Preparation Of Aggregation-induced-emission Type Fluorescent Probes And Their Application For Detecting Metabolic Enzymes

Metabolic enzymes including nitroreductase and alkaline phosphatase catalyze various metabolic reactions in the human body and also maintain the metabolic balance of substances in the body.Real-time and accurate detection of the activity/level of metabolic enzymes is vital to our understanding of physiological and pathological processes.Fluorescent probes with aggregation-induced emission（AIE）exhibit many advantages,such as high detection sensitivity and excellent photostability.In this study,two AIE fluorescent probes are designed and prepared for the detection of nitroreductase and alkaline phosphatase respectively.The main contents are as follows:（1）Design and preparation of AIE fluorescent probe TPA-Py-NO₂ based on triphenylamine for the detection of nitroreductase（NTR）.On the basis of methoxytriphenylamine,the fluorophore TPA-Py is synthesized by Suzuki,Knoevenagel condensation and other reactions,and then the recognition group 2-benzyl-5-nitrofuran for nitroreductase is introduced to obtain the fluorescent probe TPA-Py-NO₂.The structure of the probe TPA-Py-NO₂ has been characterized and its detection performance has been investigated.When the probe reacts with nitroreductase,the nitro group on TPA-Py-NO₂ is reduced to an amino group,which causes the elimination of the recognition group and releases the fluorophore molecule TPA-Py,which emits strong fluorescence at 650 nm.The probe can also be used for fluorescence imaging of nitroreductase in living cells and mouse tumors.（2）Design and preparation of AIE fluorescent probe TPE-CN-pho based on tetraphenylethylene for the detection of alkaline phosphatase（ALP）.On the basis of tetraphenylethylene,the fluorophore TPE-CN-OH is synthesized through reactions such as Suzuki reaction,and then the fluorophore is modified with the phosphate group,which is alkaline phosphatase response group,to make it to fluorescent probe TPE-CN-pho.The structure of the probe TPE-CN-OH has been characterized and its detection performance has been investigated.In the aqueous phase,after the fluorescent probe reacts with alkaline phosphatase,the hydrophilic phosphate group on the probe molecule is cleaved to generate the poorly water-soluble enzymatic product TPE-CN-OH,which quickly aggregates in water and emits green fluorescence（emission wavelength is 543 nm）.After the probe responds to alkaline phosphatase,the fluorescence intensity increases by 9-fold.The probe can be applied to fluorescence imaging of endogenous alkaline phosphatase in living cells.