ABSTRACTEthylbenzene dehydrogenase (EbDH) catalyzes the initial step in anaerobic degradation of ethylbenzene in denitrifying bacteria, namely, the oxygen-independent hydroxylation of ethylbenzene to (S)-1-phenylethanol. In our study we investigate the kinetic properties of 46 substrate analogs acting as substrates or inhibitors of the enzyme. The apparent kinetic parameters of these compounds give important insights into the function of the enzyme and are consistent with the predicted catalytic mechanism based on a quantum chemical calculation model. In particular, the existence of the proposed substrate-derived radical and carbocation intermediates is substantiated by the formation of alternative dehydrogenated and hydroxylated products from some substrates, which can be regarded as mechanistic models. In addition, these results also show the surprisingly high diversity of EbDH in hydroxylating different kinds of alkylaromatic and heterocyclic compounds to the respective alcohols. This may lead to attractive industrial applications of ethylbenzene dehydrogenase for a new process of producing alcohols via hydroxylation of the corresponding aromatic hydrocarbons rather than the customary procedure of reducing the corresponding ketones.

Propargyl acrylate-based functionalizable monolithic stationary phase (FMSP) for capillary electrochromatography, easily modifi able by alkyne-azide 1,3-dipolar cycloaddition, is developed. Optimization of FMSP is focused on (i) physical properties: rigidity, stability, and pore size and (ii) the ease of capillary preparation and grafting. The effect of grafting is demonstrated on the separation of eleven polycyclic aromatic hydrocarbons (PAHs), three fl avonols, and chlorpheniramine before and after derivatization. Two azides are synthesized: the fi rst, cinnamyl azide increases the retention of PAHs and improves separation of fl avonols, the second, 6-azido-6-deoxy-β -cyclodextrin allows the chiral separation of warfarin, improves the effi ciency of PAHs separation and increases retention of chlorpheniramine.

Quantum chemical modeling is used to predict the reactivity of ethylbenzene dehydrogenase (EBDH), a molybdenum enzyme that catalyzes the stereoselective oxidation of alkylaromatic and alkylheterocyclic hydrocarbons to the (S)-enantiomers of secondary alcohols. The reaction mechanism is studied for four different substrates: ethylbenzene, 4-ethylphenol, allylbenzene, and 4-ethylpyridine with a cluster model of the active site. The modeling predicts radical CAH activation followed by the formation of a radical intermediate product. Then another electron is transferred to form a carbocation species in TS2, followed by a tightly associated OH rebound step. The modeling study allows qualitative correlation of energy barriers with the results of kinetic assays and identifies factors influencing the chemical reactivity of EBDH with different substrates.