Alkaline phosphatase (ALP) is a family of hydrolase enzymes that play crucial roles in a wide range of biological processes. Its primary function is to catalyze the hydrolysis of phosphate group from various molecules, a process known as dephosphorylation. It regulates a diverse cellular functions such as bone metabolism, liver function, and alteration of ALP can be a biomarker for pathological conditions. Hence, the development of fluorescent probes for the ALP detection is essential for studying dynamic dephosphorylation processes in living organisms. Herein, we report a ratiometric fluorescent probe based on a 1,8-naphthalimide derivative functionalized with an ALP-reactive phosphate group and a chalcogen or halogen atom as recognition moieties to enhance the probe internalization in mammalian cells. Upon ALP-mediated cleavage of the phosphate group, the photophysical properties of these compounds undergo significant changes, enabling ratiometric fluorescence detection. Although all the synthesized compounds demonstrated excellent biocompatibility, as well as strong selectivity and sensitivity towards ALP, the selenium-based compound exhibited superior performance in cell-based studies due to its rapid cellular internalization. This compound enables monitoring of ALP activity within a shorter time frame and can be used to identify new inhibitors of ALP. Furthermore it was observed that chalcogen-containing compounds are internalized by cells primarily through macropinocytosis and are likely recognized via chalcogen bonding, with compounds exhibiting stronger chalcogen bond-forming abilities demonstrating higher cellular uptake.
