Noncovalent-Interaction-Driven Single-Molecule Exchange Enables Enantiomer Discrimination of Selected Amino Acids in a Nanopore.

Chiral discrimination of amino acids and short peptides remains challenging due to their subtle stereochemical differences and the limited resolution of conventional analytical techniques. Here, we report a single-molecule nanopore sensing strategy in which noncovalent-interaction-driven molecular exchange enables robust discrimination of specific amino acid enantiomers and diastereomeric peptides. By integrating cucurbit[7]uril with an engineered α-hemolysin nanopore, transient molecular pairs are formed and undergo configuration-dependent exchange, converting enantiomeric analytes into pseudo-diastereomeric complexes that generate distinct ionic current signatures. We demonstrate enantiomeric discrimination for selected proteinogenic, and unnatural amino acids bearing aromatic or aliphatic side chains and provide mechanistic insight through symmetry analysis and density functional theory calculations. Beyond amino acids, this platform resolves enzymatically cleaved diastereomeric peptides and enables quantitative monitoring of enzymatic conversion via enantiomeric excess measurements. This work establishes a mechanistic framework for stereochemical recognition under nanoscale confinement and offers a rational design principle for next-generation nanopore sensors targeting chiral biomolecules.