CH–π Interactions Elucidated at the Single-Molecule Level

CH−π interactions represent a weak yet significant molecular force that is prevalent in diverse biological and chemical systems. Despite compelling evidence supporting the existence of CH−π interactions, experimentally measuring these weak interactions remains challenging due to the coexistence of numerous stronger noncovalent interactions. Here, we construct a refined system to accurately compare the strength of CH−π interactions between phenylalanine derivatives and aliphatic amino acids through molecular exchange processes inside a protein nanopore. Based on a quantitative comparison of binding strengths, we found that CH−π interactions are primarily governed by dispersive attraction, and a more acidic C–H bond results in stronger CH−π interactions, both of which are consistent with the literature. Most importantly, we resolved the long-standing debate regarding the deuterium isotope effect on CH−π interactions and confirmed that deuterium forms weaker CD−π interactions compared to protic CH−π interactions.