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 N 3 ‐methyluridine (m 3 U) and 2′‐ O ‐alkyl/2′‐fluoro‐ N 3 ‐methyluridine (2′‐ O ‐alkyl/2′‐F‐m 3 U) phosphoramidites as well as their incorporation into a 14‐mer DNA and RNA oligonucleotide sequence. Synthesis of the 2′‐ O ‐alkyl‐m 3 U phosphoramidite starts with commercially available uridine to achieve a tritylated m 3 U intermediate, followed by 2′‐ O ‐alkylation and finally phosphitylation. Synthesis of the 2′‐F‐m 3 U 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 m 3 U 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‐ N 3 ‐methyluridine analogs and their corresponding phosphoramidites Alternate Protocol 1 : Synthesis of 2′‐ O ‐TBDMS‐ N 3 ‐methyluridine and its phosphoramidite Alternate Protocol 2 : Synthesis of 2′‐fluoro‐ N 3 ‐methyluridine and its phosphoramidite Basic Protocol 2 : Solid‐phase synthesis of N 3 ‐methyluridine‐modified DNA and RNA oligonucleotides