Ester s of 5-Ca r boxyl-5-m eth yl-1-p yr r olin e N-Oxid e: A F a m ily of
Sp in Tr a p s for Su p er oxid e
Pei Tsai,† Kazuhiro Ichikawa,‡,§, Colin Mailer,‡,§ Sovitj Pou,† Howard J . Halpern,‡,§
Bruce H. Robinson,| Robert Nielsen,| and Gerald M. Rosen*,†,§
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy,
Baltimore, Maryland 21201, Medical Biotechnology Center, University of Maryland Biotechnology
Institute, Baltimore, Maryland 21201, Department of Radiation and Cellular Oncology, The University of
Chicago, Illinois 60637, Center for Low-Frequency EPR Imaging for In Vivo Physiology, The University of
Chicago, Chicago, Illinois 60637, University of Maryland, Baltimore, Baltimore, Maryland 21201, and
Department of Chemistry, University of Washington, Seattle, Washington 98195
grosen@umaryland.edu
Received J uly 18, 2003
Apparent rate constants, at acidic pH and neutral pH for the reaction of a family of ester-containing
5-carboxyl-5-methyl-1-pyrroline N-oxides with superoxide (O2•-) were estimated, using ferricyto-
chrome c as a competitive inhibitor. It was of interest to note that the rate constants were similar
among the different nitrones and not that significantly different from that found for 5-(diethoxy-
•-
phosphoryl)-5-dimethyl-1-pyrroline N-oxide. At acidic pH, the rate constant for spin trapping O2
was 3-fold greater than that at physiological pH. Subsequent experiments determined the half-life
of aminoxyls, derived from the reaction of these nitrones with O2•-. The EPR spectra were modeled
by using a global analysis method. The results clearly demonstrated that EPR spectra of all the
aminoxyls were inconsistent with a model that included a single γ-hydrogen splitting. A better
interpretation modeled them as two diastereomers with identical nitrogen splittings and slightly
different â-hydrogen splittings. Detailed line width analyses slightly favored an equal line width-
unequal population ratio for the two diastereomers.
In tr od u ction
3.5b,6 Aminoxyl 3 can also arise from the reaction of
nitrone 1 with HO• (Scheme 1). Further, reaction of
Spin trapping/EPR spectroscopy is one of the most
reliable methods for identifying free radicals, especially
in biological systems.1 In fact, were it not for spin
trapping, the observation that nitric oxide synthase
nitrone 1 with O2 is slow, at ∼15 M-1 s-1 at pH 7.0.6a
•-
Finally, nitrone 1 is susceptible to a variety of oxidative
reactions,6b,7 thereby further limiting its utility as a spin
•-
trap for O2 in biological milieu.
•-
(NOS; EC 1.14.13.39)2 generates O2 might have re-
Attempts to improve the reactivity of nitrones toward
mained undiscovered for many years,3 and H2O2 would
have been considered the sole NOS reduction product of
O2.4
•-
O2 through the inclusion of different substituents on
(3) (a) Pou, S.; Pou, W. S.; Bredt, D. S.; Snyder, S. H.; Rosen, G. M.
J . Biol. Chem. 1992, 267, 24173-24176. (b) Xia, Y.; Roman, L. J .;
Masters, B. S. S.; Zweier, J . L. J . Biol. Chem. 1998, 273, 22635-22639.
(c) Va´squez-Vivar, J .; Kalyanaraman, B.; Marta´sek, P.; Hogg, N.;
Masters, B. S. S.; Karoui, H.; Tordo, P.; Pritchard, K. A., J r. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 9220-9225. (d) Xia, Y.; Tsai, A.-L.; Berka,
V.; Zweier, J . L. J . Biol. Chem. 1998, 273, 25804-25808. (e) Pou, S.;
Keaton, L.; Surichamorn, W.; Rosen, G. M. J . Biol. Chem. 1999, 274,
9573-9580. (f) Va´squez-Vivar, J .; Hogg, N.; Marta´sek, P.; Karoui, H.;
Pritchard, K. A., J r.; Kalyanaraman, B. J . Biol. Chem. 1999, 274,
26736-26742. (g) Tsai, P.; Porasuphatana, S.; Pou, S.; Rosen, G. M.
J . Chem. Soc., Perkin Trans. 2000, 2, 983-988. (h) Yoneyama, H.;
Yamamoto, A.; Kosaka, H. Biochem. J . 2001, 360, 247-253. (i)
Va´squez-Vivar, J .; Marta´sek, P.; Whitsett, J .; J oseph, J .; Kalyanara-
man, B. Biochem. J . 2002, 362, 733-729.
(4) (a) Mayer, B.; J ohn, M.; Heinzel, B.; Werner, E. R.; Wachter,
H.; Schultz, G.; Bo¨hme, E. FEBS Lett. 1991, 288, 187-191. (b) Heinzel,
B.; J ohn, M.; Klatt, P.; Bo¨hme, E.; Mayer, B. Biochem. J . 1992, 281,
627-630.
(5) (a) Harbour, J . R.; Chow, V.; Bolton, J . R. Can. J . Chem. 1974,
52, 3549-3553. (b) Finkelstein, E.; Rosen, G. M.; Rauckman, E. J . Mol.
Pharmacol. 1979, 16, 676-685.
Since the late 1970s, 5,5-dimethyl-1-pyrroline N-oxide
1 has been the primary spin trap for O2•-, owing this
status to the unique 12-line EPR spectrum of aminoxyl
2, arising from the reaction of O2 with nitrone 1.5
•-
Unfortunately, aminoxyl 2 has a short lifetime, decom-
posing into several compounds, one of which is aminoxyl
* Address correspondence to Gerald M. Rosen at the University of
Maryland School of Pharmacy. Phone: 410-706-0514. Fax: 410-706-
8184.
† University of Maryland School of Pharmacy and University of
Maryland Biotechnology Institute.
‡ Department of Radiation and Cellular Oncology, The University
of Chicago.
§ Center for Low-Frequency EPR Imaging for In Vivo Physiology,
The University of Chicago, and the University of Maryland.
On leave from Kyushu University, Fukuoka, J apan.
| University of Washington.
(1) (a) There are a large number of review articles on this subject.
For a classical paper on this topic, see: J anzen, E. G. Acc. Chem. Res.
1971, 4, 31-40. (b) Finkelstein, E.; Rosen, G. M.; Rauckman, E. J .
Arch. Biochem. Biophys. 1980, 200, 1-16.
(2) Stuehr, D. J .; Kwon, N. S.; Nathan, C.; Griffith, O. W.; Feldman,
P. L.; Wiseman, J . J . Biol. Chem. 1991, 266, 6259-6263.
(6) (a) Finkelstein, E.; Rosen, G. M.; Rauckman, E. J . J . Am. Chem.
Soc. 1980, 102, 4994-4999. (b) Makino, K.; Hagi, A.; Ide, H.; Mu-
rakami, A.; Nishi, M. Can. J . Chem. 1992, 70, 2818-2827. (c) Hanna,
P. M.; Chamulitrate, W.; Mason, R. P. Arch. Biochem. Biophys. 1992,
296, 640-644.
10.1021/jo0350413 CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/30/2003
J . Org. Chem. 2003, 68, 7811-7817
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