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

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'-(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.

Fluorine in Peptide Design and Protein Engineering

“…[29] As a result, fluorine is often used as an oxygen and hydroxy mimic in organic chemistry. [30] Interestingly, the steric demand of fluorocarbon groups seems to unproportionally increase with the extent of fluorine substitution. For example, the trifluoromethyl group is often described to be at least as large as isopropyl.…”

Influence of a fluorobenzene nucleobase analogue on the conformational flexibility of RNA studied by molecular dynamics simulations

“…Chemical modification of the backbone and the nucleobases of RNA can be used to probe the secondary and tertiary structure of RNA and to study the energetic contributions that stabilize or destabilize double-strand formation (1). Recently, fluorobenzene and fluorobenzimidazol derivatives have been suggested as nucleic acid base analogues and universal bases (2)(3)(4)(5)(6) and to decompose and analyse the contributions of base stacking and hydrogen bonding to RNA secondary structure stability (5)(6)(7). In the case of DNA, such base analogues have been used to study the mechanism of DNA replication [reviewed in (8)].…”

Rnai Activity of Sirnas Modified with 2′-Aminoalkyl-Substituted Fluorinated Nucleobases

“…[1] The siRNA containing the universal nucleoside at pos. 7 in the antisense strand was analyzed for silencing activity by TaqMan-PCR and compared to the mismatch siRNA ( Figure 3).…”

“…Moreover, ribozymes modified with this nucleoside analog showed a better or at least equal catalytic activity relative to Watson-Crick mismatches. [1] Due to these data, we investigated the ability of this compound to tolerate WatsonCrick mismatches in order to avoid HIV escape mutations in RNA interference. The influence of this nucleoside analog on siRNA efficiency was analyzed with a proven siRNA targeting GFP.…”