Synthesis and preliminary evaluation of pro-RNA 2'-O-masked with biolabile pivaloyloxymethyl groups in an RNA interference assay.

The cellular delivery of bioactive nucleic acid-based drugs such as small interfering RNA (siRNA) represents a major technical hurdle for their pharmaceutical application. Prodrug-like approaches provide an attractive concept to address the delivery problem. With the aim to prepare RNA-based prodrugs bearing biolabile protections which facilitate cellular uptake and are prone to be removed enzymatically inside cells in order to release functional RNA, we synthesized pro-RNA totally or partially masked in 2'-OH position with pivaloyloxymethyl (PivOM) groups. A suitable strategy has been developed to synthesize and to purify base-sensitive mixed 2'-OH/2'-O-PivOM oligoribonucleotides, and to include them in siRNA. In this strategy, the fluoride labile [(triisopropylsilyl)oxy]-benzyloxycarbonyl group (tboc) as nucleobase protection (for A and C), the TBS group as 2'-OH protection and the Q-linker to solid-support were compatible with the PivOM groups masking some 2'-OH. We have taken advantage of the specific stability of the PivOM group to apply selected acidic, basic, and fluoride ions treatment for the deprotection and release of pro-RNA. This kind of pro-siRNA was studied in a human cell culture-based RNAi assay and preliminary promising data are discussed.

First evaluation of acyloxymethyl or acylthiomethyl groups as biolabile 2'-O-protections of RNA.

[reaction: see text] Short oligo-U sequences containing 2'-O-acyloxymethyl or acylthiomethyl groups as biolabile 2'-O-protections of RNA have been synthesized. These modified homouridylates are deprotected upon cellular esterase activation to release the parent RNA. They exhibit exceptional resistance to nuclease degradation, and the evaluation of their pairing properties shows that the 2'-acyloxymethyl groups do not prevent the duplex dsRNA formation. These biolabile 2'-modifications overcome the first hurdle to turn oligoribonucleotides into candidates for RNA interference drugs.