title: Synthesis of 2'-modified nucleotides and their incorporation into hammerhead ribozymes.
abstract: Several 2'-modified ribonucleoside phosphoramidites have been prepared for structure-activity studies of the hammerhead ribozyme. The aim of these studies was to design and synthesize catalytically active and nuclease-resistant ribozymes. Synthetic schemes for stereoselective synthesis of the R isomer of 2'-deoxy-2'-C-allyl uridine and cytidine phosphoramidites, based on the Keck allylation procedure, were developed. Protection of the 2'-amino group in 2'-deoxy-2'-aminouridine was optimized and a method for the convenient preparation of 5'-O-dimethoxytrityl-2'-deoxy-2'-phthalimidouridine 3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) was developed. During the attempted preparation of the 2'-O-t-butyldimethylsilyl-3'-O-phosphoramidite of arabinouridine a reversed regioselectivity in the silylation reaction, compared with the published procedure, was observed, as well as the unexpected formation of the 2,2'-anhydronucleoside. A possible mechanism for this cyclization is proposed. The synthesis of 2'-deoxy-2'-methylene and 2'-deoxy-2'-difluoromethylene uridine phosphoramidites is described. Based on a '5-ribose' model for essential 2'-hydroxyls in the hammerhead ribozyme these 2'-modified monomers were incorporated at positions U4 and/or U7 of the catalytic core. A number of these ribozymes had almost wild-type catalytic activity and improved stability in human serum, compared with an all-RNA molecule.
authors.affiliation: Department of Chemistry and Biochemistry, Ribozyme Pharmaceuticals Inc., Boulder, CO 80301, USA.
authors.forename: L
authors.initials: L
authors.lastname: Beigelman
authors.forename: A
authors.initials: A
authors.lastname: Karpeisky
authors.forename: J
authors.initials: J
authors.lastname: Matulic-Adamic
authors.forename: P
authors.initials: P
authors.lastname: Haeberli
authors.forename: D
authors.initials: D
authors.lastname: Sweedler
authors.forename: N
authors.initials: N
authors.lastname: Usman
mesh_terms: D003562:Cytidine / Q000031:analogs & derivatives; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D004795:Enzyme Stability; D006801:Humans; D009711:Nucleotides / Q000138:chemical synthesis* / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000138:chemical synthesis* / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014529:Uridine / Q000031:analogs & derivatives
chemical_list: D009711:Nucleotides; D009943:Organophosphorus Compounds; D016337:RNA, Catalytic; D003562:Cytidine; D003857:Deoxyuridine; D014529:Uridine