Rigid 2′,4′-Difluororibonucleosides: Synthesis, Conformational Analysis, and Incorporation into Nascent RNA by HCV Polymerase

Abstract

We report on the synthesis and conformational properties of 2'-deoxy-2',4'-difluorouridine (2',4'-diF-rU) and cytidine (2',4'-diF-rC) nucleosides. NMR analysis and quantum mechanical calculations show that the strong stereoelectronic effects induced by the two fluorines essentially "lock" the conformation of the sugar in the North region of the pseudorotational cycle. Our studies also demonstrate that NS5B HCV RNA polymerase was able to accommodate 2',4'-diF-rU 5'-triphosphate (2',4'-diF-rUTP) and to link the monophosphate to the RNA primer strand. 2',4'-diF-rUTP inhibited RNA synthesis in dinucleotide-primed reactions, although with relatively high half-maximal inhibitory concentrations (IC50 > 50 μM). 2',4'-diF-rU/C represents rare examples of "locked" ribonucleoside mimics that lack a bicyclic ring structure.

Locked 2′-Deoxy-2′,4′-Difluororibo Modified Nucleic Acids: Thermal Stability, Structural Studies, and siRNA Activity

“…The therapeutic potential use of LNA (27) has led to the synthesis of a wide number of related analogues known as BNAs (bicyclic nucleic acids) (28). Our laboratory has had a long standing interest in nucleosides modified with fluorine at C2´ (11,(29)(30)(31)(32)(33) and more recently at both the C2´ and C4´ positions (34,35). 4´-Fluoro nucleosides are relatively unexplored in the literature, investigated in only a handful of studies (36)(37)(38)(39)(40), most of which encountered acid instability of the glycosidic linkage, which precluded these molecules from further studies and incorporation within oligonucleotides.…”

Synthesis and Properties of 2′-Deoxy-2′,4′-difluoroarabinose-Modified Nucleic Acids

“…As previously reported, quantum mechanical calculations indicate a clear preference for the North pucker in 2′,4′-diF-rU with a very high interconversion barrier to the destabilized South conformation ( P ≈ 180°). 17 For 2′-F-araU, quantum mechanical calculations predict similar energies for the North and South puckers with a low activation barrier. 22 The South conformation is shifted toward the East region ( P ≈ 140).…”

“…17 Thus, reaction of commercially available 2′-F-araU ( 1 ) with triphenylphosphine and iodine afforded the corresponding 5′-iodo derivative 2 in 93% yield. The 4′,5′-exocyclic double bond in 3 was installed by an elimination reaction of the 5′-iodo with sodium methoxide in 53% yield.…”

“…Because the reactivity of the 5′-iodine is considerably decreased due to the presence of the 4′-fluorine, we placed a 3′-benzoyl group in nucleoside 4 to assist in the substitution reaction. 17,25,26 The actual substitution was carried out in the next step by activating the protected 5′-iodo,4′-fluoro analogue 5 to the corresponding hypoiodite with m CPBA in water-saturated dichloromethane with concomitant migration of the benzoyl group from the 3′ to the 5′ position to obtain 5 in 68% yield. 17,24 Deprotection of the 5′- O -benzoyl group with methanolic ammonia proceeded in nearly quantitative yields to afford the novel nucleoside monomer 2′,4′-diF-araU ( 7 ).…”

“…For example, we recently reported the effect of introducing a 4′-fluorine on 2′-deoxy-2′-fluororibouridine. 17 The strong gauche and anomeric effects 18–21 imparted by both the 2′- and 4′-fluorines, respectively, strongly favor the North conformation, thus locking the sugar in the North region of the pseudorotational cycle. In the case of 2′,4′-difluoroarabinonucleosides, the 2′-fluorine is expected to promote a [O4′–C1′–C2′–F2′] gauche interaction that favors the South sugar conformation, whereas the 4′-fluorine should impart a strong anomeric effect [O4′(p-type)/ σ * C4′–F4′ ] that drives the pseudorotational equilibrium toward the North conformation (Figure 1B).…”