Multicomponent kinetic analysis and theoretical studies on the phenolic intermediates in the oxidation of eugenol and isoeugenol catalyzed by laccase

Abstract

Laccase catalyzes the oxidation of natural phenols and thereby is believed to initialize reactions in lignification and delignification. Numerous phenolic mediators have also been applied in laccase-mediator systems. However, reaction details after the primary O-H rupture of phenols remain obscure. In this work two types of isomeric phenols, EUG (eugenol) and ISO (trans-/cis-isoeugenol), were used as chemical probes to explore the enzymatic reaction pathways, with the combined methods of time-resolved UV-Vis absorption spectra, MCR-ALS, HPLC-MS, and quantum mechanical (QM) calculations. It has been found that the EUG-consuming rate is linear to its concentration, while the ISO not. Besides, an o-methoxy quinone methide intermediate, (E/Z)-4-allylidene-2-methoxycyclohexa-2,5-dienone, was evidenced in the case of EUG with the UV-Vis measurement, mass spectra and TD-DFT calculations; in contrast, an ISO-generating phenoxyl radical, a (E/Z)-2-methoxy-4-(prop-1-en-1-yl) phenoxyl radical, was identified in the case of ISO. Furthermore, QM calculations indicated that the EUG-generating phenoxyl radical (an O-centered radical) can easily transform into an allylic radical (a C-centered radical) by hydrogen atom transfer (HAT) with a calculated activation enthalpy of 5.3 kcal mol(-1) and then be fast oxidized to the observed eugenol quinone methide, rather than an O-radical alkene addition with barriers above 12.8 kcal mol(-1). In contrast, the ISO-generating phenoxyl radical directly undergoes a radical coupling (RC) process, with a barrier of 4.8 kcal mol(-1), while the HAT isomerization between O- and C-centered radicals has a higher reaction barrier of 8.0 kcal mol(-1). The electronic conjugation of the benzyl-type radical and the aromatic allylic radical leads to differentiation of the two pathways. These results imply that competitive reaction pathways exist for the nascent reactive intermediates generated in the laccase-catalyzed oxidation of natural phenols, which is important for understanding the lignin polymerization and may shed some light on the development of efficient laccase-mediator systems.

Aerobic Oxidation of Isoeugenol to Vanillin with Copper Oxide Doped Reduced Graphene Oxide

“…Vanillyl methyl ketone (B) is formed via hydration of the double bond of isoeugenol to an alcohol, followed by oxidation of the alcohol intermediate . Dehydrodiisoeugenol (C) is formed via radical coupling reaction between radical intermediate and isoeugenol, which is stable and hence remains in the solution . The formation of such by‐product can be reduced by using protecting phenolic derivative such as O‐methyl ethers, oacetyl esters, and glucosides, indicating that the –OH group of aromatic ring of isoeugenol participates in dehydrodiisoeugenol formation.…”

Large-Scale Comparative Analysis of Eugenol-Induced/Repressed Genes Expression in Aspergillus flavus Using RNA-seq

“…Eugenol can be biotransformed by some microbial enzymes. A few eugenol-converting enzymes have been reported, including vanillyl-alcohol oxidase from Penicillium simplicissimum (de Jong et al, 1992 ), 4-thylphenol methylenehydroxylase from Pseudomonas putida DJ1 (Reeve et al, 1989 ), eugenol dehydrogenase from Pseudomonas fluorescens E118 (Furukawa et al, 1998 ), laccase from fungi (Qi et al, 2015 ), eugenol oxidase from Rhodococcus sp. strain RHA1 (Jin et al, 2007 ), and lipase from Candida antarctica and Staphylococcus aureus (Horchani et al, 2010 ; Chiaradia et al, 2012 ).…”

Understand the Specific Regio- and Enantioselectivity of Fluostatin Conjugation in the Post-Biosynthesis

“…The deacyloxylation serves to generate the active species p -QM [ 58 , 59 , 60 ], which is the key to formation of pre-reaction complex for the following C–C coupling [ 61 , 62 ]. The formation of p -QM is the rate-determining step of the dimerization.…”

“…The calculated results are in good agreement with the experimental observation, where the acyl FSTs were stable in aprotic organic solvents and under acidic conditions (pH < 4), but the reaction significantly accelerates under basic conditions (pH > 6) (see Supplementary Figure 116 in [1]). The deacyloxylation serves to generate the active species p-QM [58][59][60], which is the key to formation of pre-reaction complex for the following C-C coupling [61,62]. The formation of p-QM is the rate-determining step of the dimerization.…”