Among the various classes of modified nucleotides and oligonucleotides, phosphorothioate analogs, in which the sugar-phosphate backbone is modified by the substitution of a sulfur atom for one of the nonbridging oxygen atoms, have been most extensively studied in both in vitro and in vivo experiments. However, this substitution induces P-chirality of the dinucleoside phosphorothioate moiety. Consequently, even short phosphorothioate oligonucleotides synthesized using standard chemical methods exist as mixtures of many diastereoisomers. In our laboratory, the oxathiaphospholane (OTP) method has been developed for a stereocontrolled synthesis of oligo(deoxyribonucleoside phosphorothioate)s. Recently, this approach has been extended to ribonucleoside derivatives, and stereodefined phosphorothioate diribonucleotides were incorporated into oligomers suitable for mechanistic studies on deoxyribozymes. Next, it was found that the OTP ring can be opened with nucleophiles as weak as the phosphate or pyrophosphate anion, giving rise to nucleoside α-thiopolyphosphates. Surprisingly, the reaction between nucleoside OTP and O,O-dialkyl H-phosphonate or O,O-dialkyl H-phosphonothioate led to nucleoside 5'-O-(α-thio-β-O,O-dialkyl-hypophosphate) or 5'-O-(α,β-dithio-β-O,O-dialkyl-hypophosphate), respectively, i.e., derivatives containing a direct P-P bond.

Separate diastereomers of suitably protected dithymidine (3',5')-phenylphosphonates and dithymidine (3',5')-phenylphosphonothioates were obtained via a palladium(0) catalysed stereospecific cross-coupling reaction between separate diastereomers of the corresponding dinucleoside H-phosphonates and dinucleoside H-phosphonothioates with iodobenzene. These compounds were converted into the corresponding phosphoramidite building blocks and used for incorporation of P-stereodefined dithymidine phenylphosphonate and phenylphosphonothioate units TxT into DNA oligonucleotide chain. Dodecathymidylates with centrally positioned one (T 9 TxTT 9 ) or two modified units (T 8 TxTTxTT 8 ) exhibited different affinity towards complementary DNA (dA 20 ) or RNA (A 20 ) strands depending on stereochemistry at the phosphorus center, as determined by UV melting temperature studies. Oligonucleotides containing the R P -phenylphosphonate or R P -phenylphosphonothioate internucleotide linkages exhibited higher binding affinity to the complementary strands than their S P -counterparts, but slightly lower than the non-modified reference T 20 . All phenylphosphonothioate-modified oligonucleotides formed less stable than their oxo-counterparts duplexes. As expected, the cleavage of the oligonucleotides investigated with 3'-and 5'-exonucleases was stalled at the modification site, independently of P-chirality at the modification site.

Organic chemistry Z 0200 1,3,2-Oxathiaphospholane Approach to the Synthesis of P-Chiral Stereodefined Analogues of Oligonucleotides and Biologically Relevant Nucleoside Polyphosphates -[60 refs.]. -(NAWROT, B.; REBOWSKA, B.; MICHALAK, O.; BULKOWSKI, M.; BLAZIAK, D.; GUGA, P.; STEC, W.