Computational insights into the oxidation of mono- and 1,4 disubstituted arenes by the Toluene Dioxygenase enzymatic complex

Toluene Dioxygenase (TDO) enzymatic complex has been widely used for preparation of enantiopure cis-cyclohexadienediols for synthetic applications. Along the last 30 years, a variety of mono- and di-substituted arenes have been studied as substrates for this enzyme, and some interesting observations have been reported regarding the yield and selectivity of the biotransformation. Nevertheless, none of them has been explained considering the active site’ structural and electronic features. In this work we present the first computational model of TDO’s active site, with a description of its architecture and interactions with the substrate to understand and predict substrate orientation. Our findings indicate that in the O₂-free TDO, the iron(II) is stabilized by the coordination of an oxygen atom from the neighboring Gln215 residue. Besides, the active site is comprised by four pockets with different relative affinities for the substrates’ substituents. Monosubstituted arenes adopt a pose in which the alkyl chains maximize the London dispersion interactions with minimal steric clashes, giving an explanation for the observed trend in the benzylic hydroxylation yields. Finally, the computational results allowed us to rationalize the enantiomeric excess of 1,4-disubstituted arenes and the regioselectivity of the dihydroxylation of fluorobenzene. These data were used to develop and validate an affinity index (A_I), as a quantitative indicator of the orientation preference for these substrates. This practical and easy-to-use tool can be applied to successfully predict the orientation of the para-disubstituted benzenes into the TDO active site.