title: 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 (3)H(2) and (2)H(3) conformations, similar to the C3'-endo/C2'-endo conformation equilibrium seen in natural furanose nucleosides. In the (2)H(3) 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 (3)H(2) (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.
authors.affiliation: Isis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, California 92010, United States. pseth@isisph.com
authors.forename: Punit P
authors.initials: PP
authors.lastname: Seth
authors.forename: Jinghua
authors.initials: J
authors.lastname: Yu
authors.forename: Ali
authors.initials: A
authors.lastname: Jazayeri
authors.forename: Pradeep S
authors.initials: PS
authors.lastname: Pallan
authors.forename: Charles R
authors.initials: CR
authors.lastname: Allerson
authors.forename: Michael E
authors.initials: ME
authors.lastname: Østergaard
authors.forename: Fengwu
authors.initials: F
authors.lastname: Liu
authors.forename: Piet
authors.initials: P
authors.lastname: Herdewijn
authors.forename: Martin
authors.initials: M
authors.lastname: Egli
authors.forename: Eric E
authors.initials: EE
authors.lastname: Swayze
mesh_terms: D002384:Catalysis; D018360:Crystallography, X-Ray; D053138:Cyclohexenes / Q000138:chemical synthesis* / Q000737:chemistry; D008958:Models, Molecular; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D009696:Nucleic Acids / Q000138:chemical synthesis* / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D013402:Sugar Alcohols / Q000737:chemistry*
chemical_list: D053138:Cyclohexenes; D009696:Nucleic Acids; D009841:Oligonucleotides; D013402:Sugar Alcohols; C543192:hexitol; D012313:RNA