title: Affinity-labeling-based introduction of a reactive handle for natural protein modification.
abstract: A new chemical method to site-specifically modify natural proteins without the need for genetic manipulation is described. Our strategy involves the affinity-labeling-based attachment of a unique reactive handle at the surface of the target protein, and the subsequent selective transformation of the reactive handle by a bioorthogonal reaction to introduce a variety of functional probes into the protein. To demonstrate this approach, we synthesized labeling reagents that contain: 1) a benzenesulfonamide ligand that directs specifically to bovine carbonic anhydrase II (bCA), 2) an electrophilic epoxide group for protein labeling, 3) an exchangeable hydrazone bond linking the ligand and the epoxide group, and 4) an iodophenyl or acetylene handle. By incubating the labeling reagent with bCA, the reactive handle was covalently attached at the surface of bCA through epoxide ring opening. Either after or before removing the ligand by a hydrazone/oxime-exchange reaction, which restores the enzymatic activity, the reactive handle incorporated could be derivatized by Suzuki coupling or Huisgen cycloaddition reactions. This method is also applicable to the target-specific multiple modification in a protein mixture. The availability of various (photo)affinity-labeling reagents and bioorthogonal reactions should extend the flexibility of this strategy for the site-selective incorporation of many functional molecules into proteins.
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan.
authors.forename: Haruto
authors.initials: H
authors.lastname: Wakabayashi
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Miyagawa
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Koshi
authors.forename: Yousuke
authors.initials: Y
authors.lastname: Takaoka
authors.forename: Shinya
authors.initials: S
authors.lastname: Tsukiji
authors.forename: Itaru
authors.initials: I
authors.lastname: Hamachi
mesh_terms: D000114:Acetylene; D000345:Affinity Labels / Q000737:chemistry*; D000818:Animals; D024402:Carbonic Anhydrase II / Q000737:chemistry; D002417:Cattle; D006835:Hydrazones; D015336:Molecular Probe Techniques; D011506:Proteins / Q000737:chemistry*; D013449:Sulfonamides; D000096926:Benzenesulfonamides
chemical_list: D000114:Acetylene; D000345:Affinity Labels; D024402:Carbonic Anhydrase II; D006835:Hydrazones; D011506:Proteins; D013449:Sulfonamides; D000096926:Benzenesulfonamides