A Versatile Synthetic Strategy Enables the Discovery of Regioisomer‐Dependent in Vivo Performance in Self‐Immobilizing Hemicyanine Dyes

Self-immobilizing near-infrared (NIR) fluorogenic probes based on hemicyanine dyes show great promise for in vivo imaging due to their superior retention efficiency at target sites. However, their broader development and application have been hampered by tedious synthetic routes and insufficient understanding of how structural variations govern performance in complex biological systems. Herein, we report a versatile and efficient late-stage condensation strategy that provides easy access to a series of self-immobilizing hemicyanine dyes, enabling the first systematic comparison between 5- and 7-regioisomers. Although both probes showed strong fluorogenic responses to alkaline phosphatase in vitro, there in vivo behavior differed remarkably. Unexpectedly, the 5-substituted probe (P5) exhibited significantly lower tumor retention and imaging contrast in live mice than the 7-substituted analogue (P7). Density functional theory (DFT) calculations and mechanistic studies attributed this divergence to the higher reactivity and slower formation kinetics of the quinone methide intermediate generated from P5, leading to nucleophilic attack by water and rapid diffusion from the detection site in highly dynamic in vivo environments. This study not only offers a practical synthetic route but also establishes regiochemical tuning of QM reactivity and formation kinetics as a crucial design strategy for developing imaging agents suited for in vivo applications.