Assignment of the EPR Spectrum of 5,5-Dimethyl-1-pyrroline
N-Oxide (DMPO) Superoxide Spin Adduct
†
Jean-Louis Cle ´ment,
‡
Nicolas Ferre ´,
§
Didier Siri,
§
Hakim Karoui,
‡
Antal Rockenbauer,
|
and
Paul Tordo*
,‡
UMR 6517, CNRS and Aix-Marseille Universities, Centre de Saint Je ´ ro ˆ me, 13397 Marseilles,
Cedex 20, France, and Chemical Research Center, Institute for Chemistry, P.O. Box 17,
H-125 Budapest, Hungary
ptordo@srepir1.univ-mrs.fr
Received August 24, 2004
Spin trapping consists of using a nitrone or a nitroso compound to “trap” an unstable free radical
as a long-lived nitroxide that can be characterized by electron paramagnetic resonance (EPR)
spectroscopy. The formation of DMPO-OOH, the spin adduct resulting from trapping superoxide
(O
2
•-
) with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), has been exploited to detect the generation
of superoxide in a wide variety of biological and chemical systems. The 12-line EPR spectrum of
DMPO-OOH has been either reported or mentioned in more than a thousand papers. It has been
interpreted as resulting from the following couplings: A
N
= 1.42 mT, A
H
= 1.134 mT, and A
H
γ
(1H) = 0.125 mT. However, the DMPO-OOH EPR spectrum has an asymmetry that cannot be
reproduced when the spectrum is calculated considering a single species. Recently, it was proposed
that the 0.125 mT splitting was misassigned and actually results from the superimposition of two
individual EPR spectra associated with different conformers of DMPO-OOH. We have prepared
5,5-dimethyl-[3,3-
2
H
2
]-1-pyrroline N-oxide (DMPO-d
2
), and we showed that the EPR spectrum of
the corresponding superoxide spin adduct is composed of only six lines, in agreement with the
assignment of the 0.125 mT splitting to a γ-splitting from a hydrogen atom bonded to carbon 3 of
DMPO. This result was supported by DFT calculations including water solvation, and the asymmetry
of the DMPO-OOH EPR spectrum was nicely reproduced assuming a chemical exchange between
two conformers.
Introduction
Since the discovery
1
of superoxide dismutase (SOD),
the role of superoxide (O
2
•-
) has been investigated in a
myriad of biological processes. Spin trapping coupled with
electron paramagnetic resonance (EPR) is intensively
used to characterize free radicals in biological milieu,
with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) being a
popular spin trap for these studies. The formation of the
spin adduct (DMPO-OOH) resulting from trapping su-
peroxide with DMPO (Scheme 1) has been exploited to
detect the generation of O
2
•-
in a wide variety of biological
and chemical systems.
* Address correspondence to this author. Phone: + 33 491 632 851.
Fax: +33 491 288 758.
†
Part of this work was presented at the annual meeting of the Royal
Chemical Society ESR group (Warwick U.K., March 29, 2004).
‡
Laboratoire “Structure et Re ´activite ´ des Espe `ces Paramagne ´tiques”
(SREP), CNRS and Aix-Marseille Universities.
§
Laboratoire de Chimie The ´orique et Mode ´lisation Mole ´culaire
(LCT2M), CNRS and Aix-Marseille Universities.
|
Chemical Research Center, Institute for Chemistry.
(1) McCord, J. M.; Fridovich, I. J. Biol. Chem. 1969, 244, 6049-
6055.
1198 J. Org. Chem. 2005, 70, 1198-1203
10.1021/jo048518z CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/13/2005