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.
Imaging GPCR internalization using near-infrared Nebraska red-based reagents
