Yamamoto J et al. (2006), Chemical synthesis of oligodeoxyribonucleotides...

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Nucleic Acids Res 2006 Jan 01;3416:4406-15. doi: 10.1093/nar/gkl572.

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Chemical synthesis of oligodeoxyribonucleotides containing the Dewar valence isomer of the (6-4) photoproduct and their use in (6-4) photolyase studies.

Yamamoto J , Hitomi K , Todo T , Iwai S .

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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.

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	Scheme 1. The (6–4) photoproduct (2) and its Dewar valence isomer (3) formed at the TT site (1).
	Scheme 2. Synthesis of the building block of the Dewar photoproduct.
	Figure 1. (A) Formation of the Dewar photoproduct. The dinucleoside monophosphate of the (6–4) photoproduct (2) was irradiated with Pyrex-filtered light from a 100 W high-pressure mercury lamp for 0 h (a), 1 h (b), 2 h (c) and 4 h (d), and the samples were analyzed by HPLC, using an acetonitrile gradient from 0 to 5% for 16 min. (B) Absorption spectra of 2 (a) and the irradiation product (b). The absorption spectra of the two peaks of trace b in (A) were extracted with the data processing software for the photodiode array detector.
	Figure 2. (A) Conversion of 4a and 4b to 5a and 5b. Compounds 4a (a) and 4b (c) were irradiated with a 450 W high-pressure mercury lamp for 1 h, and the starting materials (a and c) and the reaction mixtures (b and d) were analyzed by HPLC, using an acetonitrile gradient from 11 to 17% for 20 min. The thick and thin lines show the chromatograms monitored at 325 and 245 nm, respectively. (B) Deprotection of 5a and 5b. Aliquots of 5a and 5b were treated with aqueous ammonia at room temperature for 2 h, and were analyzed by HPLC after evaporation (a and b, respectively). (c) A mixture of the deprotected compounds was co-injected with the authentic Dewar photoproduct (trace d in Figure 1A). The acetonitrile gradient was the same as that described in the legend to Figure 1A.
	Figure 3. Reversed-phase HPLC analysis of crude oligonucleotides after deprotection. (A and B) The 20mer (A) and the 30mer (B) synthesized by the ordinary method using tetrazole. (C) The 20mer synthesized by the BIT-activation method. The HPLC conditions are described in Materials and Methods.
	Figure 4. (A) Reversed-phase HPLC analysis of the Dewar 12mer. (a) The 12mer synthesized in this study, (b) co-injection with the Dewar photoproduct-containing 12mer prepared by irradiation of the (6–4) photoproduct-containing 12mer and (c) co-injection with the (6–4) photoproduct-containing 12mer with the same sequence context. The thick and thin lines show the chromatograms monitored at 325 and 254 nm, respectively, and the 325 nm chromatogram is magnified by a factor of 5. (B) Analysis of the (6–4) photolyase reaction with the (6–4) 12mer (a) and the Dewar 12mer (b and c). The reaction times were 3 h (a and b) and 24 h (c). The HPLC conditions are described in Materials and Methods.
	Figure 5. (A and B) Fluorescence emission spectra of the Dewar (red), (6–4) (blue) and TT (green) 20mers hybridized to a complementary strand containing 2-aminopurine in the absence (A) and in the presence (B) of the (6–4) photolyase. (C and D) Fluorescence emission spectra of the (6–4) 20mer hybridized to a complementary strand without 2-aminopurine in the absence (C) and in the presence (D) of the (6–4) photolyase.

References [+] :

Christine, Cyclobutylpyrimidine dimer base flipping by DNA photolyase. 2002, Pubmed