Identification of metabolically stable 5'-phosphate analogs that support single-stranded siRNA activity.
Bottom Line: Our results indicate that the electronic and spatial orientation of the 5'-phosphate analog was critical for ss-siRNA activity.Moreover, ApoC III ss-siRNAs were able to reduce the triglyceride and LDL cholesterol in transgenic mice demonstrating pharmacological effect of ss-siRNA.Our study provides guidance to develop surrogate phosphate analog for ss-siRNA and demonstrates that ss-siRNA provides an alternative strategy for therapeutic gene silencing.
Affiliation: Isis Pharmaceuticals Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA email@example.com.Show MeSH
Mentions: First we synthesized of 5′-methoxymethyl, 5′-fluormethyl and 5′-aminomethyl 2′-O-MOE-thymidine phosphoramidites 30b, 31b and 32b (Scheme 4). An orthogonally protected 2′-O-(2-methoxyethyl)thymidine nucleoside 23 (Scheme 3) with 5′-hydroxymethyl substitution was identified as a versatile synthon to prepare all modified phosphoramidites 30b, 31b and 32b. Synthesis of compound 23 was accomplished according to Scheme 3 (Supplementary data). Compound 23 was converted to 5′-methoxymethyl derivative 24 (Scheme 4; Supplementary data) with methyl iodide and NaH in DMF in good yield. Treatment of compound 23 with diethylaminosulfur trifluoride (DAST) in dichloromethane yielded 5′-fluoromethyl thymidine derivative 25 (Scheme 4; Supplementary data). The synthesis of 5′-aminomethyl thymidine analog 26 was also synthesized from compound 23 (Supplementary data). The thymidine analogs 24–26 were converted into their corresponding 3′-phosphoramidites 30b, 31b and 32b according to Scheme 4 (Supplementary data). The 5′-methylcarboxylate-thymidine 3′-phosphoramidite 36 (Scheme 5) was used for the synthesis of 5′-carboxylate ss-siRNA 17 (Figure 7). The synthesis of phosphoramidite 36 was achieved as described in Scheme 5 (Supplementary data).
Affiliation: Isis Pharmaceuticals Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA firstname.lastname@example.org.