Controllable <i>in‐situ</i> Synthesis of <scp>NIR</scp>‐<scp>II</scp> Hemicyanine in Living Organisms for Efficient Tumor Imaging

Comprehensive Summary Second near‐infrared region (NIR‐II) small‐molecule fluorophores hold great promise for biomedical imaging because of their well‐defined chemical structures, superior metabolic properties, deep tissue penetration, and unprecedented clarity. However, it is impossible to apply conventional synthesis methods for the in‐situ generation of NIR‐II fluorophores in living organisms because of the need for high temperatures, strong acids or bases, and anhydrous conditions. Herein, we report an innovative strategy to create NIR‐II hemicyanine ( NIR‐II‐Hcy ) with high conversion efficiency (up to 86%) under mild conditions, using β ‐chloroacroleins derivatives ( Pre‐Hcy ) and meta ‐aminothiophenol as precursors without harsh conditions. Interestingly, this method can be extended to live mice, and the acidic microenvironment is conducive to the rapid production of NIR‐II‐Hcy ( k pH = 6.0 / k pH = 8.0 = 10). Furthermore, by leveraging the formation/cleavage of disulfide bonds and the acidic microenvironment, the in‐situ generation of NIR‐II‐Hcy enables the activation to be controlled by glutathione (GSH) and H + , thereby enabling high‐contrast and discriminative in vivo tumor imaging, and this work establishes the first paradigm for tumor microenvironment‐driven NIR‐II fluorogenesis. This is a novel finding that will facilitate simpler and faster synthesis of NIR‐II dyes with broad applications in biomedical research and clinical diagnostics.