Design And Synthesize Of A Fluorescent Probe For Detecting Activities Of Catechol-O-Methyltransferase And Its Biological Applications

Catechol-O-methyltransferase(COMT),one of the most important phase ? drug metabolizing enzymes,plays important roles in the metabolism of endogenous and xenobiotic catechols.Meanwhile COMT also plays an important role to the human body's defenses.The catalytic function or activity of COMT is the prerequisite which play important functions in the body.Catalytic function disorder of COMT lead to various body disease even death.Therefore,the evaluation and characterization of COMT activity is very important.The method based on the probe of specific molecular to detect the target enzyme's activity has been generally recognized all around the world.But currently,the characterization of COMT activity by probe and detection system is conditioned by the following conditions or has the following defects:1,Quantitative analysis need to detect the specificity products,especially a single product which is generated by metabolic enzyme,rather than the consumption of the substrates;2,COMT has two forms.And the substrates' selectivity of two forms almost have no difference under the physiological condition.So evaluate the COMT activity have to detect both two forms of COMT activity at the same time;3,COMT,known as membrane protein and soluble protein,catalytic need cofactors and membrane structural integrity or the existence of the cytoplasm.It makes the COMT reaction system become complex.And the stability and anti-interference ability of COMT catalytic function are affected by different biological analytes or/and endogenous substrates.4,COMT often need to supervise the level of activity in living cells,therefore,the probe or/and metabolites should not form the cytotoxicity;5,The COMT probe neither have been reported detect by fluorescent,nor have had rational design and optimization.Detection system needs by using high performance liquid chromatography with UV or MS and other large valuable devices to detect products.And the probes reported before have many defects such as biological analytes interference,process complex,low testing flux,high cost.Aim at solve the probe characterization in selectivity,stability,sensitivity,anti-interference ability,practical challenges and other various factors mentioned above.This paper 1,describe a high sensitivity and good anti-interference ability of coumarin fluorescent molecule as probe,develop eight different 4-substituted with different polarity small molecular fluorescent compounds,eventually found COMT selectively catalyzes the conversion of the probe(7,8-Dihydroxy-4-methylcoumarin,DHMC)to 7-hydroxy-8-methoxy-4-methylcoumarin(HMMC);2,use recombinant enzyme studied the probe selectivity of system in vitro;3,according to the COMT substrate structure characteristics,found that the 7,8-hydroxy coumarin metabolized by COMT are only produce one product;4,verified the performance of substrates and it methylation products(including the fluorescence quantum yield,pH stability,excitation and emission wavelength,etc.)and COMT enzymatic reaction kinetics characteristics of system evaluation.The resuls demonstrated that the fluorescent probes have good stability and anti-interference ability;5,found the new probe and detection system applied to 1)detect COMT enzyme activity in biological samples,the results found that the quantitative evaluation of COMT by activity are consistent with the traditional COMT quantitative method;2)COMT biological imaging.DHMC can be used in vitro biological imaging in tumor cells;3)high throughput screening of COMT inhibitors.All the results demostrated that the COMT fluorescent probe DHMC has great prospects in drug discovery and biological application.