1512
L. S. Chen et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1509–1512
This work also has illustrated that KFꢀ mediated DNA
synthesis undergoes premature chain termination at
sites of 8-Cl-dAdo in the template strand. Thus,
although 8-Cl-dAdo pairs normally with thymidine,
chain termination at 8-Cl-dAdo sites may have bio-
chemical consequences. For example, decreased DNA
replication efficiency may affect cell division and has
been shown to contribute to the cytotoxic effects of
nucleoside antimetabolites.29 Additional studies with
8-Cl-Ado and 8-Cl-dAdo will be conducted to assess the
effects of 8-modified derivatives on RNA biochemistry
and will explore the potential of 8-modified nucleotides
for the treatment of human disease.
(400 MHz, CD3CN) d: 9.28 (s, 1H, NH), 8.52 (s, 1H, H2), 7.98
(d, 1H, J=7.6 Hz, Bza), 7.63 (dd, 2H, J=7.2 Hz, J=7.2 Hz,
Bzb), 7.53 (t, 2H, J=7.6 Hz, Bzc), 7.17 (m, 9H, DMT, ꢀOPh),
6.74 (m, 4H, DMT), 6.42 (m, 1H, H10), 5.19 (m, 1H, H20), 4.19
(q, 1H, J=5.2 Hz, 30OH), 3.72 (d, 6H, J=4.0 Hz, –OCH3),
3.70-3.58 (m, 1H, H30), 3.34 (m, 1H, H40), 3.19 (m, 2H, H50),
2.54 (m, 1H, ꢀOCH2CH2CN), 2.32 (m, 1H, ꢀOCH2CH2CN),
2.17 (s, 1H,-NH(CH3)2), 1.19 (t, 12H, J=6.4 Hz, –NH(CH3)2).
31P NMR (CD3CN) d: 148.6.
17. Caruthers, M. H.;Barone, A. D.;Beaucage, S. L.;Dodds,
D. R.;Fisher, E. F.;McBride, L. J.;Matteucci, M.;Stabinsky,
Z.;Tang, J. Y. Methods Enzymol. 1987, 154, 287.
18. Dey, S.;Sheppard, T. L. Org. Lett. 2001, 3, 3983.
Unmodified oligonucleotides 7, 8, 10–13 were synthesized by
Integrated DNA Technologies, purified using standard meth-
ods, and characterized by MALDI-TOF MS.
19. Characterization of oligonucleotide 9. MALDI-TOF MS
(m/z): [M]ꢀ calculated, 12261.4;found, 12261.5.
Acknowledgements
20. Stability of 8-Cl-dAdo DNA oligonucleotide. Chlorine
substitution at C-8 enhances the rate of acid-catalyzed depur-
ination of 8-Cl-dAdo (J. Am. Chem. Soc. 1978, 100, 7620.)
However, after DNA synthesis and purification, no abasic site
formation was observed either by MALDI-TOF MS or by
radiolabeling and PAGE analysis of oligonucleotides contain-
ing 8-Cl-dAdo.
We thank our collaborators Dr. Steven Rosen, Dr.
Nancy Krett, and Dr. Varsha Gandhi for insightful
discussions. We acknowledge the Keck Biophysics
Facility and the Analytical Services Laboratory of
Northwestern University, and the University of Illinois,
Urbana-Champaign Mass Spectrometry Laboratory.
This work was supported by the National Institutes of
Health (RO1 CA85915-02) and the Leukemia and
Lymphoma Society (6505-00).
21. Morales, J. C.;Kool, E. T. J. Am. Chem. Soc. 2000, 122,
1001.
22. Single nucleotide insertion with KFꢀ. Radiolabeled
(50–32P) primer 7 was annealed to the appropriate template in
a solution containing 100 mM Tris–HCl (pH 7.5), 20 mM
MgCl2, 2 mM DTT and 0.1 mg/mL BSA. A solution contain-
ing a single dNTP in the same reaction buffer was added to the
annealed primer, and primer extension was initiated by adding
KFꢀ. Aliquots were removed from the reaction at specific
times, and the products were analyzed by 20% dPAGE. Final
extension conditions were 4 mM primer/template duplex, 50
nM KFꢀ, 10 mM dNTP, 150 mM Tris–HCl (pH 7.5), 20 mM
MgCl2, 3 mM 2-mercaptoethanol, 1 mM DTT and 0.05 mg/
mL BSA.
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16. Characterization of phosphoramidite 6. 1H NMR