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A fluorescent probe for detecting carboxylesterase and its preparation method and application

A technology of fluorescent probe and carboxylesterase, which is applied in the field of analysis and detection, can solve the problems of difficult enzyme accurate analysis, fluorescent probe is easily interfered by external factors, cumbersome steps, etc., and achieves good anti-interference, intuitive detection mode, The effect of improving detection precision and accuracy

Inactive Publication Date: 2019-01-18
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Under the irradiation of excitation light at 442nm, fluorescence can be emitted in the wavelength range of 460-650nm, thereby realizing the fluorescence-enhanced detection of carboxylesterase; Detection applications in the field of industrial production; in addition, the steps to realize the detection of enzymes using this method are cumbersome
A research paper (RSC Analyst, 2012, 137, 716-721) reported a fluorescent probe based on a derivative of resorufin (9-hydroxy-3-isophenoxazinone), which has almost no fluorescence at 585nm; When carboxylesterase exists in the system, it can catalyze the cleavage of the carboxylate bond of the probe molecule, resulting in the cleavage of the benzyl group and release of resorufin. Under the irradiation of 550nm laser, the probe molecule will It emits orange-red fluorescence, realizing the fluorescence-enhanced detection of carboxylesterase; however, this fluorescent probe is easily interfered by external factors, and sometimes it is difficult to achieve accurate analysis of enzymes

Method used

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  • A fluorescent probe for detecting carboxylesterase and its preparation method and application
  • A fluorescent probe for detecting carboxylesterase and its preparation method and application
  • A fluorescent probe for detecting carboxylesterase and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0067] (1) Dissolve 2304mg of 2-methyl-4H-1-benzofuran-4-one (14.4mmol) and 1320mg of malononitrile (20mmol) in 20mL of acetic anhydride, stir and heat at 143°C for 12 hours After the reaction, spin dry under reduced pressure to remove the solvent, then add 20mL of water, heat to reflux for 1 hour, and control the temperature to 100°C; after cooling to room temperature, extract with dichloromethane, and the obtained organic layer is then separated with water and saturated sodium chloride. Washed once, the organic phase was dried with anhydrous sodium sulfate, filtered; the organic solvent was removed by rotary evaporation, and the resulting solid was purified by silica gel column chromatography (petroleum ether: dichloromethane, V / V=2:3) to obtain the white solid product 2 -(2-Methyl-4H-dihydrobenzopyran-4-yl)-malononitrile 1857mg (yield rate is 62%); This product is characterized by H NMR spectrum, H NMR spectrum figure is as follows figure 2 shown;

[0068] (2) Dissolve 24...

Embodiment 2

[0077] (1) Dissolve 5552mg of 2-methyl-4H-1-benzofuran-4-one (34.7mmol) and 3262mg of malononitrile (49.4mmol) in 40mL of acetic anhydride, stir and heat to control the temperature at 140°C for 10 hours, after the reaction, spin dry under reduced pressure to remove the solvent, then add 48mL of water, heat to reflux for 0.5 hours, and control the temperature to 95°C; Wash each once, dry the organic phase with anhydrous sodium sulfate, filter; remove the organic solvent by rotary evaporation, and purify the obtained solid by silica gel column chromatography (petroleum ether: dichloromethane, V / V=2:3) to obtain a white solid product 2-(2-Methyl-4H-dihydrochromen-4-yl)-malononitrile 5485 mg (76% yield);

[0078] (2) Dissolve 1500mg of 4-aminobenzyl alcohol (12.18mmol) in 7.3mL of 1,4-dioxane, 7.3mL of water and 12mL of 1M sodium hydroxide solution, cool to -5°C, add 2930mg of dioxane Di-tert-butyl carbonate (13.4mmol), stirred at room temperature for 1.5 hours, spin-dried under ...

Embodiment 3

[0084] (1) Dissolve 2825mg of 2-methyl-4H-1-benzofuran-4-one (17.65mmol) and 1631mg of malononitrile (24.7mmol) in 25mL of acetic anhydride, stir and heat to control the temperature at 141°C for reaction 11 hours, after the reaction, spin dry under reduced pressure to remove the solvent, then add 24mL of water, heat to reflux for 0.7 hours, and control the temperature to 98°C; Wash each once, dry the organic phase with anhydrous sodium sulfate, filter; remove the organic solvent by rotary evaporation, and purify the obtained solid by silica gel column chromatography (petroleum ether: dichloromethane, V / V=2:3) to obtain a white solid product 2-(2-Methyl-4H-dihydrochromen-4-yl)-malononitrile 2525 mg (68.8% yield);

[0085] (2) Dissolve 1355mg of 4-aminobenzyl alcohol (11mmol) in 7.15mL of 1,4-dioxane, 7.15mL of water and 11.55mL of 1M sodium hydroxide solution, cool to -3°C, add 3127mg of dioxane Di-tert-butyl carbonate (14.3mmol), stirred at room temperature for 3.5 hours, spi...

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Abstract

The invention belongs to the technical field of analysis and detection, and discloses a fluorescent probe used for detecting carboxylesterase and a preparation method and an application thereof. The fluorescent probe is 2-{2-[4-(4'-benzyl acetate benzyloxy-formamido)-styryl]-4H-benzopyran-4-yl}-malononitrile, and has a structural formula in a formula (I). and the fluorescent probe is used for detecting carboxylesterase. Compared with the current detection technique of fluorescence, the fluorescent probe has high selective response for carboxylesterase, strong anti-interference performance, and effective and visual detection mode, low investment cost, and simple synthesis route and method, and is suitable for enlarge production and practical application.

Description

technical field [0001] The invention belongs to the technical field of analysis and detection, and in particular relates to a fluorescent probe, a preparation method thereof and an application in carboxylesterase detection. Background technique [0002] Carboxylesterase is an enzyme in the serine esterase superfamily that catalyzes the hydrolysis of many exogenous substrates, such as catalyzing the hydrolysis of fatty acid esters to acids, alcohols, and water. Because carboxylesterase can catalyze many substrates, it is widely used in organic synthesis and industrial production, and is also an important drug target and prodrug activator. In addition, carboxylesterase also plays an important role in the detoxification of organophosphorus compounds, the modification or removal of drugs. Developing a new method that can be applied to the detection and monitoring of carboxylesterase is a research hotspot in various fields such as drug synthesis, detoxification and industrial pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D311/04C09K11/06G01N21/64
CPCC07D311/04C09K11/06C09K2211/1088G01N21/6486
Inventor 曾钫夏细桃吴水珠
Owner SOUTH CHINA UNIV OF TECH
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