A Building Block for the Sequence-Specific Introduction of Cis-Syn Thymine Dimers into Oligonucleotides. Solid-Phase Synthesis of TpT[c,s]pTpT

We report the synthesis of a synthetic intermediate for the sequence specific incorporation of the cis—syn thymine dimer into oligonucleotides via phosphoramidite-based solid-phase DNA synthesis technology. The required phosphoramidite la was obtained in four steps from the known, pT-0₃/-(fe/7-butyldimethylsilyl)thymidylyl-(3'—'^50-thymidine, R,S-O-methyl phosphate, compounds 6a,b. Compounds 6a,b were photolyzed with Pyrex-filtered light in the presence of the triplet sensitizer, acetophenone. The resulting four products, 12a, 12b, 8a, and 8b, were separated by a combination of flash chromatography and preparative reverse phase gradient HPLC. The structures of the four products were determined by complete deprotection and correlation with authentic cis—syn and trans-syn cyclobutane dimers of TpT, 9 and 13, obtained from the photosensitized irradiation of thymidylyl-(3'-^5')-thymidine 7. Compound 8a, one of the two protected cis-syn isomers epimeric at phosphorus, was converted to the Tp-195 derivative 10a, followed by removal of the tert-butyldimethylsilyl group to give 11a. Compound 11a was converted to the target pT-03 morpholinylphosphonite la which was purified by flash chromatography. The cis-syn thymine dimer containing phosphoramidite was coupled to thymidine derivatized controlled pore glass support and further extended by an additional thymidine. The methyl phosphate protecting groups were removed, and the resulting thymine dimer containing tetranucleotide 14 was obtained after cleavage from the support. Tetrathymidylate 15 was prepared in a similar fashion for comparison purposes. Photolysis of 14 at 254 nm gave 15 as the major photoreversion product, thus establishing the integrity of the cis-syn thymine dimer unit. Both oligonucleotides were characterized by *H and³¹P NMR spectroscopy, C-18 and anion exchange HPLC chromatography, high resolution gel electrophoresis, and by a chemical degradation sequence. It was also established that the cis—syn cyclobutane dimer of TpT was stable to the conditions required to remove the standard amino protecting groups of A, C, and G. This makes the building block la applicable to the synthesis of cis—syn thymine dimer containing oligonucleotides of heterogeneous sequence.