Synthesis of 4'-C-(aminoethyl)thymidine and 4'-C-[(N-methyl)aminoethyl]thymidine by a new synthetic route and evaluation of the properties of the DNAs containing the nucleoside analogs

Abstract

A gapmer-type antisense oligonucleotide is an oligonucleotide therapeutic that targets pathogenic mRNA directly, and it is expected to be a next-generation therapeutic drug. In this study, we designed and synthesized 4'-C-[(N-methyl)aminoethyl]-thymidine (4'-MAE-T) as a novel nucleoside analog and compared its properties with those of 4'-C-aminoethyl-thymidine (4'-AE-T). Furthermore, we designed a new synthetic route for 4'-C-aminoethyl-modified nucleosides and accomplished the synthesis of 4'-AE-T via a novel pathway with high total yield. DNA containing 4'-MAE-T analogs decreased RNA affinity slightly more than unmodified DNA and DNA containing 4'-AE-T, but significantly improved nuclease resistance compared to unmodified DNA in a solution containing bovine serum. In addition, the impact of 4'-MAE-T on DNA stability was higher than that of 4'-AE-T. Also, DNA containing these analogs can activate Escherichia coli-derived RNase H. Thus, 4'-MAE-T has the potential to be used in gapmer-type antisense nucleic acids as a suitable candidate for the development of therapeutic antisense oligonucleotides.

Keywords: 4′-aminoalkyl group; Antisense oligonucleotide; Nuclease resistance; RNase H; Thermal stability.