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

Abstract

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 β-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.

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

“…In the literature there exist several procedures to construct an α,α-difluoroketone from a corresponding siloxydifluorocyclopropane [38–40]. However, based on previous observations in the nucleoside synthesis of the 6’F-bc 4,3 -T [37] we thought to construct the 6’-diF-bc 4,3 building block in utilizing this methodology. Along this synthesis, the glycal 1 was treated with N -iodosuccinimide (NIS) in the presence of persilylated thymine to produce the iodine intermediates 2α/β (Scheme 1, Table 1, entry 1).…”

“…To further study the preferred sugar pucker of the 6’-diF-bc 4,3 -T nucleoside, a potential energy profile versus pseudorotation phase angle of nucleoside 7 was calculated using quantum mechanical methods. For the calculations we used the Gaussian 09 software package [47] at the second order Møller–Plesset (MP2) level of theory, the 6-311G* basis set, and the same methodology as for the 6’F-bc 4,3 - T [37]. The obtained energy profile of nucleoside 7 (Fig.…”

“…The phosphoramidite building block 9 was incorporated into oligonucleotides and duplexed to complementary DNA and RNA to determine the effect of the 6’-diF-bc 4,3 -T modification on the helical structure and duplex thermostability. The sequences chosen for the investigation were the same as previously used for the 6’F-bc 4,3 -DNA ( ON1 : 5’-d(GGA TGT TCt CGA)-3’, ON2 : 5’-d(GGA tGT TCT CGA)-3’, ON3 : 5’-d(GGA TGt tCT CGA)-3’, ON4 : 5’-d(GCA ttt ttA CCG)-3’) [37]. UV-melting experiments of the modified duplexes were recorded to assess the affinity of the 6’-diF-bc 4,3 -T modified oligonucleotides towards complementary DNA and RNA (Table 3).…”

“…Moreover, we investigated the substrate recognition of the 6’-diF-bc 4,3 -T analog by RNase H. In addition, the RNase H experiment was also performed with the previously report 6’F-bc 4,3 -DNA (Fig. 1) [37].…”

Asymmetric Transfer Hydrogenation of gem‐Difluorocyclopropenyl Esters: Access to Enantioenriched gem‐Difluorocyclopropanes

“…gem-Difluorocyclopropanes are classically synthesized by cyclopropanation of alkenes with difluorocarbene, which can be generated from various precursors. [6,11,12] Examples of diastereoselective difluorocyclopropanations of alkenes possessing adjacent stereocenters have been reported, [13] but to date only a few methods are available for the synthesis of enantioenriched gem-difluorocyclopropanes. Diastereoselective auxiliary-based approaches involve Michael addition of lithium enolate IV (derived from a chiral N-acyl imidazolidinone) to mesityl 4-bromo-4,4-difluorocrotonate, followed by radical-induced ring closure, to afford difluorocyclopropane V. [14] Ring-closure induced by Michael addition of glycine-derived enolate VI to N-(difluorobromocrotonyl)oxazolidinone VII was also disclosed to access a-amino acid derivative VIII [15] (Scheme 1 A).…”