Visible-light Responsive Defluorination-Acyl Fluoride Exchange for Photoclick Labeling Based on Phenoxazine Chromophore

Photoclick chemistry represents an integration of photo- and click chemistry, enabling spatiotemporal control, high selectivity, and efficient conjugation. Photo-induced defluorination acyl fluoride exchange (photo-DAFEx), as a novel photoclick reaction, has emerged as a promising tool for the flourishing field of photoaffinity labeling (PAL) for drug discovery and the exploration of protein interactions. Currently, the first-generation photo-DAFEx reaction relies on the photo-defluorination of a monocyclic m-trifluoromethylaniline, and consequently the excitation wavelength (λ_ex.) falls within the UV-B band (311 nm), limiting its widespread applications. Therefore, there is a high demand for the discovery of innovative cores that can expedite visible-light-induced photo-DAFEx reactions and for the exploration of their crosslinking capabilities. Herein, we report that the combination of phenoxazine chromophores with dialkylated amine auxochromes expands the excitation wavelength (λ_ex.) of the photo-DAFEx into the visible region (405 nm), enabling the multifunctional design of photoaffinity probes for in-situ identification of drug-target interactions. By employing the phenoxazine-based photo-DAFEx reagent, we successfully developed potent PAL probes targeting hCA-II and BRD4, which can be activated with controllability using a 405 nm LED, thereby underscoring the potential of photo-DAFEx in advancing our understanding of protein-ligand interactions.