4′-C-Methoxy-2′-deoxy-2′-fluoro Modified Ribonucleotides Improve Metabolic Stability and Elicit Efficient RNAi-Mediated Gene Silencing

Abstract

We designed novel 4'-modified 2'-deoxy-2'-fluorouridine (2'-F U) analogues with the aim to improve nuclease resistance and potency of therapeutic siRNAs by introducing 4'-C-methoxy (4'-OMe) as the alpha (C4'α) or beta (C4'β) epimers. The C4'α epimer was synthesized by a stereoselective route in six steps; however, both α and β epimers could be obtained by a nonstereoselective approach starting from 2'-F U. H NMR analysis and computational investigation of the α-epimer revealed that the 4'-OMe imparts a conformational bias toward the North-East sugar pucker, due to intramolecular hydrogen bonding and hyperconjugation effects. The α-epimer generally conceded similar thermal stability as unmodified nucleotides, whereas the β-epimer led to significant destabilization. Both 4'-OMe epimers conferred increased nuclease resistance, which can be explained by the close proximity between 4'-OMe substituent and the vicinal 5'- and 3'-phosphate group, as seen in the X-ray crystal structure of modified RNA. siRNAs containing several C4'α-epimer monomers in the sense or antisense strands triggered RNAi-mediated gene silencing with efficiencies comparable to that of 2'-F U.

Structural basis for the synergy of 4′- and 2′-modifications on siRNA nuclease resistance, thermal stability and RNAi activity

“…The Ufo and Ufob ( 16 ) and dTo ( 35 ) phosphoramidite building blocks were synthesized as reported, and the synthetic procedures for Ufme, uo and uob are described in the Methods section. All modified oligonucleotides were synthesized via standard solid phase phosphoramidite chemistry and products were characterized with mass spectrometry ( Supplementary Table S1 ).…”

“…Percentage of N -type conformation (% N ) was calculated by applying the empirical equation % N = 100 - 10 × ( J H1′ - J H2′ ). b Values from ( 16 ). c Values from ( 35 ).…”

“…In regard to protection against nucleases, the ( S )-epimers with various 2′-substituents were more effective than the corresponding ( R )-epimers. Motivated by these findings and given the importance of metabolic stability in the development of oligonucleotide (ON) therapeutics, we embarked on an investigation of the effect of 4′- C -modification on the stability of siRNAs ( 16 ). We found that similar to 5′- C -methyl moieties ( 15 ), 4′- C -methoxy substituents pose a challenge to nucleases in that they insert themselves between phosphates and in close vicinity of two conserved metal ions (typically Mg 2+ ) at the active site.…”

“…The thermodynamic stability of RNA duplexes containing such nucleotides is similar to that of the native counterparts ( 21 ). Indeed, we found that incorporation of 2′-F,4′- Cα -OMe-U ( Ufo , Figure 1 ) caused only minor changes to the melting temperature ( T m ) of RNA duplexes, whereby the modified ribose adopted a North ( N ) C3′- endo conformation in the crystal structure of a modified octamer ( 16 ). Conversely, insertion of the β epimer of 2′-F,4′- C -OMe-U ( Ufob , Figure 1 ) with an inverted stereocenter at C4′ strongly destabilized an RNA duplex ( 16 ).…”

“…Here, we report the syntheses of three novel C4′-modified nucleotide ( uo, uob and Ufme ) building blocks (Figure 1 ), their incorporation into oligonucleotides and the resulting consequences of these modifications for oligonucleotide thermal stability and nuclease resistance. Crystal structures of self-complementary RNA octamers 5′-r(CGAA X UCG)-3′ ( X = uo, uob, Ufme, Ufo ( 16 ) and Ufob ) at high resolution allow insights into the conformational preferences of nucleotides with 2′-,4′- C -disubstituted ribose moieties with either α or β stereochemistry at the C4′-position. The structural data allow a rationalization of the observed thermodynamic and nuclease stability data and point to steric (hydrophobic 4′- Cα/β substituent) and electrostatic origins (phosphate-phosphate spacing) of the increased protection against nuclease degradation.…”