CN114621210A

CN114621210A - Preparation method and application of novel fluorescent molecular probe for detecting L-cysteine

Info

Publication number: CN114621210A
Application number: CN202011432494.2A
Authority: CN
Inventors: 不公告发明人
Original assignee: Hunan Chaoji Testing Technology Co ltd
Current assignee: Hunan Chaoji Testing Technology Co ltd
Priority date: 2020-12-10
Filing date: 2020-12-10
Publication date: 2022-06-14

Abstract

The invention discloses a fluorescent molecular probe for specifically recognizing L-cysteine, which has the following chemical structural formula:

Description

Preparation method and application of novel fluorescent molecular probe for detecting L-cysteine

Technical Field

The invention belongs to the technical field of analytical chemistry, relates to a preparation method of a novel fluorescent molecular probe for detecting L-cysteine, and particularly relates to application of the fluorescent molecular probe in the aspect of detecting the L-cysteine in organisms.

Background

L-Cysteine (L-Cys) is one of important amino acids in protein, has important biochemical functions, and plays a key role in the metabolism of a plurality of essential biochemical substances such as coenzyme A, biotin, heparin and the like. Modern studies have shown that cysteine is an essential substance for cell and tissue growth, and the lack of cysteine may lead to a series of diseases such as slow growth, alopecia, edema, lethargy, neurotoxicity, hypohemopoiesis, metabolic disorders, liver damage and parkinson's disease in children. Therefore, the development of a high-sensitivity and high-selectivity detection method for detecting cysteine in cells in real time has important significance and value.

Various methods have been used for cysteine detection, including potentiometry, UV-visible absorption spectrophotometry, high performance liquid chromatography, and gas chromatography-mass spectrometry. However, these conventional analysis methods have problems of high cost, complicated sample processing and operation processes, difficulty in intracellular detection, and the like. As a novel detection method, the fluorescence probe method has the advantages of high sensitivity, strong selectivity, real-time monitoring, strong instrument operability, low detection limit, living cell imaging and the like, and becomes a common analyte detection method in biological and environmental science. At present, organic small-molecule fluorescent probes for detecting cysteine have been developed, for example, CN 103755672 a provides a fluorescent molecular probe based on a coumarin parent nucleus, which can specifically detect cysteine, but has a long response time of 60 minutes. For another example, CN 108623533 a reports a fluorescence molecular probe with ESIPT based on a mother nucleus of benzothiazole, which can achieve specific detection of cysteine within 2 minutes, but its fluorescence exhibits blue color (excitation wavelength is 405 nm), is easily interfered by background fluorescence during intracellular imaging, and has poor water solubility, and it needs to be used in an organic solvent or a mixed solvent of organic solvent/water, which all limits the application of the probe in vivo. Therefore, the invention develops the ratio type fluorescent molecular probe with ESIPT property and capable of being used for cysteine specific identification, the probe has the advantages of simple synthesis, good water solubility, red light region at the emission wavelength and the like, can realize the rapid identification of cysteine in cells, and has very important application value.

Disclosure of Invention

Aiming at the defects of the existing fluorescent probe for detecting cysteine, the first aim of the invention is to provide a fluorescent molecular probe capable of realizing rapid and specific detection of cysteine.

The second purpose of the invention is to provide a method for preparing the fluorescent molecular probe, which is simple to operate and has easily available raw materials.

In order to achieve the technical purpose, the invention provides a fluorescent probe, which has a structure shown in formula I:

formula I

The preparation method of the fluorescent probe is preferably as follows:

4-hydroxy-m-phthalaldehyde is taken as a raw material, and is refluxed with 2-aminothiophenol in absolute ethyl alcohol at room temperature, after the reaction is finished, the mixture is poured into ice water, and white solid can be obtained after filtering, washing and drying precipitates. Dissolving white solid in trifluoroacetic acid, adding hexamethyl-hydroxylamine, stirring, and heating to 90 deg.C^oC, refluxing, cooling to room temperature after the reaction is finished, dropwise adding into ice water, adjusting the pH value of the system to 6, filtering and washing the precipitate, and separating and purifying by silica gel column chromatography. Dissolving the purified product in anhydrous ethanol, adding 2-methylpyridine salt, 90^oC, refluxing. After the reaction is finished, cooling to room temperature, concentrating in vacuum, and separating and purifying by silica gel column chromatography. And dissolving the purified product in anhydrous dichloromethane under the protection of nitrogen and ice bath, dropwise adding triethylamine, slowly dropwise adding a dichloromethane solution dissolved with 1mmol of acryloyl chloride, stirring overnight at room temperature, after the reaction is finished, performing vacuum concentration, performing silica gel column chromatography separation and purification, and finally drying to obtain the target fluorescent molecular probe.

The synthesis of the invention is as follows:

the invention provides application of the fluorescent probe, which can be applied to cysteine detection. The detection principle of the probe is as follows: the ESIPT (excited-state intramolecular proton transfer) mechanism of the probe is utilized, the ESIPT function of the probe is hindered by the allyl ester group, so that the probe only generates enol emission, and the ESIPT function of the probe is recovered after cysteine is added, so that the probe recovers the keto emission, and the ratio type detection of the cysteine is finally realized. The detection mechanism is as follows:

the invention provides a method for determining cysteine by using the fluorescent probe. The determination method comprises the following steps: the fluorescent probe was dissolved in PBS buffer solution at room temperature, and the concentration was set to 5. mu.M-30. mu.M. Cysteine water solution with different concentrations is added into a probe system, fluorescence intensity is measured, and quantitative detection of cysteine is realized through the linear relation between the fluorescence intensity and the cysteine concentration.

In the above detection method, preferably, the solvent system is PBS buffer solution.

Preferably, the pH of the detection method is 7.4.

In the above detection method, the concentration of the fluorescent probe is preferably 10. mu.M.

The fluorescent probe can be applied to the detection of cysteine in cells. The specific detection method comprises the following steps: 10 μ M of fluorescent probe solution was added to HeLa medium and placed at 37^oC, 5% CO₂Incubate in the incubator for 30 minutes under an atmosphere, then wash three times with PBS buffer, remove probe molecules that have not entered the cells, the cells appear green fluorescence, then change the medium and incubate for 30 minutes with cysteine buffer (25 μ M), the cells emit intense red fluorescence. Experiments show that the fluorescent probe has a good imaging effect on cysteine in cells and can be used for detecting cysteine in organisms.

Compared with the prior art, the technical scheme of the invention has the following beneficial technical effects:

the fluorescent molecular probe for detecting cysteine has the following advantages:

(1) the fluorescent probe has the advantage of good selectivity, can specifically identify cysteine, and cannot be interfered by amino acids such as homocysteine, glutathione, leucine, isoleucine, methionine, tyrosine, tryptophan, threonine, serine, alanine, phenylalanine, histidine, aspartic acid, glycine, glutamic acid, glutamine, lysine, arginine and the like;

(2) the fluorescent probe has the advantage of high sensitivity, the fluorescence intensity of the probe solution and the concentration of cysteine have good linear relation in the range of 0-10 mu M, and the detection line is as low as 0.68 mu M;

(3) the probe has stronger red light emission, good water solubility, small cytotoxicity and good imaging effect, and can detect cysteine in living cells.

Drawings

FIG. 1 is a fluorescence emission spectrogram of fluorescence intensity of a fluorescent probe varying with cysteine concentration in the practice of the present invention;

FIG. 2 is a graph of the linear relationship between the fluorescence intensity of the fluorescent probe and the concentration of cysteine in the practice of the present invention;

FIG. 3 is a graph showing the selectivity of a fluorescent probe for cysteine in the practice of the present invention;

FIG. 4 is a confocal image of fluorescence of a fluorescent probe in HeLa cells in the practice of the present invention.

Detailed Description

The following embodiments are intended to further illustrate the present invention and are not intended to limit the present invention.

Example 1

Synthesis and structural characterization of Compound 2

4-Hydroxyisophthalaldehyde (1200 mg, 8 mmol) was dissolved in 20mL of absolute ethanol in a 25mL round-bottomed flask at room temperature, and 2-aminothiophenol (500 mg, 4mmol) and 30% H were added to the solution₂O₂(4mmol) and 37% HCl (2mmol), after the reaction is completed, the reaction system is poured into ice water, and the filter cake is filtered, collected and washed, and dried to obtain 1180.4 mg of white solid with the yield of 85.2%.¹H NMR (300 MHz, CDCl₃)TM (ppm): 12.95 (s, 1H), 8.38 (d, J = 2.1 Hz, 1H), 8.05 (d, J = 8.1 Hz, 1H), 8.01-7.93 (m, 2H), 7.87 (t,J = 7.7 Hz, 2H), 7.48 (m, 2H), 7.44-7.31 (m, 2H), 7.15 (d, J = 8.7 Hz, 1H). ¹³C NMR (75 MHz, CDCl₃) TM(ppm): 168.51, 166.68, 160.16, 154.04, 151.44, 134.76, 132.62, 131.71, 127.27, 126.76, 126.26, 125.76, 125.34, 124.91, 122.86, 122.18, 121.53, 121.48, 118.48, 117.02. HRMS (m/z): 361.0461 [M+H]+, calcd. for C₂₀H₁₂N₂OS₂ = 360.4472

Synthesis and structural characterization of Compound 3

Compound 2 (903 mg, 2.5mmol) and hexamethyl-hydroxylamine (1400 mg, 10mmol) were placed in a 25mL round-bottomed flask, and 25mL of trifluoroacetic acid was added to the reaction system, and the temperature was raised to 90%^oC, heating and refluxing, after the reaction is finished, taking down the reaction, cooling, pouring the cooled reaction product into 500mL of ice water, adjusting the pH value to be about 6 by using a 1N NaOH solution, separating out a large amount of precipitate, carrying out suction filtration, collecting and washing a filter cake, carrying out vacuum drying, and purifying by column chromatography to obtain 831.8 mg of a product, wherein the yield is 83.6%. 1H NMR (300 MHz, CDCl)₃)TM (ppm):13.01 (s, 1H)，10.49 (s, 1H)，8.26 (d, J = 2.1 Hz, 1H)，8.12 (d, J = 8.1 Hz, 1H), 8.05 (s, 1H), 7.94 (s, 1H), 7.73 (t,J = 7.7 Hz, 2H), 7.34 (m, 2H), 7.29-7.22 (m, 2H), 7.12 (d, J = 8.7 Hz, 1H).

Synthesis and structural characterization of Compound 4

Compound 3 (697 mg, 2.0mmol) was added to 10mL of ethanol in a 25mL round-bottomed flask, and after sufficient dissolution, 2-methylpyridinium salt (702 mg, 3mmol) and 1mmol of piperidine were added to the reaction system, and the temperature was raised to 90%^oC, heating and refluxing. After the reaction is finished, the mixture is cooled and concentrated, and the product is purified by column chromatography, so that 678.8 mg of the product is obtained, and the yield is 56.1%.¹H NMR (500 MHz, DMSO-d₆): δ（ppm）12.7 (s, 1H), 8.27(d, J = 6.5 Hz, 2H), 8.19 (d, J = 8.0 Hz, 1H), 8.14 (d, J = 7.0 Hz, 1H), 8.09(d, J = 8.0 Hz, 1H), 7.99 (m, 2H), 7.78 (s, 1H), 7.65 (t,J = 7.1 Hz, 2H), 7.59 (t, J = 6.5 Hz, 1H), 7.51 (t, J = 7.0 Hz, 1H), 7.41 (d, J = 15.5 Hz, 1H), 7.34-7.21 (m, 2H), 4.24(s, 3H).

Synthesis and structural characterization of target molecular probes

Compound 4 (605 mg,1 mmol) was dissolved in 10m of a solution under nitrogen protection in an ice bathL Anhydrous dichloromethane in a 25mL round-bottom flask, triethylamine was added dropwise to the reaction system and a solution of 1mmol of acryloyl chloride in dichloromethane, 0mmol, was slowly added dropwise^oStirring under C, and monitoring the reaction by TLC until the reaction is complete. Respectively using water and saturated NaHCO₃The solution and saturated saline were extracted, and the organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated to obtain 350.6 mg of the objective fluorescent probe with a yield of 52.3%.¹H NMR (400 MHz, DMSO-d₆): δ（ppm）8.36(d, J = 7.1 Hz, 2H), 8.29 (d, J = 8.5 Hz, 1H), 8.24-8.12 (m, 2H), 8.04(d, J = 7.0 Hz, 1H), 8.01 (m, 2H), 7.76 (s, 1H), 7.68 (t,J = 7.0 Hz, 2H), 7.65 (t, J = 6.6 Hz, 1H), 7.56-7.42 (m, 2H), 7.31 (d, J = 8.2 Hz, 1H), 7.12 (d, J = 11.2 Hz, 1H), 7.05 (dd, J = 11.6, 2.0 Hz, 1H), 6.76 (d, J = 9.2 Hz, 1H),6.36 (dd, J = 17.2, 10.8 Hz, 1H)，4.24(s, 3H). HRMS-ESI(C₃₁H₂₂IN₃O₂S₂):m/z calcd. for[M-I]⁺:532.1153; found: 532.1071.

Example 2

Preparation of fluorescent probe mother liquor

659 mg of target fluorescent molecular probe with the purity of more than 99% is accurately weighed, carefully transferred into a 50mL volumetric flask, added with acetonitrile solution at room temperature, and completely dissolved and then fixed to a scale mark to obtain 1mM probe mother liquor. During the test, 20 μ L of the above solution was taken out by a microsyringe each time and dissolved in the test system, and the total volume of the solution was 2mL each time, at which time the concentration of the fluorescent probe in the test system was 10 μ M.

Example 3

Preparation of cysteine mother liquor

Cysteine was prepared in 5mL stock solutions in different concentration gradients (0 mM, 0.1mM, 0.3mM, 0.6mM, 1mM, 1.5mM, 2mM, 2.5mM, 3.0 mM) in PBS buffer. The remaining amino acids required for the tests were prepared separately in PBS buffer to a 3mM stock solution.

Example 4

Change in fluorescence intensity of fluorescent probe and concentration of cysteine

4.900mL of PBS buffer solution was measured, 50. mu.L of 1mM probe stock solution was dissolved therein, and 50. mu.L of cysteine stock solutions of different concentrations were transferred so that the concentration of the probe in the entire detection system was 10. mu.M and the concentrations of cysteine were 0. mu.M, 1. mu.M, 3. mu.M, 6. mu.M, 10. mu.M, 15. mu.M, 20. mu.M, 25. mu.M and 30. mu.M, respectively. After incubation at room temperature for 20 min, the fluorescence spectra of the different systems were tested in 10mm cuvettes, respectively. The fluorescence emission spectrum change is shown in figure 1. The result shows that the fluorescence emission intensity of the system at 506nm gradually decreases with the increase of the added cysteine concentration, a new fluorescence emission peak is generated at 617nm, and the fluorescence intensity gradually increases with the increase of the cysteine concentration.

Example 5

Linear relationship between fluorescence intensity of fluorescent probe and concentration of cysteine

The fluorescence intensity in each detection system in example 4 was calculated to establish a standard curve of fluorescence intensity versus cysteine concentration, as shown in FIG. 2, and the results show that when the concentration of cysteine was in the range of 0-10. mu.M, I_{617 nm}/I_{506 nm}Has good linear relation with the concentration of cysteine, and the regression linear equation is y =0.22543x +0.40987, and the linear correlation coefficient R²= 0.99579. Through calculation of a formula, the detection limit of the probe to the response of the cysteine is 0.68 mu M, and the fluorescent probe has good sensitivity.

Example 6

Selectivity of fluorescent probes for different substances

Measuring 4.900mL PBS buffer solution, dissolving 50 μ L of probe mother liquor with the concentration of 1mM therein, transferring 50 μ L of homocysteine, glutathione, leucine, isoleucine, methionine, tyrosine, tryptophan, threonine, serine, alanine, phenylalanine, histidine, aspartic acid, pyroglutamic acid, glycine, glutamic acid, glutamine, lysine and arginine mother liquor with the concentration of 3mM into a detection system respectively, incubating at room temperature for 20 min, detecting fluorescence spectra of different systems respectively, recording the fluorescence intensity at 617nm and 507nm and calculating I_{617 nm}/I_{506 nm}The value of (c). As shown in FIG. 4, it was found that the fluorescence of the fluorescent probe was significantly enhanced only by the addition of cysteine, while the probe had only a slight change in fluorescence when other small test molecules were added. The fluorescent probe is shown to have good selectivity.

Example 7

Response of fluorescent probes to cysteine in cells

First, a probe solution was added to HeLa medium and the mixture was placed in a bath of 37^oC, 5% CO₂Incubating in an incubator for 30 min under the atmosphere, washing with PBS buffer solution for three times to remove probe molecules which do not enter cells,

the cells were transferred to a fluorescence microscope for imaging, the medium was replaced, and the cells were incubated with a cysteine buffer solution (25. mu.M) for 30 minutes, washed three times with a PBS buffer solution, and placed under a fluorescence microscope to observe the change in fluorescence. Experiments show that the probe molecules entering the cell body react with cysteine, and the fluorescence is changed from green to red, so that the fluorescent probe has a good imaging effect on the cysteine in the cell and can be used for detecting the cysteine in the organism.

Although the present invention has been described with reference to the specific embodiments shown in the drawings, it is not intended to limit the scope of the present invention, and various modifications or variations can be made by those skilled in the art from the disclosure of the present invention without inventive efforts.

Claims

1. A fluorescent molecular probe for specifically recognizing L-cysteine, which is characterized by having a structure shown in formula (I):

formula I.

2. The method for preparing a novel fluorescent probe for cysteine detection according to claim 1, wherein the method comprises the following steps: with 4-hydroxym-benzeneUsing diformaldehyde as a raw material, refluxing the diformaldehyde and 2-aminothiophenol in absolute ethyl alcohol at room temperature, pouring into ice water after the reaction is finished, filtering and washing precipitates, and drying to obtain a white solid; dissolving white solid in trifluoroacetic acid, adding hexamethyl-hydroxylamine, stirring, and heating to 90 deg.C^oC, refluxing, cooling to room temperature after the reaction is finished, dropwise adding into ice water, adjusting the pH value of the system to 6, filtering and washing the precipitate, and separating and purifying by silica gel column chromatography; dissolving the purified product in anhydrous ethanol, adding 2-methylpyridine salt, 90^oC, refluxing. After the reaction is finished, cooling to room temperature, concentrating in vacuum, and separating and purifying by silica gel column chromatography; and dissolving the purified product in anhydrous dichloromethane under the protection of nitrogen and ice bath, dropwise adding triethylamine, slowly dropwise adding a dichloromethane solution dissolved with 1mmol of acryloyl chloride, stirring at room temperature overnight, after the reaction is finished, carrying out vacuum concentration, carrying out silica gel column chromatography separation and purification, and finally drying to obtain the target fluorescent molecular probe.

3. Use of a fluorescent probe according to claims 1 and 2 for the detection of cysteine.

4. Use according to claim 3, characterized in that it is used for the detection of cysteines in the environment and in cells.

5. Use of a fluorescent probe according to any of claims 1 to 4, characterized in that the cysteine detection conditions are: the excitation wavelength is 365 nm, the fluorescence emission spectrum detection is carried out between 400-700 nm, the solvent of the detection system is PBS (10 mM, pH = 7.4) buffer solution, and the pH is 5.8-8.4.

6. The use according to claims 3 and 4, characterized in that the solution to be tested is added to the fluorescent probe detection solution, and the fluorescence intensity of the detection solution is measured as an evaluation index for determining whether cysteine is contained or not and for quantitatively detecting the concentration of cysteine.