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Specific fluorescent probe for catechol-O-methyltransgerase (COMT) and application thereof

A methyltransferase and fluorescent probe technology, applied in the field of biomedicine, can solve the problems of poor compound stability, complex sample pretreatment, inconvenient quantitative detection, etc., and achieve high sensitivity, good fluorescence emission spectral characteristics, and simple and easy synthesis process. row effect

Active Publication Date: 2017-03-01
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

More importantly, the above-mentioned substrates and their metabolites can only be quantitatively detected by mass spectrometry, and the two products are usually difficult to separate on liquid chromatography due to their similar structure and physical and chemical properties; The pre-treatment is complicated, which brings inconvenience to its quantitative detection

Method used

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  • Specific fluorescent probe for catechol-O-methyltransgerase (COMT) and application thereof
  • Specific fluorescent probe for catechol-O-methyltransgerase (COMT) and application thereof
  • Specific fluorescent probe for catechol-O-methyltransgerase (COMT) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] .3-Benzothiazole-7,8-dihydroxycoumarin synthesis

[0046] The synthetic route of 3-benzothiazole-7,8-dihydroxycoumarin is as follows figure 2 As indicated, weigh 1.0 g of 2,3,4-trihydroxybenzaldehyde and dissolve it in 25 mL of methanol, add 1.5 mL of 2-(2-benzothiazole) ethyl acetate and 0.1 mL of piperidine, and stir at room temperature for 20 minutes. Heating to 65° C., reacting for 4 h, and TLC detected the end of the reaction. After the reaction solution was cooled, 10 mL of water was added, filtered, and the filter cake was collected, which was the crude product. The crude product was added to 20 mL of acetonitrile and refluxed for 1 h, filtered after cooling, and dried in vacuo to obtain 1.8 g of the product 3-benzothiazole-7,8-dihydroxycoumarin as a brownish yellow solid with a yield of 88%. 1 H NMR spectrum and 13 C NMR spectrum as image 3 and Figure 4 shown;

[0047] 1 H NMR (400MHz, DMSO-d 6 )δ: 6.95(d, J=4.4Hz, 1H), 7.45(m, 2H), 7.56(t, J=8.0Hz, 1...

Embodiment 2

[0051] Determination of the lower limit of detection of COMT in vitro

[0052] The experiment was carried out on a microplate reader using a 96-well plate, 3-benzothiazole-7,8-dihydroxycoumarin 5μM, S-adenosylmethionine 200μM, dithiothreitol 2mM, MgCl 2 5mM, COMT single enzyme 20ng / ml~200μg / ml, pH 7.4 Tris-HCl buffer 50mM, the total volume is 200μL, incubated at 37℃ for 5min and analyzed by microplate reader, the average value of each group is the same as that without COMT Compared with the control group, it was shown that the COMT of 40ng / ml was statistically significant (P Figure 8 ).

Embodiment 3

[0054] COMT time standard curve determination

[0055] The experiment was carried out on a microplate reader using a 96-well plate, 3-benzothiazole-7,8-dihydroxycoumarin 5μM, S-adenosylmethionine 200μM, dithiothreitol 2mM, MgCl 2 5mM, COMT single enzyme 20ng / ml~200ng / ml, pH 7.4 Tris-HCl buffer 50mM, total volume 200μL, incubate at 37℃ for 5min, analyze with a microplate reader every 1 minute, the fluorescence intensity of the product and the incubation time Make a standard curve, the R of each standard curve 2 >0.99 (see Figure 9 ), indicating that the standard curve has a wide linear range and can accurately quantify the content of COMT.

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Abstract

The invention provides a specific fluorescent probe for COMT and application thereof. A specific probe substrate provided by the invention is 3-benzothiazole-7,8-dihydroxycoumarin (3-BTD); the substrate can be specifically catalyzed by COMT to produce an 8-methoxy product and presents a fluorescence signal at a position of 520 nm; and the activity of COMT can be quantitatively determined by detecting changes of fluorescence intensity. The probe is applicable to quantitative assessment of the activity of COMT in biological samples from different sources, in-vitro rapid screening of inhibitors for COMT and evaluation of the inhibitory capability of the inhibitors. A method provided by the invention can be used for quantitative assessment of the actual activity of COMT in a variety of in-vitro biological samples and realizes rapid screening of inhibitors and quantitative assessment of the inhibitory capability of the inhibitors; the method is also applicable to evaluation of the catalytic activity of COMT of different species and COMT mutants with different amino acid sequences, so the method can assess the capability of COMT in metabolization of catechol drugs; and the method has the advantages of high sensitivity, high accuracy, good environment interference resisting capability, convenience in high-flux detection and inhibitor screening, etc.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a catechol-O-methyltransferase specific fluorescent probe and its application. Background technique [0002] Catechol-O-methyltransferase (Catechol-O-methyltransferase, COMT) is an important phase II transferase in the human body, widely distributed in human tissues such as liver, kidney, lung, breast, red blood cells and brain middle. In the human body, COMT is responsible for removing active and toxic catechol compounds, such as neurotransmitters dopamine, epinephrine and norepinephrine, and catechol estrogen compounds such as estradiol (Pharmacol Rev.1999; 51 (4):593-628). In addition, COMT enzymes are also involved in exogenous catechol drugs (such as carbidopa, benserazide, apomorphine, dobutamine, fenoldopam, α-methyl-L-DOPA, Metabolic clearance of isoproterenol and rimiterol, etc.). [0003] The COMT enzyme in the human body is polymorphic, and it is lo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06G01N21/64C12Q1/48
Inventor 杨凌葛广波夏杨柳王平钱星凯窦同意
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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