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

Identifieur interne : 003118 ( Main/Exploration ); précédent : 003117; suivant : 003119

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

Auteurs : Astrid E. Klöpffer ; Joachim W. Engels [Allemagne]

Source :

RBID : ISTEX:3E9E819D7F9B665CF64963FA2876A5D9D78EA7E6

Descripteurs français

English descriptors

Abstract

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.
Fluorinated nucleoside analogues and modified RNAs: The optimised synthesis of 2′‐aminoethyl‐substituted fluorinated benzene and benzimidazole nucleosides (see scheme) and their incorporation into oligoribonucleotides is reported. The influence of these universal bases on the thermodynamic stability of RNA 12‐mer duplexes was investigated by UV/Vis melting studies. The effect of these artificial nucleobases on the reaction rate of ribozyme‐catalysed RNA cleavage was studied by kinetic analyses to evaluate their tolerance against point mutations in biological systems. Owing to their improved properties for RNA recognition and their easy incorporation into hammerhead ribozymes, the nucleosides are applicable to a variety of different mRNA targets.

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DOI: 10.1002/cbic.200300809


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Le document en format XML

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<div type="abstract" xml:lang="en">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.</div>
<div type="abstract" xml:lang="en">Fluorinated nucleoside analogues and modified RNAs: The optimised synthesis of 2′‐aminoethyl‐substituted fluorinated benzene and benzimidazole nucleosides (see scheme) and their incorporation into oligoribonucleotides is reported. The influence of these universal bases on the thermodynamic stability of RNA 12‐mer duplexes was investigated by UV/Vis melting studies. The effect of these artificial nucleobases on the reaction rate of ribozyme‐catalysed RNA cleavage was studied by kinetic analyses to evaluate their tolerance against point mutations in biological systems. Owing to their improved properties for RNA recognition and their easy incorporation into hammerhead ribozymes, the nucleosides are applicable to a variety of different mRNA targets.</div>
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