Synthesis of 4′‐ <i>C</i> ‐α‐Aminoethoxy‐2′‐ <i>O</i> ‐Methyl‐5‐Methyl‐ and 5‐Propynyl‐Uridine Phosphoramidites for Structurally Optimized Antisense Oligonucleotides

Chemical modifications are fundamental to improving the physicochemical and biological performance of oligonucleotide (ON) therapeutics by enhancing their hybridization affinity, nuclease resistance, and metabolic stability. Among the various sugar modifications developed, 4'-carbon (4'-C) substitutions have attracted significant attention for their ability to reinforce the sugar-phosphate backbone while preserving the natural C3'-endo conformation and Watson-Crick base pairing. The 4'-C-α-aminoethoxy (4'AEo) framework, in particular, provides steric protection against nuclease degradation without impairing RNase H activity, making it a valuable scaffold for next-generation antisense oligonucleotides (ASOs). This article presents optimized and reproducible synthetic protocols for two novel 4'-C-modified uridine phosphoramidite building blocks, 4'AEo^mU (4'-C-α-aminoethoxy-2'-O-methyl-5-methyluridine) and 4'AEo^pU (4'-C-α-aminoethoxy-2'-O-methyl-5-(1-propynyl)uridine), both fully compatible with automated solid-phase oligonucleotide synthesis. The synthesis of 4'AEo^mU begins with commercially available 2'-O-methyl-5-methyluridine, which undergoes iodination of the 5'-hydroxyl group, 4',5'-reductive dehalogenation, and 3'-O-silyl protection, followed by 4',5'-epoxidation and ZnCl₂-mediated nucleophilic epoxide opening with N-trifluoroacetylaminoethanol to afford the α-configured product as the major isomer, as confirmed by NOESY NMR analysis. In the 4'AEo^pU synthetic route, regioselective O-silyl deprotection of the 4'-C-α-aminoethoxy-2'-O-methyluridine derivative (compound 10) is followed by 5-iodination and subsequent Sonogashira-Hagihara coupling to introduce the 5-propynyl substituent. The resulting intermediate then undergoes 5'-O-DMTr protection and phosphitylation to yield the desired phosphoramidite. Both protocols exhibit high stereochemical control, reproducibility, and efficiency, yielding analytically pure intermediates validated by TLC, NMR, and mass spectrometry. Compared with previously reported synthetic approaches relying on glycal or radical intermediates, these methods minimize reaction complexity, improve yields, and maintain compatibility with standard phosphoramidite chemistry. Collectively, the described synthetic routes provide a reliable and scalable platform for generating structurally defined 4'-C-modified uridine derivatives. The resulting phosphoramidites enable the construction of antisense oligonucleotides with enhanced duplex stability, nuclease resistance, and favorable pharmacological properties, thereby advancing the rational design of next-generation ON therapeutics with improved safety and efficacy. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 4'-C-α-aminoethoxy-2'-O-methyl-5-methyluridine phosphoramidite (4AEo^mU) (9) Basic Protocol 2: Synthesis of 4'-C-α-aminoethoxy-2'-O-methyl-5-(1-propynyl) uridine phosphoramidite (4AEo^pU) (15).