Electrochemical-ESR detection of hydrogen atom adducts of 5-membered ring nitrone spin traps

Article abstract of DOI:10.1246/bcsj.75.149

In order to make the spin trapping technique more useful in biological free radical studies, using an specially designed ESR cell for in situ electrochemical reactions, five 5-membered ring nitrones, which are analogues of 5,5-dimethyl-1-pyrroline N-oxide

Full text of DOI:10.1246/bcsj.75.149

Bull. Chem.
Soc. Jpn.
75
1
Bull. Chem. Soc. Jpn., 75, 149–150 (2002)
149
The Chemical
Society of Ja-
Short Articles
pan
The Chemical
Society of Ja-
pan
OB
Electrochemical-ESR Detection of
Hydrogen Atom Adducts of 5-Mem-
bered Ring Nitrone Spin Traps
1
15
2002
149
N.Endo et al.
Nobuyuki Endo, Kousuke Higashi,† Kenji Kanaori,†
Kunihiko Tajima,† and Keisuke Makino*
150
BCSJA8
0009-2673
K01022
6
12
2001
9
Fig. 1. Chemical structure of 5-membered ring nitrone spin
traps used in the present study and their spin adducts. The
conventional numbering systems and substituted groups
are also shown.
Institute of Advanced Energy, Kyoto University,
Gokasho, Uji, Kyoto 611-0011
†Department of Applied Biology,
Kyoto Institute of Technology,
Matsugasaki, Sakyo-ku, Kyoto 606-8585
Research Inc., in which the working and counter electrodes are
made of gold wire (0.5 mm o.d.) and silver wire is used as the ref-
erence electrode. The ESR spectrometer used was a JEOL-TE300
(JEOL) operated at 100 kHz field modulation. A magnetic field
adjustment was performed using Mn2+ in MgO as a reference:
The peak distance between g = 1.981 and 2.034 was 8.69 mT.
The spin traps used in the present study are summarized in Fig.
1. 2-Methyl-1-pyrroline N-oxide (2-MPO),7 2,4,4-trimethyl-1-
pyrroline N-oxide (2,4,4-M3PO), and 3,3,5,5-tetramethyl-1-pyrro-
line N-oxide (3,3,5,5-M4PO)8–9 were synthesized according to pre-
viously reported methods. 2-MPO and 2,4,4-M3PO were purified
by vacuum distillation (67 °C at 0.5 mmHg and 85 °C at 5 mmHg,
respectively) immediately prior to use, and 3,3,5,5-M4PO by re-
crystallization from pentane. DMPO (purchased from LABOTEC
Co.) as well as 5,5-dimethyl-4-phenyl-1-pyrroline N-oxide (4-Ph-
DMPO) and 5-methyl-5-phenyl-1-pyrroline N-oxide (5-Ph-5-
MPO) (purchased from Radical Research Inc.) were used without
further purification. Spectroscopy grade CH3CN was obtained
from Nacalai Tesque and purified by double distillation over P2O5.
Supporting electrolytes, (n-Bu)4NBF4, used in CH3CN were also
from Nacalai Tesque.
10
2001
(Received June 12, 2001)
2.6
4.7
In order to make the spin trapping technique more use-
ful in biological free radical studies, using an specially de-
signed ESR cell for in situ electrochemical reactions, five 5-
membered ring nitrones, which are analogues of 5,5-dimeth-
yl-1-pyrroline N-oxide (DMPO), one of the most convention-
ally used spin traps, have been explored for their hydrogen
atom (OH) adducts formation. ESR spectra consisting of only
OH adducts and the resulting ESR parameters are discussed.
Spin trapping with several nitrone analogues, such as
DMPO (5,5-dimethyl-1-pyrroline N-oxide) and α-phenyl-t-bu-
tyl nitrone (N-benzylidene-t-butylamine N-oxide), is known to
be an exclusively useful tool for investigations on the charac-
terization and biological implication of short-lived free radical
intermediates arising from biological functions.1 Several stan-
dard methods for the spin trapping of such short-lived radicals
as hydroxyl, superoxide anion, and alkyl radicals have already
been reported.2–4 In the case of the hydrogen atom (OH),
which is known to be generated biologically, however, no prac-
tical method has so far been established. Although DMPO
traps OH, in many cases it is hard to identify OH formation
from the ESR spectra because of a very low OH adduct concen-
tration; therefore other major signals disturb the identifica-
tion.5
The aim of the present work, therefore, was to obtain ESR
spectra of the OH spin adducts of several potent ring nitrone
spin traps and their ESR parameters. Our previous study has
demonstrated a method to observe an ESR spectrum composed
of only DMPO-H, thus allowing us to apply it to other spin
trapping systems.6 In this work, an electrochemical reaction
was conducted in a helical electrochemical ESR cell especially
designed for this purpose, and in situ ESR measurements were
performed. This technique is useful because, whenever a new
spin trap is developed, one can readily obtain the ESR spec-
trum of its OH spin adduct and its ESR parameters.
Results and Discussion
Detection of Hydrogen Atom Adducts of Spin Traps.
We have reported that the ESR spectrum due to the hydrogen
adduct of DMPO was observed after electrolysis upon supply-
ing −1400 mV (vs Ag/Ag+) for an aqueous DMPO solution
(10.0 mM) containing KClO4 (0.1 M). By monitoring the ESR
signal intensity of the DMPO-H adduct, the optimum condi-
tion was confirmed to be follows: applied potential, −1400
mV; time for electrolysis, 30 s. By the same procedure, ESR
spectra ascribable to the OH and OD radical adducts of DMPO
derivatives, except for 2-MPO and 2,4,4-M3PO, were record-
ed; the obtained hyperfine coupling constants (hfcc’s) are list-
ed in Table 1. ESR signals due to the 2-MPO-H and 2,4,4-
M3PO-H radical adducts, however, were never detected, even
by prolonging the time for electrolysis (2 min) and the poten-
tial (below −1800 mV (vs Ag/Ag+)).
Further electrochemical ESR measurements were continued
for CH3CN solutions of DMPO (10 mM) containing 3.0% H2O
in order to obtain the ESR spectra of hydrogen radical adducts.
After supplying −1400 mV (vs Ag/Ag+) to a solution of
DMPO for 30 s, the observed ESR spectrum showed a well re-
solved hyperfine structure, and the hfcc values of nitrogen and
methylene protons at the 2-position were estimated to be aN =
Experimental
The used ESR electrochemical cell was a REL-001 of Radical

150 Bull. Chem. Soc. Jpn., 75, No. 1 (2002)
© 2002 The Chemical Society of Japan
Table 1. Hfcc Values (in mT) of Hydrogen Atom Adducts of
DMPO and Its Derivatives
H hyperfine coupling constant in mT
Spin trap Solvent
aN aH(2) c) aH(2) c) aH(5) d) aH(5) d)
DMPO
H2O
1.66 2.28
1.66 2.27
1.52 2.01
1.52 2.02
2.28
0.35 f) N.R.e) N.R.e)
2.01
N.R.e) N.R.e)
0.31f) N.R.e) N.R.e)
N.R.e) N.R.e)
D2O
AC/Ha)
AC/Db)
2-MPO
H2O
not detected
1.51 2.11
1.52 0.33 f)
AC/Ha)
AC/Db)
—
—
1.84
1.85
2.11
2.19
2,4,4-
M3PO
H2O
not detected
1.48 2.22
1.52 0.35 f)
AC/Ha)
AC/Db)
—
—
1.48
1.51
2.38
2.48
3,3,5,5-
M4PO
H2O
1.68 2.20
1.66 2.18
1.53 1.93
1.53 1.94
2.20
N.R.e) N.R.e)
D2O
0.33 f) N.R.e) N.R.e)
Fig. 2. ESR spectra obtained for DMPO and DMPO deriva-
tives in CH3CN containing 3.0% H2O or D2O and
(n-Bu)4NBF4 (0.1 M), by the in situ electrochemical ESR
method at 25 °C. (a) was observed for CH3CN solution
DMPO (10.0 mM), containing D2O (3.0%), and (b), and
(c) were observed for 2-MPO and 2,4,4-M3PO, in the pres-
ence of 3.0% H2O. Electrochemical reduction was per-
formed at −1,400 mV (vs Ag/Ag+) for 30 s and the spectra
were obtained after turning it off. The stick diagrams illus-
trated below the ESR spectrum correspond to the estimated
hfcc values.
AC/Ha)
AC/Db)
1.93
N.R.e) N.R.e)
0.30 f) N.R.e) N.R.e)
4-Ph-
DMPO
H2O
1.64 1.82
1.64 1.81
1.51 1.62
1.52 1.63
2.68
N.R.e) N.R.e)
0.41 f) N.R.e) N.R.e)
2.37
N.R.e) N.R.e)
0.36 f) N.R.e) N.R.e)
D2O
AC/Ha)
AC/Db)
5-Ph-
5-MPO
H2O
1.62 2.21
1.59 2.18
1.46 1.91
1.46 1.91
2.28
N.R.e) N.R.e)
0.34 f) N.R.e) N.R.e)
2.25
N.R.e) N.R.e)
0.31 f) N.R.e) N.R.e)
D2O
AC/Ha)
AC/Db)
time on the ESR spectra of the OH spin adducts of 2,4,4-M3PO,
2-MPO, 4-Ph-DMPO, and 5-Ph-5-MPO. This method allows
us to detect the hydrogen adducts of newly developed nitrone
spin traps. In addition, the present procedure is suitable for in-
vestigating of biologically important hydrogen atom transfer
reaction processes, such as auto-oxidation of unsaturated fatty
acids. Further investigations on the molecular structure of
short-lived 5-membered nitroxide radicals are in progress.
a) CH3CN containing 3.0% water; b) CH3CN containing
3.0% D2O; c) proton hfcc at 2-position; d) proton hfcc at 5-
position; e) not resolved; f) deuteron hfcc.
1.52, and aH = 2.01 mT, respectively. When 3.0% H2O was re-
placed by D2O, a triplet splitting due to a deuteron atom at the
2-position was observed, as depicted in Fig. 2a. These results
indicate that the electrochemical reduction of H2O or D2O gave
short-lived OH or OD radicals in CH3CN. In fact, no detectable
amount of hydrogen radical adducts of DMPO derivatives
were recorded in the absence of water in CH3CN.
Under the same condition, ESR measurements were per-
formed for CH3CN solutions of DMPO derivatives in the pres-
ence of 3.0% H2O and D2O. An ESR spectrum ascribed to the
OH adducts of 2-MPO was obtained, as shown in Fig. 2b; the
estimated hfcc’s are listed in Table 1. By a comparison of the
hfcc of OH and OD adducts of 2-MPO (Table 1), hfcc’s of aH =
1.84, and aH = 2.11 mT were assigned to the non-equivalent
methylene protons at the 5-position. A similar ESR spectrum
was also observed for 2,4,4-M3PO, as represented in Fig. 2c.
The present results suggest that the lifetime of the hydrogen
adducts 2-MPO and 2,4,4-M3PO may be prolonged in less po-
lar solvents. By the same procedure, ESR spectra were ob-
served for the OH adducts of 3,3,5,5-M4PO, 4-Ph-DMPO and
5-Ph-MPO; the evaluated hfcc’s are listed in Table 1.
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