A Visible and Near-Infrared Light Activatable Diazo-Coumarin Probe for Fluorogenic Protein Labeling in Living Cells

28 July 2020, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Chemical modification of proteins in living cells permits valuable glimpses into the molecular interactions that underpin dynamic cellular events. While genetic engineering methods are often preferred, selective labeling of endogenous proteins in a complex intracellular milieu with chemical approaches represents a significant challenge. In this study, we report novel diazo-coumarin compounds that can be photo-activated by visible (430‒490 nm) and near-infrared light (800 nm) irradiation to photo-uncage reactive carbene intermediates, which could subsequently undergo insertion reaction with concomitant fluorescence “turned-on”. With these new molecules in hand, we have developed a new approach for rapid, selective and fluorogenic labeling of endogenous protein in living cells. By using CA-II and eDHFR as model proteins, we demonstrated that subcellular localization of proteins can be precisely visualized by live-cell imaging and protein levels can be reliably quantified in multiple cell types using flow cytometry. Dynamic protein regulations such as hypoxia induced CA-IX accumulation can also be detected. In addition, by two-photon excitation with an 800 nm laser, cell-selective labeling can also be achieved with spatially controlled irradiation. Our method circumvents the cytotoxicity of UV light and obviates the need for introducing external reporters with “click chemistries”. We believe that this approach of fluorescence labeling of endogenous protein by bioorthogonal photo-irradiation opens up exciting opportunities for discoveries and mechanistic interrogation in chemical biology.


protein labeling
photo-cross-linkable coumarin
Live cell fluorescence imaging


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