Force Reveals Hidden Conformations and Dissociation Pathways in Individual π‐Interacting Dimers

Harnessing mechanical force to control molecular structure is a central strategy in the design of mechano-responsive materials. Noncovalent interactions are particularly attractive in this context because of their reversibility and tunable mechanical stability, yet the conformational energy landscapes of such motifs often remain inaccessible to conventional ensemble techniques. Here, we use atomic force microscopy-based force spectroscopy to probe individual π-interactions within a perylene diimide dimer. Single-molecule pulling experiments combined with molecular dynamics simulations reveal two distinct long-lived conformers with parallel and anti-parallel perylene diimide orientations that are indistinguishable by ensemble techniques. The parallel conformer exhibits greater mechanical stability and ruptures through a sequential pathway in which the dimer converts to an anti-parallel arrangement before π-π dissociation. Passive force spectroscopy resolves both conformers in real-time, validates the force-induced interconversion pathway predicted by steered molecular dynamics simulations, and quantifies their mechanical resistance and lifetime under constant load. Together, these results show that combining passive force spectroscopy with molecular simulations can reveal hidden conformational states in noncovalent assemblies and map their force-dependent energy landscape. Our findings provide molecular-level insight into the mechanics of π-π interactions and highlight single-molecule force spectroscopy as a powerful approach to uncover hidden structural states in supramolecular systems.