One-Pot Synthesis of Functionalized Coumarin Fluorophores Enables Rapid Access to Live-Cell Bioorthogonal Labeling and Microenvironmental Sensing Agents

Coumarins are robust and versatile organic scaffolds with broad applications in fluorescence imaging. Herein, we report a one-pot methodology for functionalizing coumarins from commercially available and readily accessible intermediates. Using this streamlined approach, we synthesized a library of 7-aminocoumarins with diverse substituents and varied photophysical properties in good yields and on gram scale. To demonstrate the versatility of this methodology, we developed two functional imaging agents: CouPyC6, a mitochondrially targeted viscosity sensor for fluorescence intensity and lifetime imaging, and CouTz, the first zero-distance tetrazinyl-coumarin, which can participate in fluorogenic IEDDA reactions with a range of dienophiles. The latter enables wash-free, real-time bioorthogonal imaging of labeled proteins and glycan targets on the surface of live cells, offering simple access to functional coumarins for advanced imaging applications.