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Development of a Versatile Protein Labeling Tool for Live‐Cell Imaging Using Fluorescent β‐Lactamase Inhibitors

A versatile protein labeling system was developed using non-β-lactam β-lactamase inhibitors. Fluorophore-diazabicyclooctane prodrug conjugates efficiently and stably labeled β-lactamase tag-fused proteins localized in various intracellular compartments in living cells. Furthermore, use of a pH-activatable fluorophore allowed visualization of lysosomal protein trafficking at the late stage of autophagy under cellular starvation.


To understand the function of protein in live cells, real-time monitoring of protein dynamics and sensing of their surrounding environment are important methods. Fluorescent labeling tools are thus needed that possess fast labeling kinetics, high efficiency, and long-term stability. We developed a versatile chemical protein-labeling tool based on fluorophore-conjugated diazabicyclooctane β-lactamase inhibitors (BLIs) and wild-type TEM-1 β-lactamase protein tag. The fluorescent probes efficiently formed a stable carbamoylated complex with β-lactamase, and the labeled proteins were visualized over a long period of time in live cells. Moreover, use of an α-fluorinated carboxylate ester-based BLI prodrug enabled the probe to permeate cell membranes and stably label intracellular proteins after unexpected spontaneous ester hydrolysis. Lastly, combining the labeling tool with a pH-activatable fluorescent probe allowed visual monitoring of lysosomal protein translocation during autophagy.

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