From a mechanistic point of view, the two proposed compounds could potentially react in different manners in the active site of the enzyme, also because their key electrophilic centers not only possess a different chemical environment but they are not topologically equivalent. Thus, as shown in Scheme 2, after the formation of the ion pair E (+/-) :I reactant complex with B3, the attack of the sulfur of Cys145 to the b-carbon of the substrate can take place, followed by the proton transfer from the protonated His41 to the a-carbon, leading to a stable covalent product E-I. Nevertheless, considering the nature of the warhead in B3, the final proton transfer could also take place to the carbonyl oxygen atom (E-I ' ). In the case of the inhibition with B4, this dual possibility of the final proton transfer does not appear after the acylation of the enzyme because the proton from His41 can only be transferred to the carbonyl oxygen atom of the inhibitor (Scheme 3). The present study is focused on the computational study of the mechanism of inhibition of M pro by B3 and B4. The reaction mechanisms for each inhibitor were initially explored by nudged elastic band calculation of the minimum energy paths. Then, two free energybased methodologies, such as the umbrella sampling (US) and free energy perturbation (FEP) methods, both at density functional theory level combined with classical force fields, were employed to explore the full inhibition process.