Synthesis of <i>N</i>³‐Methyluridine‐ and 2′‐<i>O</i>‐Alkyl/2′‐Fluoro‐<i>N</i>³‐Methyluridine‐Modified Phosphoramidites and Their Incorporation into DNA and RNA Oligonucleotides

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.