Tuning probe permeability chalcogen and halogen atom substitution for monitoring alkaline phosphatase activity in mammalian cells.

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 groups from various molecules, a process known as dephosphorylation. It regulates diverse cellular functions such as bone metabolism and liver function. Alteration of ALP can be a biomarker for pathological conditions. Hence, the development of fluorescent probes for 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, chalcogen-containing compounds were found to be internalized by cells primarily through macropinocytosis, likely mediated by chalcogen bonding, with those capable of forming stronger chalcogen bonds demonstrating greater cellular uptake.