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Identification of metabolically stable 5'-phosphate analogs that support single-stranded siRNA activity.

Prakash TP, Lima WF, Murray HM, Li W, Kinberger GA, Chappell AE, Gaus H, Seth PP, Bhat B, Crooke ST, Swayze EE - Nucleic Acids Res. (2015)

Bottom Line: The ss-siRNA activity in vivo requires a metabolically stable 5'-phosphate analog.Our results indicate that the electronic and spatial orientation of the 5'-phosphate analog was critical for ss-siRNA activity.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.

View Article: PubMed Central - PubMed

Affiliation: Isis Pharmaceuticals Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA tprakash@isisph.com.

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Synthesis of phosphoramidites 30b–32b.
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Figure 20: Synthesis of phosphoramidites 30b–32b.

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).


Identification of metabolically stable 5'-phosphate analogs that support single-stranded siRNA activity.

Prakash TP, Lima WF, Murray HM, Li W, Kinberger GA, Chappell AE, Gaus H, Seth PP, Bhat B, Crooke ST, Swayze EE - Nucleic Acids Res. (2015)

Synthesis of phosphoramidites 30b–32b.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4381071&req=5

Figure 20: Synthesis of phosphoramidites 30b–32b.
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).

Bottom Line: The ss-siRNA activity in vivo requires a metabolically stable 5'-phosphate analog.Our results indicate that the electronic and spatial orientation of the 5'-phosphate analog was critical for ss-siRNA activity.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.

View Article: PubMed Central - PubMed

Affiliation: Isis Pharmaceuticals Inc., 2855 Gazelle Ct, Carlsbad, CA 92010, USA tprakash@isisph.com.

Show MeSH