Cross-linking of chitosan with ferulic acid in the presence of bacterial laccase: Investigation of the mechanism – the Michael addition reaction or the formation of Schiff bases?

We demonstrate for the first time the feasibility of using bacterial laccase as a biocatalyst for oxidizing ferulic acid (FA) to enable nucleophilic crosslinking of chitosan under neutral conditions, targeting future applications in medicine and the food industry. We have established optimal conditions achieving a high degree of chitosan crosslinking (up to 85%). Employing quantum-chemical modeling and spectral analysis techniques, it was determined that the predominant mechanism for the nucleophilic attack of chitosan amino groups on the phenoxyl radical – the oxidation product of FA – involves Michael addition, followed by radical recombination. This process results in the formation of crosslinks consisting of β-β’ coupled FA dimers while retaining the phenoxyl functionalities.