ortho-Substituents govern aryl aldehyde reactivity: toward lysine-targeted, tunable inhibitors of glucose-6-phosphate...

Certain drugs achieve highly potent and long-lasting enzyme inhibition by forming covalent bonds with nucleophilic amino acid functionalities. Aldehyde-containing inhibitors are able to react with lysine ε-amino functionalities to form reversible-covalent imine (Schiff base) adducts. ortho-Substituted aryl aldehydes have been used to further stabilize these imines through intramolecular interactions. However, relationships between aryl aldehyde structure, reactivity, and enzyme inhibition remain poorly defined. To address this gap, we characterized imine formation across a diverse panel of aryl aldehydes in aqueous solution and tested whether reactivity trends will predict covalent inhibition mechanisms. First, we assessed imine yield, reaction rate, and apparent binding affinity by NMR and UV-vis spectroscopy using a lysine surrogate. We then evaluated inhibitory activity against an enzyme using dose-response and dilution assays to determine potency and reversibility. Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (G6PD), which contains a lysine-rich active site, served as the enzyme model. The reactivity of aryl aldehydes in our panel was primarily governed by ortho-substituents, particularly their ability to stabilize the imine and coordinate the reacting partners. Reactivity generally correlated with inhibitor potency, but not with reversibility. We also identified previously unreported aryl aldehyde G6PD inhibitors bearing boronic acid, ethynyl, and phosphonate substituents, spanning irreversible-covalent, reversible-covalent, and rapid-equilibrium inhibition mechanisms. These findings establish structure-reactivity-activity relationships for aryl aldehydes and demonstrate their potential for tunability in rational inhibitor design. Collectively, this work strengthens the foundation for lysine-targeted covalent inhibitor development.