Previously used, e.g., to investigate the hydrolysis of formamide in basic conditions, here we relied on ab initio well-tempered metadynamics (WT-MetaD) simulations to study the reactivity of 2 as free in solution vs. confined in the cage cavity. Considered the computational cost of these simulations, and the complexity of our systems, we employed a semi-empirical density-functional tight-binding (DF-TB) method, in its self-consistent charge corrected variant SCC-DFTB. Recently shown to provide comparable accuracy to DFT with large basis sets in terms of prediction of barrier heights and reaction energies for organic molecules, 43 SCC-DFTB guarantees satisfactory accuracy in our case at an affordable computational cost. We simplified our models by studying a system with an OH nearby the amide (Figure ), and constraining the C-N-C(O)-C dihedral (!) of amide 2 to representative values, in order to simulate attack to different conformers. We compared 2 conformers with ! equal to 0 (cis conformer), ⇡ (trans conformer), and ⇡/4 (a twisted cis The blue profile refers to 2 in trans conformation, the red one refers to the reaction when 2 is cis, while the violet profile refers to the free energy profile of a cis-distorted configuration of 2 with ! = ⇡/4. A relative reactivity score for each amide conformers ( ! ), normalized based on the maximum measured value (i.e., that for !=⇡/4, set to 1), is associated to the simulated conformers of 2 (right secondary y axis). (c) Isomerization of 2 in the cage, co-encapsulated with 3 (left: trans-2, right: cis-2). (d) Free energy profiles for the isomerization of 2 (i) when this is free in solution (dotted curve, cis in pink), (ii) when 2 is encapsulated in 1 (dashed curve, cis in dark pink), (iii) when this is co-encapsulated with another (trans) 2 guest in cage 1 (dot-dashed curve, cis in light red), and (iv) when 2 is encapsulated in 1 together with the co-guest 3 (solid curve, cis state in light red). The data show that an increasing crowding stabilizes more and more the reactive 2 conformations (e.g., cis) in the cage cavity. Right secondary y axis: relative probabilities (P conf ! ) for the different conformations (!) of 2 in the various host-guest systems calculated based on the G values extracted from WT-MetaD simulations. conformer). We employed replica infrequent ab initio WT-MetaD simulations to obtain information on the reaction coefficients (rate of hydroxide attack and TI formation) for the various conformers of 2 (see Methods section in the SI for details). The reaction barrier of ! = ⇡/2 has been also tested, but this conformer was found too unstable to compute meaningful kinetic data. From multiple infrequent WT-MetaD runs activating/biasing the transition (R!TI, and the TI!R processes), we reconstructed the unbiased kinetics for the transition events and could estimate the characteristic transition times, ⌧ of f and ⌧ on . The kinetic constant for hydroxyde release can be calculated as k of f = 1/⌧ of f . The kinetic constant for hydroxyde attack (k on ) can be obtained in similar way from the ⌧ on and accounting for the OH concentration in the system (in our simplified setup, where OH is constrained in close proximity of the amide -perfectly basic conditions -, the theoretical OH concentration to be considered is equal to that of pure water: 55.6 M -see Methods for details).