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 p hosphoramidite building units, several masked phosphoryl groups have b een examined as 2'-phosphate precursors, which should not be migrated to the 3' position when the 3' hydroxy protecting group must be remove d to introduce a phosphoramidite residue into the 3'-position. As a co nsequence, bis(2-cyano-1,l-dimethylethoxy)thiophosphoryl (BCMETP) was found to be the most suitable 2'-phosphate precursor. This thiophospho ryl group could be introduced into the 2'-hydroxyl of 3',5'-silylated uridine derivative 7 by phosphitylation with 2-cyano-1,l-dimethylethox y)(diethylamino)phosphine followed by sulfurization. Treatment of the 2'-thiophosphorylated product 15 with (HF)(x) . Py in THF gave exclusi vely the 3',5'-unprotected uridine derivative 16a. Compound 16a was co nverted to the phosphoramidite unit 22 via a two-step reaction. This b uilding 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-cyan oethyl groups were effectively removed from the fully protected deriva tive 25 by treatment with DBU in the presence of N,O-bis(trimethylsily l)acetamide (BSA). The resulting 2'-thiophosphoryl group was successfu lly 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-la beled product 35 having a pyrophosphate bridge at the 2' position. Rea ction of 30 with monobromobimane gave the 2'-S-alkylated product 33 in aqueous solution. Application of the phosphoramidite unit 22 to the s olid phase synthesis using aminopropyl CPG gel gave successfully [U(2' -p)p]U-n (n = 1, 3, 5). It was found that stability of the succinate l inker between the CPG and oligouridylates was unaffected by the treatm ent with DBU when BSA was present. Several enzymatic properties of the synthetic 2'-phosphorylated and 2'-thiophosphorylated oligouridylates are also described.