A novel ESIPT fluorescent probe based on 3-hydroxyphthalimide derivative for detection of alkaline phosphatase...

Alkaline phosphatase (ALP) is a family of enzymes that play significant roles in various physiological and pathological processes. In this study, a new ALP fluorescent probe was designed utilizing N-alkylated 3-hydroxyphthalimide as the fluorophore, while incorporating a phosphate ester group at the position of the 3-hydroxyl group to serve as the enzyme recognition site. Initially, a series of N-alkylated 3-hydroxyphthalimide compounds were designed and synthesized. The fluorophore with a methylester of phenylalanine substituent attached to the imine nitrogen atom exhibited enhanced fluorescence emission at 515 nm. Subsequently, phosphorylation at the 3-hydroxy group yielded the probe which could be hydrolyzed by ALP, leading to increased fluorescence intensity through excited-state intramolecular proton transfer (ESIPT). Moreover，the fluorescent intensity at 515 nm increases linearly with ALP concentration (R² = 0.995), with a LOD of 0.21 U/L. The probe was almost nontoxic to the cultured cells and was successfully applied in bioimaging in living cells. The results suggest that it has potential for detecting alkaline phosphatase in cellular environments.