NMR Investigation of Enzymatic Coupling of Sulfonamide Antimicrobials with Humic Substances

Phenoloxidases mediate the oxidative transformation of soil phenolic constituents, contributing to the formation of humic substances and the chemical incorporation of some xenobiotic organic compounds into natural organic matter. We previously demonstrated phenoloxidase-mediated covalent coupling of sulfonamide antimicrobials with model humic constituents. Here, we investigate fungal peroxidase-mediated covalent coupling of ¹³C-sulfamethazine and ¹⁵N-sulfapyridine to humic substances. ¹H–¹³C heteronuclear single quantum correlation (HSQC) nuclear magnetic resonance spectroscopy provided an initial indication of peroxidase-mediated covalent binding of ¹³C-sulfamethazine to humic acid. To confirm the role of the sulfonamide anilinic nitrogen in coupling to humic acid and to determine the nature of the covalent linkage, we incubated ¹⁵N-sulfapyridine with humic acid and peroxidase and examined reaction products in ¹H–¹⁵N heteronuclear multiple bond (HMBC) experiments. The HMBC spectra revealed the presence of Michael adducts (i.e., anilinohydroquinones, anilinoquinones) and possibly other covalent linkages. No evidence for Schiff base formation was observed. Analogous experiments with the model humic constituent catechol provided corroborating evidence for these assignments. Michael adducts are expected to exhibit greater environmental stability than imine linkages that can form between sulfonamides and 2,6-dimethoxyphenols. Because the free anilinic nitrogen is required for the bioactivity of sulfonamide antimicrobials, nucleophilic addition occurring through this moiety could result in the biochemical inactivation of these compounds.