Convenient synthesis of nucleoside 5′-(α-P-thio) triphosphates and phosphorothioate nucleic acids (DNA and RNA)

Modified deoxy-and ribo-nucleoside triphosphates are chemically synthesized in multiple steps due to the protection and deprotection of the nucleoside functionalities. To conveniently synthesize the S-modified triphosphates for enzymatically preparing phosphorothioate DNAs and RNAs (PS-DNA and PS-RNA) as potential therapeutics, herein we report a one-pot strategy to synthesize the deoxy-and ribo-nucleoside 5'-(alpha-P-thio) triphosphates (dNTP alpha S and NTP alpha S) without protecting any nucleoside functionalities. This facile synthesis is achieved by treating the nucleosides with a mild phosphitylating reagent, reacting selectively with the 5'-hydroxyl group of each unprotected nucleoside, followed by sulfurization and hydrolysis to afford the crude dNTP alpha S and NTP alpha S analogs (mixtures of S-p and R-p diastereomers). We also demonstrated that after just simple precipitation (without HPLC and ion-exchange purification), the quality of the synthesized dNTPaS and NTPaS analogs is excellent for direct DNA polymerization and RNA transcription, respectively. Since Klenow DNA polymerase and T7 RNA polymerase accept the S-p diastereomers of dNTP alpha S and NTP alpha S analogs, respectively, while the R-p diastereomers are neither substrates nor inhibitors, the diastereomerically-pure PS-DNAs and PS-RNAs can be conveniently synthesized enzymatically.