2012/01/05 by Tomonori Tamura, Shinya Tsukiji, Itaru Hamachi · 96 citations
Biochemistry, Genetics and Molecular Biology · Chemistry · Medicine · #Biochemistry #Biophysics #Biotin and Related Studies #Chemical biology #Chemistry #Click Chemistry and Applications #Combinatorial chemistry #Covalent bond #Diazirine #In vitro #Ligand (biochemistry) #Monoclonal and Polyclonal Antibodies Research #Organic chemistry #Piperazine #Protein ligand #Reagent #Receptor #Stereochemistry #Target protein #Tosyl
paper · doi:10.1021/ja209641t
published in Journal of the American Chemical Society 134(4), 2216-2226 (American Chemical Society)
openalex publication_date 2012/01/05 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/26
The ability to modify target "native" (endogenous) proteins selectively in living cells with synthetic molecules should provide powerful tools for chemical biology. To this end, we recently developed a novel protein labeling technique termed ligand-directed tosyl (LDT) chemistry. This method uses labeling reagents in which a protein ligand and a synthetic probe are connected by a tosylate ester group. We previously demonstrated its applicability to the selective chemical labeling of several native proteins in living cells and mice. However, many fundamental features of this chemistry remain to be studied. In this work, we investigated the relationship between the LDT reagent structure and labeling properties by using native FK506-binding protein 12 (FKBP12) as a target protein. In vitro experiments revealed that the length and rigidity of the spacer structure linking the protein ligand and the tosylate group have significant effects on the overall labeling yield and labeling site. In addition to histidine, which we reported previously, tyrosine and glutamate residues were identified as amino acids that are modified by LDT-mediated labeling. Through the screening of various spacer structures, piperazine was found to be optimal for FKBP12 labeling in terms of labeling efficiency and site specificity. Using a piperazine-based LDT reagent containing a photoreactive probe, we successfully demonstrated the labeling and UV-induced covalent cross-linking of FKBP12 and its interacting proteins in vitro and in living cells. This study not only furthers our understanding of the basic reaction properties of LDT chemistry but also extends the applicability of this method to the investigation of biological processes in mammalian cells.