Aminophosphonic Acid Derivatives <b>3r</b> and <b>3s</b> with Dual Herbicidal Activity and Crop Selectivity Targeted 5-Enolpyruvylshikimate-3-phosphate Synthase

In this study, a series of aminophosphoric acid derivatives were synthesized with 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) as a potential target, and their herbicidal activities were evaluated. At 600 g ai/ha, compounds <b>3r</b> and <b>3s</b> exhibited good herbicidal efficacy against <i>Digitaria sanguinalis</i> (DIGSA) and <i>Setaria viridis</i> (SETVI), with inhibition rates of 80% and 90%, and 80% and 95%, respectively. Additionally, both compounds <b>3r</b> and <b>3s</b> demonstrated significant enzyme inhibition activity against <i>S. viridis</i> EPSPS (<i>Sv</i>EPSPS). Expression and purification of <i>Sv</i>EPSPS and <i>Portulaca oleracea</i> EPSPS (<i>Po</i>EPSPS) proteins followed by microscale thermophoresis (MST) revealed that compounds <b>3r</b> and <b>3s</b> had excellent binding affinities for both <i>Sv</i>EPSPS and <i>Po</i>EPSPS, with dissociation constants (<i>K</i>_d) of 0.65 and 1.27 μM, and 0.41 and 0.42 μM, respectively. Isothermal titration calorimetry (ITC) further validated the MST results, which were consistent with expectations. Molecular docking and molecular dynamics (MD) simulations further demonstrated that compounds <b>3r</b> and <b>3s</b> formed strong and stable complexes with <i>Sv</i>EPSPS and <i>Po</i>EPSPS. Moreover, neither compound <b>3r</b> nor <b>3s</b> showed specific binding to the <i>Sv</i>EPSPS^K355A and <i>Po</i>EPSPS^K355A mutant proteins, confirming that K355 is a key amino acid residue for binding. These findings suggest that compounds <b>3r</b> and <b>3s</b> have the potential to act as selective EPSPS inhibitors for the control of six species of grass weeds in crop fields.