Mechanistic Evidence for a Radical-Radical Recombination Pathway of Flavin-based Photocatalytic Tyrosine Labeling

We recently introduced flavin-based photocatalysts such as riboflavin tetraacetate (RFT) as a robust platform for light- mediated protein labeling via phenoxyl radical-mediated tyrosine-biotin phenol coupling on live cells. To gain insight into this coupling reaction, we conducted detailed mechanistic analysis for RFT-photomediated activation of phenols for tyrosine labeling. Contrary to previously proposed mechanisms, we find that the initial covalent binding step between the tag and tyrosine is not radical addition, but rather radical-radical recombination. Radical addition to a phenol, while often proposed in the literature, is prohibitively unfavorable according to DFT calculations. Radical-radical recombination, followed by rearomatization, is the preferred pathway for the phenol-phenol coupling presented herein, but may also explain the mechanism of other tyrosine-tagging approaches described in the literature. Competitive kinetics experiments show that phenoxyl radicals are generated by the reaction of phenols with several reactive intermediates in the proposed mechanism: primarily with the riboflavin-photocatalyst excited state or singlet oxygen, but also possibly with the semi-reduced photocatalyst or hydroperoxyl radicals produced at key steps in the mechanism.