Design,Synthesis And Application Evaluation Of Nitroxyl And Lipases Fluorescent Probes

Fluorescent probe is one of the visualization methods for the detection of bioactive molecules in vivo.Due to its high sensitivity,high selectivity and good biocompatibility,fluorescent probe has been widely used to detect protein,nucleic acid,enzyme and bioactive small molecules in vitro and in vivo.Based on the important physiological roles of nitroxyl and hydrolases in vivo,fluorescence probes for detecting nitroxyl,lipase and diacylglycerol lipase were studied in this thesis.This thesis includes the following three parts.Part 1:A rapid-response and near-infrared fluorescent probe for imaging of nitroxyl in living cells.Nitroxyl（HNO）is the one-electron reduction product of nitric oxide and plays a unique biological role in physiological processes.HNO plays an important role in the treatment of heart failure,alcoholism and the prevention of IR injury.However,the generation pathway and physiological mechanism of endogenous HNO are still unclear due to its short lifetime in vivo and the lack of tools for its rapid detection.In this work,a new fluorescent probe CPN for detecting HNO was prepared by employing 2-（diphenylphosphino）benzoate as a recognition group and CP-OH as a near-infrared fluorophore.The hydroxyl group of CP-OH reacted with the carboxyl group of 2-（diphenylphosphino）benzoic acid to form an ester bond,which inhibited the intramolecular charge transfer（ICT）effect and quenched the fluorescence of CPN.When probe CPN interacted with HNO,the ester of CPN was destroyed to release the fluorescent group CP-OH,which emitted strong fluorescence in the near-infrared region（670 nm）.In vitro and in vivo performance evaluation of probe CPN showed that it could rapidly detect HNO in vitro and in vivo with the detection limit of 1.0×10^-6 M,which was very important for monitoring the dynamic changes of HNO in biological systems.Therefore,probe CPN has great potential to study the role of HNO in biological systems.Part 2:A ratio fluorescent probe for detecting lipase activity in human serum.Acute pancreatitis is a common gastrointestinal disease with high morbidity and high mortality.Serum lipase mainly comes from pancreas.It is one of the important biomarkers in the diagnosis of acute pancreatitis,and its sensitivity and accuracy are much higher than serum amylase.Therefore,the detection of serum lipase activity is of great significance for the early diagnosis of acute pancreatitis.However,only a few single emission fluorescent probes have been developed to detect lipase activity in two-phase solution,and the accuracy of their test results is easily affected by the environment.Therefore,there is still a demand for developing an environment friendly fluorescence probe with self-calibration for the detection of lipase activity.In this study,a ratio fluorescent probe（CARA）was designed and synthesized to detect lipase activity based on the hydrolysis of lipase to ester bond.Using alanine as the core,its amino-group was linked with the carboxyl group of coumarin acid by amide bond to form energy donor（CA）,and the carboxyl group of CA was linked with the energy acceptor rhodamine alcohol（RA）by ester bond.Fluorescent Resonance Energy Transfer（FRET）effect was established between CA and RA.When the probe CARA interacted with lipase,the FRET process between CA and RA was altered by the hydrolysis of its eater bond by lipase,resulting in the changes of fluorescence signal,so that CARA could be used to detect lipase activity.In vitro evaluation of probe CARA showed that it had good selectivity and anti-interference ability to lipase in human serum（HS）and aqueous solution.The detection limits of CARA probe in phosphate buffered saline（PBS）and HS/PBS solution were 48.5 U/L and 69.88 U/L,respectively.Therefore,the probe CARA has potential application values in the early clinical diagnosis of acute pancreatitis.Part 3:A natural substrate-based ratio fluorescent probe for detecting diacylglycerol lipase activity.Diacylglycerol lipase（DAGL）is a key enzyme for endogenous cannabinoid 2-arachidonoylglycerol（2-AG）biosynthesis.DAGL participates in various physiological actions in the central nervous system by regulating the level of 2-AG.DAGL is reported to play important roles in neuropsychiatric diseases,including epilepsy,anxiety,depression and fear.However,the dynamic activity of DAGL in various neuropsychiatric diseases is still unclear due to the lack of tools for the in-situ detection of DAGL activity.In this work,a fluorescent probe（DCR）was designed and synthesized for the specific detection of DAGL activity based on the structure of DAGL substrate-diacylglycerol and the specific hydrolysis of 1-ester bond of diacylglycerol by DAGL.Using glycerol as the core,coumarin acid（CR）and rhodamine derivative（LR）were linked with 1-OH group and 2-OH group of glycerol through ester bond,respectively,which established FRET system between the two fluorophores.When the probe DCR interacted with DAGL,the FRET system was destroyed due to the hydrolysis of its 1-ester bond,resulting in changes in the FRET signals between two fluorophores,so that DCR can be applied to detect the activity of DAGL.In vitro and in vivo performance evaluation of probe DCR showed that it had a good selectivity to DAGL,and could be applied to the visual detection of DAGL activity in tissues and in vivo.