Solid-Phase Synthesis of Oligonucleotide 5′-(α-P-Thio)triphosphates and 5′-(α-P-Thio)(β,γ-methylene)triphosphates

A robust solid-phase synthesis was developed to obtain original oligonucleotides (ONs) functionalized at their 5' end with modified triphosphate (TP) moieties, in which a nonbridging oxygen atom of the a phosphorus atom was replaced by a sulfur atom and the labile P-O-P linkage was changed into a methylene bridge between the beta and gamma phosphorus atoms. The efficient method is based on solid-supported ON assembly followed by 5'-H-phosphonylation, oxidation to the thiophosphate subsequently activated as a phosphoan-hydride with diphenyl phosphoryl chloride, then nucleophilic substitution with the alkylammonium salt of pyrophosphate or its beta,gamma-methylene analogue. After deprotection and release from the solid support under basic conditions, 5'-(alpha-P-thio)TP and 5'-(alpha-P-thio)(beta,gamma-methylene) TP oligonucleotides were obtained in satisfactory yields, and they were isolated with high purity. These hydrolysis-resistant 5'-TP ONs will be useful in biological research to elucidate the mechanism of enzymes involved in mRNA processing and maturation.