Cross-Polarization of Insensitive Nuclei from Water Protons for Detection of Protein–Ligand Binding

Hyperpolarization derived from water protons enhances the NMR signal of ¹⁵N nuclei in a small molecule, enabling the sensitive detection of a protein-ligand interaction. The water hyperpolarized by dissolution dynamic nuclear polarization (D-DNP) acts as a universal signal enhancement agent. The ¹⁵N signal of benzamidine was increased by 1480-fold through continuous polarization transfer by <i>J</i>-coupling-mediated cross-polarization (<i>J</i>-CP) via the exchangeable protons. The signal enhancement factor favorably compares to factors of 110- or 17-fold using non-CP-based polarization transfer mechanisms. The hyperpolarization enabled detection of the binding of benzamidine to the target protein trypsin with a single-scan measurement of ¹⁵N <i>R</i>₂ relaxation. <i>J</i>-CP provides an efficient polarization mechanism for ¹⁵N or other low-frequency nuclei near an exchangeable proton. The hyperpolarization transfer sustained within the relaxation time limit of water protons additionally can be applied for the study of macromolecular structure and biological processes.