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Nucleic Acids Research 2006 34(5):1540-1551; doi:10.1093/nar/gkl061
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Published online 17 March 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

Article

Synthesis and characterization of oligonucleotides containing 2'-fluorinated thymidine glycol as inhibitors of the endonuclease III reaction

Yusuke Doi1, Atsushi Katafuchi2, Yoshie Fujiwara1, Kenichi Hitomi1,3, John A. Tainer3, Hiroshi Ide2 and Shigenori Iwai1,*

1Division of Chemistry, Graduate School of Engineering Science, Osaka University 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan 2Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University Higashi-Hiroshima, Hiroshima 739-8526, Japan 3Department of Molecular Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

*To whom correspondence should be addressed. Tel: +81 6 6850 6250; Fax: +81 6 6850 6240; Email: iwai{at}chem.es.osaka-u.ac.jp

Received February 3, 2006. Revised February 28, 2006. Accepted February 28, 2006.

Endonuclease III (Endo III) is a base excision repair enzyme that recognizes oxidized pyrimidine bases including thymine glycol. This enzyme is a glycosylase/lyase and forms a Schiff base-type intermediate with the substrate after the damaged base is removed. To investigate the mechanism of its substrate recognition by X-ray crystallography, we have synthesized oligonucleotides containing 2'-fluorothymidine glycol, expecting that the electron-withdrawing fluorine atom at the 2' position would stabilize the covalent intermediate, as observed for T4 endonuclease V (Endo V) in our previous study. Oxidation of 5'- and 3'-protected 2'-fluorothymidine with OsO4 produced two isomers of thymine glycol. Their configurations were determined by NMR spectroscopy after protection of the hydroxyl functions. The ratio of (5R,6S) and (5S,6R) isomers was 3:1, whereas this ratio was 6:1 in the case of the unmodified sugar. Both of the thymidine glycol isomers were converted to the corresponding phosphoramidite building blocks and were incorporated into oligonucleotides. When the duplexes containing 2'-fluorinated 5R- or 5S-thymidine glycol were treated with Escherichia coli endo III, no stabilized covalent intermediate was observed regardless of the stereochemistry at C5. The 5S isomer was found to form an enzyme–DNA complex, but the incision was inhibited probably by the fluorine-induced stabilization of the glycosidic bond.


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Nucleic Acids Res., March 12, 2007; 35(5): 1601 - 1611.
[Abstract] [Full Text] [PDF]


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