Synthesis, biophysical properties, and RNase H activity of 6'-difluoro[4.3.0]bicyclo-DNA.

Here we present the synthesis, the biophysical properties, and the RNase H profile of 6'-difluorinated [4.3.0]bicyclo-DNA (6'-diF-bc4,3-DNA). The difluorinated thymidine phosphoramidite building block was synthesized starting from an already known gem-difluorinated tricyclic glycal. This tricyclic siloxydifluorocyclopropane was converted into the [4.3.0]bicyclic nucleoside via cyclopropane ring-opening through the addition of an electrophilic iodine during the nucleosidation step followed by reduction. The gem-difluorinated bicyclic nucleoside was then converted into the corresponding phosphoramidite building block which was incorporated into oligonucleotides. Thermal denaturation experiments of these oligonucleotides hybridized to complementary DNA or RNA disclosed a significant destabilization of both duplex types (ΔT m/mod = -1.6 to -5.5 °C). However, in the DNA/RNA hybrid the amount of destabilization could be reduced by multiple insertions of the modified unit. In addition, CD spectroscopy of the oligonucleotides hybridized to RNA showed a similar structure than the natural DNA/RNA duplex. Furthermore, since the structural investigation on the nucleoside level by X-ray crystallography and ab initio calculations pointed to a furanose conformation in the southern region, a RNase H cleavage assay was conducted. This experiment revealed that the oligonucleotide containing five modified units was able to elicit the RNase H-mediated cleavage of the complementary RNA strand.

6'-Fluoro[4.3.0]bicyclo nucleic acid: synthesis, biophysical properties and molecular dynamics simulations.

Here we report on the synthesis, biophysical properties and molecular modeling of oligonucleotides containing unsaturated 6'-fluoro[4.3.0]bicyclo nucleotides (6'F-bc4,3-DNA). Two 6'F-bc4,3 phosphoramidite building blocks (T and C) were synthesized starting from a previously described [3.3.0]bicyclic sugar. The conversion of this sugar to a gem-difluorinated tricyclic intermediate via difluorocarbene addition followed either by a NIS-mediated or Vorbrüggen nucleosidation yielded in both cases the ß-tricyclic nucleoside as major anomer. Subsequent desilylation and cyclopropane ring opening of these tricyclic intermediates afforded the unsaturated 6'F-bc4,3 nucleosides. The successful incorporation of the corresponding phosphoramidite building blocks into oligonucleotides was achieved with tert-butyl hydroperoxide as oxidation agent. Thermal melting experiments of the modified duplexes disclosed a destabilizing effect versus DNA and RNA complements, but with a lesser degree of destabilization versus complementary DNA (ΔT m/mod = -1.5 to -3.7 °C). Molecular dynamics simulation on the nucleoside and oligonucleotide level revealed the preference of the C1'-exo/C2'-endo alignment of the furanose ring. Moreover, the simulation of duplexes with complementary RNA disclosed a DNA/RNA-type duplex structure suggesting that this modification might be a substrate for RNase H.