Structural Basis of Duplex Thermodynamic Stability and Enhanced Nuclease Resistance of 5‿C-Methyl Pyrimidine-Modified Oligonucleotides

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• 作者：Alexander V. Kel′in ; Ivan Zlatev ; Joel Harp ; Muthusamy Jayaraman ; Anna Bisbe ; Jonathan O′Shea ; Nate Taneja ; Rajar M. Manoharan ; Saeed Khan ; Klaus Charisse ; Martin A. Maier ; Martin Egli ; Kallanthottathil G. Rajeev ; Muthiah Manoharan
• 刊名：Journal of Organic Chemistry
• 出版年：2016
• 出版时间：March 18, 2016
• 年：2016
• 卷：81
• 期：6
• 页码：2261-2279
• 全文大小：728K
• ISSN：1520-6904

文摘

Although judicious use of chemical modifications has contributed to the success of nucleic acid therapeutics, poor systemic stability remains a major hurdle. The introduction of functional groups around the phosphate backbone can enhance the nuclease resistance of oligonucleotides (ONs). Here, we report the synthesis of enantiomerically pure (R)- and (S)-5′-C-methyl (C5′-Me) substituted nucleosides and their incorporation into ONs. These modifications generally resulted in a decrease in thermal stability of oligonucleotide (ON) duplexes in a manner dependent on the stereoconfiguration at C5′ with greater destabilization characteristic of (R)-epimers. Enhanced stability against snake venom phosphodiesterase resulted from modification of the 3′-end of an ON with either (R)- or (S)-C5′-Me nucleotides. The (S)-isomers with different 2′-substituents provided greater resistance against 3′-exonucleases than the corresponding (R)-isomers. Crystal structure analyses of RNA octamers with (R)- or (S)-5′-C-methyl-2′-deoxy-2′-fluorouridine [(R)- or (S)-C5′-Me-2′-FU, respectively] revealed that the stereochemical orientation of the C5′-Me and the steric effects that emanate from the alkyl substitution are the dominant determinants of thermal stability and are likely molecular origins of resistance against nucleases. X-ray and NMR structural analyses showed that the (S)-C5′-Me epimers are spatially and structurally more similar to their natural 5′ nonmethylated counterparts than the corresponding (R)-epimers.