1460 J. Am. Chem. Soc., Vol. 121, No. 7, 1999
Gadosy and McClelland
Scheme 1
by protonation by the solvent of the initially formed singlet
nitrene 10a. A similar approach has been described with
2-azidofluorene24 and with p-methoxyphenyl azide 9b.25 Singlet
nitrenes are highly reactive species,26,27 but they are also very
basic24 and thus can be trapped by protonation.24,25,27,28
In this paper we report a study of the aqueous solution
photochemistry of 2-azido-1-methylimidazole 12. Our objective
was to see if this system produces the cation 2+, in the same
manner as the aryl azides 9. There is indeed evidence from both
product analysis and flash photolysis experiments that this does
happen, although cation formation competes with a ring opening
typical of heterocyclic azides.29
however serve as a source of glyoxal, forming appropriate
derivatives with reagents capable of reacting with glyoxal,
including guanosine.13,17 A product capable of reacting like
glyoxal is seen in cells18 and in ViVo in man19 following
treatment with the 2-nitroimidazole misonidazole.
A kinetic study has suggested that 1 reacts by initial formation
of the cationic intermediate 2+.14 At pH 7, this occurs by simple
N-O bond heterolysis, although under appropriate conditions
there are pathways involving catalysis by H+ and added buffer
acids. Low concentrations of thiols such as GSH divert 2+ to 6
and the cis:trans forms of 7.13d,20 This occurs with no change
in the rate constant for the disappearance of 1,14 classic evidence
for a reaction where the nucleophile reacts after the rate-limiting
step. Even phosphate buffer is capable of trapping the cation,
forming the cis:trans adducts 8.13d
Although theoretical calculations suggest that the structure
more closely resembles 2+(b),21 the cation can also be viewed
as a nitrenium ion when written as 2+(a). Such electrophiles
have been implicated in the DNA binding observed with car-
cinogenic amines such as 4-aminobiphenyl and 2-aminofluo-
rene.22 Reduction products of nitroimidazoles have also been
found to bind to DNA.8 Although the species responsible for
this has not been identified, the cation 2+ is an obvious
candidate.
Results
Acidity Constant. A spectroscopic titration curve was
constructed working at 290 nm. At this wavelength, there is a
factor of 2 difference in the extinction coefficients of the imi-
dazole and imidazolium forms of 12. Fitting with the appropriate
equation gave pKa ) 4.05 ( 0.05. A value in satisfactory
agreement of 3.95 was obtained from the pH of an 0.1 M
solution of the HCl salt of 12 that had been half-neutralized
with NaOH.30
Products. (a) Acid Solution. These experiments were carried
out directly in an NMR tube, with solutions of the HCl salt of
12 in D2O containing small amounts of DCl. Upon irradiation,
the characteristic imidazole ring protons give way to upfield
signals at 4.5-5.5 ppm, accompanied by three new methyl
singlets also upfield from the original. As summarized below,
two of these match the methyl peaks of cis-trans 3; the doublets
for the ring protons are also present.13d The third methyl peak
is at 2.54 ppm and corresponds to the methylammonium ion,
as was verified in the 13C NMR spectra. There is no change in
the position or ratio of the three methyl peaks with time, as is
also true for the ring protons. Recording immediately after
irradiation, however, results in a pair of doublets at 4.55 and
4.91 ppm that on standing convert to a singlet at 4.75 ppm.
The latter corresponds to glyoxal bis-hydrate 4, whose presence
was also confirmed in the 13C NMR. The total area of the signals
at 4.55, 4.75, and 4.91 ppm remains constant, equal to two-
thirds the methylammonium peak. A final piece of the puzzle
is the observation in the 13C NMR of a weak peak at 159 ppm,
matching the carbon of cyanamide.
The technique of flash photolysis has recently been used to
generate and directly study arylnitrenium ions,23 including 11a
derived from 4-aminobiphenyl.24 This cation was observed in
aqueous solution upon irradiation of the azide 9a, being formed
(16) Panicucci, R.; McClelland, R. A. Can. J. Chem. 1989, 67, 2128.
(17) (a) Varghese, A. J.; Whitmore, G. F. Cancer Res. 1983, 43, 78. (b)
Raleigh, J. A.; Liu, S. F. Biochem. Pharmac. 1983, 32, 1444. (c) Int. J.
Radiat. Oncol. Biol. Phys. 1984, 10 , 1337. (d) Varghese, A. J.; Whitmore,
G. F. Chem. Biol. Interact. 1985, 56, 269. (e) Silver, A. R. J.; McNeill, S.
S. Biochem. Pharmac. 1985, 34, 3537. (f) Silver, A. R. J.; McNeill, S. S.;
O’Neill, P.; Jenkins, T. C.; Ahmed, I. Biochem. Pharmac. 1986, 35, 3923.
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1984, 10, 1361.
(20) (a) Varghese, A. J. Biochem. Biophys. Res. Commun. 1983, 112,
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Biochem. Pharmac. 1988, 37, 363. (c) Farah, S. Ph.D. Thesis, University
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(21) Bolton, J. L.; McClelland, R. A. J. Mol. Struct., Theochem. 1988,
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(22) (a) Beland, F. A.; Kadlubar, F. F. In Chemical Carcinogenesis and
Mutagenesis; Cooper, C. S., Grover, P. L., Eds.; Springer-Verlag: Berlin,
1990; Vol. 1, pp 267-325. (b) Kadlubar, F. F. In DNA Adducts,
Identification and Biological Significance; Hemminki, K., Dipple, A.,
Shuker, D. E. G., Kadlubar, F. F., Segerback, D., Bartsch, H., Eds.; IARC
Publication No. 125; IARC: Lyon, 1994; pp 199-216. (c) Kennedy, S.
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(26) Schuster, G. B.; Platz, M. S. AdV. Photochem. 1992, 17, 143.
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(28) (a) Michalak, J.; Zhai, H. B.; Platz, M. S. J. Phys. Chem. 1996,
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