Synthesis of uridine phosphoramidite analogs: reagents for site-specific incorporation of photoreactive sites into RNA sequences.

The synthesis of three new photoactive RNA phosphoramidites, 5-bromouridine, 5-iodouridine, and O4-triazolouridine, is reported. The 5' OH of bromouridine and iodouridine were protected as dimethoxytrityl ether using dimethoxytrityl chloride and pyridine. Selective protection of 2' OH was achieved as the corresponding tert-butyldimethylsilyl ether. Protected ribonucleosides were converted to phosphoramidites using 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. O4-Triazolouridine phosphoramidite monomer was prepared in one step from uridine phosphoramidite. These phosphoramidites were used to incorporate photoprobes at any chosen sites in the RNA sequences during chemical syntheses. The modified monomers were incorporated into RNA oligomers with coupling yields > 98%. After chemical synthesis, O4-triazolouridine was converted to 4-thiouridine by the addition of thiolacetic acid during standard deprotection methods. The extent of thiation and incorporation of modified nucleotides into RNA sequences were confirmed by nuclease digest, HPLC, and gel electrophoresis.