The reaction mechanism of chiral hydroxylation of p-OH and p-NH2 substituted compounds by ethylbenzene dehydrogenase

Abstract

Ethylbenzene dehydrogenase (EbDH; enzyme commission (EC) number: 1.17.99.2) is a unique biocatalyst that hydroxylates alkylaromatic and alkylheterocyclic compounds to (S)-secondary alcohols under anaerobic conditions. The enzyme exhibits a high promiscuity catalyzing oxidation of over 30 substrates, inter alia, para-substituted alkylphenols and alkylanilines. Secondary alcohols with OH and NH2 substituents in the aromatic ring are highly valuable synthons for many biologically active compounds in the fine chemical industry. EbDH hydroxylates most of the studied compounds highly enantioselectively, except for five substrates that harbour OH and NH2 groups in the para position, which exhibit a significant decrease in the percent enantiomeric excess (% ee). This phenomenon is inconsistent with the previously suggested enzyme mechanism, but it may be linked to a stabilization of the carbocation intermediate by deprotonation of the OH or NH2 substituent in the active site that yields a transient quinone (imine) ethide species. This would initiate an alternative reaction pathway involving the addition of a water molecule to a C=C double bond. This hypothesis was cross-validated by density functional theory (DFT) cluster modelling of the alternative reaction pathway with 4-ethylphenol, as well as by experimental assessment of the pH dependency of enantiomeric excesses. The results reported herein suggest that the alternative reaction pathway may significantly contribute to the overall reaction if the carbocation intermediates are stabilized by deprotonation.