CN113637048B - Two-photon fluorescent probe of gamma-glutamyl transpeptidase and preparation method and application thereof - Google Patents
Two-photon fluorescent probe of gamma-glutamyl transpeptidase and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a two-photon fluorescent probe of gamma-glutamyl transpeptidase, which is characterized by having a structure shown in a general formula (IA) or (IB), and being obtained by modifying a targeting group GSH which specifically reacts with gamma-glutamyl transpeptidase GGT onto a simple fluorescent molecule. In the process of identifying GGT, the fluorescent probe can form a six-membered ring compound, and the rigid structure of the six-membered ring compound enables the probe to have a two-photon effect, and when the fluorescent probe acts with the GGT which is over-expressed in cancer cells, the optical signal is obviously changed, so that the purpose of distinguishing the cancer cells from normal cells through targeted detection is achieved. Meanwhile, the two-photon fluorescence has the advantages of good water solubility, high sensitivity, quick response and two-photon excitation, and has a good application prospect.
Description
Technical Field
The invention relates to the technical field of fluorescent marking. More particularly, relates to a two-photon fluorescent probe of gamma-glutamyl transpeptidase and a preparation method and application thereof.
Background
Gamma-glutamyl transpeptidase (GGT) is a cell surface binding enzyme that selectively catalyzes the cleavage of gamma-glutamine bonds in Glutathione (GSH) and gamma-glutamyl compounds, playing an important role in regulating cellular GSH and cysteine homeostasis. GGT is used as a 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 proliferation, metastasis and metabolism processes of tumor cells, and can be used as an important cancer diagnosis biomarker and a treatment target. Therefore, by detecting GGT enzyme activity, it is of great importance to early diagnosis of tumor and prediction of the degree of metastasis of tumor.
Traditional methods for detecting GGT enzyme include colorimetric assays, high Performance Liquid Chromatography (HPLC), electrochemistry and the like. Among them, the colorimetric assay has low detection accuracy, while HPLC assay has higher accuracy, it is relatively time-consuming and the detection cost is high; electrochemical detection has high sensitivity and speed for GGT detection, but the operation is relatively complex, and none of these methods can image GGT in living cells in real time. The fluorescence imaging method is considered as an ideal method for GGT detection based on the advantages of high sensitivity, quick response, nondestructive detection, real-time space imaging and the like. To date, some single photon fluorescent probes have been used for detecting GGT activity, but practical application in deep tissue imaging is limited due to problems such as autofluorescence, photobleaching phenomenon, interference of shallow penetration depth, damage of short wavelength excitation light to cell tissues, and the like. The two-photon fluorescent probe can minimize the fluorescent background, reduce the photodamage, and has better three-dimensional space positioning and increased penetration depth. The two-photon technology has great application potential and wide application prospect in the fields of future photoelectron integration, biomolecular 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
The invention aims to provide a two-photon fluorescent probe of gamma-glutamyl transpeptidase, which has high sensitivity and selectivity, low cytotoxicity and detection limit for detecting GGT in cells and blood.
The second object of the present invention is to provide a method for preparing a two-photon fluorescent probe of gamma-glutamyl transpeptidase.
The third object of the present invention is to provide an application of a two-photon fluorescent probe of gamma-glutamyl transpeptidase.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a two-photon fluorescent probe of gamma-glutamyl transpeptidase having a structure represented by general formula (IA) or (IB):
in the formulae (IA) and (IB), the substituent R 1 -R 5 Each independently is-H, -F, -Cl, -Br, -I, cyano, nitro, sulfonic acid group; x is hydroxy or glycinyl.
The compound (IA) and the compound (IB) are obtained by modifying a GGT reaction specific targeting group Glutathione (GSH) to a simple small molecular probe, and under the action of gamma-transglutaminase, intramolecular cyclization cascade reaction is carried out to form a six-membered ring compound:
the fluorescent probe provided by the invention has a strong electron withdrawing group and a strong electron donating group on the product molecule after the reaction of the fluorescent probe and GGT, and the annular structure increases the rigidity of the molecular conformation of the fluorescent probe, so that the fluorescent probe has two-photon characteristics.
In a second aspect, the invention provides a method for synthesizing a two-photon fluorescent probe, comprising the following steps: dissolving a compound (IIA) and glutathione GSH in an organic solvent according to a certain proportion, stirring at room temperature for reaction for 0.5 hour, adding a triethylamine solution, and continuing stirring at room temperature for reaction to obtain a monosubstituted product (IA) and a disubstituted product (IB);
wherein the substituents R 1 -R 5 Each independently is-H, -F, -Cl, -Br, -I, cyano, nitro, sulfonic acid group; x is hydroxy or glycinyl.
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 the compound (IIA) to glutathione in the organic solvent is 1:1-4.
Optionally, the concentration of the compound (IIA) in the organic solvent is 1-3mM, and the concentration of glutathione is 1-12mM.
Alternatively, the concentration of the triethylamine solution is 1-12mM. The addition of triethylamine solution can provide basic conditions.
Alternatively, after adding the triethylamine solution, the reaction was continued with stirring at room temperature for 1 to 8 hours.
Preferably, the molar ratio of the compound (IIA) to the glutathione in the organic solvent is 1:1-2, and after the triethylamine solution is added, the reaction is continued for 1-4 hours at room temperature, and the product is mainly the compound (IA).
Preferably, the molar ratio of the compound (IIA) to the glutathione in the organic solvent is 1:3-4, and after the triethylamine solution is added, the reaction is continued for 5-8 hours at room temperature, and 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 the specific targeting group Glutathione (GSH) of GGT reaction to a simple small molecular 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 use of the two-photon fluorescent probe.
Optionally, the two-photon fluorescent probe of the gamma-glutamyl transpeptidase is used for measuring the content of the gamma-glutamyl transpeptidase.
The two-photon fluorescent probe provided by the invention can selectively identify gamma-glutamyl transpeptidase GGT in a solution, has the advantages of high reaction speed and high sensitivity, and also 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 two-photon fluorescent probe of the gamma-glutamyl transpeptidase is applied to the preparation of a reagent for identifying cancer cells.
The two-photon fluorescent probe detects the activity of gamma-glutamyl transpeptidase by the change of fluorescence intensity in a solution, can observe the change of the color of the solution before and after the change of the color of the solution from colorless to yellow-green under visible light, and can distinguish cancer cells (GGT over-expressed cells) and normal cells on the cell level through single-photon and two-photon fluorescent imaging respectively. Meanwhile, the two-photon fluorescent probe has specificity on GGT selection, and other anions and cations, amino acids and in-vivo enzymes hardly affect the luminous capacity of the compound.
The beneficial effects of the invention are as follows:
the two-photon fluorescent probe of gamma-glutamyl transpeptidase is obtained by modifying a targeting group GSH which specifically reacts with gamma-glutamyl transpeptidase GGT onto a simple fluorescent molecule. In the process of identifying GGT, the fluorescent probe can form a six-membered ring compound, and the rigid structure of the six-membered ring compound enables the probe to have a two-photon effect, and when the fluorescent probe acts with the GGT which is over-expressed in cancer cells, the optical signal is obviously changed, so that the purpose of distinguishing the cancer cells from normal cells through targeted detection is achieved. Meanwhile, the two-photon fluorescence has the advantages of good water solubility, high sensitivity, quick response and two-photon excitation, and has a good application prospect.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows (A) ultraviolet absorbance spectra and (B) fluorescence emission spectra of fluorescent probe (IA-1) of example 1 after interaction with GGT in PBS buffer (pH=7.4).
FIG. 2 shows (A) ultraviolet absorbance spectra and (B) fluorescence emission spectra of fluorescent probe (IB-1) of example 6 after the fluorescent probe was reacted with GGT in PBS buffer (pH=7.4).
FIG. 3 shows the selective recognition of GGT by the fluorescent probe (IA-1) in example 1 (Pho (1 mM), apr (1 mM), glu (1 mM), try (1 mM); GGsTOP (200. Mu.M) +GGT (300U/L), GGT (300U/L), mg 2+ (2mM),Na + (20mM),K + (20mM),Zn 2+ (2mM),Al 3+ (2mM),Ca 2+ (2mM),Cu 2+ (2 mM) and NH 4+ (2mM))。
FIG. 4 shows the selective recognition of GGT by the fluorescent probe (IB-1) in example 6 (Pho (1 mM), apr (1 mM), glu (1 mM), try (1 mM); GGsTOP (200. Mu.M) +GGT (300U/L), GGT (300U/L), mg 2+ (2mM),Na + (20mM),K + (20mM),Zn 2+ (2mM),Al 3+ (2mM),Ca 2+ (2mM),Cu 2+ (2 mM) and NH 4+ (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 cell imaging of fluorescent probe (IA-1) of example 1 in HUVEC, OVCAR3 and SKOV-3 cells.
FIG. 8 shows a single photon cell imaging of fluorescent probe (IB-1) in HUVEC, OVCAR3 and SKOV-3 cells of example 6.
FIG. 9 shows a two-photon cell imaging of fluorescent probe (IA-1) in OVCAR3 (A) and HUVEC (B) cells in example 1.
FIG. 10 shows a two-photon cell imaging of fluorescent probe (IB-1) in OVCAR3 (A) and HUVEC (B) cells in example 6.
Detailed Description
In order to make the technical scheme and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail with reference to the accompanying drawings.
Example 1: synthesis of two-photon fluorescent Probe (IA-1)
Will be 2X 10 -5 mol of tetrafluoro terephthalonitrile and 3X 10 -5 mol L-glutathione was dissolved in 20mL DMF. Stirred at room temperature for 0.5h, then slowly added 4X 10 -5 The triethylamine solution was stirred at room temperature for 3 hours, and the reaction solution turned yellow. Solvent was removed under vacuum by reverse phase silica gel C 18 Separating the product to obtain a pale yellow solid, namely the compound (IA-1). 1 H NMR(D 2 O,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)
Will be 2X 10 -5 mol of tetrafluoroterephthalyl dichloride and 3X 10 -5 mol L-glutathione was dissolved in 20mL DMF. Stirred at room temperature for 0.5h, then slowly added 4X 10 -5 Solution of triethylamine in mol to reactionIn the reaction system, stirring was carried out at room temperature for 2.5 hours, and after the reaction was stopped, the solvent was removed under vacuum, and the reaction was carried out by reversing the silica gel C 18 The product was isolated to give compound (IA-2). ESI-MS: [ M-H ]] - ＝504.01.
Example 3: synthesis of two-photon fluorescent Probe (IA-3)
Will be 2X 10 -5 mol of tetrafluoro-p-benzene dibromo and 3X 10 -5 mol L-glutathione was dissolved in 20mL DMF. Stirred at room temperature for 0.5h, then slowly added 4X 10 -5 Adding triethylamine solution into the reaction system, stirring at room temperature for 3.5 hr, removing solvent under vacuum condition after stopping reaction, and passing through reverse silica gel C 18 The product was isolated to give compound (IA-3). ESI-MS: [ M-H ]] - ＝591.91.
Example 4: synthesis of two-photon fluorescent Probe (IA-4)
Will be 2X 10 -5 mol of tetrafluoro-p-benzene dinitro and 3X 10 -5 mol L-glutathione was dissolved in 20mL DMF. Stirred at room temperature for 0.5h, then slowly added 4X 10 -5 Adding triethylamine solution into the reaction system, stirring at room temperature for 2 hr, removing solvent under vacuum condition after stopping the reaction, and passing through reverse silica gel C 18 The product was isolated to give compound (IA-4). ESI-MS: [ M-H ]] - ＝526.06.
Example 5: synthesis of two-photon fluorescent Probe (IA-5)
Will be 2X 10 -5 mol of tetrafluoroterephthalonitrile and 3X 10 -5 The mol 5-L-glutamyl-L-cysteine was dissolved in 20 mM LDMF. Stirred at room temperature for 0.5h, then slowly added 4X 10 -5 The triethylamine solution was stirred at room temperature for 3 hours, and the reaction solution turned yellow. Solvent was removed under vacuum by reverse phase silica gel C 18 The 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 -5 mol of tetrafluoro terephthalonitrile and 9X 10 -5 mol L-glutathione was dissolved in 30mL DMF. Stirred at room temperature for 0.5h, then slowly added to 9X 10 -5 The triethylamine solution was stirred at room temperature for 6 hours, and the reaction solution was changed from colorless to yellow. Solvent was removed under vacuum by reverse phase silica gel C 18 The product was isolated as a pale yellow solid, which was the compound (IB-1). 1 H NMR(D 2 O,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 -5 mol of tetrafluoroterephthalyl dichloride and 9X 10 -5 mol L-glutathione was dissolved in 30mL DMF. Stirred at room temperature for 0.5h, then slowly added to 9X 10 -5 Adding triethylamine solution into the reaction system, stirring at room temperature for 7 hours, removing solvent under vacuum condition after stopping the reaction, and passing through reverse silica gel C 18 The 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 -5 mol of tetrafluoro-p-benzene dibromo and 9X 10 -5 mol L-glutathione was dissolved in 30mL DMF. Stirred at room temperature for 0.5h, then slowly added to 9X 10 -5 Adding triethylamine solution into the reaction system, stirring at room temperature for 8 hr, removing solvent under vacuum condition after stopping reaction, and passing through reverse silica gel C 18 The 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 -5 mol of tetrafluoro-p-benzene dinitro and 9X 10 -5 mol L-glutathione was dissolved in 30mL DMF. Stirred at room temperature for 0.5h, then slowly added to 9X 10 -5 The solution of triethylamine in the reaction system was stirred at room temperature for 5 hours, the solvent was removed under vacuum, and the reaction mixture was passed through reverse silica gel C 18 The 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 -5 mol of tetrafluoroterephthalonitrile and 9X 10 -5 mol 5-L-glutamyl-L-cysteine was dissolved in 30mL DMF. Stirred at room temperature for 0.5h, then slowly added to 9X 10 -5 The triethylamine solution was stirred at room temperature for 6 hours, and the reaction solution was changed from colorless to yellow. Solvent was removed under vacuum by reverse phase silica gel C 18 The product was isolated to give compound (IB-5). ESI-MS: [ M-H ]] - ＝659.11.
Test example 1
Changes in absorption spectra and fluorescence spectra of fluorescent probe molecules (IA-1) and (IB-1) after interaction with GGT, respectively.
The obtained probe was dissolved in DMSO to prepare 1X 10 probe -2 Stock solution of M was diluted to 100. Mu.M with PBS. The gamma-glutamyl transpeptidase was weighed and dissolved in PBS to prepare a 300U/L stock solution. And (3) taking more than 2mL of probe and GGT by using a cuvette, and testing ultraviolet absorption change and fluorescence intensity change under the constant temperature condition, wherein the excitation wavelength is 405nm. The results are shown in figures 1 and 2.
From FIG. 1, it can be seen that the absorption band of the fluorescent probe molecule (IA-1) at 340nm gradually decreases in the presence of GGT, while a new absorption peak appears at 405nm and gradually increases with time. In the fluorescence spectrum, the fluorescence intensity of the fluorescent probe molecule (IA-1) itself is very weak, and after GGT is added, the fluorescence intensity is remarkably enhanced. The same phenomenon can be observed in FIG. 2, from which it can be seen that fluorescent probe molecules (IA-1) and (IB-1) have a very high sensitivity to GGT recognition.
Test example 2
Probe molecules (IA-1) and (IB-1) were added to GGT solution, ggstp solution (inhibitor of GGT), various metal ion aqueous solutions, and various enzyme-containing aqueous solutions, respectively, to examine stability and selection specificity of probes.
As can be seen from fig. 3 and 4, the fluorescence of the solution of the probe incubated with GGT was significantly enhanced, but in the presence of ggsotp, the fluorescence was completely inhibited, indicating that the enhancement of the fluorescence of the probe was indeed caused by GGT specificity. The probe molecules have no obvious fluorescence enhancement in aqueous solutions of various metal ions and enzymes, which indicates 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 probe pair GGT were tested by measuring the fluorescence intensity after reaction with 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 the probe molecule (IA-1) to GGT is 1-60U/L, and the linear detection range of the probe molecule (IB-1) to GGT is 1-50U/L; pass of detection limitThe 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 the blank detection and k is the slope of the fitted line.
Test example 4
Identification detection of GGT in tumor cells by fluorescent probe molecules (IA-1) and (IB-1) is realized through single photon cell imaging.
Fluorescent probe molecules (IA-1) and (IB-1) were incubated with HUVEC cells, which were normal cells that did not express GGT, for 1h, respectively, and cell imaging was collected in the green channel at 405nm excitation wavelength. And then taking human ovarian cancer cells OVCAR3 and SKOV-3 cells over-expressed by GGT as experimental groups, incubating fluorescent probe molecules with the two cells for 1h, and collecting cell imaging of a green channel under the excitation wavelength of 405nm.
From FIGS. 7 and 8, the control cells imaged very weakly green signals, because there was no GGT present in the cells and they were not able to react with the probe molecules to generate fluorescence. Bright fluorescence can be observed in the green channel by cell imaging of the experimental group, and the experimental results show that the probe molecule (IA-1) and the probe (IB-1) have the capability of distinguishing normal cells from tumor cells over-expressing GGT.
Test example 5
The identification detection of GGT in tumor cells by fluorescent probe molecules (IA-1) and (IB-1) is realized 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, performing cell imaging by a two-photon laser scanning confocal microscope, and collecting cell imaging of blue, green and red channels at an excitation wavelength of 800 nm. And then taking human ovarian cancer cells OVCAR3 and SKOV-3 with over-expressed GGT as experimental groups, incubating fluorescent probe molecules with the two cells for 1h, and collecting blue, green and red channel cell imaging at an excitation wavelength of 800 nm.
From fig. 9 and 10, cell imaging of the ovarian cancer cells OVCAR3 of the experimental group can observe fluorescent signals in three channels, especially bright fluorescence in the red channel, as shown in fig. a. In contrast, no fluorescent signal was observed in HUVEC cells, as shown in panel B. In summary, these confocal imaging experiments demonstrate that probe molecules (IA-1) and (IB-1) can effectively distinguish normal cells from cancer cells by single-photon and two-photon fluorescence imaging.
It should be understood that the foregoing examples of the present invention are provided merely for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention, and that various other changes and modifications may be made therein by one skilled in the art without departing from the spirit and scope of the present invention as defined by the appended claims.
Claims (2)
1. An application of a two-photon fluorescent probe of gamma-glutamyl transpeptidase in determining the content of gamma-glutamyl transpeptidase, wherein the application is for the purpose of non-disease diagnosis; the two-photon fluorescent probe is characterized by having a structure shown in a general formula (IA) or (IB):
in the formulae (IA) and (IB), the substituent R 2 、R 4 Is cyano, R 1 、R 3 、R 5 Each independently is-F, -Cl, -Br or-I; x is hydroxy or glycinyl.
2. Application of a two-photon fluorescent probe of gamma-glutamyl transpeptidase in preparing a reagent for identifying cancer cells; the cancer cell is lung cancer cell, cervical cancer cell, breast cancer cell or ovarian cancer cell, and is characterized in that the two-photon fluorescent probe has a structure shown in a general formula (IA) or (IB):
in the formulae (IA) and (IB), the substituent R 2 、R 4 Is cyano, R 1 、R 3 、R 5 Each independently is-F, -Cl, -Br or-I; x is hydroxy or glycinyl.
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