.11 (40%) 2.54 (60%) 2.67 (60%) 3.15 (40%) 2.43 (60%) 2.74 (60%) 3.19 (60%) 2.52 (60%) 2.74 (60%) 3.15 (60%) 2.57 (60%) 2.79 (60%) 3.95 (50%)
N
H
Best fit for
a
a
15.13
3.15
15.28
2.56
15.26
2.69
15.18
3.20
15.40
2.45
15.13
2.77
15.02
3.16
15.22
2.52
15.07
2.76
15.08
3.18
15.26
2.59
15.08
2.82
15.48
3.45
single species
a
Slow degradation (loss of isopropyl group).
Data from Stolze et al. [2].
b

192
K. Stolze et al. / Biochemical Pharmacology 68 (2004) 185–193
single species (assuming distorted lines and a high line
width) is also given below in Table 5.
(a)
On the other hand, a contribution of spin adducts formed
with the primary alkoxyl radical can be excluded since
practically no change was observed even when concen-
trated solutions of the spin traps (around 100–150 mM)
were used. In analogy to the EPPN series [2], also the PPyN
derived spin traps form lipid radical adducts, which are
considerably more stable (ca. 10–30 min) than those with
DMPO or DEPMPO. Similar EPR spectra were obtained
from the other spin traps tested (see Table 5).
(
b)
(
c)
4. Discussion
Twelve novel compounds derived from the spin traps
(d)
PBN and EPPN were synthesized and fully characterized
in this study. These substances form rather stable super-
oxide adducts (t1=2 ¼ 4–11 min), which are at least ten
times more stable than the respective adducts with DMPO
2
0 G
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
CPPyN-2, CPPyN-3, CPPyN-4 and CPPN. (a) To a nitrogen-bubbled
solution of peroxidized linoleic acid (2 mM) and CPPyN-2 (20 mM) in
(
of some hydrophilic derivatives of EMPO (t1 > 20 min
t1=2 ¼ 45 s) [16] or PBN [3]. Only the superoxide adducts
=2
[7]) or DEPMPO (t1/2 ¼ ca. 7–15 min [10,11,13]) are even
phosphate buffer (20 mM, pH 7.4, containing 1.5% acetonitrile) FeSO
0.2 mM) was added and the spectrum was recorded with the following
4
more stable.
The half-lives of the superoxide adducts were deter-
mined using a first order exponential decay approxima-
(
spectrometer settings: sweep width, 50 G; modulation amplitude, 0.3 G;
5
microwave power, 20 mW; time constant, 0.16 s; receiver gain, 2 ꢁ 10 ;
2
tion (Pearson correlation coefficient r > 0:99 except for
scan rate, 17.9 G/min. The bars represent 10,000 arbitrary units. (b) Same
conditions, except that CPPyN-3 was used. (c) Same conditions, except that
CPPyN-4 was used. (d) Same conditions, except that CPPN was used.
2
2
CPPN (r ¼ 0:97) and CPPyN-2 (r ¼ 0:98)). Under the
experimental conditions used a second order decay of the
superoxide adducts was negligible. An important aspect
which has to be considered is the fact that the observed
spectral intensity not only depends on the half life of the
spin adducts but also from 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. Preliminary tests, based on the spectral intensity of
the respective superoxide spin adducts after 5 min, have
shown that the performance of the PPyN compounds is
between DMPO and DEPMPO, decreasing from the more
hydrophilic to the more lipophilic compounds due to
steric hindrance.
anaerobic setup, obtained by flushing with nitrogen for
several minutes, was chosen. The reaction system con-
sisted of peroxidized linoleic acid and the respective spin
trap dissolved in 20 mM phosphate buffer, pH 7.4, contain-
ing 1% acetonitrile. To this solution Fe , dissolved 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 [11,13] or EPPN
2] derivatives.
In Fig. 5, the radical adducts derived from linoleic acid
hydroperoxide and the spin traps CPPyN-2 (Fig. 5a),
CPPyN-3 (Fig. 5b), CPPyN-4 (Fig. 5c), and CPPN
2þ
[
(
Fig. 5d) generated in the anaerobic Fenton system are
All novel compounds form also methoxyl and ethoxyl
radical spin adducts. n-Octyloxyl adducts were detected
with some of the tested compounds, e.g. with iPPPyN-2
and iPPPyN-3, which turned out to form the most stable
alkoxyl radical adducts. Unfortunately, no alkoxyl radical
spin adducts derived from peroxidized linoleic acid were
found, even while employing higher concentrations of the
spin trap. A severe limitation for the detection of such
radicals is the fact that alkoxyl radicals undergo rapid b-
shown. The EPR spectra are typical of a partially immo-
bilized carbon-centered radical adduct. The spectral para-
meters were obtained by computer simulation assuming
the contribution of two different species to the observed
EPR spectra (see also Table 5). In contrast to the two
different stereoisomers of the a-hydroxyethyl radical
adduct where the two nitrogen splittings were identical,
the spectra of the LOOH-derived spin adducts could only
be simulated using two different values for the nitrogen
splitting. This makes the contribution of secondary, struc-
turally different radicals likely, most probably stemming
from degradation processes of the primary oxidation pro-
ducts. For comparison, the best fit for the contribution of a
6
scission with a rate constant of around 10 , which effi-
ciently competes with the spin trapping reaction. This
applies especially as the maximum concentration of the
different PPyN derivatives in aqueous solution did not
exceed values about 100 mM. On the other hand, the spin

K. Stolze et al. / Biochemical Pharmacology 68 (2004) 185–193
193
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5] Zhang H, Joseph J, Vasquez-Vivar J, Karoui H, Nsanzumuhire C,
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adducts formed from secondary carbon centered radicals,
which are formed according to several pathways [17] in
ꢀ
ꢀ
secondary reactions from LOO [18–23] and LO , are very
stable and were readily detected. The structure of the
various secondary radical adduct could not be determined,
but the oxygen in the primary alkoxyl radical formed from
peroxidized linoleic acid is most likely situated in position
2
000;473:58–62.
[
[
6] 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.
7] 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.
9
or 13. Depending on the reaction conditions, a variety of
carbon-centered radicals can be formed from these primary
radicals.
[8] Sankuratri N, Janzen EG. Synthesis and spin trapping chemistry of a
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In conclusion, the four PPyN-3 derivatives can be
recommended for the trapping of superoxide radicals, since
the half-life of their superoxide adducts is 10 min or more.
Hydroxyl radical adducts are not stable. The stability of
alkoxyl radical adducts decreases with increasing chain
length. Although the possibility to detect the primary
alkoxyl radical formed during lipid peroxidation of fatty
acids seems to be rather low, low-molecular weight sec-
ondary alkoxyl radicals should be detectable with iPPPyN-
[
10] Fr e´ javille C, Karoui H, Tuccio B, Le Moigne F, Culcasi M, Pietri S,
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2
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[
[
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stable for several minutes. In addition, all novel spin traps
investigated can be recommended as possible alternatives
to PBN for the detection of carbon-centered radicals.
For this purpose, a homologous series of spin traps
with increasing lipophilicity can readily be synthesized
from commercially available compounds in two or three
steps.
[
15] Hawkes GE, Herwig K, Roberts JD. Nuclear magnetic resonance
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Acknowledgments
8
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RP. Detection of free radicals produced from the reaction of cyto-
chrome P-450 with linoleic acid hydroperoxide. Biochem J 1997;328:
The authors wish to thank R. Stadtm u¨ 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 F o¨ rderung der wissenschaftlichen
Forschung.
5
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[
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