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 α phosphorus atom was replaced by a sulfur atom and the labile P–O–P linkage was changed into a methylene bridge between the β and γ 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 phosphoanhydride with diphenyl phosphoryl chloride, then nucleophilic substitution with the alkylammonium salt of pyrophosphate or its β,γ-methylene analogue. After deprotection and release from the solid support under basic conditions, 5′-(α-P-thio)TP and 5′-(α-P-thio)(β,γ-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.