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.本研究报道了咯嗪（alloxazine）的固态结构性质——咯嗪是存在于诸多具有重要生物活性分子中的三环环系，并直接基于粉末X射线衍射（powder X-ray diffraction, XRD）数据完成了其晶体结构解析。由于包含咯嗪互变异构体与异咯嗪（isoalloxazine）互变异构体的晶体结构在里特维尔德精修中均能与粉末XRD数据实现高质量拟合，因此需要借助其他技术手段确定固态下的互变异构体形式。具体而言，通过对比实验15N核磁共振谱中的各向同性化学位移，与包含咯嗪及异咯嗪互变异构体的晶体结构所计算得到的对应数值，高分辨率固态15N NMR数据佐证了咯嗪互变异构体的存在。此外，在PBE0-MBD级别下开展的周期性色散校正密度泛函理论（DFT-D）计算表明，包含咯嗪互变异构体的晶体结构具有显著更低的能量。本研究同时报道了对晶体结构中互变异构体形式之间相互转换的计算研究——该转换通过沿两条分子间N–H···N氢键的质子转移实现；在PBE0-MBD级别下开展的DFT-D计算表明，对于沿两条氢键发生协同（而非分步）质子转移的反应机制，该互变转换对应的过渡态能量更低。然而，基于包含咯嗪与异咯嗪互变异构体的晶体结构的相对能量，经估算可知：在室温热平衡条件下，晶体结构中超过99.9%的分子将以咯嗪互变异构体形式存在。