SYNTHESIS OF DNA CONTAINING MODIFIED BASES BY POSTSYNTHETIC SUBSTITUTION - SYNTHESIS OF OLIGOMERS CONTAINING 4-SUBSTITUTED THYMINE - O4-ALKYLTHYMINE, 5-METHYLCYTOSINE, N4-(DIMETHYLAMINO)-5-METHYLCYTOSINE, AND 4-THIOTHYMINE

A strategy is described for synthesis of oligomers modified in the 4-position of thymine by postsynthetic substitution. 4-Triazolothymine phosphoramidite monomer has been prepared in one step from thymine amidite monomer and incorporated into a 12 mer AGCGAAXTCGCT using a DNA-synthesizer. The fully protected oligomer containing 4-triazolothymine, while still bound to CPG-support, was treated at 25-degrees-C with either alcohol/DBU, dilute aqueous NaOH, concentrated aqueous ammonia, 1,1-dimethylhydrazine, or thiolacetic acid, to produce essentially pure oligodeoxynucleotides containing O4-alkylthymine, thymine, 5-methylcytosine, N4-(dimethylamino)-5-methylcytosine [i.e., 4-(2,2-dimethylhydrazino)-5-methylpyrimid-2-one (T(DH))], or 4-thiothymine respectively. This first and efficient synthesis of T(DH) oligomers indicates that this may be a general route to the synthesis of oligomers containing thymine with a reactive group at the 4-position. The melting temperature (Tm) of a DNA duplex containing T(DH):G or T(DH):A pairs was similar to that of a duplex with A:C mismatch.