Chalcogenated phosphine derivatives as urease inhibitors for agricultural: soil application and biophysical studies

Urease, a metalloenzyme that catalyzes urea hydrolysis, is associated with nitrogen losses in soils. In agricultural settings, although the commercial inhibitor N-(butyl)thiophosphoric triamide (NBPT) is widely used, its low stability under adverse environmental conditions has prompted the search for new, more selective, and stable inhibitors. In this study, triphenylphosphine derivatives functionalized with chalcogens (SF1-SF3) and the corresponding precursor (SF4) were investigated for the ability to transiently inhibit urease catalytic activity. These compounds were tested against urease from Canavalia ensiformis and against soil with varying physicochemical properties. Classic kinetic assays and biophysical studies of urease-ligand interactions were carried out to investigate the mechanisms of urease inhibition. Even in the presence of humic substances, the selenium-containing derivative SF3 was the most effective urease inhibitor among the tested compounds, regardless of soil type. SF3 works as a typical uncompetitive inhibitor, likely by interacting with free cysteine residues located in the flap region near the active site. In situ spectroscopic evidence shows that SF3 may react with cysteine residues to form SF1 and H₂Se. Molecular fluorescence approaches demonstrated that SF3 spontaneously interacts with urease and with urease-SF3 via static quenching, driven by electrostatic interactions. These findings highlight SF3 as a promising candidate for application as a urease inhibitor in enhanced-efficiency fertilizers.