this lesion may be well-bypassed by polymerases and muta-
genic, as suggested by in vitro primer extension assays.7 NI
therefore may contribute to the mutagenic spectrum of DNA
damage induced by ONOO-, and its measurement may be
useful as a specific biomarker of ONOO--mediated oxida-
tion.
Scheme 1
Evaluation of the chemical and biological properties of
oxidative DNA lesions is facilitated by the availability of
oligodeoxynucleotides (ODN) and, eventually, genomes con-
taining these lesions site-specifically.1 Most commonly,
oxidative lesions are prepared site-specifically by damaging
a specific base within an ODN.10-12 This approach facilitates
and accelerates the initial characterization of these lesions
while circumventing the common problem of lesion instabil-
ity, a major obstacle to incorporation of these damaged bases
by automated DNA synthesis. However, low yields and the
formation of multiple products that are often difficult to
separate limit the use of ODN sequences to those containing
only one G residue. The present methods for preparing NI-
containing ODNs are hindered by both of these character-
istics.7,8 In advance of biological evaluation of NI mutage-
nicity and genotoxicity, we sought to develop a synthetic
method for the preparation of NI lesions site-specifically
within ODNs that is high-yielding and independent of base
sequence.
A convertible nucleoside phosphoramidite offers a con-
venient route to incorporating an unnatural nucleoside at a
defined site within an ODN.13 This approach has been used
successfully to prepare ODNs containing nucleosides that
are unstable to DNA synthesis conditions,14,15 containing
structural analogues of a given base, and with additional
functionality.16-22 In the present case, the use of the con-
vertible nucleoside approach would avoid potential protec-
tion-deprotection schemes with the guanidino functionality
of NI, enabling an efficient synthesis of a phosphoramidite
building block. Herein, we describe the synthesis of a
convertible nucleoside phosphoramidite for the site-specific
incorporation of an NI lesion into an ODN by postsynthetic
substitution and the effect of this lesion on duplex stability.
Although we only present the synthesis of the biologically
relevant NI lesion, the method allows access to other
potentially useful alternative structures.
5(4)-Bromo-4(5)-nitroimidazole (2) was prepared in 91%
yield by nitrating 1.23 Treatment of 2 with NaH in CH3CN,
followed by condensation with 3,5-di-O-toluoyl-R-1-chloro-
2-deoxy-D-ribofuranose gave exclusively two â-nucleosides,
the 5-bromo-4-nitroimidazole isomer 3 in 50% yield and the
4-bromo-5-nitroimidazole isomer in 35% yield. The struc-
tures of these isomers were assigned by comparison of the
UV spectra with those of literature analogues.24,25 Interest-
ingly, 4-halo-5-nitroimidazoles have been reported to be
resistant to nucleophilic displacement of the halo substitu-
ent.26,27 In the present study, the 5-bromo-4-nitroimidazole
isomer readily underwent reaction at the site of the bromo
substituent, whereas the 4-bromo-5-nitroimidazole isomer
was resistant to reaction at the bromo position under the same
conditions.
Saponification of the toluoyl esters of 3 was achieved in
90% yield by treatment with guanidine in MeOH for 3.75 h
at 0 °C. Initial attempts to deprotect 3 using reagents such
as NaOMe in MeOH, NH4OH, and K2CO3 in MeOH were
unsuccessful because of the decomposition of 3, presumably
as a result of displacement of the bromo substituent. The â
conformation of 4 was confirmed by NOE NMR spectros-
copy. Irradiating at the 1′ proton, we observed an NOE
enhancement at the 4′ proton, and by irradiating the imida-
zolic proton NOE enhancements were observed at the 3′
proton and 5′ protons, thus confirming the structure. It is
anticipated that 4 will serve as a branching point for
generating libraries of 5-substituted-4-nitroimidazole nucleo-
sides and, when incorporated into DNA, 5-substituted-4-
nitroimidazole-containing ODNs, since previous studies have
shown the bromo substituent of 5-bromo-4-nitroimidazoles
to be displaced by carbon,25 sulfur and oxygen,27 and
nitrogen9 nucleophiles.
The convertible nucleoside 4 was synthesized in three steps
beginning with 5(4)-bromo-1H-imidazole (1) (Scheme 1).
(10) Henderson, P. T.; Delaney, J. C.; Gu, F.; Tannenbaum, S. R.;
Essigmann, J. M. Biochemistry 2002, 41, 914-921.
(11) Kino, K.; Sugiyama, H. Chem. Biol. 2001, 8, 369-378.
(12) Shafirovich, V.; Mock, S.; Kolbanovskiy, A.; Geacintov, N. E.
Chem. Res. Toxicol. 2002, 15, 591-597.
(13) Verma, S.; Eckstein, F. Annu. ReV. Biochem. 1998, 67, 99-134.
(14) MacMillan, A. M.; Chen, L.; Verdine, G. L. J. Org. Chem. 1992,
57, 2989-2991.
(15) Ikeda, H.; Saito, I. J. Am. Chem. Soc. 1999, 121, 10836-10837.
(16) Xu, Y. Z.; Zheng, Q.; Swann, P. F. J. Org. Chem. 1992, 57, 3839-
3845.
(17) Gao, H.; Fathi, R.; Gaffney, B. L.; Goswami, B.; Kung, P. P.; Rhee,
Y.; Jin, R.; Jones, R. A. J. Org. Chem. 1992, 57, 6954-6959.
(18) Allerson, C. R.; Chen, S. L.; Verdine, G. L. J. Am. Chem. Soc.
1997, 119, 7423-7433.
(19) Haginoya, N.; Ono, A.; Nomura, Y.; Ueno, Y.; Matsuda, A.
Bioconjugate Chem. 1997, 8, 271-280.
(20) Kohgo, S.; Shinozuka, K.; Ozaki, H.; Sawai, H. Tetrahedron Lett.
1998, 39, 4067-4070.
For later use as an analytical standard, the 2′-deoxynucleo-
side of NI (dNI, 5) was synthesized from 4 in nearly
(21) MacMillan, A. M.; Verdine, G. L. J. Org. Chem. 1990, 55, 5931-
5933.
(22) MacMillan, A. M.; Verdine, G. L. Tetrahedron 1991, 47, 2603-
2616.
(23) Del Carmen, M.; Barrio, G.; Scopes, D. I. C.; Holtwick, J. B.;
Leonard, N. J. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 3986-3988.
(24) Gallo, G. G.; Pasqualucci, C. R.; Radaelli, P.; Lancini, G. C. J.
Org. Chem. 1964, 29, 862-865.
(25) Rousseau, R. J.; Robins, R. K.; Townsend, L. B. J. Am. Chem. Soc.
1968, 90, 2661-2668.
(26) Carbon, J. A. J. Org. Chem. 1961, 26, 455-461.
(27) Hasan, A.; Lambert, C. R.; Srivastava, P. C. J. Heterocycl. Chem.
1990, 27, 1877-1883.
246
Org. Lett., Vol. 6, No. 2, 2004