Protein Chemical Labeling Using Biomimetic Radical Chemistry

Abstract

Chemical labeling of proteins with synthetic low-molecular-weight probes is an important technique in chemical biology. To achieve this, it is necessary to use chemical reactions that proceed rapidly under physiological conditions (i.e., aqueous solvent, pH, low concentration, and low temperature) so that protein denaturation does not occur. The radical reaction satisfies such demands of protein labeling, and protein labeling using the biomimetic radical reaction has recently attracted attention. The biomimetic radical reaction enables selective labeling of the C-terminus, tyrosine, and tryptophan, which is difficult to achieve with conventional electrophilic protein labeling. In addition, as the radical reaction proceeds selectively in close proximity to the catalyst, it can be applied to the analysis of protein-protein interactions. In this review, recent trends in protein labeling using biomimetic radical reactions are discussed.

Keywords: bioinspired chemical catalysis; biomimetic radical reaction; protein labeling.

Figure 1

Generation of tyrosyl radical and tyramide radical. (a) Mechanism of dityrosine generation via single-electron transfer (SET). (b) Tyramide, a labeling agent that mimics tyrosine (c) Mechanism of oxidation in the active site of horseradish peroxidase (HRP).

Figure 2

Tyrosine labeling with PTAD and N-methylated luminol derivatives. (a) Tyrosine labeling with PTAD and side reaction with amine group via isocyanate generation. (b) Tyrosine labeling with N-methylated luminol derivative in the presence of HRP and H₂O₂.

Figure 3

Immunohistochemical signal amplification using HRP-proximity protein labeling. Tyramide and N’-acyl-N-methylphenylenediamine were reported as HRP-proximity protein labeling agents.

Figure 4

Ascorbate peroxidase (APEX) -proximity labeling of endogenous proteins in living cells.

Figure 5

Proposed reaction mechanism for C-terminal labeling with flavin photocatalyst.

Figure 6

Proposed reaction mechanism for tryptophan β-position labeling with iridium photocatalyst.

Figure 7

Proposed reaction mechanism for tyrosine labeling with ruthenium photocatalyst.

Figure 8

Target selective labeling by proximity labeling using ligand-conjugated photocatalysts 26 and 27.

Figure 9

Photocatalyst-proximity tyrosine labeling. (a) Model substrate 28 was labeled with 29. (b) Structure of labeling agent MAUra 29. (c) Model substrate with a rigid proline linker with a distance of several nanometers between ruthenium and tyrosine.

Figure 10

Photocatalyst-proximity labeling with MAUra.

Figure 11

Electrochemical tyrosine labeling. SCE: saturated calomel electrode.

Figure 12

Tryptophan labeling with keto-ABNO and the electrochemical activation of tryptophan labeling.