Enzymatic Coupling of the Herbicide Bentazon with Humus Monomers and Characterization of Reaction Products

Abstract

To elucidate the binding mechanism of the herbicide bentazon (3-isopropyl-1H-2,1,3-benzothiadiazine-4(3H)-one 2,2-dioxide) with humic monomers in the presence of an oxidative enzyme, the reaction of bentazon with catechol, caffeic acid, protocatechuic acid, and syringaldehyde was investigated. In the presence of a laccase from the fungus Polyporus pinsitus, catechol was the most reactive humic monomer; bentazon with catechol in the presence of the laccase was completely transformed in 30 min at pH 4.0. The reactivity of bentazon decreased with increasing pH, but reactivity of bentazon decreased with increasing pH, but complete transformation of bentazon could be achieved even at high pH if the concentration of catechol was increased. When bentazon was incubated with humic acid (extract of leonardite) in the presence of the laccase, a reaction of the two substrates was observed, as indicated by bentazon disappearance. Two metabolites that result from the reaction of bentazon with catechol were isolated by TLC and HPLC and identified by mass spectrometry and NMR spectroscopy. A product with a molecular weight of 348 was characterized by 1-D, 2-D 1H-, and 13C-NMR spectroscopy and identified as a dimer composed of one catechol and one bentazon molecule. A second reaction product with a molecular weight of 586 appeared to be a trimer, consisting of one catechol molecule and two bentazon molecules. The analyses also showed that catechol was bound to the protonated nitrogen of the heterocyclic ring and not to a carbon of the aromatic ring of bentazon; this incorporation results from nucleophilic addition of the o-quinone to the nitrogen.