Two-photon fluorescent probe of gamma-glutamyl transpeptidase, and preparation method and application thereof
阅读说明：本技术 一种γ-谷氨酰转肽酶的双光子荧光探针及其制备方法和应用 (Two-photon fluorescent probe of gamma-glutamyl transpeptidase, and preparation method and application thereof ) 是由 霍瑞锦 刘卫敏 吴加胜 汪鹏飞 于 2020-04-27 设计创作，主要内容包括：本发明公开了一种γ-谷氨酰转肽酶的双光子荧光探针,其特征在于,具有通式(IA)或(IB)所示的结构,是将对γ-谷氨酰转肽酶GGT特异性反应的靶向基团GSH修饰到简单荧光分子上得到的。该荧光探针在识别GGT的过程中,会形成六元环状化合物,其具有的刚性结构使探针具有双光子效应,与癌细胞中过表达的GGT作用时,光学信号发生显著变化,从而达到了靶向检测区分癌细胞和正常细胞的目的。同时,该双光子荧光具有水溶性好、灵敏度高、反应快速、且具有双光子激发的优势,具有较好的应用前景。(The invention discloses a two-photon fluorescent probe of gamma-glutamyl transpeptidase, which is characterized in that the two-photon fluorescent probe has a structure shown in a general formula (IA) or (IB), and is obtained by modifying a targeting group GSH which reacts specifically with gamma-glutamyl transpeptidase GGT onto a simple fluorescent molecule. The fluorescent probe can form a six-membered cyclic compound in the process of identifying GGT, the rigid structure of the six-membered cyclic compound enables the probe to have a two-photon effect, and when the six-membered cyclic compound acts with the over-expressed GGT in cancer cells, optical signals are obviously changed, so that the aim of distinguishing the cancer cells from normal cells through targeted detection is fulfilled. Meanwhile, the two-photon fluorescence has the advantages of good water solubility, high sensitivity, quick reaction, two-photon excitation and good application prospect.)
1. A two-photon fluorescent probe for gamma-glutamyl transpeptidase, characterized by having a structure represented by general formula (IA) or (IB):
in the formulae (IA) and (IB), the substituent R1-R5independently-H, -F, -Cl, -Br, -I, cyano, nitro and sulfonic group; x is hydroxyl or glycine.
2. A method for synthesizing the two-photon fluorescent probe according to claim 1, which comprises the following steps: dissolving the compound (IIA) and glutathione in an organic solvent according to a certain proportion, stirring for reaction for 0.5 hour at room temperature, adding triethylamine, and continuously stirring for reaction at room temperature to obtain a mono-substituted product (IA) and a di-substituted product (IB);
wherein, the substituent R1-R5independently-H, -F, -Cl, -Br, -I, cyano, nitro and sulfonic group; x is hydroxyl or glycine.
3. The method according to claim 2, wherein the molar ratio of the compound (IIA) to glutathione in the organic solvent is 1: 1-4.
4. The method according to claim 2, wherein the concentration of the compound (IIA) in the organic solvent is 1-3mM, and the concentration of glutathione is 1-12 mM.
5. The method according to claim 2, wherein the concentration of triethylamine is 1 to 12 mM.
6. The method according to claim 2, wherein the time for the reaction to continue stirring at room temperature after the triethylamine solution is added is 1 to 8 hours.
7. The method according to claim 2, wherein the organic solvent is one or more selected from the group consisting of dimethylsulfoxide, N-dimethylformamide, tetrahydrofuran, triethylamine, methanol, ethanol, acetone, and toluene.
8. Use of a two-photon fluorescent probe for a gamma-glutamyl transpeptidase according to claim 1 for determining the content of gamma-glutamyl transpeptidase.
9. Use of a two-photon fluorescent probe of the gamma-glutamyl transpeptidase according to claim 1 for preparing a reagent for identifying cancer cells.
Technical Field
The invention relates to the technical field of fluorescence labeling. More particularly, relates to a two-photon fluorescent probe of gamma-glutamyl transpeptidase, a preparation method and application thereof.
Background
Gamma-glutamyl transpeptidase (GGT) is a cell surface-bound enzyme that selectively catalyzes the cleavage of the gamma-glutamine bond in Glutathione (GSH) and gamma-glutamyl compounds, and plays an important role in regulating cellular GSH and cysteine homeostasis. GGT is taken as glutamyl transpeptidase which is over-expressed in various malignant tumor cells such as lung cancer, cervical cancer, breast cancer, ovarian cancer and the like, plays an important biological function in the processes of proliferation, metastasis and metabolism of the tumor cells, and can be taken as an important cancer diagnosis biomarker and a treatment target. Therefore, by detecting GGT enzyme activity, it is of great significance to early diagnosis of tumors and prediction of the degree of metastasis of tumors.
Conventional methods for detecting GGT enzyme include colorimetric assay, High Performance Liquid Chromatography (HPLC), and electrochemistry. The colorimetric assay method has low detection accuracy, and HPLC assay has high accuracy, but is relatively time-consuming and high in detection cost; electrochemical detection has high sensitivity and speed for GGT detection, but the operation is relatively complex, and the methods cannot image GGT in living cells in real time. The fluorescence imaging method is considered to be an ideal method for GGT detection based on the advantages of high sensitivity, quick response, nondestructive detection, real-time space imaging and the like. Some single-photon fluorescent probes have been used for the detection of GGT activity so far, but practical application thereof in deep tissue imaging is limited due to problems of autofluorescence, photobleaching phenomenon and interference of shallow penetration depth, and damage of short-wavelength excitation light to cell tissue. Two-photon fluorescent probes can minimize fluorescent background, reduce photodamage, have better three-dimensional positioning and increased penetration depth. The two-photon technology has huge application potential and wide application prospect in the fields of future photoelectron integration, biomolecule detection, medical diagnosis and the like.
Therefore, the development of two-photon fluorescent probes for detecting GGT in living cells and deep tissues has important scientific and practical significance.
Disclosure of Invention
An object of the present invention is to provide a two-photon fluorescent probe for gamma-glutamyl transpeptidase, which has high sensitivity and selectivity for the detection of GGT in cells and blood, and low cytotoxicity and detection limit.
The second purpose of the invention is to provide a preparation method of the two-photon fluorescent probe of gamma-glutamyl transpeptidase.
The third purpose of the invention is to provide the application of the two-photon fluorescent probe of the gamma-glutamyl transpeptidase.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a two-photon fluorescent probe for γ -glutamyl transpeptidase, having a structure represented by general formula (IA) or (IB):
in the formulae (IA) and (IB), the substituent R1-R5independently-H, -F, -Cl, -Br, -I, cyano, nitro and sulfonic group; x is hydroxyl or glycine.
The compound (IA) and the compound (IB) provided by the invention are obtained by modifying a targeting group Glutathione (GSH) with GGT reaction specificity to a simple small molecule probe, and generate intramolecular cyclization cascade reaction under the action of gamma-glutamine transferase to form a six-membered ring compound:
the product molecule of the fluorescent probe reacted with GGT has strong electron withdrawing group and strong electron donating group, and the rigidity of the molecular conformation is increased by the ring structure, so that the fluorescent probe has two-photon characteristic.
In a second aspect, the present invention provides a method for synthesizing a two-photon fluorescent probe, comprising the following steps: dissolving the compound (IIA) and glutathione GSH in an organic solvent according to a certain proportion, stirring for reaction for 0.5 hour at room temperature, adding triethylamine solution, and continuously stirring for reaction at room temperature to obtain a mono-substituted product (IA) and a di-substituted product (IB);
wherein, the substituent R1-R5independently-H, -F, -Cl, -Br, -I, cyano, nitro and sulfonic group; x is hydroxyl or glycine.
In the preparation method provided by the invention, the compound (IA) and the compound (IB) are generated simultaneously, and different main products of the compound (IA) or the compound (IB) can be obtained by controlling the molar ratio of the raw material compound (IIA) to glutathione and the reaction time.
Optionally, the molar ratio of compound (IIA) to glutathione in the organic solvent is 1: 1-4.
Optionally, the concentration of compound (IIA) in the organic solvent is 1-3mM and the concentration of glutathione is 1-12 mM.
Alternatively, the concentration of the triethylamine solution is 1-12 mM. Addition of triethylamine solution can provide basic conditions.
Alternatively, the time for continuing the reaction with stirring at room temperature after adding the triethylamine solution is 1 to 8 hours.
Preferably, when the molar ratio of the compound (IIA) to glutathione in the organic solvent is 1:1-2, the triethylamine solution is added, and the reaction is continued for 1-4 hours at room temperature, the product is mainly the compound (IA).
Preferably, when the molar ratio of the compound (IIA) to glutathione in the organic solvent is 1:3-4, the triethylamine solution is added, and the reaction is continued for 5-8 hours at room temperature, the product is mainly the compound (IB).
Optionally, the organic solvent is selected from one or more of dimethyl sulfoxide, N-dimethylformamide, tetrahydrofuran, triethylamine, methanol, ethanol, acetone and toluene.
The two-photon fluorescent probe provided by the invention is obtained by modifying targeting group Glutathione (GSH) with GGT reaction specificity to a simple small molecule probe, and has the advantages of few synthesis steps, short reaction time, convenient purification and simple process.
The third aspect of the invention provides the application of the two-photon fluorescent probe.
Optionally, the application of the two-photon fluorescent probe of the gamma-glutamyl transpeptidase in determining the content of the gamma-glutamyl transpeptidase.
The two-photon fluorescent probe provided by the invention can selectively recognize gamma-glutamyl transpeptidase GGT in solution, has the advantages of high reaction speed and high sensitivity, and simultaneously has the advantages of two-photon detection, wherein the linear detection range of the concentration is 0-60U/L, and the detection limit is 0.115U/L.
Optionally, the application of the two-photon fluorescent probe of the gamma-glutamyl transpeptidase in preparing a reagent for identifying cancer cells.
The two-photon fluorescent probe detects the activity of gamma-glutamyl transpeptidase according to the change of fluorescence intensity in the solution, and can observe the change of the solution color before and after the change of the solution color (the solution color changes from colorless to yellow green) under visible light, and can distinguish cancer cells (GGT over-expressed cells) and normal cells by single-photon and two-photon fluorescence imaging respectively at the cell level. Meanwhile, the two-photon fluorescence probe has specificity on the selection of GGT, and other anions and cations, amino acids and enzymes in organisms have almost no influence on the luminous capacity of the compound.
The invention has the following beneficial effects:
the two-photon fluorescent probe of gamma-glutamyltranspeptidase in the invention is obtained by modifying a targeting group GSH which reacts specifically to gamma-glutamyltranspeptidase GGT onto a simple fluorescent molecule. The fluorescent probe can form a six-membered cyclic compound in the process of identifying GGT, the rigid structure of the six-membered cyclic compound enables the probe to have a two-photon effect, and when the six-membered cyclic compound acts with the over-expressed GGT in cancer cells, optical signals are obviously changed, so that the aim of distinguishing the cancer cells from normal cells through targeted detection is fulfilled. Meanwhile, the two-photon fluorescence has the advantages of good water solubility, high sensitivity, quick reaction, two-photon excitation and good application prospect.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
Fig. 1 shows (a) an ultraviolet absorption spectrum and (B) a fluorescence emission spectrum of the fluorescent probe (IA-1) after interaction with GGT in a PBS buffer solution (pH 7.4) in example 1.
Fig. 2 shows (a) an ultraviolet absorption spectrum and (B) a fluorescence emission spectrum of the fluorescent probe (IB-1) after interaction with GGT in a PBS buffer solution (pH 7.4) in example 6.
FIG. 3 shows the selective recognition of GGT by the fluorescent probe (IA-1) in example 1 (Pho (1mM), Apr (1mM), Glu (1mM), Try (1 mM); GGsTOP (200. mu.M) + GGT (300U/L), GGT (300U/L), Mg2+(2mM),Na+(20mM),K+(20mM),Zn2+(2mM),Al3+(2mM),Ca2+(2mM),Cu2+(2 mM) and NH4+(2mM))。
FIG. 4 shows the selective recognition of GGT by the fluorescent probe (IB-1) in example 6 (Pho (1mM), Apr (1mM), Glu (1mM), Try (1 mM); GGsTOP (200. mu.M) + GGT (300U/L), GGT (300U/L), Mg2+(2mM),Na+(20mM),K+(20mM),Zn2+(2mM),Al3+(2mM),Ca2+(2mM),Cu2+(2 mM) and NH4+(2mM))。
FIG. 5 shows the linear relationship between the fluorescence intensity of the fluorescent probe (IA-1) and the GGT concentration in example 1.
FIG. 6 shows the linear relationship between the fluorescence intensity of the fluorescent probe (IB-1) and the GGT concentration in example 6.
FIG. 7 shows a single photon cytographic image of the fluorescent probe (IA-1) in HUVEC, OVCAR3 and SKOV-3 cells in example 1.
FIG. 8 shows the image of a single photon cell with the fluorescent probe (IB-1) in HUVEC, OVCAR3 and SKOV-3 cells in example 6.
FIG. 9 shows the two-photon cytographic image of the fluorescent probe (IA-1) in OVCAR3(A) and HUVEC (B) cells in example 1.
FIG. 10 shows the two-photon cytographic image of the fluorescent probe (IB-1) in OVCAR3(A) and HUVEC (B) cells in example 6.
Detailed Description
In order to make the technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
Example 1: synthesis of two-photon fluorescent Probe (IA-1)
2 x 10 to-5mol of tetrafluoroterephthalonitrile and 3X 10-5mol L-glutathione is dissolved in 20mL DMF. Stirring at room temperature for 0.5h, then slowly adding 4X 10-5And adding a triethylamine solution into the reaction system by mol, and stirring at room temperature for 3 hours to ensure that the reaction solution turns yellow. Removing the solvent under vacuum through reverse phase silica gel C18Separating the product to obtain light yellow solid, i.e. compound (IA-1).1H NMR(D2O,400MHz):δ4.40(S,1H),3.92(m,3H), 3.62-3.58(m,1H),3.29-3.23(m,1H),2.60-2.57(m,2H),2.22-2.19(m,2H).ESI-MS: [M-H]-＝486.07.
Example 2: synthesis of two-photon fluorescent Probe (IA-2)
2 x 10 to-5mol tetrafluoro-p-phenylene dichloride and 3X 10-5mol L-glutathione is dissolved in 20mL DMF. Stirring at room temperature for 0.5h, then slowly adding 4X 10-5Adding mol triethylamine solution into the reaction system, stirring for 2.5 hours at room temperature, removing the solvent under vacuum after the reaction is stopped, and passing through reverse silica gel C18The product was isolated to give compound (IA-2). ESI-MS: [ M-H ]]-＝504.01.
Example 3: synthesis of two-photon fluorescent Probe (IA-3)
2 x 10 to-5mol tetrafluoro-p-dibromo and 3X 10-5mol L-glutathione is dissolved in 20mL DMF. Stirring at room temperature for 0.5h, then slowly adding 4X 10-5Adding the triethylamine solution into the reaction system by mol, stirring for 3.5 hours at room temperature, removing the solvent under the vacuum condition after the reaction is stopped, and passing through reverse silica gel C18The product was isolated to give compound (IA-3). ESI-MS: [ M-H ]]-＝591.91.
Example 4: synthesis of two-photon fluorescent Probe (IA-4)
2 x 10 to-5mol tetrafluoro-p-phenylene dinitro and 3X 10-5mol L-glutathione is dissolved in 20mL DMF. Stirring at room temperature for 0.5h, then slowly adding 4X 10-5Adding mol triethylamine solution into the reaction system, stirring for 2 hours at room temperature, removing the solvent under vacuum after the reaction is stopped, and passing through reverse silica gel C18The product was isolated to give compound (IA-4). ESI-MS: [ M-H ]]-＝526.06.
Example 5: synthesis of two-photon fluorescent Probe (IA-5)
2 x 10 to-5mol of tetrafluoro-p-phenylene dicyano group and 3X 10-5mol 5-L-glutamyl-L-cysteine was dissolved in 20mL DMF. Stirring at room temperature for 0.5h, then slowly adding 4X 10-5And adding a triethylamine solution into the reaction system by mol, and stirring at room temperature for 3 hours to ensure that the reaction solution turns yellow. Removing the solvent under vacuum through reverse phase silica gel C18The product was isolated to give compound (IA-5). ESI-MS: [ M-H ]]-＝429.06.
Example 6: synthesis of two-photon fluorescent Probe (IB-1)
Will be 3X 10-5mol of tetrafluoroterephthalonitrile and 9X 10-5mol L-glutathione was dissolved in 30mL DMF. Stirring at room temperature for 0.5h, then slowly adding 9X 10-5And adding a triethylamine solution into the reaction system by mol, stirring at room temperature for 6 hours, and changing the reaction solution from colorless to yellow. Removing the solvent under vacuum through reverse phase silica gel C18Separating the product to obtain light yellow solid (IB-1).1H NMR(D2O,400MHz):δ4.43(S,2H),3.96(m,6H), 3.75-3.64(m,2H),3.34-3.28(m,2H),2.64-2.52(m,4H),2.22-2.17(m,4H).ESI-MS: [M-H]-＝773.14.
Example 7: synthesis of two-photon fluorescent Probe (IB-2)
Will be 3X 10-5mol tetrafluoro-p-phenylene dichloride and 9X 10-5mol L-glutathione was dissolved in 30mL DMF. Stirring at room temperature for 0.5h, then slowly adding 9X 10-5Adding mol triethylamine solution into the reaction system, stirring for 7 hours at room temperature, removing the solvent under vacuum after the reaction is stopped, and passing through reverse silica gel C18The product was isolated to give compound (IB-2). ESI-MS: [ M-H ]]-＝791.09.
Example 8: synthesis of two-photon fluorescent Probe (IB-3)
Will be 3X 10-5mol tetrafluoro-p-dibromo and 9X 10-5mol L-glutathione was dissolved in 30mL DMF. Stirring at room temperature for 0.5h, then slowly adding 9X 10-5mol triethylamine solventAdding the solution into a reaction system, stirring at room temperature for 8 hours, removing the solvent under vacuum after the reaction is stopped, and passing through reverse silica gel C18The product was isolated to give compound (IB-3). ESI-MS: [ M-H ]]-＝878.99.
Example 9: synthesis of two-photon fluorescent Probe (IB-4)
Will be 3X 10-5mol tetrafluoro-p-phenylene dinitro and 9X 10-5mol L-glutathione was dissolved in 30mL DMF. Stirring at room temperature for 0.5h, then slowly adding 9X 10-5Adding the triethylamine solution into the reaction system by mol, stirring for 5 hours at room temperature, removing the solvent under vacuum condition, and passing through reverse silica gel C18The product was isolated to give compound (IB-4). ESI-MS: [ M-H ]]-＝813.13.
Example 10: synthesis of two-photon fluorescent Probe (IB-5)
Will be 3X 10-5mol of tetrafluoro-p-phenylene dicyano group and 9X 10-5mol 5-L-glutamyl-L-cysteine was dissolved in 30mL DMF. Stirring at room temperature for 0.5h, then slowly adding 9X 10-5And adding a triethylamine solution into the reaction system by mol, stirring at room temperature for 6 hours, and changing the reaction solution from colorless to yellow. Removing the solvent under vacuum through reverse phase silica gel C18The product was isolated to give compound (IB-5). ESI-MS: [ M-H ]]-＝659.11.
Test example 1
The change of the absorption spectrum and the fluorescence spectrum of the fluorescent probe molecules (IA-1) and (IB-1) after the interaction with GGT respectively.
The obtained probe was dissolved in DMSO to prepare a solution of 1X 10-2Stock solution of M, diluted to 100. mu.M with PBS. Weighing gamma-glutamyl transpeptidase, dissolving in PBS solution, and preparing 300U/L stock solution. And (3) taking a probe with a concentration of more than 2mL and GGT by using a cuvette, and testing ultraviolet absorption change and fluorescence intensity change under a constant temperature condition, wherein the excitation wavelength is 405 nm. See fig. 1 and 2 for results.
As can be seen from FIG. 1, in the presence of GGT, the absorption band of the fluorescent probe molecule (IA-1) at 340nm gradually decreases, while a new absorption peak at 405nm appears and gradually increases with time. In the fluorescence spectrum, the fluorescence intensity of the fluorescent probe molecule (IA-1) is very weak, and the fluorescence intensity is obviously enhanced after the GGT is added. The same phenomenon can be observed in FIG. 2, from which it can be seen that the fluorescent probe molecules (IA-1) and (IB-1) have a high sensitivity to GGT recognition.
Test example 2
Probe molecules (IA-1) and (IB-1) were added to GGT solution, GGsTOP solution (inhibitor of GGT), aqueous solutions of various metal ions, and aqueous solutions containing various enzymes, respectively, to examine the stability and selection specificity of the probes.
From fig. 3 and 4, it can be concluded that the solution fluorescence of the probe incubated with GGT is significantly enhanced, but in the presence of ggsotop the fluorescence is completely suppressed, indicating that the fluorescence enhancement of the probe is indeed caused by GGT specificity. The probe molecules have no obvious fluorescence enhancement in aqueous solutions of various metal ions and enzymes, which shows that the probe molecules (IA-1) and (IB-1) have good selectivity and stability.
Test example 3
The linear detection range and sensitivity of the detection probe to the GGT are detected by testing the fluorescence intensity after the reaction with the fluorescent probe molecules (IA-1) and (IB-1) at different GGT concentrations.
As can be seen from FIGS. 5 and 6, the linear detection range of GGT by the probe molecule (IA-1) was 1-60U/L, and the probe molecule (IB-1)The linear detection range of GGT is 1-50U/L; limit of detection passThe detection limit of the probe molecule (IA-1) to GGT is 0.115U/L and the detection limit of the probe molecule (IB-1) to GGT is 0.223U/L. Where σ is the standard deviation of blank detection and k is the slope of the fitted line.
Test example 4
The fluorescent probe molecules (IA-1) and (IB-1) realize the recognition and detection of GGT in tumor cells through single photon cell imaging.
Taking normal HUVEC cells without GGT expression as a control group, respectively incubating fluorescent probe molecules (IA-1) and (IB-1) with the HUVEC cells for 1h, and collecting cell images of green channels at an excitation wavelength of 405 nm. And then using human ovarian cancer cells OVCAR3 and SKOV-3 cells with GGT over-expression as experimental groups, incubating the fluorescent probe molecules and the two cells for 1h, and collecting cell images of a green channel under the excitation wavelength of 405 nm.
From FIGS. 7 and 8, it can be seen that the green signal of the control cells was very weak, because the cells did not have GGT and could not react with the probe molecules to generate fluorescence. The imaging of the cells in the experimental group, which observed bright fluorescence in the green channel, indicates that the probe molecule (IA-1) and the probe (IB-1) have the ability to distinguish between normal cells and tumor cells overexpressing GGT.
Test example 5
The fluorescent probe molecules (IA-1) and (IB-1) realize the recognition and detection of GGT in tumor cells through two-photon cell imaging.
Taking HUVEC cells as a control group, respectively incubating fluorescent probe molecules (IA-1) and (IB-1) with the HUVEC cells for 1h, and carrying out cell imaging by using a two-photon laser scanning confocal microscope at an excitation wavelength of 800nm to collect cell imaging of blue, green and red channels. And then using human ovarian cancer cells OVCAR3 and SKOV-3 cells with GGT over-expression as experimental groups, incubating the fluorescent probe molecules and the two cells for 1h, and collecting cell images of blue, green and red channels at the excitation wavelength of 800 nm.
From fig. 9 and 10, it was observed that cell imaging of the experimental group of ovarian cancer cells OVCAR3 allowed for the observation of fluorescent signals in three channels, particularly bright fluorescence in the red channel, as shown in fig. a. In contrast, no fluorescence signal was observed in HUVEC cells, as shown in panel B. In conclusion, the confocal imaging experiments show that the probe molecules (IA-1) and (IB-1) can effectively distinguish normal cells from cancer cells through single-photon fluorescence imaging and two-photon fluorescence imaging.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.