Synthesis of N3-Methyluridine- and 2'-O-Alkyl/2'-Fluoro-N3-Methyluridine-Modified Phosphoramidites and Their Incorporation into DNA and RNA Oligonucleotides.

Abstract

In this article, we describe the synthesis of N3-methyluridine (m3U) and 2'-O-alkyl/2'-fluoro-N3-methyluridine (2'-O-alkyl/2'-F-m3U) phosphoramidites as well as their incorporation into a 14-mer DNA and RNA oligonucleotide sequence. Synthesis of the 2'-O-alkyl-m3U phosphoramidite starts with commercially available uridine to achieve a tritylated m3U intermediate, followed by 2'-O-alkylation and finally phosphitylation. Synthesis of the 2'-F-m3U phosphoramidite is obtained from a commercially available 2'-F-uridine nucleoside. These phosphoramidite monomers are compatible with DNA and RNA oligonucleotide synthesis using conventional phosphoramidite chemistry. This strategy offers efficient synthetic access to various modifications at the 2'-position of m3U that can be employed in numerous nucleic acid-based therapeutic applications, including antisense technologies, small interfering RNAs, CRISPR-Cas9, and aptamers. The data presented in this article are based on our previously published reports. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 2'-O-alkyl-N3-methyluridine analogs and their corresponding phosphoramidites Alternate Protocol 1: Synthesis of 2'-O-TBDMS-N3-methyluridine and its phosphoramidite Alternate Protocol 2: Synthesis of 2'-fluoro-N3-methyluridine and its phosphoramidite Basic Protocol 2: Solid-phase synthesis of N3-methyluridine-modified DNA and RNA oligonucleotides.