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Thioredoxin reductase fluorescent probe, and preparation method and application thereof

A technology of thioredoxin and fluorescent probes, applied in the field of fluorescent probes, to achieve the effects of simple synthetic route, rapid action and easy preparation

Active Publication Date: 2015-10-14
SHENZHEN HANKANG TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Currently, only one fluorescent probe molecule containing a cyclic disulfide bond has been reported in the literature for the detection of thioredoxin reductase

Method used

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  • Thioredoxin reductase fluorescent probe, and preparation method and application thereof
  • Thioredoxin reductase fluorescent probe, and preparation method and application thereof
  • Thioredoxin reductase fluorescent probe, and preparation method and application thereof

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Effect test

preparation example Construction

[0028] The preparation method of the thioredoxin reductase fluorescent probe of the embodiment of the present invention is characterized in that it comprises the following steps:

[0029] Step 1: After dissolving 4-dihexylamino salicylaldehyde in a solvent, adding ethyl acetoacetate and piperidine, stirring and heating to reflux, cooling and filtering to obtain an intermediate compound;

[0030] Step 2: taking the intermediate compound, dissolving it in a solvent, adding 5-methylfuran aldehyde and piperidine, stirring and heating to reflux, cooling and separating by column chromatography to obtain the fluorescent probe for thioredoxin reductase.

[0031] Its preparation route is as follows:

[0032]

[0033] Specifically, the mass ratio of the 4-dihexylamino salicylaldehyde, ethyl acetoacetate and piperidine is 1:1.0~3.5:0.1~1.2; the intermediate compound, 5-methylfuran aldehyde, piperidine The mass ratio of pyridine is 1:0.4-1.6:0.2-1.6. The heating reflux temperature in...

Embodiment 1

[0042] Embodiment 1 Preparation of Fluorescent Probe for Thioredoxin Reductase

[0043]

[0044] Dissolve 1 g of 4-diethylamino salicylaldehyde (5.17 mmol) and 1.35 g of ethyl acetoacetate 4-diethylamino salicylaldehyde (10.38 mmol) in 20 ml of absolute ethanol, add 100 μl of piperidine as catalyst. The reaction solution was heated to reflux for 5 hours, cooled to room temperature, and a large number of yellow solids were precipitated. The yellow solid was suction filtered and washed with absolute ethanol to obtain the pure intermediate 1a (1.2 g, 4.6 mmol), with a yield of 90%. 1H NMR (400MHz, CDCl3) δ8.46(s, 1H), 7.40(d, J=9.0Hz, 1H), 6.63(dd, J=9.0, 2.3Hz, 1H), 6.48(d, J=2.2Hz , 1H), 3.47(q, J=7.1Hz, 1H), 2.69(s, 1H), 1.25(t, J=7.1Hz, 1H).

[0045] Intermediate 1a (250.0 mg, 0.96 mmol) and 5-methylfuran aldehyde (106.0 mg, 0.96 mmol) were dissolved in 20 ml of absolute ethanol, and 100 μl of piperidine was added as a catalyst. The reaction solution was heated to reflu...

Embodiment 2

[0046] Thioredoxin reductase inhibitory activity assay of embodiment 2 fluorescent probe

[0047] The DTNB method was used for the determination of fluorescent probe inhibition of thioredoxin reductase activity. All experiments were performed at 25°C. The total volume of the sample to be tested was 40 μl, which contained 0.3 μl of TrxR. The sample to be tested was added to 35.5 μl of buffer solution, which contained 1M phosphate (pH 7.0), 500 mM EDTA (pH 7.4), NADPH (0.48 mM) and 1 μl of fluorescent probes at different concentrations. After incubation for five minutes, 3.2 μl of DTNB was added to the reaction solution. The same volume of DMSO was added to the control group. Measure the increase process of the ultraviolet absorption intensity at 412nm within 120 seconds with a microplate reader, so as to calculate the inhibition of the thioredoxin reductase activity by the fluorescent probe. Measured IC 50 = 0.52 μM.

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Abstract

The invention discloses a thioredoxin reductase fluorescent probe, and a preparation method and application thereof. The thioredoxin reductase fluorescent probe is a compound containing a 7-diethylaminocoumarin fluorophore and a furyl acryloyl fluorescent quenching group. The fluorescent probe is covalently combined to the thioredoxin reductase through Michael addition reaction, thereby emitting fluorescence, wherein the magnitude of fluorescence intensity is directly proportional to the thioredoxin reductase content. The fluorescent probe has higher inhibition activity and selectivity for thioredoxin reductase, has very low responsiveness for other high-content mercapto small molecules in the biological system, can be widely used for detecting the thioredoxin reductase content in a sample, and is especially suitable for detecting the thioredoxin reductase content in tumor cells of a living body.

Description

technical field [0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a fluorescent probe for thioredoxin reductase and its preparation method and application. Background technique [0002] Thioredoxin reductase (thioredoxin reductase, TrxR) is a dimeric selenoenzyme, which belongs to the family of pyridine nucleotide disulfide redox proteases. Thioredoxin reductase, thioredoxin (Trx) and reduced nicotinamide adenine dinucleotide phosphate (triphosphopyridine nucleotide, NADPH) together constitute the thioredoxin system, the main function of which is to regulate cell internal redox balance. A large number of studies have shown that thioredoxin reductase is overexpressed in a variety of human primary tumors, and it is a key regulatory enzyme for abnormal tumor proliferation. It can be used as a tumor marker to detect its activity in the human body, thereby monitoring the occurrence and development of tumors in vivo. develop....

Claims

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Application Information

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IPC IPC(8): C09K11/06C07D407/06G01N21/64
Inventor 卜宪章吴海强黄磊陈雨
Owner SHENZHEN HANKANG TECH CO LTD
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