Laccase-Mediated Michael Addition of ¹⁵N-Sulfapyridine to a Model Humic Constituent

Chemical incorporation of sulfonamide antimicrobials into natural organic matter may represent an important process influencing the fate of these synthetic, primarily agents in soil and sediment environments. We previously demonstrated that a fungal peroxidase mediates covalent coupling of sulfonamide antimicrobials to model humic constituents; reactions with the 2,6-dimethoxyphenol syringic acid produced Schiff bases (Bialk et al. Environ. Sci. Technol. 2005, 39, 4436−4473). Here, we show that fungal laccase-mediated reaction of sulfapyridine with the ortho-dihydroxyphenol protocatechuic acid yields a Michael adduct. We synthesized ¹⁵N-enriched sulfapyridine to facilitate determination of the covalent linkage(s) formed between sulfapyridine and protocatechuic acid by NMR spectroscopy. ¹H-¹⁵N heteronuclear multiple bond correlation experiments and tandem mass spectrometry demonstrated that the sulfapyridine anilinic nitrogen engaged in a Michael addition reaction to oxidized protocatechuic acid to form an anilinoquinone. Michael adducts are more stable than the previously reported imine linkages between sulfonamides and 2,6-dimethoxyphenols. Michael addition to quinone-like structures in soil organic matter is expected to diminish the mobility and biological activity of sulfonamide antimicrobials.