Second generation PSMA-targeted turn-on probe for imaging cargo release in prostate cancer cells.

Targeted payload release in cancer cells can be modulated by tuning both the linker, spacer, and the payload chemistries. In previous studies, a PSMA-targeted probe incorporating a 7-amino-4-methylcoumarin (AMC) payload and a PEG linker resulted in predominant payload release in the lysosome (pH ∼5.0). Here, we introduce a second-generation PSMA-targeted turn-on probe with a shorter, hydrophobic linker and a 7-hydroxy-4-methylcoumarin (HMC) payload. Based on pH-dependent kinetic studies, the HMC payload exhibits faster cleavage at a slightly higher pH (pH 5.5), suggesting an earlier release-potentially more in early endosomes than lysosomes. Our results demonstrate that subtle changes in linker and payload structures can alter intracellular release kinetics, offering improved control over the cellular release site, which is critical for optimizing targeted therapeutic and imaging strategies in prostate cancer cells.