Development of diazirine–alkyne tag for protein–ligand interaction analysis with photoaffinity labeling

We have developed a diazirine–alkyne tag for target protein–ligand interaction analysis. A sulfonamide photoaffinity probe was readily prepared using copper-catalyzed azide–alkyne cycloaddition (CuAAC), which site-specifically formed a covalent bond with human carbonic anhydrase 1 (∼30% labeling yield). The photolabeled position (E58–H64 region) was determined based on the results of a tandem mass spectrometry (MS/MS) analysis of tryptic peptides and was supported by molecular modeling studies. Our method may be useful for determining unknown ligand-binding sites of various target biomolecules.