CN114957380A - Water-soluble fluorescent probe for determining glutamyl transpeptidase, and synthetic method and application thereof - Google Patents
Water-soluble fluorescent probe for determining glutamyl transpeptidase, and synthetic method and application thereof Download PDFInfo
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Abstract
The invention discloses a water-soluble fluorescent probe for determining glutamyl transpeptidase, and a synthesis method and application thereof, wherein the fluorescent probe takes 2-dicyanomethylene-3-cyano-4, 5, 5-trimethyl-2, 5-dihydrofuran (TCF) as a fluorescent signal report group, modifies a fluorescent group (TCF-OH) with an acrylate linking group, and introduces a short peptide GSH unit into an enzyme cutting site as GGT through nucleophilic addition of sulfydryl and an alpha, beta-unsaturated carbonyl compound. The probe TCF-GGT has good water solubility, glutaminebond hydrolysis is catalyzed under the action of gamma-glutamyltranspeptidase, so that an intramolecular elimination process is initiated, TCF-OH with red fluorescence is formed, and the water-soluble fluorescent probe is developed according to the property of red fluorescence increase of the fluorescent probe. The invention can not only visually detect the target glutamyltranspeptidase by naked eyes, but also quantitatively detect the target glutamyltranspeptidase by a fluorescence method, and has the advantages of good selectivity, high detection sensitivity and the like.
Description
Technical Field
The invention belongs to the technical field of enzyme activity detection, and relates to a method strategy for detecting cell endogenous gamma-glutamyl transpeptidase. More particularly, it relates to a fluorescent probe and its synthesis method and application in detecting glutamyl transpeptidase.
Background
Gamma-glutamyl transpeptidase (GGT) is an enzyme that is widely present on the surface of mammalian cells and catalyzes the hydrolysis of glutamine bonds in Glutathione (GSH) or other gamma-glutamyl compounds. GGT activity is significantly elevated in many patients with diseases such as liver cancer, ovarian cancer, colorectal cancer, and the like. Therefore, the activity of GGT is generally considered as one of important indicators for medical diagnosis. Therefore, the development of a probe for specifically and sensitively detecting γ -glutamyltranspeptidase activity has important applications in clinical diagnosis.
At present, the detection of gamma-glutamyl transpeptidase mainly depends on means such as chromatography, electrochemiluminescence, electrochemistry, surface enhanced resonance Raman scattering, colorimetry and fluorescence, and most of the methods need complicated instruments, thereby limiting the practical application of the methods. The fluorescence analysis method has the advantages of high sensitivity, low invasiveness, controllable biocompatibility and the like, is more suitable for cell/living body analysis compared with methods such as immunohistochemistry and microelectrodes, has higher sensitivity and selectivity in a spectral analysis means, has lower requirements on thresholds of instruments and equipment, has wider commercial value, and is a gamma-glutamyltranspeptidase analysis method with application value at present.
Therefore, the problem to be solved by those skilled in the art is how to provide a fluorescent probe with good water solubility, high sensitivity and high selectivity.
Disclosure of Invention
In view of the above, the present invention provides a water-soluble fluorescent probe for determining γ -glutamyl transpeptidase, which is directed to the problems of the prior art.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a water-soluble fluorescent probe for measuring gamma-glutamyl transpeptidase, the structural formula of the fluorescent probe is as follows:
the fluorescent probe takes 2-dicyanomethylene-3-cyano-4, 5, 5-trimethyl-2, 5-dihydrofuran (TCF) as a fluorescent reporter group, can visually identify a target gamma-glutamyl transpeptidase by naked eyes, and can also enter living cells to measure the cell endogenous gamma-glutamyl transpeptidase.
Another purpose of the invention is to provide a synthetic method of the water-soluble fluorescent probe for measuring gamma-glutamyl transpeptidase.
In order to achieve the purpose, the invention adopts the following technical scheme:
a synthetic method of a water-soluble fluorescent probe for determining gamma-glutamyl transpeptidase specifically comprises the following steps:
(1) using absolute ethyl alcohol as a reaction medium, using malononitrile and 3-hydroxy-3-methyl-2-butanone as reactants, using sodium ethoxide as a reaction catalyst, reacting at normal temperature for 1 hour, performing reflux reaction for 1 hour, performing suction filtration, and washing with cold diethyl ether for three times to obtain an off-white solid 2-dicyanomethylene-3-cyano-4, 5, 5-trimethyl-2, 5-dihydrofuran (TCF);
(2) adding p-hydroxybenzaldehyde into the 2-dicyanomethylene-3-cyano-4, 5, 5-trimethyl-2, 5-dihydrofuran obtained in the step (1) by taking absolute ethyl alcohol as a reaction solvent, taking piperazine as a reaction alkali, reacting for 12 hours at normal temperature, separating by 200-300-mesh silica gel column chromatography after the reaction is finished, and taking dichloromethane/methanol as an eluent to obtain orange red solid TCF-OH;
(3) adding dichloromethane serving as a solvent into TCF-OH, adding triethylamine to adjust an alkaline environment, adding acryloyl chloride to react at room temperature for 12 hours, performing water extraction, performing column chromatography separation on 200-300-mesh silica gel, and eluting with dichloromethane to obtain a yellow solid TCF-A;
(4) dissolving glutathione in PBS (phosphate buffer solution) with the pH value of 7.4, adding TCF-A dissolved in acetonitrile, reacting at normal temperature for 24 hours, carrying out reduced pressure distillation, washing residual solids with dichloromethane and methanol, and finally carrying out column chromatography separation on silica gel with 200-300 meshes by taking dichloromethane/methanol as an eluent to obtain yellow solid TCF-GGT, namely the water-soluble fluorescent probe.
The synthetic route of the water-soluble fluorescent probe is as follows:
by adopting the technical scheme, the invention has the following beneficial effects:
the invention uses the synthesized fluorescent probe for identifying and detecting gamma-glutamyltranspeptidase, because the structure of the fluorescent probe contains the glutamine bond of the identifying site of the gamma-glutamyltranspeptidase (2-dicyanomethylene-3-cyano-4, 5, 5-trimethyl-2, 5-dihydrofuran (TCF) is taken as a fluorescent signal reporting group, an acrylate linking group is used for modifying a fluorescent group (TCF-OH), a short peptide GSH unit is introduced into the enzyme cutting site which is taken as GGT through the nucleophilic addition of sulfydryl and alpha, beta-unsaturated carbonyl compound), the fluorescent probe has excellent water solubility, is yellow in aqueous solution, catalyzes glutamine bond hydrolysis in the presence of gamma-glutamyltranspeptidase, further triggering the intramolecular elimination process to form a product with red fluorescence, so that the target gamma-glutamyl transpeptidase can be visually identified by naked eyes.
Preferably, the molar ratio of the sodium ethoxide, the 3-hydroxy-3-methyl-2-butanone and the malononitrile in the step (1) is 1: (6-7): (12-14).
Preferably, the molar ratio of TCF, p-hydroxybenzaldehyde and piperazine in the step (2) is 1: 1: (2.5-3.5).
Preferably, the molar ratio of TCF-OH, acryloyl chloride and triethylamine in the step (3) is 1: (1.2-2.5): (3-5).
Preferably, the molar ratio of TCF-A to glutathione in the step (4) is 1: (1.5-2.5).
Aiming at the reaction of synthesizing the fluorescent probe, the inventor obtains various raw material ratios through creative tests, wherein the ratio of glutathione to triethylamine is particularly important, and the triethylamine influences the acid-base regulation of the reaction and is related to whether the reaction can be smoothly carried out; the content of glutathione directly influences the degree of reaction progress, and is related to the steps of complete reaction and excessive treatment.
In addition, the inventors respectively perform characterization through methods such as nuclear magnetic resonance hydrogen spectroscopy, mass spectrometry and the like, and show that the fluorescent probe has been successfully synthesized.
Preferably, the optimal reaction concentration for synthesizing the fluorescent probe is 10. mu. mol. L -1 。
In the detection process, if the concentration of the fluorescent probe used is less than 10. mu. mol. L -1 The probe itself emits light and its response signal is weak, which may impair the detection sensitivity to some extent.
Preferably, the optimal conditions for reacting gamma-glutamyl transpeptidase with ratiometric fluorescent probes are co-incubation at 37 ℃ for 60 minutes in PBS at pH 7.4.
The fluorescent probe can efficiently and selectively recognize gamma-glutamyltranspeptidase in PBS and has high sensitivity to gamma-glutamyltranspeptidase.
The invention also aims to provide a specific application of the water-soluble fluorescent probe in detecting gamma-glutamyl transpeptidase.
Preferably, the gamma-glutamyltranspeptidase is over-expressed in human liver cancer cells, and the synthesized water-soluble fluorescent probe can enter living cells to detect the cell endogenous gamma-glutamyltranspeptidase.
According to the technical scheme, compared with the prior art, the invention provides the water-soluble fluorescent probe for measuring glutamyl transpeptidase, and the synthesis method and the application thereof, and the probe has the following excellent effects:
the fluorescent probe disclosed by the invention contains an identification group of gamma-glutamyltranspeptidase, is yellow in aqueous solution, and after the gamma-glutamyltranspeptidase is added, the probe catalyzes glutamine bond hydrolysis under the action of transglutaminase to further initiate an intramolecular elimination process, so that the formed compound has red fluorescence, and the aim of detecting the gamma-glutamyltranspeptidase is fulfilled by enhancing the red fluorescence intensity. The method strategy for detecting the gamma-glutamyltranspeptidase disclosed by the invention has high market application and popularization values.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a drawing showing the preparation of intermediate TCF-A of the present invention 1 H NMR spectrum.
FIG. 2 is a diagram showing a fluorescent probe TCF-GGT according to the present invention 1 H NMR spectrum.
FIG. 3 is a diagram showing UV absorption (a) and fluorescence emission (b) spectra of a fluorescent probe of the present invention before and after reaction with gamma-glutamyl transpeptidase.
FIG. 4 is a graph showing the fluorescence intensity at 605nm and GGT activity after incubation of the fluorescent probe of the present invention with gamma-glutamyl transpeptidase of various concentrations.
FIG. 5 is a graph (a) showing fluorescence imaging of the inventive fluorescent probe on endogenous γ -glutamyl transpeptidase in DON-pretreated human hepatoma cells, a graph (b) showing fluorescence imaging of the inventive fluorescent probe on endogenous γ -glutamyl transpeptidase in human hepatoma cells, and a graph (c) showing intracellular fluorescence intensity histograms in graphs (a) and (b).
FIG. 6 is a graph (a) showing fluorescence intensity images of tumor-bearing mice without probe injection, (b-f) showing fluorescence intensity images of the fluorescent probe of the present invention injected into the tumor-bearing mice at different times (10-300min), and (g) showing fluorescence intensity values of tumor sites at different times.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention discloses a water-soluble fluorescent probe for measuring gamma-glutamyl transpeptidase with high sensitivity and high selectivity, a synthetic method and application thereof.
The present invention will be further specifically illustrated by the following examples for better understanding, but the present invention should not be construed as being limited thereto, and the insubstantial modifications and variations of the present invention as shown in the above-described summary of the invention are considered to fall within the scope of the invention by those skilled in the art.
The invention discloses a water-soluble fluorescent probe for measuring gamma-glutamyl transpeptidase, the structural formula of the water-soluble fluorescent probe is as follows:
the technical solution of the present invention will be further described with reference to the following specific examples.
Example 1
Synthesis of fluorescent probes
1. The synthesis steps are as follows:
(1) NaOEt (0.884g, 13mmol) was dissolved in 10mL of anhydrous ethanol, 3-hydroxy-3-methyl-2-butanone (9.000g, 88mmol) and malononitrile (12.000g, 181mmol) were added, and after stirring at room temperature for one hour, 30mL of anhydrous ethanol was added, and the mixture was heated to 85 ℃ and refluxed for one hour. After the reaction is finished, filtering, washing and drying by cold ether to obtain off-white TCF powder, wherein the reaction process is shown as formula (1);
(2) dissolving the TCF (0.199g, 1mmol) obtained in the step (1), p-hydroxybenzaldehyde (0.122g, 1mmol) and piperazine (0.258mg, 3mmol) in 10mL of absolute ethyl alcohol, stirring at normal temperature for 12h, after the reaction is finished, performing column chromatography separation on 200-300-mesh silica gel, and taking dichloromethane/methanol as an eluent to obtain orange-red solid TCF-OH, wherein the reaction process is shown as a formula (2);
(3) TCF-OH (0.135g,0.445mmol) and triethylamine (0.25mL,1.8mmol) were dissolved in 10mL of dry dichloromethane, and a solution of acryloyl chloride (54.3mg,0.60mmol) in dichloromethane (5mL) was added dropwise over an ice bath and stirred at room temperature overnight. Then, 30mL of dichloromethane was added to the reaction solution, washed 3 times with deionized water (3X 20mL), washed 1 time with saturated NaCl solution, and the organic phase was washed with anhydrous Na 2 SO 4 After drying, carrying out reduced pressure distillation to remove the solvent, then carrying out column chromatography separation on silica gel with 200-300 meshes, eluting with dichloromethane to obtain a yellow solid TCF-A, wherein the reaction process is shown as a formula (3);
(4) glutathione (0.086g,0.28mmol) was dissolved in 4mL of PBS buffer (10mM, pH 7.4) under N 2 A solution of TCF-A (0.083g,0.23mmol) in acetonitrile (4mL) was added dropwise with protection and the reaction stirred at room temperature for 24 h. The solvent was evaporated under reduced pressure and the residue was washed with dichloromethane and methanolAnd (3) finally, carrying out column chromatography separation on the solid through 200-300-mesh silica gel, and taking dichloromethane/methanol as an eluent to obtain a yellow solid TCF-GGT, wherein the reaction process is shown as a formula (4).
Example 2
Synthesis of fluorescent probes
1. Synthesis procedure
(1) NaOEt (0.68g, 10mmol) was dissolved in 10mL of anhydrous ethanol, 3-hydroxy-3-methyl-2-butanone (6.12g, 60mmol) and malononitrile (8.0g, 121mmol) were added, and after stirring at room temperature for one hour, 30mL of anhydrous ethanol was added, and the mixture was heated to 85 ℃ and refluxed for one hour. After the reaction is finished, filtering, washing and drying by cold ether to obtain off-white TCF powder, wherein the reaction process is shown as formula (1);
(2) dissolving the TCF (0.199g, 1mmol) obtained in the step (1), p-hydroxybenzaldehyde (0.122g, 1mmol) and piperazine (0.215mg, 2.5mmol) in 10mL of absolute ethyl alcohol, stirring at normal temperature for 12h, after the reaction is finished, performing column chromatography separation on 200-300-mesh silica gel, and taking dichloromethane/methanol as an eluent to obtain orange-red solid TCF-OH, wherein the reaction process is shown in a formula (2);
(3) TCF-OH (0.151g,0.5mmol) and triethylamine (0.277mL,2.0mmol) were dissolved in 10mL of dry dichloromethane, and a solution of acryloyl chloride (90.5mg,1.0mmol) in dichloromethane (5mL) was added dropwise over ice and stirred at room temperature overnight. Then, 30mL of dichloromethane was added to the reaction solution, washed 3 times with deionized water (3X 20mL), washed 1 time with saturated NaCl solution, and the organic phase was washed with anhydrous Na 2 SO 4 Drying, distilling under reduced pressure to remove the solvent, and performing 200-300 mesh silica gel column chromatographySeparating, eluting with dichloromethane to obtain yellow solid TCF-A, wherein the reaction process is shown as formula (3);
(4) glutathione (0.1842g,0.6mmol) was dissolved in 4mL of PBS buffer (10mM, pH 7.4) under N 2 A solution of TCF-A (0.1071g,0.3mmol) in acetonitrile (4mL) was added dropwise with protection and the reaction stirred at room temperature for 24 h. And (3) evaporating the solvent under reduced pressure, washing the residual solid with dichloromethane and methanol, and finally separating by using 200-300-mesh silica gel column chromatography, wherein dichloromethane/methanol is used as an eluent to obtain a yellow solid TCF-GGT, and the reaction process is shown as a formula (4).
The following further verifies the excellent effects of the technical scheme of the invention compared with the prior art, and the specific contents are as follows:
And (3) testing and analyzing:
fig. 1 is an HRMS spectrum of a probe, with specific spectral peaks: 1 H NMR(500MHz,DMSO-d 6 ) δ (ppm)8.00(d, J ═ 8.7Hz,2H),7.94(d, J ═ 16.5Hz,1H),7.36(d, J ═ 8.6Hz,2H),7.23(d, J ═ 16.5Hz,1H),6.58(dd, J ═ 17.2,1.1Hz,1H),6.43(dd, J ═ 17.2,10.3Hz,1H),6.20(dd, J ═ 10.3,1.1Hz,1H),1.81(s,6H), which correspond to the probe group, can prove the success of probe synthesis.
FIG. 2 shows a probe 1 H NMR spectrum, and obtaining mass spectrum data of ESI-MS [ M + H ]] + C 31 H 33 N 6 O 9 S + 665.2039 theoretical values of m/z: 665.2024. further verifies that the probe is successfully synthesized.
To sum up from 1 HNMR, HRMS demonstrated the chemical structure of the probe.
Experiment 2
Test of the ability of fluorescent probes to react with gamma-glutamyltranspeptidase in buffer solution
The synthesized fluorescent probe was adjusted to 10.0 mmol. multidot.L -1 10 μ L of the aqueous solution of (4) was added to a solution containing 1mL of PBS (10 mmol. multidot.L) -1 pH 7.4) into a centrifuge tube, 10 μ L of γ -glutamyl transpeptidase (10U/mL) was added -1 ) The final concentration was adjusted to 0.1 U.mL -1 And after 60 minutes, changes of the ultraviolet absorption spectrum and the fluorescence emission spectrum are detected.
FIG. 3 shows UV absorption (a) and fluorescence emission (b) spectra of a fluorescent probe before and after reaction with gamma-glutamyl transpeptidase. Wherein A represents a fluorescent probe (10. mu. mol. L) -1 ) B represents a fluorescent probe (10. mu. mol. L) -1 ) And gamma-glutamyl transpeptidase (0.1 U.mL) -1 ) The mixed solution of (2).
As can be seen from FIG. 3(a), a new absorption peak appeared at 575nm in the solution after the reaction. Meanwhile, it was found from FIG. 3(b) that a new emission peak occurred at 605nm due to the addition of γ -glutamyl transpeptidase.
Thus, the gamma-glutamyl transpeptidase can react with the fluorescent probe to form a compound with red fluorescence through intramolecular cyclization.
Experiment 2
Determination of minimum detection limit of gamma-glutamyl transpeptidase by fluorescent probe
Titration of the fluorescent probes with gamma-glutamyl transpeptidase at 37 deg.C by UV absorption and fluorescence emission spectroscopy over 3.3s B The minimum detection limit of the fluorescent probe to the gamma-glutamyl transpeptidase is up to 0.044 mU.mL by the calculation of/S -1 The synthetic fluorescent probe has high detection sensitivity to the gamma-glutamyl transpeptidase, and the probe has potential application value in the aspect of high-efficiency detection of the gamma-glutamyl transpeptidase in aqueous solution. And the fluorescence intensity of the fluorescent probe at 605nm and the GGT concentration at 0-50 mU.mL are shown in FIG. 4 -1 Linear dependence within range (linear correlation coefficient R) 2 ＝0.996)。
Experiment 3
Detection of human liver cancer cell endogenous gamma-glutamyl transpeptidase by fluorescent probe
The invention is used for the fluorescence imaging application of endogenous gamma-glutamyl transpeptidase in Hep G2 cells,the method comprises the following specific steps: fluorescent probe (30. mu. mol. L) -1 ) The cells were added to a culture medium containing Hep G2 cells, incubated in a carbon dioxide incubator for 60 minutes, and then imaged by a confocal microscope. As a control, firstly, diazo oxo-norleucine (DON) inhibitor was added to Hep G2 cells to inhibit the activity of intracellular gamma-glutamyl transpeptidase, and after incubation for 30 minutes, fluorescent probe (30. mu. mol. L) was added -1 ) The culture was incubated for 60 minutes, and fluorescence imaging was observed with a confocal microscope.
FIG. 5 is a photograph of a fluorescent image of a fluorescent probe against a cell's endogenous gamma-glutamyl transpeptidase. Wherein FIG. 5(b) shows the probe concentration of 30. mu. mol. L -1 And adding into Hep G2 cells, and culturing for 30 minutes. FIG. 5(a) shows the addition of diazo-oxo-norleucine (DON) inhibitor (100. mu. mol. L) -1 ) Culturing in Hep G2 cell for 30 min, and adding 5. mu. mol. L -1 Fluorescence imaging after incubation of the probe in the cells for 30 minutes.
As can be seen from FIG. 5, when the γ -glutamyl transpeptidase in Hep G2 cells was inhibited by inhibitor DON, the probe showed almost no fluorescence. In the absence of inhibitor, the probe reacts specifically with endogenous gamma-glutamyl transpeptidase in the cell to produce red fluorescence. The result shows that the fluorescent probe can be used for detecting the cell endogenous gamma-glutamyl transpeptidase.
Experiment 3
Detection of animal endogenous gamma-glutamyl transpeptidase by fluorescent probe
The invention is used for the fluorescence imaging application of the tumor-bearing mouse endogenous gamma-glutamyl transpeptidase, and the specific steps are as follows: fluorescent probes (50. mu.L, 7.5mM) were injected into the tumor sites in tumor-bearing mice, fluorescence imaging pictures were taken after 10,30,90,180 and 300min, respectively, and fluorescence intensity analysis was quantified for different incubation times.
FIG. 6 is a graph of fluorescence imaging of the fluorescent probe on the endogenous gamma-glutamyl transpeptidase in tumor-bearing mice. Wherein FIG. 6(a) is the fluorescence image of tumor-bearing mice without probe injection, (b-f) is the fluorescence intensity image of fluorescence probe pair injected into tumor-bearing mice at different times (10,30,90,180 and 300min), (g) is the fluorescence intensity value of tumor sites at different times.
As can be seen from FIG. 6, the fluorescence intensity at 605nm gradually increased when the fluorescent probe was injected into the tumor-bearing mice, and decreased after 90min, to about 40% at 300 min. The experimental result shows that the fluorescent probe can be used for detecting the activity of the endogenous gamma-glutamyltranspeptidase of the tumor-bearing mouse, and the probe can be metabolized and decomposed in the mouse after a long time (more than 300 min).
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. A water-soluble fluorescent probe for measuring gamma-glutamyl transpeptidase is characterized in that the structural formula of the water-soluble fluorescent probe is as follows:
2. the method for synthesizing a water-soluble fluorescent probe for measuring γ -glutamyl transpeptidase according to claim 1, comprising the steps of:
(1) using absolute ethyl alcohol as a reaction medium, using malononitrile and 3-hydroxy-3-methyl-2-butanone as reactants, using sodium ethoxide as reaction alkali, stirring at normal temperature for 1 hour, performing reflux reaction for 1 hour, performing vacuum filtration, and washing with cold diethyl ether for three times to obtain an off-white solid 2-dicyanomethylene-3-cyano-4, 5, 5-trimethyl-2, 5-dihydrofuran (TCF);
(2) adding p-hydroxybenzaldehyde into the 2-dicyanomethylene-3-cyano-4, 5, 5-trimethyl-2, 5-dihydrofuran (TCF) obtained in the step (1) by taking absolute ethyl alcohol as a reaction solvent, taking piperazine as a reaction catalyst, stirring at normal temperature for reaction for 12 hours, and after the reaction is finished, carrying out column chromatography separation on 200-300-mesh silica gel, taking dichloromethane/methanol as an eluent, so as to obtain orange red solid TCF-OH;
(3) dissolving TCF-OH in dichloromethane, adding triethylamine to adjust an alkaline environment, adding acryloyl chloride to react at room temperature for 12 hours, extracting with water, separating by 200-300 mesh silica gel column chromatography, and eluting with dichloromethane to obtain a yellow solid TCF-A;
(4) dissolving glutathione in PBS (phosphate buffer solution) with the pH value of 7.4, adding an acetonitrile solution of TCF-A, reacting at normal temperature for 24 hours, carrying out reduced pressure distillation, washing residual solids with dichloromethane and methanol, and finally carrying out column chromatography separation on silica gel with 200-300 meshes by taking dichloromethane/methanol as an eluent to obtain a yellow solid TCF-GGT, namely the water-soluble fluorescent probe.
3. The method for synthesizing the water-soluble fluorescent probe for measuring gamma-glutamyl transpeptidase according to claim 2, characterized in that in step (1), the molar ratio of sodium ethoxide, 3-hydroxy-3 methyl-2-butanone and malononitrile is 1: (6-7): (12-14).
4. The method for synthesizing a water-soluble fluorescent probe for the determination of γ -glutamyl transpeptidase according to claim 2, wherein in step (2), the molar ratio of TCF, p-hydroxybenzaldehyde and piperazine is 1: 1: (2.5-3.5).
5. The method for synthesizing a water-soluble fluorescent probe for the determination of γ -glutamyl transpeptidase according to claim 2, wherein in step (3), the molar ratio of TCF-OH, acryloyl chloride and triethylamine is 1: (1.5-2.5): (3-5).
6. The method for synthesizing a water-soluble fluorescent probe for the determination of γ -glutamyl transpeptidase according to claim 2, wherein in step (4), the molar ratio of TCF-A to glutathione is 1: (1.2-2.5).
7. Use of the water-soluble fluorescent probe according to claim 1 or the water-soluble fluorescent probe synthesized by the method according to claim 2 for detecting γ -glutamyl transpeptidase.
8. The use of a water-soluble fluorescent probe for detecting γ -glutamyl transpeptidase according to claim 7, wherein said glutamyl transpeptidase is overexpressed in human hepatoma cells.
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