J. Am. Chem. Soc. 1996, 118, 905-906
905
technology may fail. Albeit considerably more persistent than
most free radicals, nitroxides are nevertheless often subject to
the usual free radical destruction processes of combination,
disproportionation, and oxidation/reduction, yielding diamag-
netic products. The rapid formation of diamagnetic spin adducts
in traditional spin trapping experiments is an unwanted occur-
rence which can constitute a serious obstacle, since once such
products are formed in biological systems employing conven-
tional nitrone spin traps, they are lost among a vast number of
diverse diamagnetic molecules.1a,7 The ability to easily locate
diamagnetic spin adducts in complex mixtures offers an
appealing alternative should one be faced with technical
difficulties often encountered while attempting to isolate ni-
troxides resulting from conventional nitrone spin traps before
they decay into diamagnetic species.1a,d In spin trapping with
1, the characteristic chromophore of the diamagnetic spin
adducts arising from nitroxides via combination, disproportion-
ation, or reduction, while crucially different from the chro-
mophore of 1, is in fact the same as that of the initially formed
ESR-detectable nitroxide spin adducts. Therefore, this char-
acteristic chromophore should also expedite the purification (and
subsequent structure determination) of these paramagnetic
species from reaction mixtures amenable to nitroxide longevity.8
Nitrone 1 (R ) OEt) is a stable green solid (mp 43-45 °C)
and is readily prepared in three steps from guaiazulene (Scheme
1). Exposure of guaiazulene to oxalyl bromide in ether at room
temperature according to the method of Treibs9 yields acyl
bromide 2, which is directly esterified with EtOH to provide
the violet ethyl ester 3 in 80% yield. Oxidation of 3 with 2
equiv of DDQ in aqueous acetonitrile at room temperature in
analogy to the method of Takase10 affords a 74% yield of red
aldehyde 4. Condensation of 4 with N-tert-butylhydroxylamine
hydrochloride in pyridine at 95 °C provides 1 (R ) OEt) in
nearly quantitative yield.11
Highly Sensitive Colorimetric Detection and Facile
Isolation of Diamagnetic Free Radical Adducts of
Novel Chromotropic Nitrone Spin Trapping Agents
Readily Derived from Guaiazulene
David A. Becker
Department of Chemistry, Florida International UniVersity
UniVersity Park, Miami, Florida 33199
ReceiVed August 22, 1995
The technique of spin trapping is an important method for
garnering information on free radicals difficult or impossible
to detect by direct spectroscopic observation due to their
exceedingly short lifetimes and low concentrations.1 To date,
two classes of spin trapping agents have received the most
attention, namely, nitroso compounds and nitrones. Of these,
the latter have been more frequently used, especially in
biological systems. The most commonly cited drawbacks to
the application of spin trapping agents bearing nitroso func-
tionality are instability and toxicity.1a On account of these
undesirable characteristics, researchers often opt for nitrone spin
traps despite the fact that their nitroxide spin adducts generally
provide less structural information from ESR than do those from
nitroso-based spin traps. Furthermore, the nitroxides obtained
from the addition of certain carbon-centered radicals (tertiary
alkyl and aryl) to the most widely used nitrone spin traps (PBN,2
POBN,3 and DMPO4) are, due primarily to disproportionation,1a
less persistent than those obtained from addition of such radicals
to nitroso compounds. Several groups have described the use
of isotopically labeled spin traps5 or the application of special
equipment consisting of GC/MS or HPLC-interfaced ESR
spectrometers1a,6 designed to detect, isolate, and characterize
free radical adducts of nitrone spin traps in biological systems
with varied success. Herein is reported a new and simple
colorimetric approach to the detection, isolation, and analysis
of free radical adducts of nitrones employing the novel nitrone
spin trapping agents 1 which are easily obtained from the
abundant sesquiterpene guaiazulene.
Of particular importance regarding spin trapping with 1 is
their capacity to tag free radicals by yielding characteristically
colored and highly visible diamagnetic (and paramagnetic) spin
adducts. Thus, nitrones 1 proVide the potential to implicate
the intermediacy and establish the identity of free radicals in
situations in which presently aVailable ESR detection/isolation
The obvious chromotropism that accompanies conversion of
nitrone spin traps 1 to diamagnetic free radical adducts arising
(7) (a) Iwamura, M.; Inamoto, N. Bull. Chem. Soc. Jpn. 1967, 40, 702.
(b) Janzen, E. G.; Krygsman, P. H.; Lindsay, D. A.; Haire, D. L. J. Am.
Chem. Soc. 1990, 112, 8279. (c) Even mild biological reducing agents
such as cysteine (in the presence of traces of ferric ion) and ascorbic acid
will reduce nitroxides to the corresponding hydroxylamine. See: McCo-
nnell, H. M.; McFarland, B. G. Q. ReV. Biophys. 1970, 3, 91. See also:
Sentjurc, M.; Mason, R. P. Free Radical Biol. Med. 1992, 13, 151.
(8) Even though nitroxides possess a visible chromophore of their own,
their characteristic red color is due to an absorption with a very low
extinction coefficient centered around 460 nm. For example, the visible
absorption spectrum in hexane for di-tert-butylnitroxide shows a maximum
at 465 nm with log ꢀ ) 0.95. The extinction coefficient for the absorption
giving rise to the color of the diamagnetic azulene-containing spin adducts
described herein is between 1 and 2 orders of magnitude greater. See:
Smith, P. A. S. Open-Chain Nitrogen Compounds; W. A. Benjamin, Inc.:
New York, 1965; Vol. 2, p 105 and references cited therein.
(1) (a) Janzen, E. G.; Haire, D. L. In AdVances in Free Radical Chemistry;
Tanner, D. D., Ed.; JAI Press: Greenwich, 1990; Vol. 1, p 253. (b) Knecht,
K. T.; Mason, R. P. Arch. Biochem. Biophys. 1993, 303, 185. (c)
Kalyanaraman, B. In ReViews in Biochemical Toxicology; Hodgson, E.,
Bend, J. R., Philpot, R. M., Eds.; Elsevier Biomedical: New York, 1980;
Vol. 4, p 73. (d) Perkins, M. J. AdV. Phys. Org. Chem. 1980, 17, 1. (e)
Rehorek, D. Chem. Soc. ReV. 1991, 20, 341. (f) Mason, R. P.; Chignell,
C. F. In Free Radical Damage and Its Control; Rice-Evans, C. A., Burdon,
R. H., Eds.; Elsevier Science: New York, 1994; p 319.
(2) Janzen, E. G. Acc. Chem. Res. 1971, 4, 31.
(3) Janzen, E. G.; Wang, Y. Y.; Shetty, R. V. J. Am. Chem. Soc. 1978,
100, 2923.
(4) Iwamura, M.; Inamoto, N. Bull. Chem. Soc. Jpn. 1967, 40, 703.
(5) (a) Barasch, D.; Krishna, M. C.; Russo, A. J. Am. Chem. Soc. 1994,
116, 7319. (b) Janzen, E. G.; Zhang, Y. K.; Haire, D. L. J. Am. Chem.
Soc. 1994, 116, 3738. (c) Keana, J. F. W.; Lex, L.; Mann, J. S.; May, J.
M.; Park, J. H.; Pou, S.; Prabhu, V. S.; Rosen, G. M.; Sweetman, B. J.;
Wu, Y. Pure Appl. Chem. 1990, 62, 201. (d) Pou, S.; Rosen, G. M.; Wu,
Y.; Keana, J. F. W. J. Org. Chem. 1990, 55, 4438. (e) Haire, D. L.; Oehler,
U. M.; Krygsman, P. H.; Janzen, E. G. J. Org. Chem. 1988, 53, 4535. (f)
Motten, A. G.; Levy, L. A.; London, R. E. J. Magn. Reson. 1988, 80, 112.
(g) Janzen, E. G.; Oehler, U. M.; Haire, D. L.; Kotake, Y. J. Am. Chem.
Soc. 1986, 108, 6858.
(6) (a) Kominami, S.; Rokushika, S.; Hatano, H. Int. J. Radiat. Biol.
1976, 30, 525. (b) Janzen, E. G.; Towner, R. A.; Krygsman, P. H. Free
Radical Res. Commun. 1990, 9, 353. (c) Iwahashi, H.; Albro, P. W.;
McGown, S. R.; Tomer, K. B.; Mason, R. P. Arch. Biochem. Biophys. 1991,
285, 172.
(9) Treibs, W.; Orttmann, H.; Schlimper, R.; Lindig, C. Chem. Ber. 1959,
92, 2152.
(10) Amemiya, T.; Yasunami, M.; Takase, K. Chem. Lett. 1977, 587.
(11) Spectral data for 1 (R ) OEt): 1H NMR (300 MHz, CDCl3) 9.74
(s, 1H), 8.36 (s, 1H), 8.17 (s, 1H), 7.54 (d, J ) 11 Hz, 1H), 7.43 (d, J )
11 Hz, 1H), 4.37 (q, J ) 7 Hz, 2H), 3.17 (m, 1H), 2.97 (s, 3H), 1.71 (s,
9H), and 1.38-1.43 (m, 9H); 13C NMR (75.4 MHz, CDCl3) 167.2, 148.7,
145.8, 141.3, 141.0, 137.7, 136.8, 132.7, 132.5, 123.0, 120.7, 117.1, 69.6,
60.8, 38.3, 28.4, 27.9, 24.4, and 14.4; IR (neat) 3135, 2965, 2930, 2905,
2870, 1715, 1580, 1560, 1460, 1335, 1300, 1245, 1195, 1150, 1105, 1040,
920 cm-1; UV-vis λmax (hexane) 313 nm (ꢀ ) 26 071), 358 (15 526), 417
(8390), and 588 (532); exact mass (FABMS, NBA) calcd for C22H30NO3
(M+ + 1) 356.2226, found 356.2230.
0002-7863/96/1518-0905$12.00/0 © 1996 American Chemical Society