Novel Cyclopropene Probes as Chemical Reporters for Bioorthogonal Metabolic Labeling of Benzoylated Post-Translational Modification

Lysine benzoylation (Kbz) is a recently identified post-translational modification on a histone that plays a crucial role in regulating cellular processes. Current detection methods primarily rely on mass spectrometry, which limits the ability to dynamically track lysine benzoylation within living cells. Although azide/alkyne small-sized probes have enabled in vitro labeling of various protein acylated modifications, their use in dynamic tracking in cellular levels is limited. Herein, we report a novel 1-methylcyclopropene chemical reporter that undergoes an IEDDA reaction with S-tetrazine-BODIPY 8, which was evaluated for its optical properties, kinetic constants, and bio-orthogonality, revealing it to be the most efficient benzoic acid probe 2. In addition, when SIRT2 acts on the peptides labeled with probe 2, the kinetic parameters of these peptides are comparable to those of endogenously benzoylated peptides. Finally, the metabolic labeling of lysine benzoylation was successfully validated in RAW and HepG2 cells using probe 2. Furthermore, by using a SIRT2 inhibitor, it was confirmed that this metabolic labeling can be applied to dynamically detect changes in lysine benzoylation levels in cells. These findings provide a solid foundation for the development of novel metabolic labeling strategies for dynamically tracking post-translational modifications, particularly in live cells.