(¹⁵N₅)-labeled adenine derivatives: Synthesis and studies of tautomerism by ¹⁵N NMR spectroscopy and theoretical calculations

Since the nitrogens of nucleosides and nucleotides play an important role in the molecular recognition of these compounds, ¹⁵N NMR became a method of choice in this field. Fully ¹⁵N-labeled adenine, required in the latter studies, was obtained in four synthetic steps, in a good yield. Likewise, (¹⁵N₅)-2-hexylthioether-adenine and (¹⁵N₅)-8-Br-adenine were obtained in five synthetic steps from the relatively inexpensive ¹⁵N sources: ¹⁵N-NH₄Cl, ¹⁵N-NH₄OH, ¹⁵N-NaNO₂. Full ¹⁵N labeling of these adenine prototypes enabled to obtain high-resolution ¹⁵N NMR spectra of these bases at 60.8 MHz. Furthermore, the spectra suggested the existence of the N3-H species in the tautomeric mixtures of these compounds in solution, in addition to the well-reported N9-H (major) and N7-H (minor) tautomers. These observations were also supported by quantum mechanical calculations of the tautomeric equilibria in the gas phase and in solution of the above-mentioned adenine compounds. The gas-phase tautomeric equilibria were estimated using density functional theory and second-order perturbation theory methods. Solvent effects were included by means of both continuum and discrete solvation models. The observation of the existence of the N3-H tautomer has a clear impact on the possible H-bonding patterns of these adenine prototypes and on their molecular recognition by various biological macromolecules. The above ¹⁵N-labeled analogues are expected to find use as ¹⁵N NMR probes for numerous biochemical studies.