π-Extended COUPY Fluorophores for Targeted Near-Infrared Fluorescence and Lifetime Imaging in Live Cells.

Fluorescent probes operating in the near-infrared (NIR) region offer deep-tissue visualization with minimal background interference, reduced phototoxicity, and enhanced signal-to-noise ratios. However, most conventional NIR fluorophores exhibit poor photostability, low fluorescence quantum yields, and limited chemical versatility, which constrain their use in demanding biological environments. Here, we report a new family of π-extended COUPY dyes (3-7), rationally engineered to deliver efficient far-red/NIR performance through vinylogation of the exocyclic double bond of the coumarin scaffold. This single structural modification induces a bathochromic shift exceeding 100 nm in both absorption and emission spectra while preserving the compact architecture and synthetic accessibility of the parent coumarin. The resulting dyes combine high molar absorptivity, moderate-to-high quantum yields, and exceptional photostability under biologically-relevant conditions. Their performance was validated in live-cell imaging using both intensity-based and fluorescence lifetime imaging microscopy (FLIM), demonstrating compatibility with advanced imaging techniques. Furthermore, site-specific bioconjugation to a clinically relevant peptide confirmed their suitability for targeted imaging. Collectively, π-extended COUPY dyes represent a promising class of synthetically modular, chemically robust, and biologically compatible far-red/NIR fluorophores for high-resolution imaging, targeted strategies, and advanced bioimaging modalities, and provide a foundation for future studies aimed at evaluating their performance in more complex biological models.