CN107652220B

CN107652220B - Preparation method and application technology of fluorescent probe for detecting cysteine

Info

Publication number: CN107652220B
Application number: CN201710854736.9A
Authority: CN
Inventors: 韩得满; 陈逢灶
Original assignee: Taizhou University
Current assignee: Taizhou University
Priority date: 2017-09-20
Filing date: 2017-09-20
Publication date: 2021-03-16
Anticipated expiration: 2037-09-20
Also published as: CN107652220A

Abstract

The invention belongs to the technical field of analytical chemistry and relates to a preparation method of a novel fluorescent probe and application of the novel fluorescent probe in cysteine (Cys) detection. The preparation method comprises the step of carrying out the knoevenagel condensation reaction on the probe by using 3-cyanoacetylindole and chlorinated phenylacrolein derivatives, and the probe is simple in preparation method and stable in structure. The probe can be applied to high-selectivity, high-sensitivity and rapid visual detection of cysteine by a fluorescence spectrophotometer. In addition, the probe is also successfully applied to the imaging of biological living cells. The invention provides a preparation method of a novel fluorescent probe for detecting cysteine, and the fluorescent probe has a good application prospect.

Description

Preparation method and application technology of fluorescent probe for detecting cysteine

Technical Field

The invention relates to the technical field of fluorescent probe sensing in analytical chemistry, in particular to a preparation method and application of a fluorescent probe for rapidly detecting cysteine with high selectivity and high sensitivity.

Background

With the development of life science research, the detection of small biological molecules is particularly important. For example, biological thiol small molecules, including cysteine (Cys), homocysteine (Hcy), and Glutathione (GSH), play important roles in both physiological and pathological processes, such as: cys is closely linked to the body's detoxification, metabolism and protein synthesis, and abnormal Cys content may cause various diseases, such as: growth retardation, skin lesions, hematopoietic disorders, hair discoloration, and the like. Hcy, a precursor of Cys, is implicated in diseases such as cardiovascular disease, alzheimer's disease, and psychiatric disorders. GSH is associated with aids, cancer, cardiovascular disease, and other diseases. In addition, Cys has important applications in medicine, cosmetics and food, such as: the product can be used for detoxification after poisoning of acrylonitrile and aromatic acid; a medicament for the treatment of bronchitis; the cosmetics are mainly used for hair perm liquid, beauty water, sunscreen cream and the like; the food is mainly used in bread materials and natural fruit juice. Therefore, it is very important for the detection of these biological thiol small molecules. However, Cys/Hcy is difficult to distinguish from GSH due to structural similarity. Since the content of Cys is ten times or more the content of Hcy in vivo, the main focus is currently on the differential detection of Cys and GSH. The conventional measurement methods mainly rely on liquid chromatography, spectrophotometry, isotope methods, immunological methods, and the like. Although these methods can achieve qualitative or quantitative determination, they have certain disadvantages, such as complicated sample preparation, complicated operation, expensive instrument, long testing time, and difficulty in popularization and application. Therefore, the development of a method capable of rapidly and conveniently detecting Cys with high selectivity and high sensitivity is of great significance to the initial clinical diagnosis of some serious diseases.

The invention relates to a preparation method and application technology of a fluorescent probe for detecting cysteine. The fluorescent probe is obtained by the knoevenagel condensation reaction of 3-cyanoacetylindole and chlorinated phenylacrolein derivative, and the preparation method is simple and has a stable structure. In vitro tests show that the fluorescent probe can be used for quickly and visually detecting cysteine with high selectivity and high sensitivity. In addition, the fluorescent probe is also successfully applied to the imaging of biological living cells, and experiments show that the fluorescent probe can monitor endogenous and exogenous Cys of the cells. Therefore, the probe has potential application prospect.

Disclosure of Invention

The invention aims to provide a preparation method and an application technology of a fluorescent probe for detecting cysteine.

According to the invention, the fluorescent probe is obtained by condensation reaction of 3-cyanoacetylindole and chlorinated phenylacrolein derivative, and the synthesis steps specifically comprise: (1) dissolving indole and cyanoacetic acid in anhydride, heating and refluxing for reaction for 4 hours under the protection of argon, and treating to obtain a white solid product, namely 3-cyanoacetylindole after the reaction is finished; (2) placing N, N-dimethylformamide into an ice bath for pre-cooling to 0-5 ℃, then slowly adding phosphorus oxychloride, stirring for 1 hour in the ice bath, then slowly adding 3,4, 5-trimethoxyacetophenone, stirring for 3 hours at room temperature, after the reaction is finished, pouring a reaction solution into crushed ice, adjusting the pH value to 7-8 by using sodium carbonate, generating yellow precipitates in the reaction solution, filtering, separating and purifying to obtain a yellow solid product, namely a chlorinated phenylacrolein derivative; (3) dissolving the 3-cyanoacetylindole and the chlorinated phenylacrolein derivative in the steps (1) and (2) in absolute ethyl alcohol, adding an alkaline catalyst, stirring at room temperature for reaction, separating out yellow solid in reaction liquid, filtering, separating and purifying to obtain a final product, namely the fluorescent probe for detecting cysteine.

Compared with the prior art, the patent technology has the following advantages:

1. high selectivity, on which the relevant substances have little effect;

2. the sensitivity is high, and the detection of a trace target object can be realized;

3. the response is quick, and quick detection can be realized;

3. simple synthesis, low cost and no pollution.

Drawings

FIG. 1 is a fluorescence titration chart of cysteine detection by probe

FIG. 2 is a fluorescence spectrum diagram of the response time of cysteine detection by a probe

FIG. 3 is a fluorescence spectrum of a probe for selective experiments

FIG. 4 is a pH optimized fluorescence spectrum of a detection environment

Detailed Description

The present invention will be further described with reference to the following examples, which are only for illustrating the technical solutions of the present invention and are not to be construed as limiting the present invention.

Example 1

Dissolving indole (2.00g,17.02mmol) and cyanoacetic acid (1.59g,18.69mmol) in 15mL of acid anhydride, heating under the protection of argon gas for reflux reaction for 4 hours, cooling the reaction liquid to room temperature after the reaction is finished, pouring the reaction liquid into 100mL of water, extracting with ethyl acetate, collecting an organic phase solution, drying with anhydrous sodium sulfate, carrying out rotary evaporation to obtain a crude product, and carrying out column chromatography separation to obtain a final product, namely 3-cyanoacetylindole (2.1 mmol)3g, 68% yield). Nuclear magnetic characterization data:¹H NMR(400MHz,DMSO-d6,TMS):δ12.18(s,1H),8.38(d,J＝4.0Hz,1H),8.14(dd,J₁＝2.0Hz,J₂＝7.1Hz,1H),7.51(m,1H),7.24(m,2H),4.50(s,2H)。

example 2

Placing 5mL of N, N-dimethylformamide in an ice bath for pre-cooling to 0-5 ℃, then slowly adding 2mL of phosphorus oxychloride, stirring for 1 hour in the ice bath, then slowly adding 3,4, 5-trimethoxyacetophenone (1.00g,4.76mmol), stirring for 3 hours at room temperature, after the reaction is finished, pouring the reaction liquid into crushed ice, adjusting the pH value to 7 by using sodium carbonate, generating yellow precipitate, filtering to obtain a crude product, and then carrying out column chromatography separation to obtain a final product, namely a chlorinated phenylacrolein derivative (588mg, the yield is 48%). Nuclear magnetic characterization data:¹H NMR(400MHz,CDCl₃,TMS):δ10.20(d,J＝7.0Hz,1H),6.98(s,2H),6.63(d,J＝6.9Hz,1H),3.91(s,9H)。

example 3

Dissolving the 3-cyanoacetylindole (150mg,0.82mmol) and the chlorinated phenylacrolein derivative (209mg,0.82mmol) in 10mL of absolute ethanol, adding three drops of pyridine, stirring at room temperature for reaction, separating out yellow solid in the reaction solution, filtering, separating and purifying to obtain a final product (152mg, yield 44%), namely the fluorescent probe for detecting cysteine. Nuclear magnetic characterization data:¹H NMR(400MHz,CDCl₃Δ 8.85(s,1H),8.60(d, J ═ 11.52Hz,1H),8.53(d, J ═ 3.2Hz,1H),8.47(d, J ═ 5.0,1H),7.46(d, J ═ 4.0Hz,1H),7.36(m,3H),7.04(s,2H),3.94(d, J ═ 7.2Hz, 9H). High resolution mass spectrometry: [ M + H ]]⁺＝423.1102(Calcd＝423.1111)。

Application example 1: as shown in FIG. 1, the fluorescence titration chart of the probe for detecting cysteine. When 5 mu M of probe is added into a mixed solution of dimethyl sulfoxide and water, the fluorescence of the probe is very weak under the excitation of light with the wavelength of 430nm, however, after the probe responds to cysteine with different concentrations, the fluorescence intensity at 655nm has obvious change, and the fluorescence intensity is enhanced along with the increase of the cysteine concentration and has good linear relation (R)²0.994), the detection limit reaches 240 nmol/L. Fruit of Chinese wolfberryThe test proves that the probe can be applied to the detection of cysteine with high sensitivity.

Application example 2: as shown in FIG. 2, the probe detects the response time fluorescence spectrum of cysteine. When the probe (5. mu.M) was added to a mixed solution of dimethyl sulfoxide and water, the fluorescence of the probe itself was very weak under excitation with light having a wavelength of 430nm, but after addition of 50. mu.M cysteine, the fluorescence intensity at 480nm began to gradually increase with the lapse of time, and the response was complete within 10 minutes, and the fluorescence intensity did not change any more. Thus proving that the probe can realize the rapid detection of the cysteine.

Application example 3: FIG. 3 shows the fluorescence spectra of the probe in the selectivity experiment. Adding different interferents (1-27 are respectively probe, alanine, arginine, aspartic acid, glycine, glutamic acid, proline, phenylalanine, threonine, leucine, histidine, serine, tryptophan, methionine and S) under the same test system and test conditions₂O₃ ^2-、SO₄ ^2-、Mg² ⁺、Ca²⁺、Ba²⁺、K⁺、CO₃ ^2-、NO₃ ^-、NO₂ ^-、HS^-GSH, Cys, Hcy). Experimental results prove that the probe has high selectivity to cysteine/homocysteine, and shows that the fluorescence intensity at 480nm is obviously enhanced. However, the fluorescent probe hardly responds to other related interferents and shows that the fluorescence intensity is kept unchanged

Application example 4: FIG. 4, pH optimized fluorescence spectra of the detection environment. Separately, 5 μ M of probe and 50 μ M of cysteine were added to dimethyl sulfoxide/water buffers with different pH (pH 3-10), and the probe response to cysteine was measured at different pH while keeping the other test conditions consistent. As can be seen from the figure, the probe itself maintains high stability in this different pH environment, and the fluorescence intensity hardly changes. In addition, the fluorescence intensity of the probe after responding to cysteine is gradually increased between

pH

3 and 7, the optimal effect is achieved when the pH is neutral, and the fluorescence intensity of the probe after responding to cysteine is gradually reduced between

pH

7 and 10. The comprehensive experimental data show that: the probe can keep self stability under different pH environments, can better respond cysteine in a larger pH range, achieves the best measurement effect under a neutral environment, and is beneficial to the application of the probe in biological sample detection.

Claims

1. A fluorescent probe for detecting cysteine is characterized in that the molecular structural formula is as follows:

。

2. a method for preparing a fluorescent probe for detecting cysteine according to claim 1, wherein the preparation process comprises the following steps:

a: dissolving indole and cyanoacetic acid in anhydride, heating and refluxing for reaction for 4 hours under the protection of argon, and treating to obtain a white solid product, namely 3-cyanoacetylindole after the reaction is finished;

b: placing N, N-dimethylformamide into an ice bath for pre-cooling to 0-5 ℃, then slowly adding phosphorus oxychloride, stirring for 1 hour in the ice bath, then slowly adding 3,4, 5-trimethoxyacetophenone, stirring for 3 hours at room temperature, after the reaction is finished, pouring the reaction liquid into crushed ice, adjusting the pH value by using sodium carbonate, generating yellow precipitate in the reaction liquid, filtering, separating and purifying to obtain a yellow solid product, namely a chlorinated phenylacrolein derivative;

c: dissolving the 3-cyanoacetylindole and the chlorinated phenylacrolein derivative in the a and the b in absolute ethyl alcohol, adding an alkaline catalyst, stirring at room temperature for reaction, separating out yellow solid in reaction liquid, filtering, separating and purifying to obtain a final product, namely the fluorescent probe for detecting cysteine in claim 1.

3. Use of the fluorescent probe according to claim 1 for the detection of cysteine.

4. The method for preparing the fluorescent probe for detecting cysteine according to claim 2, wherein the stoichiometric ratio of the raw indole to the cyanoacetic acid in the step a is 1: 0.1-10.

5. The method for preparing a fluorescent probe for detecting cysteine according to claim 2, wherein the pH value after the adjustment in step b is 7 to 8.

6. The method for preparing a fluorescent probe for detecting cysteine according to claim 2, wherein the basic catalyst used in step c is pyridine or triethylamine.

7. The use of the fluorescent probe according to claim 3 for detecting cysteine, wherein the optimal excitation light wavelength for detection is 420 nm to 440 nm.

8. The use of the fluorescent probe in detecting cysteine according to claim 3, wherein the solvent system used in the detection is a mixed solution of dimethyl sulfoxide and water.