Accurately Modeling the Conformational Preferences of Nucleosides

Sugar puckering of nucleosides impacts nucleic acid structures; hence their biological function. Similarly, nucleoside-based therapeutics may adopt different conformations affecting their binding affinity, DNA incorporation, and excision rates. As a result, significant efforts have been made to develop nucleoside analogues adopting specific conformations to improve bioactivity and pharmaco­kinetic profiles of the corresponding nucleoside-containing drugs. Understanding and ultimately predicting these conformational preferences would significantly help in the design of more effective structures. We report herein a computational study based on hybrid QM/MM umbrella sampling simulations that allow the accurate prediction of the sugar conformational preferences of chemically modified nucleosides in solution. Moreover, we pair these simulations with natural bond orbital (NBO) analysis to gain key insights into the role of substituents in the conformational preferences of these nucleosides

Adjusting the Structure of 2′-Modified Nucleosides and Oligonucleotides via C4′-α‑F or C4′-α-OMe Substitution: Synthesis and Conformational Analysis

We report the first syntheses of three nucleoside analogues, namely, 2′,4′-diOMe-rU, 2′-OMe,4′-F-rU, and 2′-F,4′-OMe-araU, via stereoselective introduction of fluorine or methoxy functionalities at the C4′-α-position of a 4′,5′-olefinic intermediate. Conformational analyses of these nucleosides and comparison to other previously reported 2′,4′-disubstituted nucleoside analogues make it possible to evaluate the effect of fluorine and methoxy substitution on the sugar pucker, as assessed by NMR, X-ray diffraction, and computational methods. We found that C4′-α-F/OMe substituents reinforce the C3′-endo (<i>north</i>) conformation of 2′-OMe-rU. Furthermore, the predominant C2′-endo (<i>south</i>/<i>east</i>) conformation of 2′-F-araU switches to C3′-endo upon introduction of these substituents at C4′. The nucleoside analogues were incorporated into DNA and RNA oligonucleotides via standard phosphoramidite chemistry, and their effects on the thermal stability of homo- and heteroduplexes were assessed via UV thermal melting experiments. We found that 4′-substituents can modulate the binding affinity of the parent 2′-modified oligomers, inducing a mildly destabilizing or stabilizing effect depending on the duplex type. This study expands the spectrum of oligonucleotide modifications available for rational design of oligonucleotide therapeutics

Adjusting the Structure of 2′-Modified Nucleosides and Oligonucleotides via C4′-α‑F or C4′-α-OMe Substitution: Synthesis and Conformational Analysis

We report the first syntheses of three nucleoside analogues, namely, 2′,4′-diOMe-rU, 2′-OMe,4′-F-rU, and 2′-F,4′-OMe-araU, via stereoselective introduction of fluorine or methoxy functionalities at the C4′-α-position of a 4′,5′-olefinic intermediate. Conformational analyses of these nucleosides and comparison to other previously reported 2′,4′-disubstituted nucleoside analogues make it possible to evaluate the effect of fluorine and methoxy substitution on the sugar pucker, as assessed by NMR, X-ray diffraction, and computational methods. We found that C4′-α-F/OMe substituents reinforce the C3′-endo (<i>north</i>) conformation of 2′-OMe-rU. Furthermore, the predominant C2′-endo (<i>south</i>/<i>east</i>) conformation of 2′-F-araU switches to C3′-endo upon introduction of these substituents at C4′. The nucleoside analogues were incorporated into DNA and RNA oligonucleotides via standard phosphoramidite chemistry, and their effects on the thermal stability of homo- and heteroduplexes were assessed via UV thermal melting experiments. We found that 4′-substituents can modulate the binding affinity of the parent 2′-modified oligomers, inducing a mildly destabilizing or stabilizing effect depending on the duplex type. This study expands the spectrum of oligonucleotide modifications available for rational design of oligonucleotide therapeutics

Adjusting the Structure of 2′-Modified Nucleosides and Oligonucleotides via C4′-α‑F or C4′-α-OMe Substitution: Synthesis and Conformational Analysis

We report the first syntheses of three nucleoside analogues, namely, 2′,4′-diOMe-rU, 2′-OMe,4′-F-rU, and 2′-F,4′-OMe-araU, via stereoselective introduction of fluorine or methoxy functionalities at the C4′-α-position of a 4′,5′-olefinic intermediate. Conformational analyses of these nucleosides and comparison to other previously reported 2′,4′-disubstituted nucleoside analogues make it possible to evaluate the effect of fluorine and methoxy substitution on the sugar pucker, as assessed by NMR, X-ray diffraction, and computational methods. We found that C4′-α-F/OMe substituents reinforce the C3′-endo (<i>north</i>) conformation of 2′-OMe-rU. Furthermore, the predominant C2′-endo (<i>south</i>/<i>east</i>) conformation of 2′-F-araU switches to C3′-endo upon introduction of these substituents at C4′. The nucleoside analogues were incorporated into DNA and RNA oligonucleotides via standard phosphoramidite chemistry, and their effects on the thermal stability of homo- and heteroduplexes were assessed via UV thermal melting experiments. We found that 4′-substituents can modulate the binding affinity of the parent 2′-modified oligomers, inducing a mildly destabilizing or stabilizing effect depending on the duplex type. This study expands the spectrum of oligonucleotide modifications available for rational design of oligonucleotide therapeutics

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

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′-<i>C</i>-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. ¹H NMR analysis and computational investigation of the α-epimer revealed that the 4′-OMe imparts a conformational bias toward the <i>North</i>-<i>East</i> 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