Synthesis of 2′-Aminoalkyl-Substituted Fluorinated Nucleobases and Their Influence on the Kinetic Properties of Hammerhead Ribozymes

• Published in: Chembiochem : a European journal of chemical biology Vol. 5; no. 5; pp. 707 - 716
• Main Authors: Klöpffer, Astrid E, Engels, Joachim W
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley-VCH Verlag 03.05.2004; WILEY-VCH Verlag; WILEY‐VCH Verlag
• Subjects: Base Sequence; Catalysis; Fluorine - chemistry; HIV point mutations; kinetic analysis; Kinetics; Molecular Sequence Data; Nucleic Acid Conformation; nucleobases; Ribonucleosides - chemical synthesis; Ribonucleosides - chemistry; Ribonucleosides - metabolism; ribozymes; RNA; RNA, Catalytic - chemistry; RNA, Catalytic - metabolism; Substrate Specificity; Thermodynamics
• ISSN: 1439-4227; 1439-7633; 1439-4227
• DOI: 10.1002/cbic.200300809

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′-(β-aminoethyl)-β-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 Tm 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.