Combining Photo-CIDNP and Long-Lived Spin States Enables High-Contrast Detection of Weak Protein-Ligand Interactions

Abstract

Photo-chemically induced dynamic nuclear polarization (photo-CIDNP) and long-lived spin states (LLS) are two nuclear magnetic resonance (NMR) techniques that can be combined to achieve large signal enhancements and extended lifetimes of spin order, respectively, and their combination is useful for detecting biomolecular interactions. Here, we demonstrate that integrating photo-CIDNP hyperpolarization with LLS excitation through spin-lock induced crossing (SLIC) enables the highly sensitive detection of signals of ligands at micromolar concentrations. Using indole-3-propionic acid (IPA), we show that two of its CH₂ protons simultaneously support efficient photo-CIDNP polarization and SLIC-based singlet excitation, thus allowing the conversion of hyperpolarized Zeeman order into long-lived singlet order. The resulting CIDNP-LLS approach provides a signal-to-noise enhancement of up to 30-fold over conventional LLS, enabling the detection of 25 µM IPA in a 3 mm tube within two scans. In the presence of the enzyme arylalkylamine N-acetyltransferase (AANAT) at a 100 nM concentration, CIDNP-LLS offers superior binding contrast compared to conventional CIDNP or CIDNP combined with perfect echo refocusing (CIDNP-PEARLScreen), making it a powerful method for monitoring weak protein-ligand interactions.