Influence of 5-Fluorination, 2′-Fluorination, and 2′-Stereochemical Inversion on the Structure and Glycosidic Bond Stability of Protonated Canonical Cytidine Nucleosides*

Fluorinated nucleosides are well-established as anticancer and antiviral medications. As with many pharmaceuticals, the effects of synthetic modifications are often not well understood. Here, six modified cytidine nucleoside analogues are investigated and compared to the canonical DNA and RNA cytidine nucleosides to elucidate the structural and energetic effects of fluorine substitution at the 5-position of the nucleobase and of fluorine substitution and/or stereochemical inversion at the 2′‑position of the sugar moiety. The stability of the N-glycosidic bonds of the protonated forms of these cytidine nucleoside analogues are examined using tandem mass spectrometry approaches. Increasing electronegativity at the 2′‑substituent increases stability, increasing electronegativity at the 5‑substituent decreases stability, and absence of the β 2′-hydrogen cis to the nucleobase enhances stability. The stable low-energy structures of these systems are examined using DFT methods. Predicted IR spectra enable interpretation of measured infrared multiple photon dissociation action spectra. 5-Fluorination heavily directs for and solely populates O2 protonated conformers, whereas the canonical and 2′‑modified cytidine nucleoside analogues display approximately equal populations of O2 and N3 protonated conformers. Infrared spectroscopic signatures of the 2′-fluoro substituents allow for enhanced characterization of key structural parameters of the cytidine nucleoside analogues.