Precise molecular spacer engineering for tumor-responsive pK_a tuning: a pan-cancer imaging platform for evaluating...

Precise molecular engineering for the pK_a regulation of pH-sensing probes with pan-tumor visualization capability remains attractive and challenging. Currently, most molecular engineering strategies focus directly on the fluorophore, and long-distance regulation strategies have been rarely reported. Herein, an innovative spacer-based pK_a regulation strategy was developed. Using a mesoamino-substituted sulfone-xanthene fluorophore, the spacer efficiently regulated the pK_a values to 5.87, 5.37, and 3.73 with three, two, and one spacers, respectively. Density functional theory (DFT) calculations revealed that the elongation of the N-H bond by the trifluoromethyl group (from 1.02 to 1.15 Å) promoted proton dissociation, thereby reducing pK_a. However, as the number of spacers increased, the regulation efficiency showed a decreasing trend. The optimized probe, J-S-BCF₃, successfully illuminated three orthotopic tumor models with high accuracy in depicting tumor boundaries, as well as in microlymph node metastasis and lung metastasis models. Using J-S-BCF₃, reserpine was efficiently identified as a V-ATPase inhibitor through fluorescence-based high-throughput screening of natural alkaloids. Furthermore, the probe effectively monitored tumor microenvironment reprogramming induced by the reserpine treatment. Overall, this innovative chemical strategy, which employs a unique spacer design, broadens the scope for constructing high-performance fluorescent probes with regulated properties, thereby offering high potential for detecting multiple tumors and metastases and facilitating the translation of biomedical research into clinical applications.