Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins

J Am Chem Soc. 2020 Mar 18;142(11):5078-5086. doi: 10.1021/jacs.9b12002. Epub 2020 Mar 5.

Abstract

Oxidative coupling (OC) through o-quinone intermediates has been established as an efficient and site-selective way to modify protein N-termini and the unnatural amino acid p-aminophenylalanine (paF). Recently, we reported that the tyrosinase-mediated oxidation of phenol-tagged cargo molecules is a particularly convenient method of generating o-quinones in situ. The coupling partners can be easily prepared and stored, the reaction takes place under mild conditions (phosphate buffer, pH 6.5, 4 to 23 °C), and dissolved oxygen is the only oxidant required. Here, we show an important extension of this chemistry for the activation of tyrosine residues that project into solution from the N or C-termini of peptide and protein substrates. Generating the o-quinone electrophiles from tyrosine allows greater flexibility in choosing the nucleophilic coupling partner and expands the scope of the reaction to include C-terminal positions. We also introduce a new bacterial tyrosinase enzyme that shows improved activation for some tyrosine substrates. The efficacy of several secondary amines and aniline derivatives was evaluated in the coupling reactions, providing important information for coupling partner design. This strategy was used to modify the C-termini of an antibody scFv construct and of Protein L, a human IgG kappa light chain binding protein. The use of the modified proteins as immunolabeling agents was also demonstrated.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacillus megaterium / enzymology
  • Bacterial Proteins / chemistry*
  • Monophenol Monooxygenase / chemistry*
  • Oxidative Coupling
  • Quinones / chemical synthesis
  • Single-Chain Antibodies / chemistry*
  • Tyrosine / chemistry*

Substances

  • Bacterial Proteins
  • Quinones
  • Single-Chain Antibodies
  • Tyrosine
  • Monophenol Monooxygenase