Since at room temperature (298 K) the shuttling of this system occurs on longer timescales than those efficiently sampled via classical MD simulation, we turned to Well-Tempered Metadynamics (WT-MetaD) simulations. In WT-MetaD, standard MD is modified by introducing a history-dependent bias potential properly designed to drive the system in the exploration of the most probable equilibrium conformations (and transitions) along one or more critical reaction coordinates represented by collective variables (CVs). In our case we are primarily interested in using WT-MetaD simulations to study the shuttling within rotaxane 1. Our tests demonstrated that two CVs -i.e., the location d of the geometrical center of the DB24C8 ring along the rotaxane axle (CV1), and the distance d benz-benz between the centers of the two benzene rings of the shuttling macrocycle (see Fig. and the scheme in Fig. ) -can appropriately describe and sample the shuttling mechanism in the rotaxane, allowing a recrossing between all metastable states and robust reconstruction of the freeenergy profile (see Supplementary Movie 1). A deeper inspection of the structure of these minima shows that they are quite broad along the d benz-benz coordinate. This indicates that these two broad mirror minima at d benz-benz ∼ 0.8 nm identify both states in which the macrocycle folds, stacking its benzene rings with the phenyl rings of the stoppers (state A, in Fig. ) or with the first central phenyl ring of the axle (state C, in Fig. ), and states in not maintain an open conformation and the ring shuttling is not resistance-free due, e.g., to steric effects related to the para-phenylene structure of the axle, to possible π -π interactions with the rings of the DB24C8 macrocycle, and to residual solvophobic effects. The plot of the number of contacts between the ortho-phenylene units of DB24C8 and para-phenylene units of the axle of Fig. indicates there are interactions between DB24C8 and the axle.