Real‐Time Single‐Molecule Observation of a Chemical Reaction Reshaping the Energy Landscape of a Molecular Shuttle

ABSTRACT Artificial molecular machines often rely on chemical stimuli to switch between functional states, yet the real‐time dynamics of these transitions remain largely obscured by ensemble averaging. We describe the real‐time single‐molecule tracking of a Diels–Alder reaction that irreversibly alters the co‐conformational space of a two‐station 2rotaxane. By tethering individual shuttles in optical tweezers, we observe the transformation of a fumaramide binding site into a non‐binding adduct in an aqueous environment. This covalent modification triggers a brisk shift in the macrocycle's positional preference, effectively erasing the thermodynamically favored state and leaving the secondary succinic amide‐ester station as the sole anchoring site. Our analysis allows us to reconstruct the energy landscape before and after the chemical event, revealing that the transformation reshapes the thermodynamic potential without measurably perturbing the intrinsic kinetics. This study highlights the potential of force spectroscopy for the in‐situ characterization of chemically driven changes in the function of individual molecular devices.