Synthesis and Antisense Properties of Fluoro Cyclohexenyl Nucleic Acid (F-CeNA), a Nuclease Stable Mimic of 2′-Fluoro RNA

Abstract

We report the design and synthesis of 2′-fluoro cyclohexenyl nucleic acid (F-CeNA) pyrimidine phosphoramidites and the synthesis and biophysical, structural, and biological evaluation of modified oligonucleotides. The synthesis of the nucleoside phosphoramidites was accomplished in multigram quantities starting from commercially available methyl-D-mannose pyranoside. Installation of the fluorine atom was accomplished using nonafluorobutanesulfonyl fluoride, and the cyclohexenyl ring system was assembled by means of a palladium-catalyzed Ferrier rearrangement. Installation of the nucleobase was carried out under Mitsunobu conditions followed by standard protecting group manipulations to provide the desired pyrimidine phosphoramidites. Biophysical evaluation indicated that F-CeNA shows behavior similar to that of a 2′-modified nucleotide, and duplexes with RNA showed slightly lower duplex thermostability as compared to that of the more rigid 3′-fluoro hexitol nucleic acid (FHNA). However, F-CeNA modified oligonucleotides were significantly more stable against digestion by snake venom phosphodiesterases (SVPD) as compared to unmodified DNA, 2′-fluoro RNA (FRNA), 2′-methoxyethyl RNA (MOE), and FHNA modified oligonucleotides. Examination of crystal structures of a modified DNA heptamer duplex d(GCG)-T*-d(GCG):d(CGCACGC) by X-ray crystallography indicated that the cyclohexenyl ring system exhibits both the 3H2 and 2H3 conformations, similar to the C3′-endo/C2′-endo conformation equilibrium seen in natural furanose nucleosides. In the 2H3 conformation, the equatorial fluorine engages in a relatively close contact with C8 (2.94 Å) of the 3′-adjacent dG nucleotide that may represent a pseudo hydrogen bond. In contrast, the cyclohexenyl ring of F-CeNA was found to exist exclusively in the 3H2 (C3′-endo like) conformation in the crystal structure of the modified A-form DNA decamer duplex [d(GCGTA)-T*-d(ACGC)]2. In an animal experiment, a 16-mer F-CeNA gapmer ASO showed similar RNA affinity but significantly improved activity compared to that of a sequence matched MOE ASO, thus establishing F-CeNA as a useful modification for antisense applications.

Fluorinated Nucleotide Modifications Modulate Allele Selectivity of SNP-Targeting Antisense Oligonucleotides

“…FHNA, 23 F-CeNA, 24 N-MC, 37 and F-N-MC 36 phosphoramidites were synthesized as described in the literature. ASOs were synthesized on an ABI 394 DNA/RNA Synthesizer on a 2 μmol scale using 5-methyl cytidine MOE-loaded primer support (215 μmol/g).…”

“…However, the six-membered hexitol ring in FHNA is more rigid and mimics the RNA-like C3′- endo conformation of the furanose ring (Figures 1B and 1C) 23 . In contrast, the cyclohexenyl ring in F-CeNA is more flexible and was shown to assume either the DNA- or RNA-like sugar conformation depending on the sequence context on its incorporation 24 . The 3.1.0 ring system in F-NMC is locked in the RNA-like C3′- endo conformation 25 .…”

Synthesis and Duplex‐Stabilizing Properties of Fluorinated N‐Methanocarbathymidine Analogues Locked in the C3′‐endo Conformation

“…[3] We recently characterized the antisense properties of both isomers of 2'-fluorohexitol nucleic acid (FHNA) [4,5] and 2'-fluorocyclohexenyl nucleic acid (F-CeNA). [6] We found that fluorine incorporation can indeed have a beneficial effect on the properties of modified oligonucleotides as compared to those of the parent nonfluorinated counterparts. As a continuation of that program and our interest in the synthesis of carbocyclic nucleoside analogues, [7] we investigated the synthesis and duplexstabilizing properties of both isomers of 2'-F-modified Nmethanocarbathymidine (N-MCT) oligonucleotides.…”

Synthesis and Properties of 6′-Fluoro-tricyclo-DNA

“…Copies of 1 H-, 13 C-, 19 F-, and 31 P-NMR spectra of compounds 2−18 and X-ray structural data (CIF) of compound 16. This information is available free of charge via the Internet at http://pubs.acs.org…”