Studies on Steric and Electronic Control of 2‘−3‘ Phosphoryl Migration in 2‘-Phosphorylated Uridine Derivatives and Its Application to the Synthesis of 2‘-Phosphorylated Oligouridylates

For the synthesis of 2‘-phosphorylated oligouridylates by use of new phosphoramidite building units, several masked phosphoryl groups have been examined as 2‘-phosphate precursors, which should not be migrated to the 3‘ position when the 3‘ hydroxy protecting group must be removed to introduce a phosphoramidite residue into the 3‘-position. As a consequence, bis(2-cyano-1,1-dimethylethoxy)thiophosphoryl (BCMETP) was found to be the most suitable 2‘-phosphate precursor. This thiophosphoryl group could be introduced into the 2‘-hydroxyl of 3‘,5‘-silylated uridine derivative 7 by phosphitylation with bis(2-cyano-1,1-dimethylethoxy)(diethylamino)phosphine followed by sulfurization. Treatment of the 2‘-thiophosphorylated product 15 with (HF) x ·Py in THF gave exclusively the 3‘,5‘-unprotected uridine derivative 16a. Compound 16a was converted to the phosphoramidite unit 22 via a two-step reaction. This building block was used for the solution phase synthesis of U(2‘-p)pU (29) and U(2‘-ps)pU (30). Both the 2-cyano-1,1-dimethylethyl and 2-cyanoethyl groups were effectively removed from the fully protected derivative 25 by treatment with DBU in the presence of N,O-bis(trimethylsilyl)acetamide (BSA). The resulting 2‘-thiophosphoryl group was successfully converted to a phosphoryl group by iodine treatment to give U(2‘-p)pU (29). U(2‘-ps)pU (30) was also synthesized by a modified procedure without the iodine treatment. Reaction of 29 with a new biotinylating reagent in aqueous solution in the presence of MgCl2 gave a biotin-labeled product 35 having a pyrophosphate bridge at the 2‘ position. Reaction of 30 with monobromobimane gave the 2‘-S-alkylated product 33 in aqueous solution. Application of the phosphoramidite unit 22 to the solid phase synthesis using aminopropyl CPG gel gave successfully [U(2‘-p)p] n U (n = 1, 3, 5). It was found that stability of the succinate linker between the CPG and oligouridylates was unaffected by the treatment with DBU when BSA was present. Several enzymatic properties of the synthetic 2‘-phosphorylated and 2‘-thiophosphorylated oligouridylates are also described.