Improved Asparaginyl-Ligase-Catalyzed Transpeptidation via Selective Nucleophile Quenching

The use of enzymes for the site-specific modification of proteins/peptides has become a highly accessible, widespread approach to study protein/peptide functions or to generate therapeutic conjugates. Asparaginyl endopeptidases (AEPs) that preferentially catalyze transpeptidation reactions (AEP ligases) have emerged as enticing alternatives to established approaches, such as bacterial sortases, due to their catalytic efficiency and short tripeptide recognition motifs. However, under standard conditions, a substantial excess of the nucleophile to be conjugated is needed to reach desirable yields. Herein we report a versatile approach to shift the AEP-catalyzed transpeptidation equilibrium toward product formation via selectively quenching the nucleophilicity of the competing leaving-group peptide. Our metal-complexation-based strategy enables efficient peptide/protein labeling at the N- or C-terminus with near-equimolar concentrations of nucleophile label. Furthermore, we show that this approach can enhance protein-protein ligation and facilitate the formation of transpeptidation products that are otherwise unattainable.

Automated summary

The use of enzymes, specifically asparaginyl endopeptidases (AEPs), for site-specific modification of proteins/peptides has become a popular method in studying protein/peptide functions or generating therapeutic conjugates. A novel approach using metal-complexation-based strategy has been developed to shift the AEP-catalyzed transpeptidation equilibrium towards product formation, allowing efficient peptide/protein labeling and enhancing protein-protein ligation. This method shows promise in facilitating the formation of transpeptidation products that are typically challenging to attain.