A Proton Magnetic Resonance Study of the Influence of Base Ionization on the Conformation of Pseudouridine

The proton magnetic resonance spectra of alkaline deuterium oxide solutions of α-pseudouridine (α-ψ), β-pseudouridine (β-ψ), and 1-(β-D-ribofuranosyl)-cyanuric acid (β-CAR) are analyzed to explore the possibility that conformational changes are responsible for the unusual ultraviolet spectra of β-ψ at high pH. The largest change in ribose ring conformation due to increased alkalinity is observed in β-ψ; the ribose ring, although interconverting between various puckered forms, shows a slight preference for the 2′-endo and 3′-exo conformations. There is also a preference by α-ψ and β-ψ for the gauche-gauche rotamers about the exocyclic C4′–C5′ bond; this preference is not shown by β-CAR. No change occurs in the chemical shifts of the protons of α-ψ and β-ψ on going from neutral or acidic to basic solution. Increasing temperature to 60 °C causes no significant change in either coupling constants or chemical shifts. Comparison of chemical shifts observed in β-ψ with those found in β-CAR leads us to believe that the base remains in the anti conformation with respect to the ribose ring and is therefore incapable of forming a hydrogen bond with the exocyclic hydroxyl group as had been postulated previously to explain anomalous ultraviolet spectral data. A weak hydrogen bond between the 5′-hydroxymethyl group and the 5–6 double bond remains as a plausible explanation for the unusual ultraviolet spectra.