Ligand-Dissociation-Type N,N-Dimethylaminopyrene Probes for In-Situ Site-Specific Protein Labeling.

To develop practical methods for in-situ labeling of target proteins and to analyze their binding modes with bioactive ligands, 6-N,N-dimethylaminopyrene-N-acyl-N-alkylsulfonamide-4,8-diazacyclononyne (dmpy-NASA-DACN) tags were synthesized. Strain-promoted azide-alkyne cyclization with azide-conjugated ligands (biotin and sulfonamide) gave ligand-dissociation-type dmpy probes. With these probes, specific labeling of avidin and human carboxylase 1 (hCA1) proceeded even in the presence of cell lysate proteins in ca. 10% RIPA buffer. Affinity purification, in-gel tryptic digestion on polystyrene gel, and MALDI MS analysis established the dmpy-labeled positions of target proteins. Molecular modeling studies also supported why the dmpy-labeling reactions proceeded site-specifically near ligand-binding sites on the target proteins. Our findings might contribute to the development of chemical probes that specifically label various biomacromolecules in cells.