阅读说明：本技术 一种检测半胱氨酸的荧光探针 (Fluorescent probe for detecting cysteine ) 是由 战付旭 汲静静 张占明 郑庚修 王秋芬 于 2019-07-09 设计创作，主要内容包括：本发明公开了一种检测半胱氨酸的荧光探针。本发明所述的荧光探针能够从多种氨基酸以及一些常见物质中识别区分半胱氨酸,该探针与Cys作用时,荧光由蓝色变成黄绿色,可实现比率型检测。该荧光探针对在环境中的半胱氨酸的检测和分析具有很好的应用。本发明的荧光探针在生物化学领域具有重要的应用价值。(The invention discloses a fluorescent probe for detecting cysteine. The fluorescent probe can identify and distinguish cysteine from various amino acids and some common substances, and when the probe acts with Cys, the fluorescence is changed from blue to yellow-green, so that ratio-type detection can be realized. The fluorescent probe has good application to the detection and analysis of cysteine in the environment. The fluorescent probe has important application value in the field of biochemistry.)

1. A fluorescent probe for detecting cysteine has the structure as follows:

Technical Field

The invention relates to the field of analytical chemistry, and relates to preparation of a fluorescent probe for detecting cysteine.

Background

Cysteine (Cys), a semi-essential amino acid and biological thiol, is biosynthesized in humans and is an important structural and functional unit of various polypeptides or proteins. Cysteine (Cys) plays an important role in this diverse biological processes, including biocatalysis, signal transduction, protein turnover, regulation of metabolism, maintenance of redox balance and heavy metal binding. Cysteine is not only a precursor of glutathione, acetyl coenzyme and taurine, but also a provider of sulfur ligands in the organism's pyrite complex. The lack of cysteine in human body may cause symptoms of slow growth, hair color loss, edema, lethargy, liver function damage, muscle relaxation, physical weakness, etc., and the excessive concentration of cysteine may cause diseases such as Alzheimer's disease, cardiovascular disease, cancer, etc.

At present, many methods for detecting cysteine exist, such as gel electrophoresis, raman spectroscopy, electron paramagnetic resonance, mass spectrometry, microelectrode biosensors, photoelectrochemical sensors and intracellular nuclear magnetic resonance spectroscopy, however, these methods usually require expensive instruments and complex preparation, and on the contrary, the fluorescence imaging technology has the advantages of simple operation, low cost, high sensitivity, good selectivity and the like, and has attracted extensive attention. The ratio-type fluorescent probe has a greater practical value because it can reduce the influence of the surrounding environment on the detection of cystine by using the increase and decrease value between two adjacent peaks. Therefore, it is very useful to prepare a ratiometric fluorescent probe capable of detecting cysteine.

Disclosure of Invention

One of the purposes of the invention is to provide a synthetic method of a fluorescent probe for detecting cysteine; the other purpose is to provide a fluorescent probe for detecting the cysteine.

In order to achieve the purpose, the invention adopts the following technical scheme.

A fluorescent probe for detecting cysteine has the following molecular structure:

the specific synthetic route is as follows:

the specific synthesis method comprises the following steps: (1) 4-bromo-1, 8-naphthalic anhydride and 4- (2-aminoethyl) -morpholine were dissolved in ethanol and refluxed. After the reaction is finished, cooling to the roomThe precipitate was collected by filtration and dried to give compound 1. (2) Sodium azide and compound 1 were added to DMF solution and the reaction mixture was heated to 100 ℃ for 10 h. Adding a large amount of water, CH₂Cl₂Extraction, drying of the organic phase and removal of the solvent under reduced pressure. Compound 2 is obtained and the product is used in the next reaction without purification. (3) Compound 2 and ammonium chloride were dissolved in a mixture of ethanol and water. The zinc powder was slowly added to the mixture and heated under reflux for 1 h. Adding 0.5mL ammonia water, washing with brine, extracting with ethyl acetate, drying the organic phase, removing the solvent under reduced pressure, and separating by column chromatography to obtain compound 3. (4) 4-hydroxybenzyl alcohol and imidazole were added to DMF, cooled to 0 deg.C, tert-butyldiphenylchlorosilane was added, and stirred at room temperature overnight. The reaction was washed with brine, extracted with ethyl acetate (3 × 20mL), the organic phase was dried, the solvent was removed under reduced pressure, and compound 4 was isolated by column chromatography. (5) Addition of trimethylamine and Compound 4 to CH₂Cl₂And (4) cooling to 0 ℃. Addition of acryloyl chloride to CH₂Cl₂In the reaction solution, the mixed solution of acryloyl chloride was added dropwise to the reaction solution in ice bath, and the mixture was stirred at room temperature overnight. The reaction solution is treated with 5% NaHCO₃The solution was washed with brine, the organic phase was dried, the solvent was removed under reduced pressure, and compound 5 was isolated by column chromatography. (6) Dissolving the compound 5 in methanol, adding macroporous resin-15, and stirring at room temperature for 6 h. Filtering to obtain solution, removing solvent under reduced pressure, and separating by column chromatography to obtain compound 6. (7) Dissolving Compound 3 in anhydrous CH₂Cl₂Cooling to-15 deg.C, adding 4-dimethylaminopyridine, and stirring for 10 min. The toluene phosgene solution was added dropwise to the reaction solution and stirred for 12 hours. Excess phosgene was removed and compound 6 was added, reacted at 0 ℃ and stirred overnight. Washing the reaction solution with water, CH₂Cl₂Extraction (2X 25mL), drying of the organic phase, removal of the solvent under reduced pressure, and column chromatography to obtain the probe.

The response mechanism of the fluorescent probe of the present invention is as follows: after the cysteine responds to the probe molecule, the mercapto group of the cysteine firstly generates Michael addition reaction with the double bond of the acrylic ester, then the amino group of the cysteine attacks the ester group to form a seven-membered ring, and the fluorescence of the solution of the dye molecule (compound 3) is released to change from green to blue. Thereby realizing the process of detecting cysteine in a fluorescence ratio mode. The response process of the probe molecule is as follows:

the fluorescent probe disclosed by the invention is applied to the detection of cysteine.

In the application of the fluorescent probe in detecting cysteine, the excitation wavelength is 410nm, and the detection wavelength band is 450-750 nm.

The invention has the following advantages:

the fluorescent probe for detecting cysteine has the advantages of simple synthesis, good selectivity, capability of realizing ratio type detection and important application value in the field of biochemistry.

Drawings

FIG. 1 is a diagram showing the structure of a probe molecule.

FIG. 2 shows a probe¹H NMR spectrum.

FIG. 3 is a fluorescence spectrum of the probe response to different concentrations of Cys.

FIG. 4 is a graph of probe selectivity and interference resistance.

Detailed Description