Kinetic resolution of α-bromoamides: Experimental and theoretical investigation of highly enantioselective reactions catalyzed by haloalkane dehalogenases

Haloalkane dehalogenases from five sources were heterologously expressed in Escherichia coli, isolated, and tested for their ability to achieve kinetic resolution of racemic alpha-bromoamides, which are important intermediates used in the preparation of bioactive compounds. To explore the substrate scope, fourteen alpha-bromoamides, with different C alpha- and N-substituents, were synthesized. Catalytic activity towards eight substrates was found, and for five of these compounds the conversion proceeded with a high enantioselectivity (E value >200). In all cases, the (R)-alpha-bromoamide is the preferred substrate. Conversions on a preparative scale with a catalytic amount of enzyme (enzyme: substrate ratio less 1:50 w/w) were all completed within 17-46 h and optically pure alpha-bromoamides and alpha-hydroxyamides were isolated with good yields (31-50%). Substrate docking followed by molecular dynamics simulations indicated that the high enantioselectivity results from differences in the percentage of the time in which the substrate enantiomers are bound favourably for catalysis. For the preferred (R)-substrates, the angle between the attacking aspartate oxygen atom of the enzyme, the attacked carbon atom of the substrate, and the displaced halogen atom, is more often in the optimal range (>157 degrees) for reactivity. This can explain the observed enantioselectivity of LinB dehalogenase in a kinetic resolution experiment.