Nucleic Acids Research Advance Access published online on August 26, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl572
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© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Chemistry |
Chemical synthesis of oligodeoxyribonucleotides containing the Dewar valence isomer of the (6–4) photoproduct and their use in (6–4) photolyase studies
1 Division of Chemistry, Graduate School of Engineering Science, Osaka University 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan 2 Department of Molecular Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute 10550 North Torrey Pines Road, La Jolla, CA 92037, USA 3 Radiation Biology Center, Kyoto University Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
*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 April 8, 2006. Revised July 14, 2006. Accepted July 20, 2006.
The pyrimidine(6–4)pyrimidone photoproduct, a major UV lesion formed between adjacent pyrimidine bases, is transformed to its Dewar valence isomer upon exposure to UVA/UVB light. We have synthesized a phosphoramidite building block of the Dewar photoproduct formed at the thymidylyl(3'–5')thymidine site and incorporated it into oligodeoxyribonucleotides. The diastereoisomers of the partially protected dinucleoside monophosphate bearing the (6–4) photoproduct, which were caused by the chirality of the phosphorus atom, were separated by reversed-phase chromatography, and the (6–4) photoproduct was converted to the Dewar photoproduct by irradiation of each isomer with Pyrex-filtered light from a high-pressure mercury lamp. The Dewar photoproduct was stable under both acidic and alkaline conditions at room temperature. After characterization of the isomerized base moiety by NMR spectroscopy, a phosphoramidite building block was synthesized in three steps. Although the ordinary method could be used for the oligonucleotide synthesis, benzimidazolium triflate as an alternative activator yielded better results. The oligonucleotides were used for the analysis of the reaction and the binding of Xenopus (6–4) photolyase. Although the affinity of this enzyme for the Dewar photoproduct-containing duplex was reportedly similar to that for the (6–4) photoproduct-containing substrate, the results suggested a difference in the binding mode.