We developed an AA model of 4 (Fig. , see Methods and SI for details), that was neutralized with PF - 6 counterions, immersed and equilibrated in explicit acetone solvent, to reproduce experimental conditions. As in the previous cases, we then employed WT-MetaD simulation to explore the shuttling mechanism in the rotaxane, obtaining the free-energy landscape (FES) as a function of the translational position d of the blue-box macrocycle along the axle (Fig. and Fig. ). The computed FES demonstrates a sharply "discrete I-0" shuttling mechanism in this system, in which the blue-box switches between two states corresponding to the binding of the cycle onto the two symmetric recognition sites (stations). The blue-box binding to the stations is rather strong, and the shuttling from one station to the other requires crossing a high activation barrier. 28 gets in contact with the NP groups. When this happens, the rings of the two components start to interact. Hypothesizing, for example, a CBPQT 4+ movement left-to-right along the axle, the enthalpy minima correspond to configurations in which a first pyridine rings of CBPQT 4+ stacks onto one of the axle NP rings (see, e.g., the first snapshot on the left in Fig. , bottom). This produces a local macrocycle-axle interaction increase and a favorable enthalpic change (first ∆H minimum at d ∼ -1.6 nm: a symmetrical minimum is found on the other side of the rotaxane). Proceeding left-to-right, the blue-box surrounds the NP rings: this is where we find the minimum of the free-energy. In contrast, such free-energy minima correspond to a more dynamical state, in which the bipyridine group of the blue-box fluctuates next to the NP without a well-defined stacking of the two aromatic rings. Such a "dynamic/fluctuating binding" allows preserving to some extent the ring-ring interactions while at the same time guaranteeing high mobility and preserving a high number of degrees of freedom. From this originates the entropic stabilization of these minimum free-energy states. Moving further left-to-right, a high entropic penalty thus accompanies the motion of CBPQT 4+ when this surpasses the NP recognition states (|d| < 0.6 nm). At d ∼ -0.4 nm another ∆H minimum is encountered, very similar to that previously seen at d ∼ -1.6, when the blue-box, leaving the NP, preserves its interaction with it via a one-ring stacking with it (Fig. : bottom inset on the right). It is interesting to note also how the establishment of such ring-ring stacking corresponds to a minimum in enthalpy and at the same time to an unfavorable barrier in entropy (interaction vs. blocking). The high shuttling free-energy barrier starting at d ≳ -0.6 nm in Fig. (in black) is thus first due to an entropic barrier (in blue) and then, upon breakage of the last ring-ring CBPQT 4+ -NP interaction, also by an enthalpic one -see the similar shape of the black and red barriers profiles for d ≳ -0.25 nm in Fig. (all profiles have then symmetrical feature respect to d = 0).