Design,Synthesis And Application Of Fluorescent Probe For Recognition Of Macromolecules Based On Coumarin Derivatives

Enzyme is a kind of biological macromolecule.Under the catalytic action of enzyme,various catalytic reactions can occur in the organism,which makes the complex metabolic process in the cell orderly.On the contrary,when the abnormal enzyme content,it will lead to a series of diseases.Elastase,for example,is an important proteolytic enzyme that catalyzes the decomposition of casein,hemoglobin,fibrin and albumin.Abnormal elastase content in human body can lead to various inflammatory diseases,such as acute lung injury and obstructive lung disease.Tyrosinase is an oxidase that regulates melanin production.Overexpression of tyrosinase in human body can lead to diseases such as melanoma,and its metabolic disorders can inhibit the production of melanin,resulting in skin diseases such as vitiligo.Therefore,the development of an efficient tool for the detection of biological macromolecules is of great significance for the diagnosis of related diseases.Fluorescent molecular probe technology has the advantages of simple operation,good specificity and selectivity,high sensitivity,high temporal and spatial resolution,and real-time non-damage monitoring.It has been widely used in molecular detection,tumor diagnosis,biological imaging and other fields.Among them,coumarin fluorophore has the characteristics of easy modification,large Stokes shift and stable optical properties,so it is widely used in the study of fluorescent probes.Therefore,the design and synthesis of fluorescent probes based on coumarin derivatives for the detection of biological macromolecules is an important technical means.This paper designed and synthesized the following three fluorescent probes,and completed the monitoring of enzyme level at molecular level,cellular level and in vivo level,the contents are described as follows:Firstly,a novel fluorescent probe HFC-TYR based on coumarin derivative dye was designed and synthesized.The probe can be used for detection of tyrosinase,7-Hydroxy-4-(trifluoromethyl)coumarin is fluorophore,fluorescence quenching occurs under the PET effect of m-hydroxybenzyl,when combined with tyrosinase identification,ether bond rupture,PET effects are suppressed,shows strong fluorescence at 505 nm,namely the probe for fluorescence enhanced probe.HFC-TYR has the advantages of large Stokes shift(170 nm),low fluorescence background,low fluorescence detection limit(0.12 U m L-1)and high selectivity.In addition,HFC-TYR has been successfully used to image endogenous tyrosinase in living cells and zebrafish,and can distinguish hepatoma cells(Hep G2)from other cells(293T,He La,and SKOV3).These results indicate that HFC-TYR is an effective tool for detecting and imaging tyrosinase.Second,new fluorescent probe based on coumarin derivatives synthesized(HNCOU-NE),HNCOU is fluorophore,pentafluoroethyl is he enzyme-active trigger moiety,its fluorescence background is almost zero,elastase catalytic amide bond rupture,which contain amino fluorophore release,shows strong fluorescence at 535 nm.HNCOU-NE has good detection performance,such as large Stokes shift(165 nm),high affinity for elastase(Km = 5.04 μM)and wide linear response concentration(0-10 U m L-1).In addition,we successfully monitored elastase activity in Hep G2 cells with the HNCOU-NE,and the probe achieved the detection and imaging of endogenous elastase in living zebrafish.Finally,a novel fluorescent probe HFC-NE containing self-immolative linker was designed for the detection of elastase.Under the protection of the recognition group and the self-immolative linker,the probe has no fluorescence.When the probe reacts with elastase,the amide bond breaks at first,and the self-immolative linker leaves spontaneously,and the fluorophore HFC is released.The HFC-NE probe has the advantages of low detection limit(0.0109 U m L-1),high selectivity and rapid response.In addition,HFC-NE has good water solubility and fluorescence signal enhancement 176-fold in the presence of elastase,with good signal-to-noise ratio.HFC-NE has been successfully used for the detection and imaging of endogenous elastase in living cells and zebrafish models.More importantly,HFC-NE can be used to distinguish Hep G2 cells from other cells.These results indicate that HFC-NE has potential value in the diagnosis of elastase related diseases.