Solid-phase synthesis and side reactions of oligonucleotides containing O-alkylthymine residues

As part of our studies on the molecular mechanism of mutation [Chambers, R. W. (1982) in Molecular and Cellular Mechanisms of Mutagenesis (Lemontt, J. F., & Generoso, W. M., Eds.) pp 121-145, Plenum, New York and London], we wanted to prepare specific oligonucleotides carrying O2- or O4-alkylthymidine residues. Since O-alkylthymine moieties are known to be alkali labile, side reactions were expected during the deprotection procedures used for synthesis of oligonucleotides on a solid support by the classical phosphoramidite method. We have studied these side reactions in detail. Kinetic data show the deprotection procedures displace most O-alkyl groups at rates that make these procedures inappropriate for synthesis of most oligonucleotides carrying O-alkylthymine moieties. We describe alternative deprotection procedures, using readily accessible reagents, that we have used successfully to synthesize a series of oligonucleotides carrying several different O-alkylthymine moieties. The oligonucleotides synthesized are d(A-A-A-A-G-T-alkT-T-A-A-A-A-C-A-T), where alk = O2-methyl, O2-isopropyl, O4-methyl, O4-isopropyl, and O4-n-butyl. This work extends the previously described procedure for the chemical synthesis of oligonucleotides carrying an O4-methylthymine moiety [Li, B. F., Reese, C. B., & Swann, P. F. (1987) Biochemistry 26, 1086-1093] and reports the first chemical synthesis of an oligonucleotide carrying an O2-alkylthymine. The oligonucleotides synthesized have a sequence corresponding to the minus strand that is complementary to the viral strand at the start of gene G in bacteriophage phi X174 replicative form DNA where the normal third codon has been replaced with the ocher codon, TAA.