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

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.