Quantum Mechanics/Molecular Mechanics Insights into the Enantioselectivity of the <i>O-</i>Acetylation of (<i>R,S</i>)<i>-</i>Propranolol Catalyzed by Candida antarctica Lipase B

Classical molecular dynamics (MD) simulations and combined quantum mechanics/molecular mechanics (QM/MM) calculations were used to investigate the origin of the enantioselectivity of the Candida antarctica lipase B (CalB) catalyzed <i>O-</i>acetylation of (<i>R</i>,<i>S</i>)-propranolol. The reaction is a two-step process. The initial step is the formation of a reactive acyl enzyme (AcCalB) via a tetrahedral intermediate (TI-1). The stereoselectivity originates from the second step, when AcCalB reacts with the racemic substrate via a second tetrahedral intermediate (TI-2). Reaction barriers for the conversion of (<i>R</i>)<i>-</i> and (<i>S</i>)-propranolol to <i>O-</i>acetylpropranolol were computed for several distinct conformations of TI-2. In QM/MM geometry optimizations and reaction path calculations the QM region was described by density functional theory (B3LYP/TZVP) and the MM region by the CHARMM force field. The QM/MM calculations show that the formation of TI-2 is the rate-determining step. The energy barrier for transformation of (<i>R</i>)-propranolol to <i>O</i>-acetylpropranolol is 4.5 kcal/mol lower than that of the reaction of (<i>S</i>)-propranolol. Enzyme–substrate interactions were identified that play an important role in the enantioselectivity of the reaction. Our QM/MM calculations reproduce and rationalize the experimentally observed enantioselectivity in favor of (<i>R</i>)-propranolol. Furthermore, in contrast to what is commonly suggested for lipase-catalyzed reactions, our results indicate that the tetrahedral intermediate is not a good approximation of the corresponding transition states