Bioorthogonal Photocatalytic Protein Labeling and Cross-Linking Enabled by Stabilized Ketyl Radicals

High Resolution Image Download MS PowerPoint Slide Radical reactions offer transformative potential in biological contexts but remain constrained by poor selectivity and off-target reactivity. We address these limitations through visible-light photocatalytic generation of diaryl ketyl radicals from benzophenones. This strategy circumvents traditional UV excitation pathways by suppressing triplet diradical formation─which drives nonspecific [2 + 2] cycloadditions and H atom abstraction─in favor of bioorthogonal radical–radical coupling. Our platform enables precise live-cell protein labeling with minimal cytotoxicity, including in sensitive primary neuronal cultures, and achieves site-specific modification via genetically incorporated benzophenone-based unnatural amino acids Bpa. The spatial selectivity of this approach exceeds conventional UV-based cross-linking methods, facilitating site-to-site analysis of tertiary protein interactions in structurally defined complexes. We demonstrate these capabilities by (1) quantifying dimerization interfaces of the Diels–Alderase PyrI4 and (2) resolving Bcl-X L /Bid interactions critical for apoptotic regulation. This photocatalysis-driven methodology establishes a robust alternative to cycloaddition-based bioorthogonal chemistry for spatiotemporally controlled interrogation of dynamic biomolecular processes.