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Specific fluorescent probe for catechol-o-methyltransferase and its application

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

Active Publication Date: 2019-07-12
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-methyltransferase and its application
  • Specific fluorescent probe for catechol-o-methyltransferase and its application
  • Specific fluorescent probe for catechol-o-methyltransferase and its application

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

A specific fluorescent probe for catechol-O-methyltransferase and its application. The specific probe substrate is 3‑benzothiazole‑7,8‑dihydroxycoumarin (3‑BTD), which can be specifically catalyzed by COMT enzymes to generate 8‑methoxyl products and detect at 520nm A fluorescent signal is displayed at , and the activity of COMT can be quantitatively determined by detecting the change of fluorescence intensity. The probe can not only be used for quantitative evaluation of COMT enzyme activity in biological samples from different sources, but also can be used for rapid screening of COMT inhibitors in vitro and evaluation of their inhibitory ability. This method can be used to quantitatively evaluate the real activity of COMT enzyme in various in vitro biological samples, realize rapid screening of inhibitors and quantitative evaluation of inhibitory ability; it can be used to evaluate the catalytic activity of different species of COMT and COMT mutants with different amino acid sequences , and then evaluate its ability to metabolize catechols. It has the advantages of high sensitivity, high accuracy, strong anti-environmental interference ability, easy high-throughput detection and inhibitor screening.

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 Patents(China)
IPC IPC(8): C07D311/16C09K11/06G01N21/64C12Q1/48
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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