The influence of N-alkyl chains in benzoyl-thiourea derivatives on urease inhibition: Soil studies and biophysical and theoretical investigations on the mechanism of interaction

Abstract

Ureases are enzymes produced by fungi, plants, and bacteria associated with agricultural and clinical problems. The urea hydrolysis in NH3 and CO2 leads to the loss of N-urea fertilizers in soils and changes the human stomach microenvironment, favoring the colonization of H. pylori. In this sense, it is necessary to evaluate potential enzyme inhibitors to mitigate the effects of their activities and respond to scientific and market demands to produce fertilizers with enhanced efficiency. Thus, biophysical and theoretical studies were carried out to evaluate the influence of the N-alkyl chain in benzoyl-thiourea derivatives on urease enzyme inhibition. A screening based on IC50, binding constants, and theoretical studies demonstrated that BTU1 without the N-alkyl chain (R = H) was more active than other compounds, so the magnitude of the interaction was determined as BTU1 > BTU2 > BTU3 > BTU4 > BTU5, corresponding to progressively increased chain length. Thus, BTU1 was selected for interaction and soil application essays. The binding constants (Kb) for the supramolecular urease-BTU1 complex ranged from 7.95 to 5.71 × 103 M−1 at different temperatures (22, 30, and 38 °C), indicating that the preferential forces responsible for the stabilization of the complex are hydrogen bonds and van der Waals forces (ΔH = −15.84 kJ mol−1 and ΔS = −36.61 J mol−1 K−1). Theoretical and experimental results (thermodynamics, synchronous fluorescence, and competition assay) agree and indicate that BTU1 is a mixed inhibitor. Finally, urease inhibition was evaluated in the four soil samples, where BTU1 was as efficient as NBPT (based on ANOVA two-way and Tukey test with 95% confidence), with an average inhibition of 20% of urease activity. Thus, the biophysics and theoretical studies are strategies for evaluating potential inhibitors and showed that increasing the N-alkyl chain in benzoyl-thiourea derivatives did not favor urease inhibition.