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Coumarin compound for enzymatic activity analysis and enzyme inhibitor sifting motion, and synthesizing process

A technology for screening inhibitors and coumarins, which is applied in the fields of compounds of group 5/15 elements of the periodic table, chemical instruments and methods, and material excitation analysis, etc. Soybein-based fluorescent dyes have few types and the hydroxyl group cannot be deprotonated to achieve the effects of good solubility, high photostability, and strong fluorescence intensity

Inactive Publication Date: 2011-09-14
杨得锁
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, there are mainly the following problems in use: 1. under the situation of physiological pH value (neutral), the hydroxyl group cannot be deprotonated, so the fluorescence quantum efficiency is low during use (Hamid ME; et al, Zentralbl Bakteriol 280,476- 487 (1994)
②Because many enzymes have the best enzymatic hydrolysis rate only when the neutral or pH value is less than 7, the sensitivity and accuracy of using common non-halogenated 7-hydroxycoumarin to detect enzymes is low (Obzansky DM, Rabin BR ; et al, Clin Chem 37, 1513-1518 (1991).)
③ Many protein covalent conjugates of 7-hydroxycoumarin are unstable in terms of the basic conditions required for maximum fluorescent labeling
④ At present, there are few types of coumarin-based fluorescent dyes used for enzyme detection at physiological pH

Method used

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  • Coumarin compound for enzymatic activity analysis and enzyme inhibitor sifting motion, and synthesizing process
  • Coumarin compound for enzymatic activity analysis and enzyme inhibitor sifting motion, and synthesizing process
  • Coumarin compound for enzymatic activity analysis and enzyme inhibitor sifting motion, and synthesizing process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 of the present invention: Synthesis of 6-chloro-8-fluoro-7-hydroxyl-4-methylcoumarin (compound 2)

[0024] ① Synthesis of 4-chloro-2-fluoro-resorcinol (compound 1).

[0025]

[0026]Add 100g (0.565mole) 2,3,4-trifluoronitrobenzene and 1000ml of anhydrous methanol into a 2000ml three-path bottle, cool to below 0~-5°C with an ice-salt bath, and stir electromagnetically. 335.6 g (1.243 mole) of 20% sodium methoxide in methanol was added dropwise within 2-2.5 h. Stir at room temperature for 10h. The progress of the reaction was detected by TLC, and the pH=7 was neutralized with 1 mole / L citric acid after the reaction. The above reaction system was dispersed in 2000 ml of water, and the precipitated light yellow solid was filtered, washed with water and dried. Recrystallized from absolute ethanol to obtain light yellow crystals of 2-fluoro-4-nitro-1,3-benzenediol dimethyl ether.

[0027] Into a 1000 ml round bottom flask, add 20 g of 2-fluoro-4-nitro-1,3-ben...

Embodiment 2

[0032] Example 2 of the present invention: Synthesis of 6-chloro-8 fluoro-7-hydroxyl-3-carboxycoumarin (compound 4)

[0033] 1. Synthesis of 5-chloro-3-fluoro-2,4-dihydroxybenzaldehyde (compound 3):

[0034]

[0035] 10 g of 2-fluoro-4-chloro-1,3-benzenediol, 117.2 g of hexamethylenetetramine, and 80 ml of trifluoroacetic acid were sequentially added to a 250 ml three-necked flask. Electromagnetic stirring was carried out until it was completely dissolved, and the temperature was raised to 90°C for 24 hours. After the reaction was complete, cool to room temperature, add 100 ml of 20% H 2 SO 4 , after stirring for 1 h, the reaction mixture was poured into water and extracted with ethyl acetate (500 mL×2). The organic phase was washed with saturated brine, dried over anhydrous MgSO4, concentrated, washed with ethyl acetate / n-hexane and crystallized to obtain white crystals of 3-fluoro-5-chloro-2,4-dihydroxybenzaldehyde.

[0036] ② Synthesis of 6-chloro-8-fluoro-7-hydroxy-...

Embodiment 3

[0040] Example 3 of the present invention: Synthesis of phosphatase substrate-6-chloro-8-fluoro-7-hydroxyl-4-methylcoumarin phosphate (compound 5)

[0041]

[0042] at 0°C, N 2 Under protection, the pyrimidine solution containing compound 2 was added to the POCl solution 3 (2 molar) pyrimidine solution, the reaction was followed by TLC. After the reaction, the solution was poured into a beaker filled with crushed ice, and the pH of the solution was adjusted to 5 with ammonia water. Then, pyrimidine was extracted with chloroform, and the aqueous phase was partially lyophilized to obtain a crude product.

[0043] The product was purified by Sephadex gel chromatography using a SEPHADEX LH 20 gel column with water as the eluent. The pure product was collected and frozen to obtain colorless crystals. The solid was dissolved in water, and DOWEX 50.4-200 strongly acidic cation exchange resin was added under stirring, then the resin was removed by filtration, the resin was wash...

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Abstract

The present invention relates to a coumarin compound that is used for analysis of enzymatic activity and enzyme inhibitor screening. The compound has a structure formula as shown in the right; wherein, R<1> is an H atom, ester prepared from hydrogen of OH that is substituted by aliphatic carboxyl or aromatic carboxylic acid, or ether that is prepared from hydrogen of OH that is substituted by carboxyl of sterol, monosaccharide and polysaccharide; R<2>, R<3> and R<4> can be H, alkyl containing 1 to 18 carbon atoms, perfluorinated alkyl containing 1 to 18 carbon atoms, -CN,-Ar, miscellaneous aryl, carbonyl or carboxyl amide. The coumarin compound adopts Pechmann reaction or Knovenagel reaction to introduce halogen atoms in the sixth and eighth positions of a benzene ring, to prepare the di-halogenated 7-hydroxycoumarin compound. The coumarin compound can be used as a fluorescent probe for marking analysis in the biotechnological research, and has the characteristics of simple operation, high stability, high sensitivity, high selectivity, and so on. And the coumarin compound is mainly used for the analysis of enzymatic activity, the enzyme inhibitor screening, and the detection of cell activity.

Description

technical field [0001] The invention relates to a class of coumarin compounds and synthesis methods for enzyme activity analysis and enzyme inhibitor screening, specifically including 6-chloro-8-fluoro-7-hydroxycoumarin and its derivatives as enzyme substrates synthesis and application. Background technique [0002] Fluorescent dyes are especially suitable for high-sensitivity biological detection. Hydroxy-substituted coumarins have been widely used to prepare covalent conjugates of fluorescent dyes and protein molecules and fluorescently labeled enzyme substrates (BrunM P; et al, Angew.Chem.Int.Ed., 2004, 43(26) : 3432~3436; Grant S K; et al, J.Biomol.Screen, 2002, 7(6): 531~540; Zhao Y R; et al, J.Am.Chem.Soc., 2004, 126(14): 4653~4663). 7-Hydroxy-4-methylcoumarin (also known as 4-methyl-7-hydroxycoumarin) is the parent compound for the preparation of fluorescent group-containing enzyme substrates. Phosphorylation of the 7-hydroxyl produces a series of fluorophore-cont...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D311/12C07F9/655C07H17/075G01N21/64
Inventor 杨得锁李宗孝
Owner 杨得锁
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