STRUCTURE OF A LACCASE-MEDIATED PRODUCT OF COUPLING OF 2,4-DIAMINO-6-NITROTOLUENE TO GUAIACOL, A MODEL FOR COUPLING OF 2,4,6-TRINITROTOLUENE METABOLITES TO A HUMIC ORGANIC SOIL MATRIX

This work presents laccase-mediated model reactions for coupling of re duced 2,4,6-trinitrotoluene (TNT) metabolites to an organic soil matri x. The structure of an isolated coupling product of 2,4-diamino-6-nitr otoluene (2,4-DANT) to guaiacol as humic constituent was determined, A mong several structures, the compound was identified conclusively to b e the trinuclear coupling product 5-(2-amino-3-methyl-4-nitroanilino)- 3,3' dimethoxy-4,4'-diphenoquinone. The compound has a weight of 409 g mol(-1) and may serve as a model reaction for the biogenic formation of bound residues in soil from TNT by coupling aminotoluenes (reduced TNT metabolites) to humic constituents, A linear correlation of the su bstrate consumption to the enzyme activity was detected. Based on this observation, the described reaction of 2,4-DANT coupling to guaiacol may be used for determination of laccase activity since the reaction w as not inhibited by other compounds of culture supernatants. We propos e a two-step mechanism for the coupling reaction because 2,4-DANT was not transformed by laccases in the absence of guaiacol and guaiacol ox idation was independent of the presence of 2,4-DANT, The first reactio n step is a laccase-mediated dimerization of two guaiacol monomers wit h subsequent oxidation to a diphenoquinone. The second step is the nuc leophilic addition of 2,4-DANT to the ortho position of the carbonyl g roup of the diphenoquinone structure.