CN109516969B

CN109516969B - Carboxyl-containing coumarin derivative and synthesis method and application thereof

Info

Publication number: CN109516969B
Application number: CN201811324486.9A
Authority: CN
Inventors: 阴彩霞; 解茜茜; 霍方俊
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2018-11-08
Filing date: 2018-11-08
Publication date: 2022-03-18
Anticipated expiration: 2038-11-08
Also published as: CN109516969A

Abstract

The invention discloses a coumarin derivative containing carboxyl, a synthesis method and an application thereof, wherein the derivative is named as (Z) -2-cyano-3- (4- ((E) -3- (7- (4- (ethoxycarbonyl) piperazine-1-yl) -2-oxo-2H-chromene-3-yl) -3-oxoprop-1-en-1-yl) phenyl) acrylic acid in the Chinese, is named as (Z) -2-cyano-3- (4- ((E) -3- (7- (4- (ethoxycarbonyl) piperazin-1-yl) -2-oxo-2H-chromen-3-yl) -3-oxoprop-1-en-1-yl) phenyl) acrylic acid in the English, is named XI-2. The invention provides a method for specifically detecting cysteine by using derivative XI-2, which is characterized in that the content of cysteine is quantitatively detected by a fluorescence spectrophotometer in a PBS-DMSO (4:1, v/v, pH 7.4) solution. The detection process is circulated, simple, convenient, sensitive and quick, and the detection result is accurate. The invention also provides application of the derivative XI-2 in preparation of a cell cysteine detection reagent, namely the XI-2 efficiently permeates cell membranes under the regulation and control action of NEM to realize detection of cysteine in cytoplasm.

Description

Carboxyl-containing coumarin derivative and synthesis method and application thereof

Technical Field

The invention relates to a coumarin derivative, in particular to a carboxyl-containing coumarin derivative, a synthesis method thereof and application of the carboxyl-containing coumarin derivative in efficient penetration of cell membranes and differential detection of cysteine on the cell membranes and in cytoplasm under the regulation and control action of N-ethylmaleimide (NEM).

Background

Alteration of cysteine homeostasis is associated with a variety of diseases and cellular functions, and thus dynamic real-time live-cell intracellular imaging and quantitative analysis of cysteine are very important for understanding pathological and physiological processes. Therefore, it is crucial whether the cysteine probe has high permeability.

Studies have shown that the cell membrane consists of a phospholipid bilayer and embedded proteins that cross the cell membrane and adsorb to the surface of the cell membrane. The integral membrane proteins among the membrane proteins are covalently bound to the fat molecules at cysteine residues on the cytoplasmic matrix side, inserted between the lipid bilayers, and a few proteins are covalently bound to the glycolipids. N-ethylmaleimide (NEM) is used as a covalent modification gene of protein cysteine residues, and reacts with membrane protein sulfydryl after being used for culturing cells, so that the original structure of a cell membrane is changed, the permeability of the cell membrane is improved, and the probe can efficiently detect cysteine in cytoplasm.

However, there is no report on a specific fluorescent probe for efficiently detecting cysteine in cytoplasm. This remains a challenge.

Disclosure of Invention

The invention aims to provide a coumarin derivative containing carboxyl and a preparation method thereof, wherein the coumarin derivative containing carboxyl has good selectivity and high sensitivity, and can realize quantitative circulating detection of cysteine and efficient detection of cysteine in cytoplasm under regulation and control of NEM.

The invention provides a coumarin derivative containing carboxyl, wherein the coumarin derivative is named as (Z) -2-cyano-3- (4- ((E) -3- (7- (4- (ethoxycarbonyl) piperazine-1-yl) -2-oxo-2H-chromene-3-yl) -3-oxoprop-1-ene-1-yl) phenyl) acrylic acid in Chinese, and the coumarin derivative is named as follows in English:

(Z) -2-cyano-3- (4- ((E) -3- (7- (4- (ethoxycarbonyl) piperazin-1-yl) -2-oxo-2H-chromen-3-yl) -3-oxo prop-1-en-1-yl) phenyl) acrylic acid, named XI-2. The structural formula is as follows:

the invention provides a method for synthesizing a coumarin derivative XI-2 containing carboxyl, which comprises the following steps:

1) dissolving ethyl chloroformate and sodium bicarbonate in tetrahydrofuran, slowly and dropwisely adding the solution into a mixed solution of tetrahydrofuran containing m-hydroxyphenyl piperazine and deionized water, and stirring at room temperature overnight; extracting, drying and distilling under reduced pressure to obtain a white needle crystal compound A: 4- (3-hydroxyphenyl) piperazine-1-carboxylic acid ethyl ester; wherein the molar ratio of ethyl chloroformate, sodium bicarbonate and m-hydroxyphenyl piperazine is 1.4-1.7:1.1-1.4: 1;

2) slowly dripping excessive phosphorus oxychloride into N, N-dimethylformamide under the condition of ice-water bath, stirring, adding a compound A dissolved in the N, N-dimethylformamide at 0 ℃, and stirring; pouring the system into ice water, adjusting the pH value until a large amount of white solid is separated out, and performing suction filtration to obtain a compound B: 4- (4-formyl-3-hydroxyphenyl) piperazine-1-carboxylic acid ethyl ester;

3) adding the compound B obtained in the step 2) and ethyl acetoacetate into ethanol according to the mol ratio of 1:1.5-2.0, adding a catalytic amount of piperidine, and refluxing until the reaction is complete; cooling to room temperature, precipitating yellow needle-shaped solid, and performing suction filtration to obtain a compound C: 4- (3-acetyl-2-oxo-2H-chromen-7-yl) piperazine-1-carboxylic acid ethyl ester;

4) dissolving the compound C obtained in the step 3) and terephthalaldehyde in ethanol according to the molar ratio of 1:1.5-2.0, adding a catalytic amount of piperidine, and refluxing until the reaction is complete; cooling, precipitating yellow solid, filtering to obtain yellow powdery solid, and separating by column chromatography to obtain compound D: (E) -ethyl 4- (3- (3- (4-formylphenyl) acryloyl) -2-oxo-2H-chromen-7-yl) piperazine-1-carboxylate;

5) dissolving the compound D obtained in the step 4) and 2-cyanoacetic acid in ethanol according to the mol ratio of 1:2.5-3.5, adding a catalytic amount of piperidine, and refluxing until the reaction is complete; the solvent is evaporated to dryness under reduced pressure to obtain a crude product, and the crude product is separated and purified by column chromatography to obtain a target compound XI-2.

The volume ratio of tetrahydrofuran to deionized water in the step 1) is preferably 1: 1; the molar ratio of the ethyl chloroformate to the sodium bicarbonate to the m-hydroxyphenyl piperazine is preferably 1.5:1.25: 1.

The molar ratio of the compound C to terephthalaldehyde in the step 4) is preferably 1:1.5, the volume ratio of dichloromethane to ethyl acetate of the eluent of the column chromatography is preferably 15: 1.

The molar ratio of compound D to 2-cyanoacetic acid in said step 5) is preferably 1: and 3, the volume ratio of dichloromethane to methanol is preferably 10: 1.

In the invention, the specificity detection of cysteine is realized through the fluorescence change before and after the nucleophilic substitution reaction of cysteine and a compound; efficient detection of cytoplasmic cysteines is achieved by N-ethylmaleimide (NEM) modulation.

A method for detecting cysteine comprises the following steps:

(1) preparing a PBS buffer solution with the pH of 7.4 and the concentration of 10mM, preparing a 20mM cysteine aqueous solution, and preparing a 2mM DMSO solution of XI-2;

(2) adding 2mL of PBS/DMSO (v/v,4:1, pH 7.4) solution and 10 mu L of XI-2 DMSO solution into a fluorescence cuvette, detecting on a fluorescence spectrophotometer, and gradually increasing the fluorescence intensity at 500nm with the addition of cysteine to be detected;

(3) adding 2mL of PBS/DMSO (v/v,4:1, pH 7.4) solution into 6 cuvettes respectively, adding the cysteine solutions with the volumes of 0,2, 4, 6, 8 and 10 mu L respectively, measuring the fluorescence intensity at 500nm on a fluorescence spectrometer after 5min to obtain 50.46, 285.0, 555.4, 845.7, 1087 and 1321, plotting the concentration of cysteine salt as a horizontal coordinate and the fluorescence intensity as a vertical coordinate to obtain a working curve of the concentration of cysteine; the linear regression equation is: f-12.92 c +44.92667, c having a unit of 10^-6mol/L；

(4) And when the sample solution is measured, substituting the measured fluorescence intensity into a linear regression equation to obtain the concentration of the cysteine.

In-vitro circulating detection of cysteine by carboxyl-containing coumarin derivative XI-2:

preparing 0.2M cysteine solution and 0.2M N-ethyl maleimide solution; 2mL of a PBS/DMSO (v/v,4:1, pH 7.4) solution and 10. mu.L of a DMSO solution of XI-2 were added to a fluorescence cuvette, and fluorescence intensities at 500nm were measured on a fluorescence spectrum before and after addition of 10. mu.L of a cysteine solution as 37.77 and 2669, respectively, followed by addition of 10. mu. L N-ethylmaleimide solution, and a fluorescence intensity at 500nm as 333.5. The fluorescence intensities for the subsequent 2 cycles were 2824, 350, 2720, 212.9, respectively.

The coumarin derivative XI-2 containing carboxyl can also carry out specificity detection on cysteine in water environment and cytoplasm; the detection comprises fluorescence detection and cell imaging detection.

Compared with the prior art, the invention has the following advantages and effects:

1. the coumarin derivative containing carboxyl is simple to synthesize, low in cost and convenient to operate;

2. the detection method can realize the specificity detection of cysteine, and shows high sensitivity and excellent selectivity;

3. the detection method can realize the extracorporeal circulation detection of cysteine, and has great potential in the pathology detection research center of cysteine;

4. the detection method provided by the invention realizes efficient detection of cysteine in cytoplasm under NEM regulation for the first time, and has wide application prospects in pathological and physiological researches of cysteine;

5. the invention has simple detection means, and can be realized only by means of a fluorescence spectrometer and a laser confocal microscope.

Drawings

FIG. 1 nuclear magnetic hydrogen spectrum of XI-2 prepared in example 1.

FIG. 2 nuclear magnetic carbon spectrum of XI-2 prepared in example 1.

FIG. 3 mass spectrum of XI-2 prepared in example 1.

FIG. 4 is a graph of fluorescence emission of the interaction of XI-2 with cysteine.

FIG. 5 fluorescent emission plot over time of the interaction of XI-2 with cysteine and NEM.

FIG. 6 is a graph of cysteine interaction with XI-2 and NEM cycle fluorescence.

FIG. 7 XI-2 is a bar graph of fluorescence with various analytes.

FIG. 8 XI-2 shows the working curve for cysteine determination.

FIG. 9 XI-2 shows the fluorescence emission pattern of the samples measured.

FIG. 10 XI-2 is an image of the specificity of cellular cysteines.

FIG. 11 is a graph of fluorescence intensity analysis of the cells imaged in the green channel of FIG. 10.

FIG. 12 is a graph of partial analysis of fluorescence intensity for imaging of cysteine on cell membrane in FIG. 10.

FIG. 13 XI-2 is an image of a mouse.

Detailed Description

The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.

Example 1

Preparation and characterization of XI-2

1) Ethyl chloroformate (0.488g, 4.50mmol) and sodium bicarbonate (0.315g, 3.75mmol) were dissolved in 10mL of tetrahydrofuran, and slowly added dropwise from an isopiestic dropping funnel to a 1:1 volume ratio solution (40mL) of tetrahydrofuran and deionized water in a round-bottomed flask containing m-hydroxyphenylpiperazine (0.535g, 3.00mmol), and stirred at room temperature overnight; after the reaction was completed, dichloromethane was extracted, dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain white needle crystal a: 4- (3-hydroxyphenyl) piperazine-1-carboxylic acid ethyl ester.

2) Slowly dripping phosphorus oxychloride (5mL) into 10mL of N, N-dimethylformamide under the condition of ice-water bath, stirring for 30min, slowly adding A (0.500g, 2.00mmol) dissolved in 10mL of N, N-dimethylformamide at 0 ℃ into the solution, and stirring for 2h after completely mixing; introducing the system into 100mL of ice water, adjusting the pH value to 8 by using a saturated sodium bicarbonate solution, separating out a large amount of white solid, and performing suction filtration to obtain a compound B: 4- (4-formyl-3-hydroxyphenyl) piperazine-1-carboxylic acid ethyl ester.

3) Adding the B (0.557g, 2.00mmol) obtained in the step 2) and ethyl acetoacetate (400 mu L, 3.00mmol) into 20mL of ethanol, adding 100 mu L of piperidine serving as a catalyst, and refluxing at 78 ℃ for 6h until the reaction is complete; cooling to room temperature, precipitating yellow needle-shaped solid, and performing suction filtration to obtain a compound C: 4- (3-acetyl-2-oxo-2H-chromen-7-yl) piperazine-1-carboxylic acid ethyl ester.

4) Dissolving the C (0.690g, 2.00mmol) obtained in the step 3) and terephthalaldehyde (0.400g, 3.00mmol) in 20mL ethanol, adding 100 mu L piperidine serving as a catalyst, and refluxing at 90 ℃ for 72h until the reaction is complete; cooling to separate out yellow solid, suction filtering to obtain yellow powdery solid, and purifying by silica gel column chromatography (dichloromethane/ethyl acetate, 15/1, V/V, elution) to obtain compound D: (E) -ethyl 4- (3- (3- (4-formylphenyl) acryloyl) -2-oxo-2H-chromen-7-yl) piperazine-1-carboxylate.

5) Dissolving D (0.230g, 0.500mmol) obtained in the step 4) and 2-cyanoacetic acid (0.065g, 1.50mmol) in 20mL of ethanol, adding 100 mu L of piperidine serving as a catalyst, and refluxing at 78 ℃ for 4h until the reaction is complete; the solvent was evaporated to dryness under reduced pressure to give a crude product, which was purified by silica gel column chromatography (dichloromethane/methanol, 10/1, V/V, elution) to give red powder solid XI-2.

¹H NMR (600MHz, DMSO) δ (ppm) 8.65(s,1H),7.97(t, J ═ 12.5Hz,4H),7.85(d, J ═ 8.2Hz,2H),7.76(d, J ═ 9.0Hz,1H),7.71(d, J ═ 15.7Hz,1H),7.05(dd, J ═ 9.0,2.0Hz,1H),6.90(s,1H),4.08(q, J ═ 7.1Hz,2H),3.58-3.50(m,8H),1.21(t, J ═ 7.1Hz,3H) (fig. 1).¹³C NMR(151MHz,DMSO-d₆) Delta (ppm) 186.3,160.0,158.2,155.3,155.1,148.8,141.4,132.5,130.5,129.3,126.8,117.9,112.1,109.7,98.9,61.4,46.5,15.0 (FIG. 2.) HR-MS [ probe + H]⁺M/z Calcd 528.5405, Found 528.17674 (FIG. 3).

Example 2

Preparing a PBS buffer solution with the pH of 7.4 and the concentration of 10mM, preparing a DMSO solution of 2mM XI-2, and preparing a 20mM cysteine aqueous solution; 2mL of PBS-DMSO (10mM, pH 7.4,4:1, v/v) solution and 10. mu.L of XI-2 DMSO solution were added to a fluorescence cuvette, and cysteine solution was gradually added in a volume of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45. mu.L, while the fluorescence intensity at 500nm was measured on a fluorescence spectrometer as 50.23, 898.8, 1590, 1917, 2360, 2567, 2754, 2952, 2962, 3081, and the fluorescence intensity was gradually increased. The fluorescence emission pattern is shown in FIG. 4.

Example 3

Preparing a PBS buffer solution with the pH of 7.4 and the concentration of 10mM, preparing a DMSO solution of 2mM XI-2, preparing a cysteine aqueous solution of 0.2M, and preparing an N-ethylmaleimide (NEM) solution of 0.2M; adding 10 mu L of XI-2 DMSO solution into a 2mL PBS-DMSO (10mM, pH 7.4,4:1, v/v) fluorescence cuvette, taking 10 mu L of cysteine solution, adding into the buffer solution, taking 0.5 mu L of NEM solution when the fluorescence intensity of the system is not changed any more, adding into the solution, continuing to add 2 mu L of NEM solution when the fluorescence intensity of the system is not changed any more, and repeatedly adding 2 mu L of NEM solution 4 times when the fluorescence intensity of the system is not changed any more until the fluorescence intensity of the system is not changed; addition of a further 10. mu.L of cysteine solution allowed the fluorescence of the system to rise again. The fluorescence emission pattern is shown in FIG. 5. Another fluorescence cuvette was taken, 10. mu.L of XI-2 in DMSO was added to 2mL of PBS-DMSO (10mM, pH 7.4,4:1, v/v), 10. mu.L of cysteine solution was added to the buffer solution, and when the fluorescence intensity of the system did not change any more, 10. mu. L N-ethylmaleimide (NEM) solution was added to the cuvette and detected on a spectrofluorimeter, and the fluorescence intensity at 500nm decreased with the addition of N-ethylmaleimide, the fluorescence of the system gradually returned to the fluorescence intensity of the probe itself. The fluorescence emission pattern is shown in FIG. 6.

Example 4

Preparing PBS buffer solution with pH 7.4 and concentration of 10mM, preparing DMSO solution of 2mM XI-2, preparing cysteine aqueous solution of 0.2M, and respectively preparing 0.2M Ala, Asn, Asp, Arg, Gln, Glu, Gly, Ile, Leu, Met, Pro, Ser, Thr, Trp, Tyr, Val, GSH, Hcy, K⁺，Na⁺，Mg²⁺，Mn²⁺,Ca²⁺，Cu²⁺，Cl^-，SO₄ ^2-，NO₃ ^-，Br^-，OH^-，CNS^-，CO₃ ^2-，S₂O₃ ^2-，SO₃ ^2-，S^2-An aqueous solution of (a); in 35 fluorescence cuvettes, 2mL of PBS-DMSO (4:1, pH 7.4) solution and 10. mu.L of XI-2 in DMSO solution were added, as well as 50. mu.L of each analyte: ala, Asn, Asp, Arg, Gln, Glu, Gly, Ile, Leu, Met, Pro, Ser, Thr, Trp, Tyr, Val, GSH, Hcy, K⁺，Na⁺，Mg²⁺，Mn²⁺,Ca²⁺，Cu²⁺，Cl^-，SO₄ ^2-，NO₃ ^-，Br^-，OH^-，CNS^-，CO₃ ^2-，S₂O₃ ^2-，SO₃ ^2-，S^2-And 5 μ L of Cys, were detected on a fluorescence spectrophotometer to plot a histogram of fluorescence intensity at 500nm for the different analytes (see FIG. 7). Cysteine causes the fluorescence intensity of the detection system to be obviously increased at 500nm, and other analytes do not cause the change of the fluorescence intensity of the detection system basically.

Example 5

Preparing a PBS buffer solution with the pH of 7.4 and the concentration of 10mM, preparing a solution of 2mM XI-2 by using DMSO, and preparing a 20mM cysteine aqueous solution; in 6 cuvettes, 2m of each of the cuvettes was addedAdding L PBS/DMSO (v/v,4:1, pH 7.0) solution and 10. mu.L XI-2 DMSO solution, respectively, adding cysteine solution with volume of 0,1, 2.5, 3.3, 5, 6, 7.5, 9, 10. mu.L, measuring fluorescence intensity at 500nm on a fluorescence spectrometer after 5min to obtain 50.46, 285.0, 555.4, 845.7, 1087, 1321, plotting the cysteine concentration as abscissa and the fluorescence intensity as ordinate to obtain a cysteine concentration working curve; the linear regression equation is: f-12.92 c +44.92667, c having a unit of 10^-6mol/L；

Example 6

Preparing a PBS buffer solution with the pH of 7.4 and the concentration of 10mM, preparing a solution of 2mM XI-2 by using DMSO, and preparing a 20mM cysteine aqueous solution; mu.L of a DMSO solution of XI-2 was placed in a 2mL PBS-DMSO (10mM, pH 7.4,4:1, v/v) fluorescence cuvette, 6.5. mu.L of a cysteine solution was taken and added to the cuvette by a microsyringe, and the fluorescence intensity at 500nm was 898.8 by measurement on a fluorescence spectrometer, and the value of c 66.09X 10 was determined by the linear regression equation of example 5^-6mol/L. The deviation was 1.6%. See fig. 9.

Example 7

Preparing a PBS buffer solution with the pH of 7.4 and the concentration of 10mM, preparing a DMSO solution of 2mM XI-2, preparing a 20mM cysteine solution, 15 mu M homocysteine and 1mM glutathione by using the PBS buffer solution respectively, and preparing a 1mM NEM (N-ethyl maleimide, thiol scavenger) solution by using the PBS buffer solution; add 10. mu.L of XI-2 in DMSO to 1990. mu.L of PBS; adding the probe solution into a HepG-2 cell culture solution to enable the concentration of the probe solution to be 10 mu M, incubating the probe solution and HepG-2 cells for 30min at 37 ℃, and displaying that green fluorescence is generated on cell membranes under a fluorescence imager, namely carboxyl on XI-2 and amino of membrane protein form a peptide chain, the peptide chain is stopped on the cell membranes and responds to cysteine on the cell membranes to generate green fluorescence, wherein the step (A) is shown in figure 10; adding 2mL of NEM solution into HepG-2 cell culture solution, incubating for 30min at 37 ℃, sucking the solution, washing with PBS once, adding 2mL of 10 mu M probe solution, incubating for 30min at 37 ℃, and displaying green fluorescence distribution in cytoplasm under a fluorescence imager, namely, after the action of NEM, the permeability of the cell membrane is improved, and XI-2 can efficiently detect cysteine in the cytoplasm, as shown in FIG. 10 (B); 2mL of NEM solution was added to HepG-2 cell culture, incubated at 37 ℃ for 30min, the solution was aspirated, washed once with PBS, and 20. mu.L of 20mM cysteine solution was added to 1980. mu.L of PBS; adding cysteine solution into HepG-2 cell culture solution to make the concentration of the solution 200 μ M, incubating the solution with HepG-2 cells for 30min at 37 ℃, washing the cells once with PBS, adding 2mL of 10 μ M probe solution, incubating the cells for 30min at 37 ℃, and displaying green fluorescence distributed in cytoplasm under a fluorescence imager, namely after NEM acts, XI-2 enters the cells and not only responds to cysteine in the cytoplasm, but also reacts with exogenous cysteine, as shown in FIG. 10 (C); adding 2mL of NEM solution into HepG-2 cell culture solution, incubating for 30min at 37 ℃, sucking the solution away, washing once with PBS, incubating HepG-2 cells with 2mL of 15 μ M homocysteine solution, incubating for 30min at 37 ℃, washing once with PBS, adding 2mL of 10 μ M probe solution, incubating for 30min at 37 ℃, and displaying green fluorescence distribution in cytoplasm under a fluorescence imager, comparing FIG. 10(B) with FIG. 10(C) to find that XI-2 enters the cells and only responds to cysteine in the cytoplasm, as shown in FIG. 10 (D); 2mL of NEM solution was added to HepG-2 cell culture, incubated at 37 ℃ for 30min, the solution was aspirated, washed once with PBS, 2mL of 1mM glutathione solution was incubated with HepG-2 cells, incubated at 37 ℃ for 30min, washed once with PBS, and added with 2mL of 10. mu.M probe solution, incubated at 37 ℃ for 30min, the system showed green fluorescence distribution in the cytoplasm under a fluorescence imager, and upon comparison of FIG. 10(B) and FIG. 10(C), XI-2 entered the cells and responded only to cysteine in the cytoplasm, as shown in FIG. 10 (E).

FIG. 10 is a graph showing the high-efficiency fluorescence response of the probe to cysteine in cytoplasm (green fluorescence is mainly distributed in cytoplasm) after the detection of cysteine in cell membrane by the probe under confocal fluorescence imager (green fluorescence is mainly distributed on cell membrane) and the action of NEM, and the response to cysteine in cytoplasm and exogenous cysteine after the action of NEM (green fluorescence is shown). Therefore, from the above results, it is suggested that XI-2 can efficiently and continuously monitor cysteine in cytoplasm under NEM regulation.

FIG. 11 is a graph of fluorescence intensity analysis of the imaged cells in the green channel of FIG. 10. The results of each image of the green channel in fig. 10 were analyzed as follows: the average fluorescence intensity (cell part only) of the cells in each image of green fluorescence imaging was calculated separately, i.e., the fluorescence intensity of each cell was calculated first, and then the average was calculated by summing. Comparing 5 average values, the cysteine content in cytoplasm is more than the cysteine content on cell membrane; and the results of the mean fluorescence intensity of cells treated with either homocysteine or glutathione were the same as those treated with NEM and XI-2 only, it was also possible to conclude that: NEM participates in the whole process only in the reaction with the sulfhydryl group of membrane protein to change the permeability of cell membrane, but does not scavenge the thiol in cytoplasm.

FIG. 12 is a graph showing the fluorescence distribution analysis of individual cells as a result of incubating the cells with the probe of FIG. 10. The probe directly incubates the cells, and the green fluorescence is mainly distributed on the cell membrane. It is possible that the carboxyl group on the probe forms a peptide chain with the amino group of the membrane protein, resulting in the stagnation of the probe on the surface of the cell membrane.

Example 8

Mice were anesthetized with sodium pentobarbital (100 μ L0.5 mL/0.03%) by subcutaneous injection. XI-2 solution (20. mu.L, 0.1mM) was injected subcutaneously into mice. Mice were photographed in a fluorescence imager with a single set of images of XI-2, and time-sequenced by injection of cysteine (20. mu.L, 0.2mM) at the same site. The fluorescence images were recorded in time series of 0.5, 5, 10, 20, 25, 30min, respectively. The experimental results show that when cysteine was added, an increase in fluorescence intensity was observed over a 20 minute time frame, followed by maintenance by the body, indicating that XI-2 can be successfully used for cysteine imaging in mice. In FIG. 13, (a) nude mice without any treatment, (b) XI-2 was injected subcutaneously only, and (c-h) cysteine was injected subcutaneously for 0.5, 5, 10, 20, 25, 30min, respectively.

Claims

1. A carboxyl-containing coumarin derivative XI-2, which is characterized in that the structural formula is as follows:

。

2. the method for synthesizing a coumarin derivative XI-2 containing carboxyl according to claim 1, which comprises the following steps:

3. The method for synthesizing a coumarin derivative XI-2 containing carboxyl according to claim 2, wherein in the step 1), the volume ratio of tetrahydrofuran to deionized water is 1: 1; the molar ratio of the ethyl chloroformate to the sodium bicarbonate to the m-hydroxyphenyl piperazine is 1.5:1.25: 1.

4. The method for synthesizing a coumarin derivative XI-2 containing carboxyl according to claim 2, wherein the molar ratio of C to terephthalaldehyde in the step 4) is 1:1.5, the volume ratio of dichloromethane to ethyl acetate is 15:1 as eluent of column chromatography

5. The process for the preparation of the carboxylic coumarin derivative XI-2 as claimed in claim 2, wherein in step 5) the molar ratio of compound D to 2-cyanoacetic acid is preferably 1: 3, eluting the column chromatography by using dichloromethane and methanol in a volume ratio of 10: 1.

6. A method for specifically detecting cysteine, comprising the steps of:

(1) preparing a PBS buffer solution with a concentration of 10mM at pH =7.4, preparing a 20mM aqueous solution of cysteine, preparing a 2mM DMSO solution of XI-2 according to claim 1;

(2) 2mL of a 4:1 volume mixture of PBS and DMSO, pH 7.4, was placed in a fluorescent cuvette, 10%µAdding a DMSO solution of L XI-2 into the system, detecting on a fluorescence spectrophotometer, and gradually increasing the fluorescence intensity at 500nm with the addition of cysteine to be detected;

(3) 2mL of a mixed solution of PBS and DMSO having a pH of 7.4 in a volume ratio of 4:1 was added to each of 6 cuvettes, and cysteine solutions having volumes of 0,2, 4, 6, 8, and 10 were added to each of the cuvettesµL, measuring the fluorescence intensity at 500nm on a fluorescence spectrometer after 5min to obtain 50.46, 285.0, 555.4, 845.7, 1087 and 1321, plotting the cysteine concentration as abscissa and the fluorescence intensity as ordinate to obtain a plot chartA working curve for cysteine concentration; the linear regression equation is: f =12.92 c +44.92667, c having a unit of 10^-6 mol/L；

7. The use of coumarin derivatives containing carboxyl XI-2 as claimed in claim 1 in the preparation of reagents for the detection of cysteine by extracorporeal circulation.

8. Use of the coumarin derivative XI-2 containing carboxyl groups as defined in claim 1 in the preparation of a cell cysteine detection reagent.