Identification of a Tricyclic PIII Chiral Auxiliary for Solid-Supported Synthesis of Stereopure Phosphorothioate-Containing Oligonucleotides.

Since the recognition of oligonucleotides as a therapeutic modality, significant work has been devoted to improving therapeutic properties, including nuclease stability. Phosphorothioate (PS) modifications of phosphodiesters are one of the most explored chemical modification and integral to currently approved oligonucleotide therapeutics, including antisense oligonucleotides (ASOs) and short interfering RNAs (siRNAs). Insertion of sulfur into the phosphate bridge in an n-mer leads to 2n isomeric mixtures of PSs, with different nuclease stability and protein-binding properties. Efforts to create stereopure PS-containing oligonucleotides has spurred interest in identifying new synthetic methods. Herein, work on a novel and practical tricyclic PIII chiral auxiliary and its application in solid-supported synthesis of stereopure PS-containing oligonucleotides is reported.

Impact of Phosphorothioate Chirality on Double-Stranded siRNAs: A Systematic Evaluation of Stereopure siRNA Designs.

Oligonucleotides are important therapeutic approaches, as evidenced by recent clinical successes with antisense oligonucleotides (ASOs) and double-stranded short interfering RNAs (siRNAs). Phosphorothioate (PS) modifications are a standard feature in the current generation of oligonucleotide therapeutics, but generate isomeric mixtures, leading to 2n isomers. All currently marketed therapeutic oligonucleotides (ASOs and siRNAs) are complex isomeric mixtures. Recent chemical methodologies for stereopure PS insertions have resulted in preliminary rules for ASOs, with multiple stereopure ASOs moving into clinical development. Although siRNAs have comparatively fewer PSs, the field has yet to embrace the idea of stereopure siRNAs. Herein, it has been investigated whether the individual isomers contribute equally to the in vivo activity of a representative siRNA. The results of a systematic evaluation of stereopure PS incorporation into antithrombin-3 (AT3) siRNA are reported and demonstrate that individual PS isomers dramatically affect in vivo activity. A standard siRNA design with six PS insertions was investigated and it was found that only about 10 % of the 64 possible isomers were as efficacious as the stereorandom control. Based on this data, it can be concluded that G1R stereochemistry is critical, G2R is important, G21S is preferable, and G22 and P1/P2 tolerate both isomers. Surprisingly, the disproportionate loss of efficacy for most isomers does not translate into significant gain for the productive isomers, and thus, warrants further mechanistic studies.

Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo.

Special (lipid) delivery: The role of the ionizable lipid pK(a) in the in vivo delivery of siRNA by lipid nanoparticles has been studied with a large number of head group modifications to the lipids. A tight correlation between the lipid pK(a) value and silencing of the mouse FVII gene (FVII ED(50) ) was found, with an optimal pK(a) range of 6.2-6.5. The most potent cationic lipid from this study has ED(50) levels around 0.005 mg kg(-1) in mice and less than 0.03 mg kg(-1) in non-human primates.

An immobilized and reusable Cu(I) catalyst for metal ion-free conjugation of ligands to fully deprotected oligonucleotides through click reaction.

Chelation of Cu(I) ions to an immobilized hydrophilic tris(triazolylmethyl)amine chelator on a solid support allowed synthesis of RNA oligonucleotide conjugates from completely deprotected alkyne-oligonucleotides. No oligonucleotide strand degradation or metal ion contamination was observed. Furthermore, use of the immobilized copper(I) ion overcame regioselectivity issues associated with strain-promoted copper-free azide-alkyne cycloaddition.