March 2024

Hydroxycinnamic acid amides are believed to have antioxidant, antidiabetic, cytotoxic, anticancer, neuroprotective, and antiinflammatory properties, making them interesting target molecules for potential applications in the food, cosmetics, and pharmaceutical industries. Here, we describe the synthesis of hydroxycinnamic acid amides starting from hydroxycinnamic acid esters and the corresponding amines by using variants of the promiscuous hydrolase/acyltransferase from Pyrobaculum calidifontis VA1 (PestE) in water as a solvent. Up to 97% conversion within two hours at 60 °C was achieved with methyl ferulate and tyramine as substrates. This is a promising, environmentally friendly alternative strategy to established chemical synthesis routes or enzymatic methods using lipases in nonaqueous organic solvents.

Due to their strong nucleophilicities, nucleophilic lysine and cysteine residues can be easily recognized and modified by electrophilic groups, thus, acting as the targets for covalent ligands or drugs. Therefore, the development of site-specific protein-modification chemistry for various nucleophilic residues has been explored to label proteins selectively for many biological and therapeutic applications. In this study, we constructed a series of sulfonium-based small molecules to react with the amine group of lysine residues by utilizing the strong electrophilicity of sulfonium, resulting in lysine-selective labeling via the formation of classical amide bonds under alkaline conditions (pH 9.0–11.0). After systematic optimization of the labeling conditions, this strategy was utilized for protein labeling across various bacteria’s lysates. Finally, combined with the activity-based protein profiling (ABPP) strategy, we successfully identified and analyzed hundreds of labeled lysine residues in the bacterial proteome.