Two unsymmetrical dinucleotide phosphate triesters were synthesized via transesterification from tris(2,2,2trifluoroethyl) phosphate. The protected triesters were phosphytilated to generate phosphoramidites for solid-phase oligonucleotide synthesis. Neutral phenylethyl phosphate-modified short-interfering RNAs (siRNAs) were synthesized and evaluated for their genesilencing ability, siRNA strand selection, and resistance to nucleases. These backbone-modified phosphate triester siRNAs offer many improvements compared to natural unmodified siRNAs.
A selective transesterification starting with tris(2,2,2trifluoroethyl) phosphate has been developed. This method involves a three-step substitution for 2,2,2-trifluoroethoxy groups and enables the facile synthesis of mixed unsymmetric phosphate triesters from three different alcohols. The substitution of the trifluoroethoxy group at the phosphorus proceeds selectively in the presence of DBU or lithium alkoxides. This method can be applied for the preparation of phospholipids.
Short
interfering RNAs (siRNAs) show promise as gene-silencing
therapeutics, but their cellular uptake remains a challenge. We have
recently shown the synthesis of siRNAs bearing a single neutral phenylethyl
phosphotriester linkage within the sense strand. Here, we report the
synthesis of siRNAs bearing three different hydrophobic phosphate
triester linkages at key positions within the sense strand and assess
their gene silencing in the absence of a transfection carrier. The
best siRNAs bearing hydrophobic phosphate triester tails were not
aromatic and exhibited effective gene silencing (IC50 ≈
56–141 nM), whereas the aromatic derivative with three hydrophobic
tails did not exhibit carrier-free gene silencing.
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