Nucleoside Analogues with a Seven-Membered Sugar Ring: Synthesis and Structural Compatibility in DNA-RNA Hybrids.

Herein we describe results on the pairing properties of synthetic DNA and RNA oligonucleotides that contain nucleotide analogues with a 7-membered sugar ring (oxepane nucleotides). Specifically, we describe the stereoselective synthesis of a set of three oxepane thymine nucleosides (OxT), their conversion to phosphoramidite derivatives, and their use in solid-phase synthesis to yield chimeric OxT-DNA and OxT-RNA strands. The different regioisomeric OxT phosphoramidites allowed for positional variations of the phosphate bridge and assessment of duplex stability when the oxepane nucleotides were incorporated in dsDNA, dsRNA, and DNA-RNA hybrids. Little to no destabilization was observed when two of the three regioisomeric OxT units were incorporated in the DNA strand of DNA-RNA hybrids, a remarkable result considering the dramatically different structure of oxepanes in comparison to 2'-deoxynucleosides. Extensive molecular modeling and dynamics studies further revealed the various structural features responsible for the tolerance of both OxT modifications in DNA-RNA duplexes, such as base-base stacking and sugar-phosphate H-bond interactions. These studies suggest that oxepane nucleotide analogues may find applications in synthetic biology, where synthetic oligonucleotides can be used to create new tools for biotechnology and medicine.

Exploring Atypical Fluorine-Hydrogen Bonds and Their Effects on Nucleoside Conformations.

The ability of fluorine to serve as a hydrogen-bond acceptor has been debated for many years. Short fluorine-hydrogen contacts are thought to play a key role in stabilizing some complex supramolecular systems. To directly probe the existence of fluorine-hydrogen bonds, we have performed NMR spectroscopy and computational modeling on a series of C2'-fluorinated nucleosides. Specifically, quantum mechanics/molecular mechanics (QM/MM) analysis and [19 F,1 H] HMBC NMR experiments provided direct evidence for a C-H⋅⋅⋅F hydrogen bond in a 2'-F,4'-C-α-alkyl-ribonucleoside analogue. This interaction was also supported by QTAIM and NBO analyses, which confirmed a bond critical point for the C-H⋅⋅⋅F interaction (0.74 kcal mol-1 ). In contrast, although conformational analysis and NMR experiments of 2'-deoxy-2'-fluoro-arabinonucleosides indicated a close proximity between the 2'-fluorine and the H6/8 protons of the nucleobase, molecular simulations did not provide evidence for a C-H⋅⋅⋅F hydrogen bond.

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.

Synthesis of Seven-Membered Ring Nucleosides and Serendipitous Simmons-Smith O-Glycosylation.

A series of seven-membered (oxepine) nucleosides containing various nucleobases (A, U, T, 5-FU, C) were synthesized by ring expansion of cyclopropanated glucals. We expect this new series of ring-expanded nucleic acid analogues to be useful as building blocks in the design of mixed base functional genetic systems. While exploring alternative pathways to oxepine nucleoside synthesis, we discovered an unprecedented α-stereoselective O-glycosylation of 1,2-glucals under mild Simmons-Smith conditions.

Robust silver-mediated imidazolo-dC base pairs in metal DNA: dinuclear silver bridges with exceptional stability in double helices with parallel and antiparallel strand orientation.

A new unprecedented metal-mediated base pair was designed that stabilizes reverse Watson-Crick DNA (parallel strand orientation, ps) as well as canonical Watson-Crick DNA (antiparallel strand orientation, aps). This base pair contains two imidazolo-dC units decorated with furan residues. Tm measurements and spectroscopic studies reveal that each silver-mediated furano-imidazolo-dC forms exceptionally stable duplexes with ps and aps chain orientation. This stability increase by a silver-mediated base pair is the highest reported so far for ps and aps DNA helices.

Stereocontrolled synthesis of four diastereomeric C-aryl manno- and talofuranosides.

In a chiral-pool synthesis starting from D-mannono-1,4-lactone 1a, the four diastereomeric C-aryl furanosides (1S,4R)-4a, (1S,4S)-4b, (1R,4R)-4c, and (1R,4S)-4d were obtained in a stereocontrolled manner. The key steps of the synthetic pathway comprise a stereoselective reduction of the diastereomeric hemiketals (4R)-2a and (4S)-2b as well as a stereospecific cycloetherification of the resulting diols (1R,4R)-5a, (1S,4R)-5c, and (1S,4S)-5d. This ring closure which led to the desired C-glycosides was achieved by a Mitsunobu reaction or by preparing the 1-O-benzoyl-4-O-methylsulfonyl derivative 7 which was then treated with sodium methoxide. Final hydrolysis of the 5,6-O-isopropylidene protecting group led to the diastereomeric diols (1S,4R)-4a, (1S,4S)-4b, (1R,4R)-4c, and (1R,4S)-4d, representing versatile building blocks for further synthetic transformations.