Decoupling carboxyl position and annulation site effects on ICT, pH response, and serum albumin binding in dual-regioisomeric benzoindole fluorophores.

This study reports the synthesis of three donor-acceptor (D-A) fluorescent probes (BI-1, BI-2, and BI-3) based on a benzoindole scaffold. The effects of carboxyl substitution position and benzoindole fusion pattern on their photophysical properties and protein-binding behavior were systematically examined. Spectroscopic investigations combined with molecular docking analysis demonstrate that subtle structural variations lead to pronounced differences in absorption and emission characteristics, fluorescence quantum yields, pH responsiveness, and binding preferences toward serum albumin. Docking studies, supported by site-specific competitive binding experiments, reveal distinct binding sites and interaction modes of the probes with bovine serum albumin (BSA), while complementary docking with human serum albumin (HSA) provides comparative structural insight. Notably, BI-1 exhibits a well-defined pH-dependent fluorescence response over an acidic range in vitro, with minimal perturbation from protein binding, whereas BI-2 and BI-3 display pronounced fluorescence enhancement upon protein association. These results highlight how minor modifications in the benzoindole framework can significantly alter electronic properties, protonation behavior, and protein-binding modes. Overall, this work establishes a clear structure-property-binding relationship for benzoindole-based D-A systems, offering mechanistic insight and guiding principles for the rational design of functional fluorescent molecules.