Oxidation of Benzyl Alcohols by the Laccase-Mediator System (LMS) a Comprehensive Kinetic Description

SummaryInvestigations into the reaction kinetics of the laccase-mediator system (LMS) have been carried out. Two widely used mediators, 2,2′-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS,3) and 1-hydroxybenzotriazole (HOBT,4), were compared by means of a model reaction, the oxidation of 2,4-dimethoxybenzyl alcohol (DMBA,1) to 2,4-dimethoxybenzaldehyde (DMA,2). The consumption of dioxygen was recorded electrochemically, substrate consumption and product formation were monitored by GLC.With ABTS as the mediator, the LMS reaction proceeded in two clearly distinguishable stages. The first phase is characterized by a fast decrease in oxygen with zero-order kinetics and no detectable formation of 2,4-dimethoxybenzaldehyde (2). ABTS is converted into oxidized species, the cation radical6and the dication7, respectively. In the second phase, oxygen consumption was considerably slower and followed a second-order kinetics, while the benzaldehyde was produced according to a zero-order rate law. According to the kinetic studies, the ABTS dication, but not the enzyme itself, is acting as the actual oxidant. The rate of oxidation product formation increased with increasing mediator / benzyl alcohol ratio. Less oxygen than the equivalent amount was consumed in the second reaction stage indicating that the oxidized ABTS formed in the first stage acts as an oxidant reservoir, being reduced to ABTS in turn.The LMS reaction with HOBT (4) as the mediator did not exhibit distinguishable phases, and was characterized by a comparatively slow oxygen uptake with zero-order kinetics throughout. Enzymatic oxidation of HOBT to the HOBT radical (5), which acts as the actual oxidant towards the benzyl alcohol, was the rate-determining step. The production of 2,4-dimethoxybenzaldehyde thus followed a zeroorder rate law as well. The reaction rate increased with increasing HOBT / benzyl alcohol ratios. Increasing concentrations of4caused less oxygen to be consumed per equivalent of benzaldehyde formed, indicating the occurrence of another reaction pathway at high mediator charges. At low HOBT / benzyl alcohol ratios the HOBT radical (5) acts as one-electron oxidant and is reduced to HOBT in a reversible process. In contrast, at higher HOBT / benzyl alcohol ratios5acts as a three-electron oxidant, being irreversibly reduced to benzotriazole. At commonly employed mediator concentrations, a superposition of both mechanisms results. The pure borderline cases can only be observed at HOBT / benzyl alcohol ratios below 1 and above 6, respectively.