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-bc^4,3-DNA). Two 6'F-bc^4,3 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-bc^4,3 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.

Keywords: DNA/RNA affinity; fluorinated cyclopropanes; fluorinated nucleic acids; molecular dynamics simulations; sugar modified nucleosides.

Figure 1

Chemical structure of selected nucleic acid analogs.

Scheme 1

Synthesis of the gem-difluorinated glycal 4 from the silyl enol ethers 1α/β. Reagents and conditions: a) BSA, DCM, rt, 17 h, 86%; b) BSA, DCM, rt, 18 h, 88%; c) TMSCF₃, NaI, THF, 70 °C, 2 h, 71%; d) TMSCF₃, NaI, THF, 70 °C, 4 h, 75%; e) TMSOTf, 2,6-lutidine, DCM, 0 °C to rt, 2 h, 41% (4), 39% (5α/β); f) TMSOTf, 2,6-lutidine, DCM, 0 °C to rt, 7 h, 58% (4), 29% (5α/β).

Scheme 2

Synthesis of the thymidine phosphoramidite building block 10. Reagents and conditions: a) i) thymine, BSA, NIS, DCM, 0 °C to rt, 4.5 h; ii) Bu₃SnH, AIBN, toluene, 90 °C, 30 min, 70%; b) HF-pyridine, DCM/pyridine 5:1, 0 °C to rt, 1.5 h, 71%; c) CeCl₃·7H₂O, NaBH₄, MeOH, 0 °C, 1 h, 92%; d) DMTr-Cl, pyridine, rt, 3 d, 76%; d) CEP-Cl, DIPEA, THF, rt, 4 h, 62%.

Scheme 3

Synthesis of the cytidine phosphoramidite building block 16. Reagents and conditions: a) Ac₂O, pyridine, 0 °C to rt, 17 h, 87%; b) N-benzoylcytosine, BSA, TMSOTf, ACN, 0 °C to rt, 3.5 h, 41%; c) HF-pyridine, DCM/pyridine 5:1, 0 °C, 15 min, 91%; d) i) CeCl₃·7H₂O, NaBH₄, MeOH, −78 °C, 20 min; ii) Bz₂O, DMF, rt, 7 h, 94%; e) DMTr-OTf, DCM/pyridine 1:2, rt, 19.5 h, 44%; f) CEP-Cl, DIPEA, THF, rt, 75 min, 43%.

Figure 2

Proposed mechanism for the formation of the 5’-phosphorylated fragments during the oxidation step in the synthesis of ON1 and ON2.

Figure 3

a) Potential energy profile versus pseudorotation phase angle of nucleoside 8 and its two minimal energy conformers: b) C2’-endo and c) C3’-exo.

Figure 4

Average structures of the a) 6’F-bc^4,3-DNA/DNA, b) 6’F-bc^4,3-DNA/RNA, and c) 6’F-bc^4,3-DNA/6’F-bc^4,3-DNA duplexes obtained from the last nanosecond of the simulation by firstly extracting a frame each 50 ps and secondly by doing an averaging of them.

Figure 5

Preferred sugar pucker of a) 6’F-bc^4,3-DNA/DNA, and b) 6’F-bc^4,3-DNA/RNA duplexes and torsion angles of c) 6’F-bc^4,3-DNA/DNA, and d) 6’F-bc^4,3-DNA/RNA duplexes extracted from a 100 ns molecular dynamics trajectory.