Spin trapping of C- and O-centered radicals with methyl-, ethyl-, pentyl-, and phenyl-substituted EMPO derivatives

Article abstract of DOI:10.1016/j.bmc.2005.12.051

In order to develop spin traps with an optimal ratio between hydrophilic and lipophilic properties, low toxicity, and high stability of spin adducts (especially with superoxide radicals), several EMPO-derived spin traps have recently been synthesized forming more stable superoxide adducts (t_1/2 > 20 min) than DMPO or DEPMPO. In this study, ESR-, ¹H-, and ¹³C-NMR data of several phenyl- or n-pentyl-substituted EMPO derivatives are presented with full signal assignment. Methyl groups at position 3 or 4 stabilized the superoxide adducts considerably. Spin adducts from other oxygen- and carbon-centered radicals (e.g., derived from methanol or linoleic acid hydroperoxide) are also described.

Full text of DOI:10.1016/j.bmc.2005.12.051

Bioorganic & Medicinal Chemistry 14 (2006) 3368–3376
Spin trapping of C- and O-centered radicals with methyl-,
ethyl-, pentyl-, and phenyl-substituted EMPO derivatives
Klaus Stolze,a,* Natascha Rohr-Udilova,a Thomas Rosenau,b Andreas Hofinger,b
Daniel Kolarichb and Hans Nohla,*
aResearch Institute of Biochemical Pharmacology and Molecular Toxicology, University of Veterinary Medicine Vienna,
Veterina¨rplatz 1, A-1210 Vienna, Austria
bDepartment of Chemistry, University of Natural Resources and Applied Life Sciences (BOKU),
Muthgasse 18, A-1190 Vienna, Austria
Received 12 October 2005; revised 19 December 2005; accepted 23 December 2005
Available online 24 January 2006
Abstract—In order to develop spin traps with an optimal ratio between hydrophilic and lipophilic properties, low toxicity, and high
stability of spin adducts (especially with superoxide radicals), several EMPO-derived spin traps have recently been synthesized form-
ing more stable superoxide adducts (t1/2 > 20 min) than DMPO or DEPMPO. In this study, ESR-, 1H-, and 13C-NMR data of sev-
eral phenyl- or n-pentyl-substituted EMPO derivatives are presented with full signal assignment. Methyl groups at position 3 or 4
stabilized the superoxide adducts considerably. Spin adducts from other oxygen- and carbon-centered radicals (e.g., derived from
methanol or linoleic acid hydroperoxide) are also described.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
which are carrying an additional methyl group at position
3 or 4 of the pyrroline ring (3,5-EMPtPO and 4,5-
Spin trapping of superoxide radicals using different
EMPO derivatives has recently been described by sever-
al authors.1–8 Bulky alkoxycarbonyl substituents5 or
methyl groups in position 3 or 4 of the pyrroline ring7
considerably stabilized the respective superoxide ad-
ducts (t1/2 > 20 min) when compared to commonly used
spin traps, such as DMPO (t1/2 < 1 min),9 EMPO (t1/2
ca. 8 min),1,2 or DEPMPO (t1/2 ca. 14 min).10,11 In addi-
tion to the affects exerted on spin adduct stability, alter-
ation of the 5-alkyl or 5-alkoxycarbonyl substituents
also affects the lipophilic properties of the spin trap, thus
allowing accumulation of a desired ratio of the spin trap
in a particular biological environment, for example,
within biological membranes. This can be achieved by
replacing either the alkoxycarbonyl3–5,8 or the 5-methyl
group of EMPO by an alkyl or aryl substituent.
EMPtPO). This additional substituent exerts
a
pronounced effect on the stability of the respective super-
oxide adducts. In continuation of recently published
experiments with DMPO,12 DEPMPO,10,11 EMPO,4
and 1,3,3-trimethyl-6-azabicyclo[3.2.1]oct-6-ene-N-oxide
(TRAZON),13,14 we also investigated the spin-trapping
behavior toward other oxygen- or carbon-centered radi-
cals (spin trap structures, see Fig. 1).
R1
R3
R4
R5
EMPO
EPhPO
PPhPO
EPtPO
PPtPO
3,5-EMPtPO C2H5
4,5-EMPtPO C2H5
PrMPO
sBuMPO
tBuMPO
EEPO
C2H5
C2H5
C3H7
C2H5
C3H7
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
C6H5
C6H5
n-C5H11
n-C5H11
n-C5H11
CH3 n-C5H11
C3H7
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
sec-C4H9
tert-C4H9
C2H5
The present paper describes synthesis, analytical and spin
trapping properties of a series of 5-phenyl- or 5-n-pentyl-
pyrroline derivatives with increasing lipophilicity, two of
PrEPO
C3H7
iPrEPO
BuEPO
sBuEPO
tBuEPO
iso-C3H7
n-C4H9
sec-C4H9
tert-C4H9
Keywords: EPR; Spin trapping; Superoxide; EMPO derivatives.
*
Corresponding authors. Tel.: +43 1 25077 4406; fax: +43 1 25077
4490 (K.S.); e-mail: Klaus.Stolze@vu-wien.ac.at
Figure 1. General structure of the spin traps.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.12.051

K. Stolze et al. / Bioorg. Med. Chem. 14 (2006) 3368–3376
3369
2. Results
of different incubations at 100 mM concentration,
was, however, not possible due to limited amounts
of the isolated diastereomeric forms (3,5- and 4,5-
EMPtPO) and the low yields obtained (especially from
PPhPO).
Incubation of EPhPO in the presence of xanthine/xan-
thine oxidase at pH 7.4 with a superoxide production
rate of about 2 lM/min resulted in the detection of
the EPhPO superoxide adduct (Fig. 2a). No EPR
spectrum was observed in the presence of SOD
(100 U/ml, not shown). Several scans had to be accu-
mulated to detect EPR spectra from EPtPO (Fig. 2b)
and PPtPO (not shown), whereas trans-4,5-EMPtPO
(Fig. 2c) gave a stronger and more persistent EPR sig-
nal. The half-life of superoxide adducts was deter-
mined incubating 20 mM of the respective spin trap
in oxygenated phosphate buffer (pH 7.4) for 10 min
in the above-mentioned xanthine/xanthine oxidase sys-
tem, after which SOD (100 U/mL) was added and the
decay of the EPR signal was recorded as a series of
consecutive spectra until the superoxide-related lines
disappeared. The contribution of secondary lines
(mainly hydroxyl adduct) was then subtracted from
each individual EPR spectrum before calculating the
respective half-life. With all spin traps the resulting
intensity decrease of the first two lines was approxi-
mated by a first-order exponential decay, which was
in good agreement with the experimental data (r2:
EPhPO, PPhPO, cis- and trans-4,5-EMPtPO (>0.99);
PPtPO (0.98), EPtPO (0.95), 3,5-EMPtPO (0.89
(Xa/XOD) or 0.99 (KO2-system)). In the KO2 system,
SOD (100 U/ml) and catalase (250 U/ml) were added
10 s after starting the incubation in order to decrease
possible deleterious effects exerted by the developed
peroxide. We also performed a preliminary evaluation
of the rate constant of the superoxide spin trapping
reaction for EPtPO (k ca. 10–28 Mꢀ1 sꢀ1), PPtPO
(k ca. 43–119 Mꢀ1 sꢀ1), 3,5-EMPtPO (k ca.
16–44 Mꢀ1 sꢀ1), 4,5-EMPtPO (k ca. 40-110 Mꢀ1 sꢀ1),
and EPhPO (k ca. 27–74 Mꢀ1 sꢀ1). A complete char-
acterization of the rate constants according to Weaver
et al.,15 using two independent methods and a series
Spin adduct formation with hydroxyl radicals using a
Fenton system is shown for EPhPO (Fig. 3a), EPtPO
(Fig. 3b), and trans-4,5-EMPtPO (Fig. 3c). The EPR
spectra of the hydroxyl radical adducts gradually disap-
peared within about 5 min (Ph-derivatives) to almost
20 min (Pt-derivatives). In the case of the n-pentyl deriv-
atives, a significant contribution of secondary products
to the spectral intensity was observed, possibly stem-
ming from hydroxyl radical attack at the side chain.
The two different types of spin adducts obtained from
EPhPO and methanol are shown in Figure 4a
(EPhPO/ÅOCH3, stable for a few minutes, obtained by
Fe3+-catalyzed addition of methanol according to
Dikalov et al.12) and in Figure 4b (EPhPO/ÅCH2OH, rel-
atively stable, formed using a Fenton system containing
5% methanol as described by Roubaud et al.16).
We also investigated the radicals formed in a Fenton-
type reaction from peroxidized linoleic acid18 in the
presence of Fe2+ under anaerobic conditions,12 where
a mixture of three different radical adducts could be
distinguished with EPhPO (Fig. 4c) or PPhPO (not
shown). An even more complex mixture of radicals
was observed when cis-/trans-3,5-EMPtPO, cis- or
trans-4,5-EMPtPO (spectra not shown). The spectral
parameters of lipid-derived adducts of all investigated
spin traps and the comparison with other EMPO
derivatives are listed in Table 6.
Figure 3. Formation of hydroxyl radical adducts of the spin traps
EPhPO, EPtPO, and 4,5-EMPtPO. (a) EPhPO (20 mM) was incubated
with a Fenton system containing FeSO4 (1 mM), EDTA (2 mM), and
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, 80.0 G; modulation amplitude, 0.24 G; microwave
power, 20 mW; time constant, 0.08 s; receiver gain, 1 · 104; scan rate,
57.2 G/min. (b) Incubation as in (a), except that EPtPO (20 mM) was
used. EPR parameters: sweep width, 80 G; modulation amplitude,
0.1 G; microwave power, 20 mW; time constant, 0.16 s; receiver gain,
2 · 105; scan rate, 28.6 G/min. (c) Incubation and ESR conditions as in
(a), except that trans-4,5-EMPtPO (20 mM) was used.
Figure 2. Formation of the superoxide adducts of the spin traps
EPhPO, EPtPO, and trans-4,5-EMPtPO. (a) EPhPO (40 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 0.4 mM
DTPA) were incubated and measured using the following EPR
parameters: sweep width, 60 G; modulation amplitude, 1.0 G; micro-
wave power, 20 mW; time constant, 0.16 s; receiver gain, 2.5 · 105;
scan rate, 42.9 G/min. (b) Same as in (a), except that EPtPO (20 mM)
was used. Receiver gain, 4 · 105, 5 scans. (c) Same as in (a), except that
trans-4,5-EMPtPO (20 mM) was used.

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K. Stolze et al. / Bioorg. Med. Chem. 14 (2006) 3368–3376
(1H and 13C), ESI Q-TOF MS, and IR spectroscopy
(Tables 1–5). Relatively stable superoxide adducts were
formed from EPhPO (t1/2 = 16.1 min) and the two pen-
tyl compounds having an extra methyl group at either
position 3 (3,5-EMPtPO; t1/2 = 12.1 min) or 4 (cis-
and trans-4,5-EMPtPO; t1/2 ca. 25 min) of the pyrroline
ring, slightly more stable than superoxide adducts of
DEPMPO derivatives (t1/2 = ca. 15 min)10 but less sta-
ble compared to their hydrophilic congeners.7 This sta-
bilizing effect is most probably due to steric shielding
rather than electronic influences as has already been
mentioned previously.5 Determination of the half-lives
of the superoxide adducts was done using a first-order
exponential decay approximation (Pearson’s correlation
coefficient r2 > 0.98 with two exceptions). Under the
experimental conditions used a second-order decay of
the superoxide adducts was not significant. In addition
to the half-life of the spin adducts, 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
constituents, and degradation by transition metal cata-
lyzed reactions (Fenton type reactions, oxidation by
Fe3+, etc.), play also important roles regarding the
overall efficiency of the spin trapping reaction. In view
of these facts, the half-life values given in Table 4 and
the rate constants mentioned in the text are therefore to
be interpreted only as preliminary. Further tests will be
necessary to test these compounds in different biologi-
cal systems. Hydroxyl radical adducts were stable for
about 15–20 min, except for EPhPO and PPhPO, whose
hydroxyl radical adducts were stable only for about
5 min. Sufficiently stable methoxyl radical adducts were
obtained only from the spin traps bearing a methyl
group in position 3 or 4 of the pyrroline ring, the others
decaying within about 3–5 min. 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 respective carbon-centered form or
Figure 4. Iron-dependent formation of spin adducts from EPhPO in the
presence of methanol or oxidized linoleic acid. (a) After a 10 s incubation
of EPhPO (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 ampli-
tude, 0.24 G; microwave power, 20 mW; time constant, 0.02 s; receiver
gain, 2 · 105; scan rate, 171.7 G/min. (b) EPhPO (20 mM) was incubated
with a Fenton system containing FeSO4 (1 mM), EDTA (2 mM), and
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, 80 G; modulation amplitude, 0.1 G; microwave
power, 20 mW; time constant, 0.16 s; receiver gain, 5 · 104; scan rate,
28.6 G/min. (c) To a nitrogen-bubbled solution of peroxidized linoleic
acid (5 mM) and EPhPO (100 mM) in phosphate buffer (10 mM, pH 7.4,
containing 250 units/ml catalase) FeSO4 (0.1 mM) was added and the
spectrum was recorded with the following spectrometer settings: sweep
width, 80.0 G; modulation amplitude, 1.2 G; microwave power, 20 mW;
time constant, 0.16 s; receiver gain, 2.5 · 105; scan rate, 57.2 G/min, 6
scans accumulated.
3. Discussion
Six lipophilic derivatives of the spin trap EMPO with
phenyl and n-pentyl substituents were synthesized in
this study. In contrast to the moderately lipophilic
phenyl derivatives, the n-pentyl derivatives were consid-
erably more lipophilic in comparison with the parent
compound. The structure of the compounds was com-
prehensively characterized by full NMR assignment
Table 1. 13C NMR data (ppm) of the spin traps
3a
O
4
5
O
4
3´
1´
3
3´
1´
3
O
1
N
2
O
1
N
2
1´´
2´
5´´
2´
4´´
5
6´´
2´´
1´´
O
2´´
O
3´´
5´´
4´´
3´´
0
0
0
100
200
300
400
500
600
2C
3C
3aCH3 4C
4aCH3 5C
COO 1 C
2 C
3 C
C
C
C
C
C
C
EMPOc
EPhPO
PPhPO
EPtPO
PPtPO
134.9 25.4
135.6 26.0
135.9 26.1
135.7 26.2
135.0 26.1
—
—
—
—
—
31.9
—
—
—
—
—
—
—
78.5 169.3 61.7 13.4
85.6 168.7 62.8 13.9
85.6 168.8 68.2 21.7
82.0 169.9 67.0 13.8a
82.0 170.0 67.5 21.8
82.7 170.2 62.0 13.8
82.5 169.8 62.0 13.8
85.1 168.4 61.7 13.9a
84.5 170.0 62.1 14.0a
—
—
20.3
—
—
—
—
128.5b 128.3a
34.4
34.3
28.5
28.5
37.2
36.7
135.7 128.3a 128.5b 127.0
10.2 135.6 127.0
128.4
31.7
31.7
31.5
31.5
31.8
32.1
126.2
22.4b
22.5a
22.5
128.4
13.9a
13.9
127.0
—
—
—
32.6
32.6
32.8
32.4
30.7
29.6
22.2b
22.4a
22.3
22.3
22.3
22.4
10.2
—
c3,5EMPtPO 140.1 33.7 19.0
t3,5EMPtPO 139.6 33.1 18.4
13.8
—
—
—
—
22.5
22.4
13.8
c4,5EMPtPO 136.4 34.0
t4,5EMPtPO 134.6 35.0
—
—
35.0 15.4
37.2 14.6
14.0a
14.1a
—
—
—
23.7
a Tentative assignment (reversed order also possible).
b Tentative assignment (reversed order also possible).
c Data from Stolze et al.5

Table 2. 1H NMR data [ppm] of the spin traps
3a
O
4
5
O
3´
1´
4
3
3´
1´
3
O
1
N
2
O
1
N
2
1´´
2´
2´
4´´
5
6´´
5´´
2´´
1´´
O
2´´
O
3´´
5´´
4´´
3´´
0
0
0
100
200
300
400
500
600
2CH
3CHx
3aCH3
—
4CHx
4aCH3
—
1 CH2
2 CH2
3 CH3
CHx
CHx
CHx
CH3
CHx
CH
EMPOa
6.97t
2.75m
2.16m
2.60m
4.26m
1.31t
—
1.72s
—
—
—
—
—
—
EPhPO
7.13t
7.16t
2.64–2.84m
2.64–2.82m
—
2.65m
3.06–3.16m
2.65m
—
4.34q
4.22t
1.30t
1.68m
1.29t
1.70m
1.29t
1.29t
1.30t
1.30t
—
7.34–7.43m
7.32–7.42m
1.24–1.38m
1.24–1.40m
1.29m
7.46–7.51m
7.44–7.52m
1.24–1.38m
1.24–1.40m
1.29m
7.34–7.43m
7.32–7.42m
1.24–1.38m
1.24–1.40m
1.29m
7.46–7.51m
7.44–7.52m
0.89t
7.34–7.43m
PPhPO
—
—
0.90t
—
—
7.32–7.42m
3.04–3.16m
2.26m
EPtPO
7.00t
2.74m
6.96t
2.63m
2.50m
2.63m
2.74m
3.08m
—
—
4.26m
4.17m
4.25m
4.25m
4.27m
4.27m
2.04m
2.16m
2.04m
2.16m
1.95m
2.20m
1.95m
2.20m
1.92m
2.25m
1.99m
2.10m
—
—
—
—
—
—
PPtPO
—
2.26m
2.50m
—
0.95t
—
0.89t
c3,5EMPtPO
t3,5EMPtPO
c4,5EMPtPO
t4,5EMPtPO
6.99d
6.99d
7.05t
6.98t
1.24d
1.19d
—
2.08ddc
2.49ddt
1.80ddt
2.66ddc
2.70m
—
0.90t
3.15m
—
—
1.29m
1.29m
1.29m
0.89t
2.38m
2.74m
2.26m
2.83m
1.16d
1.16d
—
1.30–1.41m
1.30–1.41m
1.30–1.41m
1.30–1.41m
1.30–1.41m
1.30–1.41m
0.87t
—
2.96m
—
0.87t
Abbreviations: s, singlet; d, doublet; t, triplet; m, multiplet; ddc, doublet of doublets (cis to ester); ddt, doublet of doublets (trans to ester).
a Data from Stolze et al.5

3372
K. Stolze et al. / Bioorg. Med. Chem. 14 (2006) 3368–3376
Table 3. ESI Q-TOF MS analysis of the spin traps
Acquired [MH]+ Calcd [MH]+ Error [ppm] mDa Acquired [MNa]+ Calcd [MNa]+ Error [ppm] mDa
Sample 1 EPhPO
Sample 2 PPhPO
Sample 3 EPtPO
Sample 4 PPtPO
Sample 5 3,5-EMPtPO
234.1201
248.1323
228.1673
242.1872
242.1808
234.1125
248.1281
228.1594
242.1751
242.1751
242.1751
32.62
16.87
34.57
50.11
23.69
50.11
7.64 256.1016
4.19 270.1157
7.89 250.1516
12.14 264.1636
5.74 264.1636
12.14 264.1702
256.0950
270.1106
250.1419
264.1576
264.1576
264.1576
25.92
18.84
38.73
22.86
22.86
47.84
6.64
5.09
9.69
6.04
6.04
12.64
Sample 6 cis-4,5-EMPtPO 242.1872
trans-4,5-EMPtPO
Table 4. Half-life of the superoxide adducts and n-octanol/buffer
partition coefficients of the spin traps
5.2. Syntheses
Synthesis and characterization of the compounds were
performed as reported previously,4,5 in analogy to the
synthesis of EMPO and its derivatives1,2 with minor
adaptations as given below.
Compound
Apparent Partition coefficient
t1/2 (min) n-octanol/phosphate buffer
(100 mM, pH 7.0)
EMPOa
EPhPO
PPhPO
EPtPO
PPtPO
8.6a
16.1
7.6
0.15a
1.79
5.03
5.2.1. a-Bromophenylacetyl chloride. a-Bromophenylace-
tic acid (25 g, 100 mmol) was refluxed for 2 h in 40 ml of
thionyl chloride. The evolving gas (sulfur dioxide and
hydrogen chloride) was absorbed into 10% sodium car-
bonate solution. Twenty milliliters of excess thionyl
chloride was recovered by distillation for reuse, the
remaining part was removed after addition of heptane
(50 ml). After removal of heptane in a rotary evapora-
tor, the crude product was used without further
purification.
2.2
10.2
15.28
35.16
44.75
32.48
33.08
0.20a
1.06a
0.80a
0.25a
0.78a
0.66a
2.88a
1.89a
1.53a
cis/trans-3,5-EMPtPOb 12.1
cis-4,5-EMPtPO
trans-4,5-EMPtPO
i-PrMPOa
s-BuMPOa
t-BuMPOa
EEPOa
25.1
25.3
18.8a
26.3a
15.7a
18.0a
20.0a
23.3a
18.0a
14.1a
18.2a
PrEPOa
i-PrEPOa
BuEPOa
5.2.2. Alkyl a-bromophenylacetate. a-Bromophenylace-
tyl chloride (70 mmol) was slowly added to a solution
of the respective alcohol (100 mmol) and pyridine
(70 mmol) in CHCl3 at 0 ꢁC (ice bath). After stirring
for 1 h, the reaction mixture was successively washed
with water (50 ml), sulfuric acid (10%, 50 ml), and con-
centrated aq NaHCO3 (50 ml), and dried over Na2SO4
overnight. Solvent and excess alcohol were removed
under reduced pressure. The crude, nearly colorless
product was used without further purification.
s-BuEPOa
t-BuEPOa
a Data from Stolze et al.5
whether the originally formed alkoxyl radicals under-
17
went rapid b-scission (rate constant ca. 106 Mꢀ1 sꢀ1
)
prior to radical trapping cannot be decided. A variety
of secondary reactions has been reported leading to
carbon-centered radicals according to different path-
ways19 involving intermediates such as LOOÅ or
LOÅ.20–25 (see Table 6).
5.2.3. Propyl 2-bromoheptanoate. Ethyl 2-bromohept-
anoate (25 ml, 128 mmol) was refluxed for 3 h in
100 ml 1-propanol containing 3 ml concentrated hydro-
chloric acid as a catalyst. The solvent was removed in a
rotary evaporator, petroleum ether was added, and trac-
es of remaining acid were removed after addition of a
small amount of CaO. After 1 h, the solution was fil-
tered and the petroleum ether was distilled off. If neces-
sary, the product was purified by distillation.
4. Conclusion
In conclusion, EPhPO and 4,5-EMPtPO can be recom-
mended for trapping of superoxide radicals, since the
half-life of their superoxide adducts is higher than
15 min. EPtPO, on the other hand, forms a rather unsta-
ble superoxide adduct and can, therefore, not be
recommended.
5.2.4. Synthesis of the alkyl 2-nitroheptanoates and alkyl
a-nitrophenylacetates. The respective alkyl 2-bromohept-
anoate or a-bromophenylacetate (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 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 a NaHCO3/Na2CO3 solu-
tion and dried over Na2SO4. After removal of the solids
by filtration, the solvent was evaporated in vacuo.
5. Experimental
5.1. Chemicals
Acrolein, a-bromophenylacetic acid, crotonaldehyde,
ethyl 2-bromoheptanoate, linoleic acid, methacrolein,
superoxide dismutase, thionyl chloride, and xanthine oxi-
dase were commercially available from Sigma–Aldrich.
Petroleum ether (high boiling, 50–70 ꢁC) was obtained
from Fluka, all other chemicals were from Merck.

K. Stolze et al. / Bioorg. Med. Chem. 14 (2006) 3368–3376
3373
The obtained colorless or pale yellow products were
used further without purification.
5.2.5. Alkyl 4-formyl-2-nitro-2-n-pentyl-butanoate (alkyl
4-formyl-2-nitro-2-phenylbutanoate). Alkyl 2-nitrohept-
anoate (23 mmol) or alkyl a-nitrophenylacetate (for EPh-
PO and PPhPO) 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 added at 0 ꢁC. The
solution was kept at 10 ꢁC for 1.5 h and then poured into
a solution of ice-cold HCl (5 ml of concentrated HCl in
150 ml water). The solution was extracted three times with
CH2Cl2 and dried over Na2SO4. After filtration, the mix-
ture was distilled under reduced pressure, and the purity
of the remaining product was assessed by thin layer chro-
matography and IR spectroscopy.
5.2.6. Ethyl 4-formyl-2-nitro-2-n-pentylpentanoate. Same
conditions as indicated above for the synthesis of alkyl
4-formyl-2-nitro-2-n-pentyl-butanoate, except that meth-
acrolein (2.5 g, 36 mmol) was used instead of acrolein.
After slow addition of methacrolein at 0 ꢁC, the solution
had to be stirred for several days at room temperature.
5.2.7. Ethyl 4-formyl-3-methyl-2-nitro-2-n-pentylbutano-
ate. Same conditions as indicated above for the synthesis
of alkyl 4-formyl-2-nitro-2-n-pentyl-butanoate, except
that crotonaldehyde (2.5 g, 36 mmol) was used instead
of acrolein. After slow addition of crotonaldehyde at
0 ꢁC, the solution had to be stirred for several days at
room temperature.
5.2.8. Synthesis of the N-oxides. Synthesis of the nitro-
nes was performed according to the procedure described
recently for the synthesis of EMPO derivatives.4,5 To a
concentrated solution of 25 mmol of the respective alkyl
4-formyl-2-nitro-2-n-pentylbutanoate (or alkyl 4-for-
myl-2-nitro-2-phenylbutanoate for EPhPO and PPhPO)
in H2O/CH3OH (v/v = 6:4), an aqueous solution of
ammonium chloride (1.87 g in 8 ml of water) was added.
While zinc dust (8.5 g, 130 mmol) was slowly added with-
in 30 min, the mixture was carefully kept at room temper-
ature. 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 and extracted four times
with 60 ml CH2Cl2. The organic phase was dried with
Na2SO4, filtered, and concentrated. 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
dark yellow oil or yellow needles (yield ca. 20% for the
pentyl derivatives, ca. 5% for the phenyl derivatives).
Additional purification was done immediately before
the EPR experiments on a 1 ml solid phase extraction col-
umn using a Chromabond C-18 100 mg column obtained
from Macherey-Nagel (Duren, Germany). The purity of
¨
the obtained products was assessed by TLC and UV spec-
troscopy. Final identification of the purified products was
performed by 1H NMR, 13C NMR, ESI Q-TOF MS, and
IR spectroscopy (see Tables 1–5).

Table 6. Comparison of the EPR parameters of radical adducts of different EPhPO, EPtPO, EMPO and EEPO derivatives
HFS (G) EMPOa
Radical
ÅOOH
EPhPO
PPhPO
EPtPO
PPtPO
c/t-3,5EMPtPO
cis-4,5EMPtPO
(74%) (15%) (11%)
trans-4,5EMPtPO
(43%) (57%) (60%) (40%)
13.28 13.28 13.00 13.05
9.67 11.89 11.65 9.00
(61%) (39%)
12.98 13.04
11.51 8.82
(55%) (45%)
13.35 13.35
9.55 11.98
(62%) (38%)
13.20 13.18
11.61 9.00
(35%) (35%) (30%)
13.40 12.75 13.40
15.03 6.80 8.10
(67%) (33%)
13.48 13.55
10.99 8.00
aN
aH
aH
13.24 13.18 13.18
11.60 9.81 7.83
—
—
—
—
—
—
—
—
0.50
—
—
—
—
—
—
—
—
0.66
ÅOH
(50%) (50%) (50%) (50%)
14.00 14.00 13.43 13.43
15.00 12.58 12.70 14.70
(40%) (34%) (26%) (52%) (48%)
13.66 13.37 13.66 13.85 14.00
12.93 15.15 11.38 12.16 15.97
(52%) (48%)
14.00 13.85
15.87 12.16
(67%) (33%)
13.75 14.30
9.30 19.25
(26%) (4%)b (25%) (24%) (21%)c (13%) (11%) (76%)d
14.30 14.00 13.85 13.82 13.80 13.85 13.87
aN
aH
aH
19.60 16.37 10.85 12.40 9.20
8.40 10.88
0.90
—
0.75 0.75
—
—
—
0.75
—
—
0.80
—
—
—
—
—
—
—
—
0.40
(100%)
15.52
22.21
20.82
—
(100%)
15.12
21.38
20.48
—
(100%)
15.06
21.37
20.47
—
(100%)
15.34
22.91.
19.87
—
(75%) (25%)
15.33 15.13
22.96 25.14
19.89 17.37
(38%) (31%) (31%)
15.43 15.34 15.03
26.08 25.08 26.28
16.15 17.21 16.68
0.60 0.55 0.50
(51%) (49%)
15.24 15.34
24.46 26.08
18.22 17.40
(100%)
15.42
26.02
17.45
—
ÅH
aN
aH
aH
aH
—
—
1.15
—
ÅCH3
(100%)
15.42
22.30
—
(100%)
14.90
21.63
—
(100%)
14.87
21.51
—
(52%) (48%)
15.08 15.15
23.80 21.22
(51%) (49%)
15.05 15.14
23.60 21.14
(68%) (16%) (16%)
15.00 15.00 15.25
15.75 18.50 26.65
(51%) (49%)
15.15 15.20
19.26 17.43
(89%) (11%)
15.20 15.00
17.43 19.30
0.45 0.70
aN
aH
aH
—
—
—
—
—
—
—
0.60
—
ÅOCH3
(50%) (50%) (53%) (47%)
13.74 13.74 13.30 13.30
10.87 7.81 9.80 7.11
0.55 0.50 0.60 0.50
(56%) (44%)
13.27 13.27
9.78 7.17
(62%) (38%)
13.48 13.48
7.70 10.49
(57%) (43%)
13.49 13.49
10.52 7.73
(50%) (35%) (15%)
13.40 13.75 13.85
5.80 14.75 17.25
(42%) (40%) (18%)
13.38 13.40 13.72
8.75 5.64 16.04
(50%) (42%) (8%)
13.60 13.60 13.88
5.38 9.02 17.92
aN
aH
aH
—
—
—
—
—
—
—
—
—
—
—
1.86
—
—
—
ÅCH2OH
(100%)
14.95
(100%)
14.54
(100%)
14.57
(100%)
14.78
(100%)
14.76
(53%) (47%)
14.86 14.66
24.69 16.68
(100%)
14.70
(100%)
14.94
aN
aH
21.25
20.85
20.62
21.40
21.08
18.85
18.18
ÅC (LOOH)
(62%) (38%) (58%) (26%) (16%) (45%) (40%) (15%) (50%) (26%) (24%) (56%) (26%) (18%) (58%) (22%) (20%)
13.45 13.45 13.40 14.10 14.95 14.90 13.67 14.18 15.00 13.85 14.00 15.30 13.85 14.00 15.20 14.25 13.65
11.45 8.55 13.50 17.40 21.60 21.70 12.68 17.25 23.00 12.16 15.97 22.90 12.16 15.87 24.90 19.25 8.80
(67%) (21%) (12%)
14.80 13.70 14.00
19.55 10.80 17.90
(33%) (29%) (15%) (14%)e
15.00 14.80 15.00 14.65
21.00 17.25 15.00 19.50
aN
aH
aH
aH
—
—
1.00
0.75
—
0.76 0.76 0.80
—
1.40 0.75
—
1.50 0.80
—
—
—
—
—
—
—
—
—
—
—
Radical
ÅOOH
i-PrMPO
s-BuMPO
t-BuMPO
EEPO
PrEPO
i-PrEPO
BuEPO
s-BuEPO
t-BuEPO
(57%)
13.30
11.88
—
(43%)a
13.30
9.66
(56%)
13.03
11.82
—
(44%)a
13.30
9.54
(56%)
13.38
11.95
—
(44%)a
13.38
9.70
(63%)
13.15
11.40
—
(37%)a
13.15
8.75
(61%)
13.23
11.52
—
(39%)a
13.23
8.80
(62%)
13.21
11.60
—
(38%)a
13.21
9.03
(67%)
13.21
11.55
—
(33%)a
(62%)
13.22
11.55
—
(38%)a
(65%)
13.27
11.65
—
(35%)a
13.27
9.07
aN
aH
aH
13.21
8.92
—
13.22
8.90
—
—
—
—
—
—
—
—

ÅOH
(72%)
(28%)a
14.25
15.35
0.5
(77%)
14.06
12.60
0.72
(23%)a
14.22
15.35
0.45
(81%)
14.13
12.71
0.70
(19%)a
14.20
15.47
0.65
(55%)
13.86
12.05
0.78
(45%)a
14.00
15.90
0.62
(59%)
13.84
12.01
0.80
(41%)a
13.98
15.90
0.58
(61%)
13.87
12.01
0.78
(39%)a
14.00
15.94
0.63
(65%)
13.80
11.97
0.78
(35%)a
13.98
15.95
0.65
(80%)
13.95
16.20
—
(20%)a
13.80
11.90
0.72
(62%)
13.90
12.16
0.80
(38%)a
14.05
16.10
0.60
aN
aH
aH
14.10
12.80
0.65/0.45
(100%)
(100%)
(100%)
(100%)
(100%)
(100%)
(100%)
(100%)
(100%)
ÅH
aN
aH
aH
15.52
22.25
20.80
15.49
22.27
20.79
15.59
22.42
20.84
15.32
22.99
19.87
15.32
22.97
19.85
15.33
23.00
19.90
15.25
23.06
19.87
15.32
22.99
19.85
15.40
23.04
20.00
ÅCH3
(100%)
15.35
(100%)
15.32
(100%)
15.44
(53%)
15.14
23.99
(47%)
15.14
21.25
(59%)
15.14
21.20
(41%)
15.08
24.17
(50%)
15.14
21.36
(50%)
15.09
24.18
(51%)
15.10
21.19
(49%)
15.09
24.13
(60%)
15.13
21.10
(40%)
15.07
24.03
(59%)
15.21
21.29
(41%)
15.11
24.25
aN
aH
22.14
22.03
21.95
ÅOCH3
(100%)
13.76
9.31
(100%)
13.62
9.12
(100%)
13.72
9.25
(100%)
13.45
9.00
(100%)
13.41
8.98
(100%)
13.42
8.92
(100%)
13.41
8.88
(100%)
13.42
8.86
(100%)
13.47
8.91
aN
aH
aH
1.28
1.43
1.40
1.45
1.45
1.47
1.47
1.47
1.47
ÅCH2OH
(100%)
14.95
21.15
—
(100%)
14.94
20.97
—
(100%)
15.01
20.98
—
(100%)f
14.76
21.70
0.87
(100%)f
14.74
21.50
0.87
(100%)f
14.77
21.60
0.90
(100%)f
14.74
21.52
0.90
(100%)f
14.78
21.35
0.92
(100%)f
14.80
21.31
0.88
aN
aH
aH
ÅC (LOOH)
(77%)
15.22
22.33
—
(23%)
14.96
20.52
—
(71%)
15.12
22.33
—
(29%)
14.96
20.62
—
(67%)
15.12
22.34
—
(33%)
14.96
20.64
—
(56%)
14.99
21.18
—
(44%)
15.05
23.82
—
(54%)
15.03
20.94
—
(46%)
15.03
23.49
—
(64%)
15.03
21.29
—
(36%)
15.13
24.30
—
(65%)
14.47
24.42
—
(35%)
14.80
21.20
—
(61%)
15.00
21.00
—
(39%)
15.05
24.02
—
(73%)
15.10
21.18
—
(27%)
15.22
24.36
—
aN
aH
aH
a Data from Stolze et al.4
b Approximate values calculated from difference spectra taken 3 min after the first scan.
c Approximate values calculated from difference spectra taken 30 min after the first scan.
d Secondary radicals from side chain attack (unresolved mixture).
Å
e Rest mainly OH adduct.
f Broad lines due to unresolved HFS.

3376
K. Stolze et al. / Bioorg. Med. Chem. 14 (2006) 3368–3376
5.2.9. Preparation of lipid hydroperoxides. Linoleic acid
hydroperoxide was synthesized according to O’Brien.18
Briefly, linoleic acid was air-oxidized for 72 h in the dark
at room temperature. The oxidation mixture was dis-
solved in petroleum ether (boiling range 60–90 ꢁC) 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 ꢁC) and evaporated under reduced pressure. The
obtained hydroperoxide was dissolved in ethanol and
stored in liquid nitrogen. The concentration of hydro-
peroxide was determined by UV spectroscopy based
on an extinction coefficient of e233 nm = 25250 Mꢀ1 cmꢀ1
in ethanol.18
Acknowledgments
The authors thank R. Stadtmuller and P. Jodl for skilful
¨
technical assistance in synthesis, purification, and char-
acterization of the spin traps. The present study was
supported by the Austrian Fonds zur Fo¨rderung der
wissenschaftlichen Forschung.
References and notes
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