CN109776390B - Preparation method and application of fluorescence molecular probe for detecting glutathione mercaptotransferase - Google Patents
Preparation method and application of fluorescence molecular probe for detecting glutathione mercaptotransferase Download PDFInfo
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Abstract
The invention belongs to the field of analytical chemistry, and discloses a fluorescent molecular probe for detecting glutathione mercaptotransferase, which is prepared by the following steps: dissolving 2,3, 3-trimethyl-3H-benzindole and iodoethane in anhydrous acetonitrile to react to obtain a product 1; mixing anhydrous N, N-dimethylformamide and anhydrous dichloromethane, dropwise adding phosphorus oxychloride and anhydrous dichloromethane, adding cyclohexanone, pouring into ice, and collecting a product 2; dissolving the product 1 and the product 2 in n-butanol and benzene solution to obtain a product 3; dissolving the product 3, methylamine hydrochloride and triethylamine in anhydrous N, N-dimethylformamide to obtain a product 4; dissolving 3, 4-dinitrobenzoic acid and triphosgene in anhydrous dichloromethane, mixing, adding N, N-diisopropylethylamine for reaction, dissolving the obtained residue in anhydrous dichloromethane, adding N, N-diisopropylethylamine and 4-dimethylaminopyridine, dissolving the product 4 in dichloromethane, and adding the mixture to obtain the molecular probe.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and relates to a preparation method and application of a fluorescence molecular probe for detecting glutathione mercaptotransferase.
Background
Glutathione mercaptotransferases are a group of phase II metabolic enzymes commonly present in various organisms, are the main detoxification systems in various organisms, are involved in the excretion of many physiological and heterologous substances, and the common substrate of glutathione mercaptotransferases is 2, 4-dinitrochlorobenzene (CDNB). The glutathione mercaptotransferase can catalyze the combination of the sulfhydryl (-SH) of glutathione with substances which are difficult to dissolve in water, such as dye, cholic acid, heme, hormone and other foreign substances with strong hydrophobicity or endogenous metabolites, so that the polarity of the substances is enhanced, the substances are easy to dissolve in water and are finally degraded and discharged out of the body. In addition, glutathione mercaptotransferase plays an important role in preventing the expansion of lipid peroxidation damage, reducing and repairing DNA damage, and the formation of drug resistance in cancer cells. Several methods of detecting GSTs have been developed, such as the analysis of 2, 4-dinitrochlorobenzene (CDNB) derivatives, ultraviolet spectroscopy, and bioluminescence analysis. These detection methods can be used to detect and track cell lysates or glutathione mercaptotransferases in vitro, however, they do not provide in situ and real-time information on glutathione mercaptotransferases in living cells and in vivo.
Disclosure of Invention
The invention aims to solve the problems in the prior art, and aims to provide a preparation method of a fluorescent molecular probe for detecting glutathione mercaptotransferase.
The invention also aims to provide the application of the fluorescent molecular probe for detecting glutathione mercaptotransferase, which is particularly suitable for large-scale data research such as large-scale sample combination screening and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of a glutathione sulfydryl transferase fluorescent molecular probe comprises the following steps:
(1) dissolving 2,3, 3-trimethyl-3H-benzindole and iodoethane in anhydrous acetonitrile, performing reflux reaction, cooling and filtering, washing and filtering precipitates, and performing vacuum drying to obtain a product 1;
(2) mixing anhydrous N, N-dimethylformamide and anhydrous dichloromethane, cooling, stirring, uniformly dripping phosphorus oxychloride and the anhydrous dichloromethane into the solution, adding cyclohexanone into the mixture until the solution is changed from colorless to yellow, heating to 45 ℃ for 3 hours, pouring the reactant into ice, standing, filtering and collecting a filtrate.
(3) Dissolving the product 1 and the product 2 in a mixed solution of n-butanol and benzene, refluxing for 3 hours, then performing vacuum drying, and purifying the obtained crude product through a silica gel column to obtain a product 3;
(4) dissolving the product 3, methylamine hydrochloride and triethylamine in anhydrous N, N-dimethylformamide, heating and stirring the mixture, and purifying the reaction crude product by using a 200-mesh and 300-mesh silica gel column to obtain a product 4;
(5) dissolving 3, 4-dinitrobenzoic acid and triphosgene in anhydrous dichloromethane, mixing at 0 ℃, adding N, N-diisopropylethylamine, reacting, removing the solvent, dissolving the obtained residue in anhydrous dichloromethane, adding N, N-diisopropylethylamine and 4-dimethylaminopyridine, dissolving the product 4 in a dichloromethane solution, adding the mixture, stirring at 25 ℃ until the raw material is exhausted, and purifying the obtained solid crude product through a 200-mesh and 300-mesh silica gel column to obtain the glutathione mercaptotransferase detection fluorescent molecular probe.
Preferably, the method comprises the following specific steps:
(1) dissolving 75mmol of 2,3, 3-trimethyl-3H-benzindole and iodoethane in 40 mL of anhydrous acetonitrile, refluxing and reacting the mixture for 12 hours, cooling to room temperature, filtering, washing the filtered precipitate with diethyl ether, and drying in vacuum to obtain a product 1;
(2) 40 mL of anhydrous N, N-dimethylformamide and 40 mL of anhydrous dichloromethane are mixed, cooled to-10 ℃, stirred for 20 minutes, 37mL of phosphorus oxychloride and 35 mL of anhydrous dichloromethane are added dropwise to the solution at a constant speed, 52.6 mmol of cyclohexanone are added to the mixture until the solution turns from colorless to yellow, the solution is heated to 45 ℃ for 3 hours, then the reaction is poured into ice and left standing for 12 hours. Collecting the filtrate and vacuum drying to obtain a product 2;
(3) dissolving 2mmol of product 1 and 1mmol of product 2 in 100 mL of a mixed solution of n-butanol and benzene, refluxing for 3 hours, and then drying in vacuum to obtain a green crude product, and purifying the crude product through a silica gel column to obtain a product 3, wherein the volume ratio of n-butanol to benzene is 7: 3;
(4) dissolving 2mmol of the product 3, 20 mmol of methylamine hydrochloride and 20 mmol of triethylamine in 50 mL of anhydrous N, N-dimethylformamide, stirring the mixture at 40 ℃ for 24 hours, and purifying the crude product by using a 200-mesh 300-mesh silica gel column to obtain a product 4;
(5) dissolving 1mmol of 3, 4-dinitrobenzoic acid and 3 mmol of triphosgene in 100 mL of anhydrous dichloromethane, mixing the mixture at 0 ℃, adding 1mL of N, N-diisopropylethylamine, reacting for 30 minutes, removing the solvent in vacuum, dissolving the obtained residue in 50 mL of anhydrous dichloromethane, adding 1mL of N, N-diisopropylethylamine and 20 mg of 4-dimethylaminopyridine, dissolving 0.2mmol of product 4 in 10 mL of dichloromethane solution, adding the mixture, stirring the mixture at 25 ℃ until the raw materials are completely consumed, purifying the obtained solid crude product by a 200-mesh 300-mesh silica gel column, and obtaining the glutathione mercaptotransferase detection fluorescent molecular probe.
Preferably, the judgment indexes of the effect of the fluorescent molecular probe for detecting glutathione mercaptotransferase are as follows:
detection sensitivity: the detection limit is 10 ng/mL;
detection response rate: the fluorescence intensity is 5 multiplied by 10 when detecting4 -3×105；
Detecting speed: 0-10 minutes;
the optical mechanism index is as follows: a glutathione mercaptotransferase fluorescent probe of a PET mechanism.
The application of the glutathione mercaptotransferase detection fluorescent molecular probe prepared by the preparation method is suitable for qualitative analysis of glutathione mercaptotransferase.
Preferably, the method for quantitatively analyzing the content of glutathione mercaptotransferase in a standard comprises the steps of:
1) preparing solution
Probe stock solution: weighing glutathione mercaptotransferase detection fluorescent molecular probes, dissolving the probes in anhydrous DMSO, and preparing a probe stock solution with the concentration of 100 mu M;
glutathione mercaptotransferase stock: weighing a target object to be detected, namely 1 mg glutathione mercaptotransferase, dissolving in 10 mL of distilled water, and preparing into glutathione mercaptotransferase stock solution with the concentration of 100 mu g/mL;
2) establishing linear equation of glutathione mercaptotransferase standard substance
Diluting the glutathione mercaptotransferase stock solution prepared in the step 1) with distilled water to obtain a glutathione mercaptotransferase standard solution with the gradient concentration of 0-1.1 mu g/mL, then respectively mixing the glutathione mercaptotransferase standard solutions with different concentrations with 10 mu M of the probe solution prepared in the step 1) and 0.1 mM of GSH solution, adding 50 mu L of Tris-hydrochloric acid buffer solution with the concentration of 10 mM and the pH value of 7.42, oscillating to uniformly mix the system, standing at 25 ℃ for 50 min, and then detecting by a fluorescence spectrophotometer to establish a linear equation of the concentration of the glutathione mercaptotransferase and the intensity of a fluorescence signal.
The method for detecting glutathione mercaptotransferase in the living cells comprises the following steps: the method for detecting glutathione mercaptotransferase in living cells comprises the following steps: culturing a to-be-detected living cell sample in a culture medium for 18-26 h, wherein the inoculation amount of the to-be-detected living cells is 2 multiplied by 107～9×107Adding the glutathione mercaptotransferase to detect a fluorescent molecular probe with the probe concentration of 1 mu M, culturing at 37 ℃ for 30 minutes, washing for multiple times by using a PBS solution, observing cell imaging under a fluorescence microscope, judging whether the living cells to be detected contain the glutathione mercaptotransferase according to the luminescence condition, wherein the judgment standard is as follows: the living cell sample contains glutathione under the condition of fluorescence enhancementA mercaptotransferase; the living cells are preferably IMR-90 cells (human embryonic lung fibroblast cell line), and the culture medium is preferably DMEM culture medium.
The method for detecting glutathione mercaptotransferase in vivo of the living mouse comprises the following steps: the method for detecting glutathione mercaptotransferase in vivo of the living mouse comprises the following steps: c57BL/6 mice (8 weeks) were housed in a 12-hour light/dark cycle with free access to food and water. 5 mg/kg of bleomycin dissolved in saline is used for pulmonary fibrosis animal molding, single intratracheal perfusion is carried out under anesthesia, and molding is finished after 28 days. A multifunctional ultrasensitive glutathione mercaptotransferase (100. mu.M 50. mu.L 1: 99 DMSO/normal saline, v/v) was subjected to intratracheal detection with a pulmonary fibrosis mouse model for 30 minutes at 37 ℃. And then observing and imaging in a Perkinelmer IVIS Lumina XRMS III series living body imager, judging whether the living body mouse to be detected contains glutathione mercaptotransferase according to the luminescence condition, wherein the judgment standard is as follows: when the fluorescence in the lung of the living mouse is enhanced, a living mouse sample contains glutathione mercaptotransferase; the living mouse is preferably a C57BL/6 mouse.
The invention prepares the high-accuracy high-precision, the invention discloses an ultrasensitive multifunctional fluorescent probe which can be used for detecting glutathione mercaptotransferase.A benzo cyanine fluorophore is synthesized firstly, and 3,4 dinitrobenzoic acid is introduced to serve as a response group for detecting the glutathione mercaptotransferase to form a multifunctional ultrasensitive fluorescent probe molecule Cy-GST (3-ethyl-2- ((E) -2- ((E) -3- ((E) -2- (3-ethyl-1,1-dimethyl-1, 3-dihydro-2H-benzo [ E ] indole-2-ylidene) ethylidene) -2- (N-methyl-3,4-dinitrobenzamido) cyclohex-1-en-1-yl) vinyl) -1,1-dimethyl-1H-benzo [ E ] indole-3-ium). When the detection is carried out, after glutathione mercaptotransferase serving as a substance to be detected is added, Cy-GST serving as a probe molecule and glutathione perform addition reaction to release a fluorescent signal. The probe has excellent specificity, sensitivity and accuracy. In addition, the probe can be applied to the dynamic detection of glutathione mercaptotransferase in the cell environment. These characteristics all make the obtained molecular probe an important tool for exploring the action process of glutathione mercaptotransferase of a life system.
The detection mechanism of the multifunctional ultrasensitive glutathione mercaptotransferase detection fluorescent molecular probe prepared by the invention (as shown in figure 1) is as follows: the fluorescence of the probe Cy-GST is changed by utilizing the electron withdrawing capability of the nitro group, the nitro group on the probe is replaced by utilizing glutathione-thiol transferase, and the electron withdrawing group of the probe disappears, so that the fluorescence signal is enhanced and the sensitivity is improved.
Advantageous effects
1) The sensitivity is high and the detection speed is high: the probe is ultrasensitive to the response of the object to be detected, and the response multiplying power can reach 6 times in about 10 minutes;
2) under the condition of not adding any other additional material, the detection sensitivity is improved, the additional material is prevented from being added, the consumption of the additional material is reduced, and the error source in the detection is reduced;
3) imaging of biological samples is diverse: the present invention successfully detects the imaging of analytes in living cells and live mice in assays that were not possible in previous methods. The realization of the imaging of the object to be tested in living cells and living mice plays a great promoting role in the deep research of the glutathione mercaptotransferase which is a biomarker.
Drawings
Drawing (A)
The invention provides a synthetic route of the ultrasensitive glutathione mercaptotransferase detection fluorescent molecular probe prepared in the embodiment 1 of the invention;
FIG. 2 is an H-spectrum of a multifunctional ultrasensitive glutathione mercaptotransferase detection fluorescent molecular probe prepared in example 1 of the present invention;
FIG. 3 is a C spectrum of a multifunctional ultrasensitive glutathione mercaptotransferase detection fluorescent molecular probe prepared in example 1 of the present invention;
FIG. 4 is a linear equation for fluorescence quantification;
FIG. 5 is a diagram of a cell imaging experiment and a live body imaging;
FIG. 6 shows the fluorescence response of a multifunctional ultrasensitive glutathione mercaptotransferase detection fluorescent molecular probe (10 μ M) and glutathione mercaptotransferase (1 μ g/mL) within a pH range of 3-9;
FIG. 7 is a fluorescence response diagram of a multifunctional ultrasensitive glutathione mercaptotransferase detection fluorescent molecular probe (10 μ M) to gradient concentration of glutathione mercaptotransferase of a target object to be detected (concentration of glutathione mercaptotransferase: 0-1.1 μ g/mL);
FIG. 8 shows a control experiment (other substances) for detecting selectivity of a multifunctional ultrasensitive glutathione mercaptotransferase to be detected and other substances by using a fluorescent molecular probe.
Detailed Description
The technical solutions of the present invention will be further described with reference to specific examples, and various alternatives and modifications according to the general technical knowledge and the technical means commonly used in the art may be substituted or modified within the scope of the present invention while keeping the technical ideas described in the present invention.
The following examples of the invention:
the fluorescence detection is carried out by using a HORIBA Scientific Fluoromax-4 fluorescence spectrometer, the excitation wavelength is 730 nm, the emission wavelength is 810 nm, the widths of the excitation slit and the emission slit are both 10.0 nm, the voltage is 400V, and the scanning speed is 2400 nm/min;
fluorescence imaging observations were performed by Olympus, FV1100(Japan) fluorescence confocal microscope;
the separation and purification of the compound are realized by adopting a thin-layer chromatography silica gel column.
(1) Dissolving 75mmol of 2,3, 3-trimethyl-3H-benzindole and iodoethane in 40 mL of anhydrous acetonitrile, refluxing and reacting the mixture for 12 hours, cooling to room temperature, filtering, washing the filtered precipitate with diethyl ether, and drying in vacuum to obtain a product 1;
(2) 40 mL of anhydrous N, N-dimethylformamide and 40 mL of anhydrous dichloromethane are mixed, cooled to-10 ℃, stirred for 20 minutes, 37mL of phosphorus oxychloride and 35 mL of anhydrous dichloromethane are added dropwise to the solution at a constant speed, 52.6 mmol of cyclohexanone are added to the mixture until the solution turns from colorless to yellow, the solution is heated to 45 ℃ for 3 hours, then the reaction is poured into ice and left standing for 12 hours. Collecting the filtrate and vacuum drying to obtain a product 2;
(3) dissolving 2mmol of product 1 and 1mmol of product 2 in 100 mL of a mixed solution of n-butanol and benzene, refluxing for 3 hours, and then drying in vacuum to obtain a green crude product, and purifying the crude product through a silica gel column to obtain a product 3, wherein the volume ratio of n-butanol to benzene is 7: 3;
(4) dissolving 2mmol of the product 3, 20 mmol of methylamine hydrochloride and 20 mmol of triethylamine in 50 mL of anhydrous N, N-dimethylformamide, stirring the mixture at 40 ℃ for 24 hours, and purifying the crude product by using a 200-mesh 300-mesh silica gel column to obtain a product 4;
(5) dissolving 1mmol of 3, 4-dinitrobenzoic acid and 3 mmol of triphosgene in 100 mL of anhydrous dichloromethane, mixing the mixture at 0 ℃, adding 1mL of N, N-diisopropylethylamine, reacting for 30 minutes, removing the solvent in vacuum, dissolving the obtained residue in 50 mL of anhydrous dichloromethane, adding 1mL of N, N-diisopropylethylamine and 20 mg of 4-dimethylaminopyridine, dissolving 0.2mmol of product 4 in 10 mL of dichloromethane solution, adding the mixture, stirring the mixture at 25 ℃ until the raw materials are completely consumed, purifying the obtained solid crude product by a 200-mesh 300-mesh silica gel column, and obtaining the glutathione mercaptotransferase detection fluorescent molecular probe.
Example 1: preparation of multifunctional ultrasensitive glutathione mercaptotransferase detection probe
(1) 75mmol of 2,3, 3-trimethyl-3H-benzindole and 75mmol of iodoethane were dissolved in 40 mL of anhydrous acetonitrile, mixed in a 250 mL round-bottom flask, and the mixture was refluxed for 12 hours, and then the heating was stopped and cooled to room temperature. Filtering the precipitate through a buchner funnel, washing the solid product with diethyl ether and vacuum drying to obtain product 1;
(2) a solution of 40 mL of anhydrous N, N-Dimethylformamide (DMF) and 40 mL of anhydrous dichloromethane was placed in a 250 mL round bottom flask, the solution was cooled to-10 ℃ and stirred for 20 minutes. 37mL of phosphorus oxychloride was then added dropwise to the above solution through a constant pressure dropping funnel along with 35 mL of anhydrous dichloromethane. 52.6 mmol of cyclohexanone were added in portions to the mixture, and the solution turned from colorless to yellow. The solution was then slowly heated to 45 ℃ for 3 hours, then the reaction was poured into a large amount of ice and left to stand overnight. Collecting yellow solid through a Buchner funnel and drying in vacuum to obtain a product 2;
(3) 2mmol of product 1 and 1mmol of product 2 were dissolved in 100 mL of a mixed solution of n-butanol and benzene (7: 3, v/v) and refluxed in a 250 mL round-bottom flask for 3 hours. And then dried in vacuo to give a green crude product. Purifying the crude product by a silica gel column to obtain a product 3;
(4) 2mmol of product 3, 20 mmol of methylamine hydrochloride and 20 mmol of triethylamine are dissolved in 50 mL of anhydrous DMF. The mixture was stirred at 40 ℃ for 24 hours. Purifying the crude product by using a 200-mesh silica gel column to obtain a product 4;
(5) 1mmol of 3, 4-dinitrobenzoic acid, 3 mmol of triphosgene are dissolved in 100 mL of anhydrous dichloromethane. The mixture was mixed at 0 ℃ and then 1mL of N, N-diisopropylethylamine was added and the reaction was continued for 30 minutes. After removal of the solvent in vacuo, the resulting residue was dissolved in 50 mL of anhydrous dichloromethane and 1mL of N, N-diisopropylethylamine and 20 mg of 4-dimethylaminopyridine were added. 0.2mmol of product 4 are dissolved in 10 mL of dichloromethane and the mixture is added, the mixture is then stirred at 25 ℃ and the reaction is monitored by silica gel chromatography until complete consumption of starting material. Purifying the obtained solid crude product by a 300-mesh silica gel column to obtain the ultrasensitive glutathione mercaptotransferase detection fluorescent molecular probe.
The C spectrum and the H spectrum of the prepared multifunctional ultrasensitive glutathione mercaptotransferase detection fluorescent molecular probe are shown in the figure 2 and the figure 3, and the effect judgment indexes are as follows:
detection sensitivity: the detection limit is 10 ng/L;
detection response rate: the fluorescence is enhanced during detection, and the maximum fluorescence can reach 6 times;
detecting speed: about 10 minutes;
the optical mechanism index is as follows: a glutathione mercaptotransferase fluorescent probe of a PET mechanism.
The feasibility of the reaction between the probe prepared in example 1 and glutathione mercaptotransferase is verified: taking an ultrasensitive glutathione mercaptotransferase solution with the concentration of 10 mu M dissolved in DMSO to detect a fluorescent molecular probe solution, adding GSH with the concentration of 0.1 mM and glutathione mercaptotransferase with the concentration of 1 mu g/mL, and stirring at room temperature for 10 min to obtain a product.
Example 2 was carried out: example 1 preparation of multifunctional ultrasensitive glutathione mercaptotransferase detection fluorescent molecular probes for quantitative analysis of glutathione mercaptotransferase in standards
1) Preparing solution
Probe stock solution: accurately weighing a multifunctional ultrasensitive glutathione mercaptotransferase detection fluorescent molecular probe, dissolving the probe in anhydrous DMSO, and preparing a probe stock solution with the concentration of 100 mu M;
glutathione mercaptotransferase stock: accurately weighing a target object to be detected, namely 1 mg of glutathione mercaptotransferase, dissolving the target object in 10 mL of distilled water, and preparing a glutathione mercaptotransferase stock solution with the concentration of 100 mu g/mL;
2) establishing linear equation of glutathione mercaptotransferase standard substance
Diluting the glutathione mercaptotransferase stock solution prepared in the step 1) with distilled water to obtain a glutathione mercaptotransferase standard solution with the gradient concentration of 0-1.1 mu g/mL, then respectively taking the glutathione mercaptotransferase standard solutions with different concentrations, mixing the glutathione mercaptotransferase standard solution with 10 mu M of the probe solution prepared in the step 1) and 0.1 mM of GSH solution, adding 50 mu L of Tris-hydrochloric acid buffer solution with the concentration of 10 mM and the pH value of 7.42, fully oscillating to uniformly mix the system, standing for 50 min at 25 ℃, and then detecting by a fluorescence spectrophotometer to establish a linear equation (figure 4) of the concentration of the glutathione mercaptotransferase and the intensity of a fluorescence signal.
Example 3 of implementation: qualitative detection of glutathione mercaptotransferase in biological samples
The method for detecting glutathione mercaptotransferase in living cells comprises the following steps: culturing IMR-90 cells in a culture medium for 18-26 h, wherein the inoculation amount of the living cells to be detected is 2 multiplied by 107～9×107Adding multifunctional ultrasensitive glutathione mercaptotransferase to detect fluorescent molecular probe with the probe concentration of 1 mu M, incubating at 37 ℃ for 30 minutes, washing with PBS solution for multiple times, and observing fine particles under a fluorescence microscopeAnd (6) imaging the cells. The cell imaging results were: the fluorescence is enhanced, so that the IMR-90 cells contain glutathione mercaptotransferase.
The method for detecting glutathione mercaptotransferase in vivo of the living mouse comprises the following steps: c57BL/6 mice (8 weeks) were housed in a 12-hour light/dark cycle with free access to food and water. 5 mg/kg of bleomycin dissolved in saline is used for pulmonary fibrosis animal molding, single intratracheal perfusion is carried out under anesthesia, and molding is finished after 28 days. A multifunctional ultrasensitive glutathione mercaptotransferase (100. mu.M 50. mu.L 1: 99 DMSO/normal saline, v/v) was subjected to intratracheal detection with a pulmonary fibrosis mouse model for 30 minutes at 37 ℃. Then observed and imaged in a Perkinelmer IVIS Lumina XRMS III series living body imager, and the imaging result is as follows: fluorescence is enhanced, so that glutathione mercaptotransferase can be judged to be contained in the pulmonary fibrosis mouse model.
FIGS. 5a and 5b are images of cells with normal and increased concentrations of glutathione mercaptotransferase, respectively. Fig. 5C and 5d are in vivo images of normal C57BL/6 mice and pulmonary fibrosis model mice, respectively.
The invention relates to experimental verification of various technical indexes of a multifunctional ultrasensitive glutathione mercaptotransferase detection fluorescent molecular probe, which comprises the following steps:
experiment condition optimization test of technical scheme of the invention
1. Optimization of reaction system pH value
In general, the pH value affects the fluorescence property of the organic molecular probe, so that the pH value is generally adjusted by using a buffer solution in the reaction so as to adapt to the requirements of the experiment. Aiming at the characteristics of glutathione mercaptotransferase to be detected, the pH value (3-9) which can be reached in a physiological environment is researched. As can be seen from FIG. 6, the fluctuation of pH value in the physiological environment range had little influence on the fluorescence intensity response exhibited by the mixed solution of the target glutathione mercaptotransferase and the probe. Therefore, in a typical biological environment system, the reaction of the probe with glutathione mercaptotransferase is optimized at a pH of 7.4.
2. Optical property and mechanism verification
The fluorescence response of the probe to the gradient concentration of the target substance glutathione mercaptotransferase to be detected is 0-1.1 mu g/mL as shown in figure 7. As can be seen from fig. 7:
3. selective analysis for detecting glutathione mercaptotransferase by probe molecule
The preparation method of the stock solution comprises the following steps: the results are shown in FIG. 8, in which horseradish peroxidase, nitroreductase, glutamyltranspeptidase, alkaline phosphatase, galactosidase and glutathione mercaptotransferase were dissolved in distilled water to obtain stock solutions of the above ions (the above solutions correspond to the reference numerals 1 to 6 in FIG. 8 in this order).
Claims (3)
1. A preparation method of a glutathione sulfydryl transferase fluorescent molecular probe is characterized by comprising the following steps: the method comprises the following steps:
(1) dissolving 2,3, 3-trimethyl-3H-benzindole and iodoethane in anhydrous acetonitrile to obtain a product 1;
(2) mixing anhydrous N, N-dimethylformamide and anhydrous dichloromethane, dropwise adding phosphorus oxychloride and anhydrous dichloromethane into the solution at a constant speed, adding cyclohexanone into the mixture, heating to 45 ℃ for 3 hours, pouring the reactant into ice, standing, filtering and collecting the filtrate to obtain a product 2;
(3) dissolving the product 1 and the product 2 in a mixed solution of n-butyl alcohol and benzene, reacting and purifying to obtain a product 3;
(4) dissolving the product 3, methylamine hydrochloride and triethylamine in anhydrous N, N-dimethylformamide, heating and stirring, and purifying a reaction crude product to obtain a product 4;
(5) dissolving 3, 4-dinitrobenzoic acid and triphosgene in anhydrous dichloromethane, mixing, then adding N, N-diisopropylethylamine, reacting, removing a solvent, dissolving the obtained residue in anhydrous dichloromethane, adding N, N-diisopropylethylamine and 4-dimethylaminopyridine, dissolving a product 4 in a dichloromethane solution, adding the mixture, stirring until the raw materials are exhausted, and purifying the obtained solid crude product to obtain the glutathione mercaptotransferase detection fluorescent molecular probe;
the specific reaction process is shown as formula I:
formula I.
2. The preparation method according to claim 1, characterized by comprising the following steps:
(1) dissolving 75mmol of 2,3, 3-trimethyl-3H-benzindole and iodoethane in 40 mL of anhydrous acetonitrile, refluxing the mixture for 12 hours, cooling to room temperature, filtering, washing the filtered precipitate with diethyl ether, and drying in vacuum to obtain a product 1;
(2) mixing 40 mL of anhydrous N, N-dimethylformamide and 40 mL of anhydrous dichloromethane, cooling to-10 ℃, stirring for 20 minutes, then dropwise adding 37mL of phosphorus oxychloride and 35 mL of anhydrous dichloromethane together at a constant speed into the solution, then adding 52.6 mmol of cyclohexanone into the mixture until the solution turns from colorless to yellow, then heating the solution to 45 ℃ and keeping for 3 hours, then pouring the reactant into ice, standing for 12 hours, collecting the filtrate and drying in vacuum to obtain a product 2;
(3) dissolving 2mmol of product 1 and 1mmol of product 2 in 100 mL of a mixed solution of n-butanol and benzene, refluxing for 3 hours, and then drying in vacuum to obtain a green crude product, and purifying the crude product through a silica gel column to obtain a product 3, wherein the volume ratio of n-butanol to benzene is 7: 3;
(4) dissolving 2mmol of the product 3, 20 mmol of methylamine hydrochloride and 20 mmol of triethylamine in 50 mL of anhydrous N, N-dimethylformamide, stirring the mixture at 40 ℃ for 24 hours, and purifying the crude product by using a 200-mesh 300-mesh silica gel column to obtain a product 4;
(5) dissolving 1mmol of 3, 4-dinitrobenzoic acid and 3 mmol of triphosgene in 100 mL of anhydrous dichloromethane, mixing the mixture at 0 ℃, adding 1mL of N, N-diisopropylethylamine, reacting for 30 minutes, removing the solvent in vacuum, dissolving the obtained residue in 50 mL of anhydrous dichloromethane, adding 1mL of N, N-diisopropylethylamine and 20 mg of 4-dimethylaminopyridine, dissolving 0.2mmol of product 4 in 10 mL of dichloromethane solution, adding the mixture, stirring the mixture at 25 ℃ until the raw materials are completely consumed, purifying the obtained solid crude product by a 200-mesh 300-mesh silica gel column, and obtaining the glutathione mercaptotransferase detection fluorescent molecular probe.
3. The application of the glutathione mercaptotransferase detection fluorescent molecular probe prepared by the preparation method of claim 1 or 2 in non-disease diagnosis purposes is characterized in that: is suitable for qualitative analysis of glutathione mercaptotransferase.
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