Design And Synthesis Of Enzyme-activated Fluorescent Probes And Its Application In Diagnosis Of Diseases

Enzymes are a group of proteins that catalyze biochemical reactions and have a crucial role in biological systems.The occurrence of many diseases is closely related to changes in enzymes.In clinical medicine,the detection of enzyme activity and other indicators can help doctors diagnose diseases and develop treatment plans.High-resolution monitoring of enzymes through in situ fluorescence imaging is important for understanding the life activities of organisms and the occurrence of diseases.Small-molecule fluorescent probes have received widespread attention because of their high sensitivity,high selectivity and the advantages of simple and rapid detection.Although a number of enzyme-activated fluorescent probes have been reported,they still have disadvantages such as low sensitivity,poor specificity,and short emission wavelength.To this end,two NIR enzyme-activated fluorescent probes with ultra-high sensitivity have been designed and synthesized in this thesis,and applied to biological imaging and early diagnosis of diseases.The main research is divided into two parts as follows:(1)An ultra-high sensitivity near-infrared(NIR) fluorescent probe YDT with a cationic indole structure was designed and synthesized as the first “turn-on”fluorescent probe with mitochondrial targeting for the detection of carboxylesterase 2(CES2).Using benzoate as the recognition group,the ester bond can be severed by CES2 and the intramolecular charge transfer process resumes,resulting in intense fluorescence at 660 nm.The probe has the advantages of high sensitivity(0.165ng/m L),large Stokes shift(150 nm),rapid response in the system(10 min),high selectivity and good biocompatibility for CES2 detection.The reaction mechanism of YDT with CES2 was systematically investigated by using spectroscopy,chromatography,mass spectrometry,theoretical calculations and molecular docking.In addition,we used the probe to distinguish Hep G2 cells from normal liver cells by fluorescence imaging,which is expected to be a new tool to guide hepatocellular carcinoma tumor resection during surgery;we also established cellular liver injury and its remediation model,inflammation model,and mouse model,revealing for the first time that CES2 can be used as a biomarker for early diagnosis of liver-related diseases,providing technical support for subsequent precise treatment.(2)MB-DPP4 was designed and synthesized as a NIR fluorescent probe for the detection of dipeptidyl peptidase(DPP-Ⅳ).The molecule uses methylene blue(MB)as a fluorophore.By destroying its conjugated structure,the binding group with selfleaving properties and the specific recognition group glycine-L-proline of DPP-Ⅳ are introduced to realize the detection of DPP-Ⅳ with high sensitivity and specificity.The probe has a low background fluorescence signal,a maximum emission wavelength of 715 nm,a long wavelength near-infrared fluorescence property,and a low detection limit of 0.29 ng/m L for DPP-Ⅳ.The reaction mechanism of MB-DPP4 with DPP-Ⅳ was systematically investigated by using spectroscopy,chromatography,mass spectrometry,theoretical calculations and molecular docking.In addition,we used the probe to distinguish thyroid cancer cells from normal thyroid cells by fluorescence imaging;we established a tumor-bearing mouse model and studied the expression level of DPP-Ⅳ in thyroid disease using fluorescent probes for the first time.This study provides a new molecular tool for the early diagnosis of thyroid diseases and screening of therapeutic drugs.