Synthesis of 2'-aminoalkyl-substituted fluorinated nucleobases and their influence on the kinetic properties of hammerhead ribozymes.
Hammerhead ribozymes are ribonucleic acids that catalyse the hydrolytic cleavage of RNA. They interfere with gene expression in a highly specific manner and recognize the mRNA target through Watson-Crick base pairing. To overcome the problem of point mutations (Watson-Crick "mismatches") occurring in viral genomes, we developed 2'-aminoethyl-substituted fluorinated nucleosides, which are universal nucleobases. The highly efficient synthetic pathway, which features a direct phthaloylamination of a primary alcohol under Mitsunobu conditions, leads to modified phosphoroamidites. The 1'-deoxy-1'-(4,6-difluoro-1H-benzimidazol-1-yl)-2'-(beta-aminoethyl)-beta-D-ribofuranose nucleoside analogue does not differentiate between the four natural nucleosides and leads to a RNA duplex that is as stable as the unmodified parent duplex. Upon incorporation into a ribozyme, the analogue's catalytic activity is equal for all four possible substrates, and the cleavage rates for the modified ribozymes are significantly higher (up to a factor of 13) than for the natural Watson-Crick "mismatch" base pairs. In agreement with the thermodynamic data obtained by measurement of the T(m) values of the RNA 12-mers, the cleavage rates for the 2'-substituted fluorinated benzimidazole derivative 4 are slightly higher than for the corresponding fluorinated benzene derivative 3.
Klöpffer
Astrid E
AE
Institute for Organic Chemistry and Chemical Biology, Johann Wolfgang Goethe University, Marie Curie Strasse 11, 60439 Frankfurt/Main, Germany.
Engels
Joachim W
JW
D001483:Base Sequence; D002384:Catalysis; D005461:Fluorine / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D012263:Ribonucleosides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
D016337:RNA, Catalytic; D012263:Ribonucleosides; D005461:Fluorine