A method for the synthesis of P-stereodefined phosphorothioate oligoribonucleotides (PS-ORNs) was developed. PS-ORNs of mixed sequence (up to 12mers) were successfully synthesized by this method with sufficient coupling efficiency (94-99%) and diastereoselectivity (≥98:2). The coupling efficiency was greatly improved by the use of 2-cyanoethoxymethyl (CEM) groups in place of the conventional TBS groups for the 2'-O-protection of nucleoside 3'-O-oxazaphospholidine monomers. The resultant diastereopure PS-ORNs allowed us to clearly demonstrate that an ORN containing an all-(Rp)-PS-backbone stabilizes its duplex with the complementary ORN, whereas its all-(Sp)-counterpart has a destabilizing effect.

In this paper, we describe the first stereocontrolled synthesis and properties of boranophosphate DNA (PB-DNA), which contains all of the four nucleobases longer than 10mer. Synthesis was accomplished via an oxazaphospholidine approach combined with acid-labile protecting groups on nucleobases. It was demonstrated that there were significant differences between all-(Rp)-and all-(Sp)-PB-DNA in terms of the duplex-formation ability, nuclease resistance, and ribonuclease H (RNase H) activity. In particular, all-(Sp)-PB-DNA was demonstrated to show a duplexformation ability with RNA and RNase H activity, both of which are necessary for antisense-type nucleic acid therapeutics.

Stereocontrolled solid-phase synthesis of phosphate/phosphorothioate chimeric oligodeoxyribonucleotides (PO/PS-ODNs) was achieved by integrating the conventional phosphoramidite method into a previously developed oxazaphospholidine method for the stereocontrolled synthesis of P-chiral oligonucleotides. P-Stereodefined PO/PS-ODNs with mixed sequences (up to 12-mers) were obtained in good yields and high stereoselectivities by reacting different combinations of monomers (conventional phosphoramidites/diastereopure nucleoside 3'-O-oxazaphospholidines), activators (ETT/CMPT), capping reagents (Pac2O/CF3COIm), and oxidizing/sulfurizing reagents (TBHP/POS) on an automated synthesizer. A thermal denaturation study examined the resultant diastereopure PO/PS-ODN 12-mers with three consecutive (Rp)- or (Sp)-PS-linkages at the internal or terminal regions of the molecules. We found that (Rp)-PO/PS-ODNs can only moderately destabilize duplexes with complementary oligoribonucleotides (ORNs) compared with their unmodified ODN counterparts (ΔTm = -0.4 °C per modification). In contrast, (Sp)-PO/PS-ODNs have larger destabilizing effects (ΔTm = -1.2 to -0.8 °C per modification). Although smaller destabilizing effects were observed when the (Sp)-PS-linkages were incorporated into the terminal regions of the molecule, there was a weaker correlation between the location of an incorporated (Rp)-PS-linkage and its destabilizing effect.

P-stereodefined boranophosphate 2′-O-Me-oligoribonucleotides (2′-O-Me-PB-ORNs) were synthesized and we revealed that an all-(Sp)-PB-backbone largely stabilized the duplex with RNA.

The combination of 5-propynyluracil and (Rp)-PS linkages in a DNA strand could significantly increase the thermal stability of a DNA/RNA hybrid duplex.