Oxidation of Non‐Phenolic Substrates with the Laccase/N‐Hydroxyacetanilide System: Structure of the Key Intermediate from the Mediator and Mechanistic Insight.

Abstract

We have investigated the reactivity and mechanistic features in the oxidation of non-phenolic substrates by the enzyme laccase under mediation by N-hydroxyacetanilide, NHA. A radical route of hydrogen-abstraction by the N–O˙ reactive intermediate of the mediator, formed in a preliminary oxidative interaction with the enzyme, seems the more viable oxidation mechanism, in keeping with analogous conclusions reached for other laccase/N–OH-type mediators. The evaluated value of the energy of the O–H bond of NHA corroborates the occurrence of hydrogen-abstraction from the benzylic substrates by the N–O˙ intermediate of NHA. The occurrence of an alternative ionic route through the oxoammonium ion (NO+) of mediator NHA is ruled out by experimental evidence acquired through an Hammett structure/reactivity correlation in the oxidation of substituted benzyl alcohols, as well as by kinetic isotope effect determinations. The NO+ species of NHA, being a one-electron oxidant of moderate strength, could in principle even be responsible for an alternative electron-transfer route of oxidation of the substrates. This hypothesis could also be dismissed through the use of probe substrates and intermolecular selectivity determinations.