Protein-Ligand Interaction Analyses with Nuclear Magnetic Resonance Spectroscopy Enhanced by Dissolution Triplet Dynamic Nuclear Polarization.

Solution-state nuclear magnetic resonance spectroscopy (NMR) is a powerful method for the analysis of intermolecular interactions within a biomolecular system. However, low sensitivity is one of the major obstacles of NMR. We improved the sensitivity of solution-state 13 C NMR for the observation of intermolecular interactions between protein and ligand using hyperpolarized solution samples at room temperature. Eutectic crystals composed of 13 C-salicylic acid and benzoic acid doped with pentacene were hyperpolarized by dynamic nuclear polarization using photoexcited triplet electrons, and a 13 C nuclear polarization of 0.72 ± 0.07% was achieved after dissolution. The binding of human serum albumin and 13 C-salicylate was observed with several hundred times sensitivity enhancement under mild conditions. The established 13 C NMR was applied for pharmaceutical NMR experiments by observation of the partial return of the 13 C chemical shift of salicylate by competitive binding with other non-isotope-labeled drugs.