title: Synthesis of uridine phosphoramidite analogs: reagents for site-specific incorporation of photoreactive sites into RNA sequences.
abstract: 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.
authors.affiliation: Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854.
authors.forename: K
authors.initials: K
authors.lastname: Shah
authors.forename: H
authors.initials: H
authors.lastname: Wu
authors.forename: T M
authors.initials: TM
authors.lastname: Rana
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001976:Bromouracil / Q000031:analogs & derivatives; D002851:Chromatography, High Pressure Liquid; D007065:Idoxuridine / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000138:chemical synthesis*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010777:Photochemistry; D012313:RNA / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D014529:Uridine / Q000031:analogs & derivatives* / Q000138:chemical synthesis
chemical_list: D009841:Oligonucleotides; D009943:Organophosphorus Compounds; C092358:triazolouridine phosphoramidite; C035991:5-iodouridine; D001976:Bromouracil; D012313:RNA; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases; C006824:5-bromouridine; D007065:Idoxuridine; D014529:Uridine