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 (north) conformation of 2′-OMe-rU. Furthermore, the predominant
C2′-endo (south/east) 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.