Although 2′-deoxy-2′-α-F-2′-β-C-methyl (2′-F/Me) uridine nucleoside derivatives are a successful class of antiviral drugs, this modification had not been studied in oligonucleotides. Herein, we demonstrate the facile synthesis of 2′-F/Me-modified pyrimidine phosphoramidites and their subsequent incorporation into oligonucleotides. Despite the C3′-endo preorganization of the parent nucleoside, a single incorporation into RNA or DNA resulted in significant thermal destabilization of a duplex due to unfavorable enthalpy, likely resulting from steric effects. When located at the terminus of an oligonucleotide, the 2′-F/Me modification imparted more resistance to degradation than the corresponding 2′-fluoro nucleotides. Small interfering RNAs (siRNAs) modified at certain positions with 2′-F/Me had similar or better silencing activity than the parent siRNAs when delivered via a lipid nanoparticle formulation or as a triantennary N-acetylgalactosamine conjugate in cells and in mice. Modification in the seed region of the antisense strand at position 6 or 7 resulted in an activity equivalent to the parent in mice. Additionally, placement of the antisense strand at position 7 mitigated seed-based off-target effects in cell-based assays. When the 2′-F/Me modification was combined with 5′-vinyl phosphonate, both E and Z isomers had silencing activity comparable to the parent. In combination with other 2′-modifications such as 2′-O-methyl, the Z isomer is detrimental to silencing activity. Presumably, the equivalence of 5′-vinyl phosphonate isomers in the context of 2′-F/Me is driven by the steric and conformational features of the C-methyl-containing sugar ring. These data indicate that 2′-F/Me nucleotides are promising tools for nucleic acid-based therapeutic applications to increase potency, duration, and safety.

中文翻译：

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“神奇”甲基的作用：2'-Deoxy-2'-α-F-2'-β-C-甲基嘧啶核苷酸通过与 5'-磷酸盐模拟物的协同作用和减轻脱靶效应来调节 RNA 干扰活性

尽管 2'-deoxy-2'-α-F-2'-β- C-甲基 (2'-F/Me) 尿苷核苷衍生物是一类成功的抗病毒药物，但尚未在寡核苷酸中研究这种修饰。在此，我们证明了 2'-F/Me 修饰的嘧啶亚磷酰胺的简便合成及其随后掺入寡核苷酸中。尽管 C3'- endo由于不利的焓，可能是由于空间效应造成的，在亲本核苷的预组织中，单次掺入 RNA 或 DNA 会导致双链体的显着热不稳定。当位于寡核苷酸的末端时，2'-F/Me 修饰比相应的 2'-氟核苷酸更能抵抗降解。当通过脂质纳米颗粒制剂或作为三触角N递送时，在某些位置用 2'-F/Me 修饰的小干扰 RNA (siRNA) 具有与亲本 siRNA 相似或更好的沉默活性-乙酰半乳糖胺缀合物在细胞和小鼠中。在位置 6 或 7 的反义链种子区域的修饰导致与小鼠中的亲本相当的活性。此外，将反义链放置在第 7 位可减轻基于细胞的测定中基于种子的脱靶效应。当 2'-F/Me 修饰与 5'-乙烯基膦酸酯结合时，E和Z异构体都具有与母体相当的沉默活性。与其他 2'-修饰如 2'- O-甲基结合，Z异构体对沉默活性有害。据推测，在 2'-F/Me 的情况下 5'-乙烯基膦酸酯异构体的等效性是由C的空间和构象特征驱动的。含-甲基的糖环。这些数据表明，2'-F/Me 核苷酸是基于核酸的治疗应用的有前途的工具，可提高效力、持续时间和安全性。