Design And Synthesis Of Enzyme-activated Fluorescent Probe Based On Phenoxazinone Structure And Its Biological Application

Metabolism is the basis of all life activities of organisms and is inseparable from the catalysis of enzymes.Therefore,enzymes play crucial role in maintaining and regulating the physiological homeostasis of living systems.Abnormal enzyme activity in living systems is often closely related to some diseases,thus enzymes are often used as important biomarkers for various diseases.Therefore,rapidly accurate analysis and real-time monitoring of enzyme activity within living systems are of great significance for the diagnosis and treatment of diseases.Fluorescent probes have been widely used in biomonitoring and clinical diagnosis for their advantages of simple operation,high sensitivity,excellent selectivity and non-invasibility,especially real-time analysis and imaging in living systems.In this paper,we took β-galactosidase(β-gal),Butyrylcholinesterase(BChE),Leucine Aminopeptidase(LAP),and Aminopeptidase N(APN)as the targets to design and synthesize four fluorescent probes based on fluorophore LAN-OH,which is characterized by high fluorescence quantum yield and near-infrared emission.The potential of LAN-OH-modification has been gradually realized through the transformation of glycosylation to esterification and subsequently to amidation for LAN-OH.This modification process has effectively expanded the application possibilities and fully exploited the application range of LAN-OH as a fluorophore.Being thoroughly studied their spectral characteristics,analytical performances and response mechanisms,the four probes were further applied to the fluorescence imaging of enzyme activity in living systems.The specific research content is as follows:(1)A highly sensitive β-gal-activated fluorescent probe LAN-βgal was developed by directly modifying the hydroxyl site of LAN-OH(ΦF = 0.47)with D-galactose residue,leading the formation of probe LAN-βgal(ΦF < 0.001)with a very low fluorescence signal.After being activated by β-gal,LAN-βgal can achieve up to a 2000-fold enhancement in fluorescence signal,which is the highest enhancement factor of β-gal-related organic fluorescence probe reported so far.LAN-βgal is characterized by high stability,good water solubility,high sensitivity,low detection limit and excellent selectivity,and can quantitatively detecte of β-gal by fluorescence and colorimetry method,and accurately detect β-gal activity in human urine and serum.In cell imaging experiments,LAN-βgal can specifically label β-gal in ovarian cancer cells,easily distinguish ovarian cancer cells from normal ovarian cells,and confirm the positive correlation between cellular senescence and intracellular β-gal levels.In in vivo imaging experiments,LAN-βgal can specifically label β-gal in ovarian cancer,achieving visual imaging of ovarian cancer tumors.Thanks to the 218-fold fluorescence ratio of tumor to normal tissue,LAN-βgal can be used effectively for fluorescenceguided surgical resection of tumors.Therefore,LAN-βgal has great potential for clinical diagnosis and surgical navigation of ovarian cancer.(2)A highly sensitive near-infrared fluorescence probe LAN-bche was developed to investigate the expression of BChE in diseases such as liver cancer,liver injury,and diabetes.LAN-bche,constructed by directly modifying the hydroxyl site of LAN-OH with cyclopropyl formate residue,is characterized by high stability,good water solubility,high sensitivity,low detection limit and excellent selectivity.LAN-bche can quantitatively detect BCh E by fluorescence and colorimetry method,and accurately detect BCh E activity in human serum.In the bioimaging experiment,LAN-bche was firstly used to study the expression of BCh E in Hep G2 cells and verified that the expression of BCh E in Hep G2 cells was down-regulated.In addition,LAN-bche can effectively distinguish between Hep G2 cells and LO2 cells.Then,LAN-bche was applied to study the expression of BChE in liver injury cells and in vivo,and confirmed the down-regulation of BCh E levels in liver injury.Finally,LAN-bche was used to study the expression of BCh E in diabetes,and verified that the expression of BCh E was upregulated in diabetes.Therefore,LAN-bche,as an efficient bioimaging tool for BCh E,is expected to provide strong support for further research on the pathogenesis of BCHErelated diseases.(3)A highly sensitive near-infrared fluorescence probe LAN-lap was developed by connecting LAN-OH with p-aminobenzyl alcohol linker and L-leucine residue.LAN-lap,characterized by high stability,excellent water solubility,high sensitivity,low detection limit and excellent selectivity,can quantitatively detect LAP by fluorescence and colorimetry method.In cell imaging experiments,LAN-lap effectively imaged endogenous LAP in cells,and confirmed that the expression of LAP was up-regulated in liver cancer and liver injury cells.In in vivo imaging,LAN-lap visualized tumors by responding to LAP,and further confirmed the up-regulation of LAP expression in liver cancer.In addition,LAN-lap was successfully applied to the imaging of LAP level in paracetamen-induced liver injury and rifampicin-induced cholestatic liver injury in vivo,and confirmed the upregulation of LAP levels in these liver injury models,visualized the degree of liver injury,further evaluated the drug hepatotoxicity.Besides,LAN-lap successfully tracked tumors in mice with peritoneal metastasis of liver cancer,and assisted surgical resection of tumors under fluorescence guidance.Therefore,LAN-lap is a LAP-mediated multifunctional molecular imaging tool that can be used for diagnosis of liver-related diseases,drug evaluation and surgical navigation.(4)A highly sensitive near-infrared fluorescence probe LAN-apn was developed to investigate the relationship between APN and thyroid cancer.LAN-apn,constructed by attaching LAN-OH with p-aminobenzyl alcohol linkers and alanine residue,is characterized by high stability,excellent water solubility,high sensitivity,low detection limit and excellent selectivity.In addition,LAN-apn can quantitatively detect APN by fluorescence and colorimetry method,and accurately detect APN activity in human urine.In bioimaging experiments,LAN-apn was successfully used to image APN in thyroid cancer cells,thyroid tumors in vivo and in vitra,and fully confirmed the overexpression of APN in thyroid cancer.Therefore,APN has the potential to be a new biomarker for thyroid cancer and provide a new strategy for the diagnosis and treatment of thyroid cancer.