Affinity-labeling-based introduction of a reactive handle for natural protein modification

Haruto Wakabayashi, Masayoshi Miyagawa, Yoichiro Koshi, Yousuke Takaoka, Shinya Tsukiji, Itaru Hamachi

Research output: Contribution to journalArticlepeer-review

18 Citations (Scopus)


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-exhange 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.

Original languageEnglish
Pages (from-to)1134-1139
Number of pages6
JournalChemistry - An Asian Journal
Issue number7
Publication statusPublished - 2008 Jul 7
Externally publishedYes


  • Affinity labeling
  • Bioorthogonal reactions
  • Protein engineering
  • Protein modifications
  • Target-specific labeling

ASJC Scopus subject areas

  • Biochemistry
  • Organic Chemistry


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