A Convenient Method for the Synthesis of 2′‐O‐Cyanoethylated Nucleotides and Their Application in the Solid‐Phase Synthesis of Related RNA Analogs

Abstract

AbstractAlthough small interfering RNA (siRNA) is a key player among gene inhibition therapeutics, there are many obstacles to the development of siRNA drugs due to inherent properties of oligonucleotides, including the unsatisfactory stability of unmodified siRNA, poor pharmacokinetic distribution, and the toxicity induced by off‐target effects. To maximize treatment potency, chemical modification of siRNA has undoubtedly been the most successful strategy by far. Widely applied modifications include phosphorothioate linkages, 2′‐O‐methyl modifications, and 2′‐fluoro modifications, among others. To extend the family of chemical modifications for oligonucleotides, 2′‐O‐cyanoethylated RNA analogs were developed through the replacement of the 2′‐hydroxyl group with a 2′‐O‐cyanoethyl group (‐OCH2CH2CN). This modification can provide several advantages over unmodified RNA, such as increased stability, improved binding affinity to complementary DNA or RNA strands, and resistance to degradation by cellular nucleases. The 2′‐O‐cyanoethyl‐modified RNAs not only are applied in RNA silencing machinery but also act as research tools for studying RNA structure and function or for developing RNA‐based diagnostics. Therefore, the efficient synthesis, deprotection, purification, and characterization of 2′‐O‐cyanoethylated RNAs deserves more attention. This protocol describes the chemical synthesis of 2′‐O‐cyanoethylated nucleotides and the solid‐phase synthesis, deprotection, and purification of 2′‐O‐cyanoethylated RNAs. © 2023 Wiley Periodicals LLC.Basic Protocol 1: Preparation of 6‐N‐dimethylformamidyl‐5′‐O‐dimethoxytrityl‐2′‐O‐cyanoethyl adenosine 3′‐(2‐cyanoethyl N,N‐diisopropyl)phosphoramiditeBasic Protocol 2: Preparation of 4‐N‐acetyl‐5′‐O‐dimethoxytrityl‐2′‐O‐cyanoethyl cytidine 3′‐(2‐cyanoethyl N,N‐diisopropyl)phosphoramiditeBasic Protocol 3: Preparation of 2‐N‐dimethylformamidyl‐5′‐O‐dimethoxytrityl‐2′‐O‐cyanoethyl guanine 3′‐(2‐cyanoethyl N,N‐diisopropyl)phosphoramiditeBasic Protocol 4: Preparation of 5′‐O‐dimethoxytrityl‐2′‐O‐2‐cyanoethyl uridine 3′‐(2‐cyanoethyl N,N‐diisopropyl)phosphoramiditeBasic Protocol 5: Solid‐phase synthesis of 2′‐O‐cyanoethylated RNA analogsBasic Protocol 6: Deprotection and purification of synthesized 2′‐O‐cyanoethyl‐RNAs