Solid-State Structural Properties of Alloxazine Determined from Powder XRD Data in Conjunction with DFT‑D Calculations and Solid-State NMR Spectroscopy: Unraveling the Tautomeric Identity and Pathways for Tautomeric Interconversion

We report the solid-state
structural properties of alloxazine,
a tricyclic ring system found in many biologically important molecules,
with structure determination carried out directly from powder X-ray
diffraction (XRD) data. As the crystal structures containing the alloxazine
and isoalloxazine tautomers both give a high-quality fit to the powder
XRD data in Rietveld refinement, other techniques are required to
establish the tautomeric form in the solid state. In particular, high-resolution
solid-state 15N NMR data support the presence of the alloxazine
tautomer, based on comparison between isotropic chemical shifts in
the experimental 15N NMR spectrum and the corresponding
values calculated for the crystal structures containing the alloxazine
and isoalloxazine tautomers. Furthermore, periodic DFT-D calculations
at the PBE0-MBD level indicate that the crystal structure containing
the alloxazine tautomer has significantly lower energy. We also report
computational investigations of the interconversion between the tautomeric
forms in the crystal structure via proton transfer along two intermolecular
N–H···N hydrogen bonds; DFT-D calculations at
the PBE0-MBD level indicate that the tautomeric interconversion is
associated with a lower energy transition state for a mechanism involving
concerted (rather than sequential) proton transfer along the two hydrogen
bonds. However, based on the relative energies of the crystal structures
containing the alloxazine and isoalloxazine tautomers, it is estimated
that under conditions of thermal equilibrium at ambient temperature,
more than 99.9% of the molecules in the crystal structure will exist
as the alloxazine tautomer.