Revealing Gasotransmitter Regulation To Phosphatase Activity In Cells By A Single-molecule Fluorescent Probe

Enzymes play a key role in almost all of metabolic processes in live cells and in vivo.Abnormal enzyme activity have been used as diagnostic markers for some diseases,and even directly related to various cancers.Therefore,it is of great significance to determine the activity of enzymes for the regulation and clinical diagnosis.Recent studies have shown that the activity of enzymes in live cells is dynamically regulated by small molecule gasotransmitters for maintaining normal physiological functions.A few novel small-molecule fluorescent probes have been innovated to individually visualize intracellular enzyme activities and small molecule gasotransmitters.But revealing the gasotransmitter regulation to enzyme activity in-situ remains a long-standing difficulty.In this dessertation,we report a three-channel imaging correlation by a single dual-reactive fluorescent probe to achieve the measurements of phosphatase activity dependent on H2S levels in cells.The main research contents are as follows:1.Based on a large number of literature studies,we designed a single-molecule fluorescent probe that can react with both H2S and phosphatase.We introduced H2S-sensitive and phosphatase-sensitive group to modify coumarin and Rhodol(an oxonium dye of mixed structure of fluorescein and rhodamine)respectively,and two fluorophores were combined to form a single fluorescent probe via a bridge linker.The probe N3-CR-PO4 was successfully synthesized by substitution reaction,azide reaction,amidation and so on.After separation and purification,the probe was characterized by mass spectrometry and nuclear magnetic resonance.2.After the probe reacted with H2S,the blue fluorescence of the coumarin was turned on;the probe reacted with phosphatase alone,the phosphate on the Rhodol was cleaved,and emitted green fluorescence.When the probe reacted with H2S and phosphatase simultaneously,resonance energy transfer can be triggered from the coumarin to the Rhodol,and the entire system emits green fluorescence.After the investigation of optical properties,we constructed a three-channel fluorescence imaging method3.By the three-channel fluorescence imaging method,the visualized detection of H2S and phosphatase in normal cells and cancer cells by the probe N3-CR-PO4 was achieved.The results showed that a slight deviation from the H2S level leads to a sharp decrease of phosphatase activity,and this was further confirmed through two commercial probes and kits.